anti-inflammatory actions of green tea catechins and ligands of peroxisome proliferator-activated...

British Society for Matrix Biology Meeting, Manchester,5–6 April 2004

Grappling with the glycome

Gavin C. Jones, Nicholas T. Seyfried and Edward

R. Bastow

The spring 2004 meeting of the BSMB was held at Hulme

Hall, University of Manchester on 5–6 April. The theme of

the meeting was recent advances in our understanding of

the structure and biological function of saccharides, with a

particular emphasis on glycosaminoglycans. The meeting was

organized by Dr Malcolm Lyon (University of Manchester)

and Dr Robert Lauder (University of Lancaster) and was

supported financially by Europa Bioproducts, Sigma-Aldrich,

Dionex and Calbiochem, who all exhibited their products.

There were 11 invited speakers: one from the USA, three from

continental Europe (Sweden, France and the Netherlands)

and seven from the UK. The meeting attracted 100 registered

delegates evenly split between 50 BSMB members (including

10 students) and 50 nonmembers (including 14 students). A

further nine short presentations were selected from the sub-

mitted poster abstracts.

Robert Haltiwanger (State University of New York at Stony

Brook, New York, USA) opened the meeting by introducing

the glycocalyx at the cell surface and its structural complexity.

He highlighted the variety of cell-surface glycans that modu-

late cellular communications, discussing their importance in

signal transduction events, before concentrating on the specific

role of O-fucose modifications in modulating the Notch sig-

nalling pathway. The extracellular domain of Notch contains

36-tandem epidermal growth factor (EGF)-like repeats many

of which are sites for both O-fucose and O-glucose type

glycosylation. Comparing the phenotypic similarities between

knockouts of O-fucosyltransferase-1 and Notch in Drosophila

and mice, he emphasized the importance of O-fucose modi-

fications in Notch function. He further developed this theme by

considering Fringe, a b1,3-N-acetylyglucosaminyltransferase,

that is involved in the elongation of the O-fucose monosac-

charide in some EGF-like repeats and that can further modu-

late Notch signalling. The sites of Fringe modification may be

encoded within the sequences of specific EGF-like repeats.

There are suggestions that these enzymatic modifications

may influence Notch–ligand interactions. Before concluding,

he identified O-glucose modification as an additional, highly

conserved modification, clustered at the central EGF-like

repeats of Notch that may additionally prove to influence

Notch biology.

Paul Crocker (University of Dundee, Dundee, UK) gave an

overview of the siglec family of transmembrane proteins,

whose members include sialoadhesin, CD22 and CD33, defin-

ing them as adhesive and signalling molecules of the immune

system that recognize sialylated proteins. He emphasized the

importance of siglec extracellular domain size in regulating

trans vs. cis interactions with cell surface ligands, using as an

illustration that observed unmasking of siglec binding sites

towards external ligands, following a sialidase treatment to

destroy cis interactions at the cell surface. He then focused on

siglec 7, commenting on its unmasked activity when expressed

on CHO cells as compared to its masked activity on natural

killer (NK) cells, where it is naturally expressed. He intro-

duced the preference of siglec 7 for masked activity on NK

cells, where it is naturally expressed. He introduced the

preference of siglec 7 for cells as compared to its sialidase

treatment to destroy signalling molecules of the immune sys-

tem that recognize sialylated proteg particular to its specificity,

the possible functions of siglec 7 were then considered. The

high levels of expression of a2–8-linked disialic acid on NK

cells, the masking of siglec-7 in NK cells by cis interactions and

the apparent rapid evolution of the sialic acid-binding

domains within the siglec family suggests that siglec-7 has

adapted to engage in cis interactions that regulate NK cell

activation.

Anne Imberty (CERMAV-CNRS, Grenoble, France)

described how PA-IL and PA-IIL, the galactose- and fucose-

binding lectins of Pseudomonas aeruginosa are associated

with the virulence of this bacterium. P. aeruginosa colonizes

the lungs of patients with cystic fibrosis (CF), where it

becomes a serious pathogen. Its specific binding is dependent

upon the increased fucosylation and high levels of Lewis a

epitopes present in CF patients. The crystal structures of

PA-IL and PA-IIL bound to their respective monosaccharide

ligands were shown. She drew particular attention to two co-

ordinated calcium ions that appear to be intimately involved in

lectin-ligand recognition. Modelling studies were used to look

at the potential binding of PA-IIL to more complex oligosac-

charide structures and these, together with the results of

enzyme-linked lectin binding assays, suggested that the Lewis

Int. J. Exp. Path. (2004), 85, A45–A77

� 2004 Blackwell Publishing Ltd A45

a epitope could be the ligand recognized by these lectins in

the lungs of CF patients.

Anne Dell (Imperial College, London, UK) gave an overview

of mass spectrometric strategies for both high throughput

glycomics and glyco-proteomics. She described the application

of both MALDI-TOF MS and ES MS/MS not only for identi-

fying carbohydrates from single proteins but also from very

complex mixtures such as cell and tissue extracts. She then

introduced a number of specific applications in which these

approaches had proven valuable and instructive. Protocols for

comparing resting and activated lymphocytes showed differ-

ences in glycosylation patterns of murine T and B cells upon

activation. Changes in the O-glycan patterns of activated

CD8+ T cells during development were also addressed. In

addition, MS provided a rapid glycan screening technique to

assess changes in both O- and N-linked glycosylation in

the organs of knockout mice and also for distinguishing core

1 and core 2 antennae O-glycans on the CA125 ovarian cancer

antigen. MS techniques have also provided valuable data on

sperm–egg interactions. Sequencing and site analysis of the

O-glycans on the murine and human forms of the zona

pellucida glycoprotein (ZP3), the putative sperm receptor

revealed identical glycosylation patterns when expressed on

mouse eggs. Interestingly, human ZP3 in transgenic mice inter-

acted with murine but not human sperm. Therefore, it appears

that host specific O-glycosylation is essential for sperm–egg

recognition.

Ten Feizi (Imperial College, London, UK) described the use

of carbohydrate microarrays as a potential high throughput

technology to assess the specific carbohydrate-binding proper-

ties of proteins. Particular emphasis was given to protein–

carbohydrate interactions in the immune system. To prepare

microarrays, carbohydrates, either synthesized or released

from proteins, cells or tissues, can be conjugated to lipids by

reductive amination forming novel, lipid-linked oligosac-

charides or neoglycolipids (NGLs). Such NGLs can easily be

immobilized onto nitrocellulose membranes, via their lipid

tails, where they can be probed for interaction with a soluble

protein. Repertoires of NGLs, either homogeneous species or

heterogeneous mixtures, can be assembled as microarrays to

allow the selection of specific ligands by carbohydrate-binding

proteins. Subsequent thin layer chromatography and mass

spectrometry can be used to characterize positive-binding

species. Specific examples used to illustrate the use of such

approaches included the interactions of the cytokine INF-aand the chemokine RANTES with sulfated carbohydrate and

chondroitin sulfate oligosaccharide microarrays.

Lan Jin (University of Edinburgh, Edinburgh, UK) discussed

the novel elucidation of the conformation of heparin oligosac-

charides by ion mobility mass spectrometry. In this gas-phase

technique, ions are impelled to travel, under the influence of a

weak electric field, through a very low density of inert gas.

Under these conditions, the time of arrival of ions at the

detector depends upon their collision frequency, which is

related to their molecular cross sections. A heparin disacchar-

ide and three different tetrasaccharides were prepared and

studied. Theoretical gas-phase conformations were generated

by molecular modelling and compared to experimental cross

sectional areas derived by ion mobility MS. A close correlation

was obtained with these small structures, and the method

clearly holds promise for probing GAG conformation.

Tim Rudd (University of Liverpool, Liverpool, UK) intro-

duced quartz crystal microbalance-dissipation (QCM-D) and

its use in the study of protein/GAG interactions. By analysing

the damping of oscillation of the crystal, after adsorption of

molecules onto its surface, QCM-D can be used to gain new

insights into complex formation. QCM-D measures both the

mass of the molecules adsorbed to a surface, in addition to the

energy dissipated by the surface. Experimentally, heparin

oligosaccharides were immobilized onto a gold-sputtered

QCM-D surface and then exposed to various protein ligands,

e.g. FGF-1, FGFR1, HGF/SF. The viscoelastic properties of the

resulting complexes could be determined from the measured

dissipation, which yields information on their shape/conform-

ation. For example, it was determined that complexes of a

heparin octasaccharide with FGF-1 were more dissipative than

complexes with HGF/SF. It was suggested that the oligosac-

charide may have flexible hinge regions and that differential

binding of specific growth factors above, or at, the hinge could

radically alter the flexibility and thus dissipation of the com-

plex. Interestingly, GAG complexes with FGFR1 were less

dissipative (more rigid) than those with FGF-1.

Joyce Taylor-Papadimitriou (Guy’s Hospital, London, UK)

introduced MUC1, the major epithelial mucin expressed by

breast carcinomas. Its extracellular domain has a tandem

repeat structure bristling with O-glycans. Joyce has shown

convincingly that MUC1 exhibits changes in O-glycoyslation

in breast cancer. Increased levels of ST3 Gal 1 enzyme in

breast cancer induces a switch from the addition of mainly

core 2-based O-glycans to Sialyl T O-glycans. Although Sialyl

T O-glycans can exist in normal cells outside the breast, in the

breast they are cancer associated, and their presence increases

mammary cancer growth and can be immunosuppressive. In

up to 30% of breast cancers, O-glycosylation is terminated

early, yielding GalNAc (Tn) or Sialyl Tn O-glycans on MUC1.

Sialyl Tn glycans, coinciding with increases in the ST6GalNac

I enzyme, are highly tumour-specific. Joyce concluded with a

very interesting discussion of how this could be exploited for

immunotherapeutic intervention in breast cancer. An immun-

ization protocol using MUC1-bearing Sialyl Tn O-glycans as

A46 British Society for Matrix Biology Meeting

� 2004 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 85, A45–A77

immunogen has been used in mice with some success to pro-

vide protection against tumour growth. This has encouraged a

presently running clinical trial of a similar strategy in breast

cancer patients.

Tony Day (University of Oxford, Oxford, UK) gave a com-

prehensive introduction to the massive GAG, hyaluronan

(HA). Though it is the simplest GAG in sequence, it never-

theless supports a wide range of diverse biological activities.

Tony suggested that a key to the behaviour of HA is that it can

adopt a variety of different conformational states in solution

with potentially fast interchanges between them. He then

summarized the many HA-binding proteins (hyaladherins)

and suggested that the hyaladherin may determine the par-

ticular HA conformation stabilized. He went on to discuss

results indicating that changes in the architecture of HA-

protein complexes may contribute to the variety of biological

functions that are attributed to HA. Particular attention was

given to TSG-6 and CD44. Molecular modelling studies indi-

cated significant differences in their HA-binding domains and

suggested that much larger perturbations in HA structure

ensue following binding to CD44 than to TSG-6. Also the

binding of TSG-6 to HA enhances the ability of HA to then

bind CD44, suggesting TSG-6 induces favourable alterations

in HA organization. Lastly, Tony discussed the role of TSG-6

in the covalent modification of HA by attachment of ‘heavy

chains’ derived from the intera-inhibitor. This cross-linking

can provide an alternative way of rearranging HA architecture

and appears to be important in cumulus cell expansion in

ovulation and fertilization.

Mark Ritchie (Waters MS Technology Centre, Manchester,

UK) gave a comprehensive overview of the practise of mass

spectrometry, before concentrating on its specific application

to the analysis of N-glycosylation in the large, gel-forming,

salivary mucin MUC5B. Though O-glycosylation dominates

in such mucins, there are abundant potential sites for

N-glycosylation, though little is known about its structure. He

then described how, experimentally, the mucin can be trypsin-

ized and resulting N-glycosylated peptides can be identified

by capillary LC Q-TOF MS. Analysis of candidate ions by MS/

MS revealed that there is N-linked occupancy of numerous

sites as well as microheterogeneity of structure.

Ulf Lindahl (Uppsala University, Uppsala, Sweden) opened

the session on complex glycosaminoglycans with a compre-

hensive overview of heparan sulfate (HS) structure and func-

tion. Pulling together the major contribution of his own lab,

with the work of other groups, on HS biosynthesis, HS-

binding specificities of various proteins and the results of bio-

synthetic enzyme knockouts, he challenged the audience with

the question: how regulated does HS structure need to be? HS

clearly is highly regulated in its synthesis and final structure,

with much structural diversity existing between HS from dif-

ferent organs. Recent evidence also demonstrates the existence

of an extracellular 6-O-sulfatase that can also modulate HS

structure postsynthetically. It has been assumed that tightly

regulated structure imparts specific protein-binding proper-

ties, though antithrombin III still remains the only case in

which a precise structure–function relationship between a

defined HS sequence and a protein has been elucidated. The

essential role of HS in development has clearly been shown

by the failure of gastrulation in mouse embryos lacking HS-

polymerizing enzymes. However, knockouts of individual enzymes

(e.g. NDST-1, 2-OST-1 or GlcA C5-epimerase) responsible for

the postpolymeric modification, and thereby the distinctive

regulated structure of HS show lethality only at late stages of

embryo development. By then, much, apparently normal, organ

development has occurred, even though this must have involved

the appropriate co-ordinated activity of known HS-binding

morphogens/growth factors. Similarly, transgenic overexpres-

sion of the heparanase enzyme, an endo-b-glucuronidase which

partially fragments HS chains, gives rise to apparently normal

and fertile animals despite their much reduced HS chain

length.

John Gallagher (University of Manchester, Manchester, UK)

spoke about the diversity in sulfation patterns within heparan

sulfate. HS has regions that are highly N/O-sulfated, termed

S-domains, interspersed with low/nonsulfated regions, enriched

in N-acetylated disaccharide units, termed NA-domains. Recent

studies have looked at the specificity of action upon HS of

the K5 lyase enzyme isolated from the K5A bacteriophage. K5

lyase breaks down the glycocalyx of Escherichia coli K5 poly-

saccharide (GlcNAc 1–4 GlcA)n, which has an identical struc-

ture to polymeric unmodified heparan, the precursor of HS.

This enzyme could partially fragment mature HS, and it has a

preference for cleaving within HS sequences containing four or

more N-acetylated units. Interestingly, N-sulfation inhibited the

enzyme. Therefore, the K5 lyase was able to protect defined and

unique regions of alternating N-acetylated and N-sulfated dis-

accharides, which are known to be common in the HS chain,

but have not been easily accessible for analysis before. These

regions would appear to occur as transition zones between the

NA-domains and S-domains of HS. Subsequently, he described

a fast gel filtration approach for analysing the stability and

stoichiometries of complex formation between heparin oligo-

saccharides of defined length and proteins. For example, as well

as being able to investigate binary heparin–FGF1 interactions, it

was possible to also investigate the formation of ternary com-

plexes between heparin-FGF1 and the receptor FGFR2A.

Tarja Kinnunen (University of Liverpool, Liverpool, UK)

switched emphasis to the Caenorhabditis elegans developmental

model, and what it can tell us about the role of HS in

British Society for Matrix Biology Meeting A47


neuronal migration. Expression of the syndecan-1 heparan

sulfate proteoglycans (HSPG) gene (sdn-1) in neuronal cells,

and the HS 2-O-sulfotransferase gene (hst-2) in various

(neuronal, hypodermis and gonad) cells, coincide with the start

of morphogenesis in mid-embryonic development. A sdn-1

deletion mutant lacking the HS attachment site had specific

neurone and axon path-finding defects. Similarly, Hst-2

mutants are also defective in neuronal migration but with a

more diverse phenotype. In both sdn-1 and hst-2 mutants,

specific serotonergic neurones (HSNs) fail to migrate to their

positions in the vulva, giving rise to a defective egg-laying

phenotype. However, in contrast to sdn-1 mutants, the hst-2

mutants have normal migration of canal-associated neurones

(CANs), suggesting that 2-O-sulfation of HS is essential for

HSN, but not CAN, migration.

Malcolm Lyon (University of Manchester, Manchester, UK)

discussed new approaches for mapping protein–GAG inter-

actions, using HGF/SF as a model protein. GAG oligosacchar-

ides can be fluorescently tagged at their reducing termini using

2-aminoacridone. After incubation with a protein, the mixture

can be run in a gel mobility shift assay on a native PAGE gel to

assess protein binding. When oligosaccharides bind to the

protein, the free oligosaccharide band becomes depleted

and a new, slower-migrating band of oligosaccharide-protein

complex appears. HGF/SF was shown to bind to a minimal

size HS tetrasaccharide, but a hexasaccharide in the case of

DS. Two highly truncated variants of HGF/SF, called NK1 and

NK2, had identical binding properties to the full-length HGF/

SF, which points to the GAG-binding site being located solely

in the N-terminal region encompassed by the smaller NK1

protein. The concept was also discussed of how zero-length

cross-linking of fluorescently tagged minimal oligosaccharides

to proteins, followed by proteolytic digestion, can potentially

liberate fluorescent neo-glycopeptides. These could be recovered,

using their anionic character, and potentially analysed to identify

the site of GAG attachment and thus binding.

Romain Vives (Institut de Biologie Structurale, Grenoble,

France) followed with a demonstration of how a similar

approach has been successfully used to map heparin-binding

sites on proteins. Proteins can be immobilized onto heparin-

linked beads, using the zero length cross-linking method. Sub-

sequent exhaustive proteolysis of the beads with thermolysin,

followed by vigorous washing, leaves specific peptides, derived

from the GAG-binding site of the protein, remaining cross-

linked to the beads. These peptides can then be identified by

direct N-terminal Edman sequencing from the beads. This

technique, for the identification of heparin-binding regions

within a protein, potentially provides an alternative to more

complex and time-consuming approaches, such as site-directed

mutagenesis, for mapping critical residues. Two examples used

to validate the approach were the chemokine RANTES and the

gC envelope protein from the pseudorabies virus.

Toin van Kuppevelt (NCLMS University Medical Centre,

Nijmegen, The Netherlands) discussed the potential for using

phage-display technology to generate antibodies capable of

distinguishing between different HS structures. He began by

emphasizing the structural complexity of HS and the challenge

this presents. The major technical barrier to acquiring a panel

of sequence-specific, anti-HS antibodies is the poor immuno-

genicity of HS. So, he has proposed the use of semisynthetic

antibody phage-display systems, expressing single chain Fvs

(scFvs), combined with ‘biopanning’ techniques, as a potential

solution. An additional advantage is that the cDNA encoding

a particularly useful scFv can be identified. He described

antibodies successfully generated through this procedure that

possess differing reactivities towards HS from various tissues.

This included antibodies with selectivity towards tumour-

associated epitopes, or reactivities with normal, but not

rejected, renal tissue. An interesting application was the trans-

fection of tumour cells with cDNA encoding for a specific

anti-HS antibody that resulted in a block of HS expression

and a reduction in tumour size when the cells were implanted

into mice. Finally, the requirement for well-defined oligo-

saccharide libraries or microarrays to aid the identification of

the epitope specificity of such antibodies was underlined.

Claire Johnson (University of Manchester, Manchester, UK)

discussed the changes in the levels and sulfation patterns of

HS, and specific HSPGs, during the differentiation of embryo-

nic stem (ES) cells into neuroectodermal precursors in vitro.

She began by introducing the process of directed differentia-

tion in vitro of ES cells into neural precursors and also a neural

differentiation assay monitored by flow cytometry. A compari-

son of ES cells and day 6-differentiated cells by FACS analy-

sis, using an HS-specific antibody, revealed an increase in

HS expression during differentiation. Parallel structural stud-

ies on the extracted HS indicated specific increases in

N- and 6-O-sulfation associated with differentiation. She also

revealed a characteristic plaque-like patterning of HS upon ES

cells, as visualized by immunocytochemistry, which was lost

upon differentiation. RT-PCR data highlighted syndecan-4

as being the only HSPG significantly increased upon differ-

entiation. Although there were also increases in NDST-4 and

3-O-ST expression, there was a surprising lack of dramatic

alterations in expression of HS biosynthetic enzymes, consider-

ing the significant HS structural changes seen.

Briedgeen Kerr (University of Cardiff, Cardiff, UK) reported

on the development of a new antibody (BKS-1) specific to a

keratanase-generated keratan sulfate (KS) neo-epitope, and its

potential applications to the structural analysis of skeletal and

corneal KS. The reactivity of BKS-1, which requires keratanase



I treatment of KS but is not generated by keratanase II, was

contrasted with that of the previously existing anti-KS antibody,

5D4, which recognizes internal linear sequences of disulfated

N-acetyl lactosamine disaccharides. Data suggests that BKS-1

recognizes nonreducing N-acetylglucosamine 6-sulfate adjacent

to a nonsulfated lactosamine disaccharide. Applications of

BKS-1 presented, included the analysis of KS substitution of

the CS attachment region of cartilage aggrecan during ageing.

James Fawcett (University of Cambridge, Cambridge, UK)

presented the Sigma-Aldrich lecture in which he considered the

roles of proteoglycans (PGs) in the regeneration and plasticity of

the central nervous system. He focused upon the chondroitin

sulfate proteoglycans (CSPGs), which are up-regulated fol-

lowing injury and appear to have predominantly inhibitory

roles in regeneration and plasticity. He illustrated this with

results from animal studies in which the injection of chondroi-

tinase ABC at the site of neural injury enabled partial recovery

of function and commented that the relatively rapid timescale

of the observed improvements pointed towards an increased

plasticity rather than regeneration as the likely cause. After

defining plasticity, he developed this theme by introducing the

concept of perineuronal nets, emphasizing the number of

CSPGs found within these structures. Referring to examples of

ocular dominance plasticity, he suggested that the develop-

mental appearance of perineuronal nets coincided with loss of

plasticity. Considering the roles of specific CS structures, he

defined chondroitin 6-sulfate (C6S) as being more inhibitory

than chondroitin 4-sulfate (C4S) and suggested that over-

sulfation may promote axon growth. He went on to discuss

the up-regulation of CS 6-O-sulfotransferase and HS 2-O-

sulfotransferase mRNA expression and concomitant increases

in C6S and HS staining, at sites of injury and described how

the treatment of cells in culture with tumour growth factor a(TGF-a) or TGF-b, cytokines known to be released following

injury, resulted in similar increases. Finally, he discussed the

PGs identified within perineuronal nets, emphasizing PG dif-

ferences between nets from different brain regions.

Gavin Brown (University of Lancaster, Lancaster, UK) con-

cluded the meeting with an ultrastructural investigation of the

corneas of dermatopontin-knockout mice. He described how

the collagen fibrils of the cornea are organized into lamellar

layers, highlighting the importance of the uniformity of both

fibril diameter and interfibrillar spacing, with the near hexa-

gonal lattice arrangement of fibrils, providing corneal trans-

parency. By reference to macular corneal dystrophies, as well

as the lumican PG-knockout mouse, which both result in

corneal opacity, he introduced the additional major contribu-

tion of KSPGs to the maintenance of corneal transparency.

Commenting on the observed KS-mediated interaction

between lumican KSPG and dermatopontin, he described the

phenotype of the dermatopontin-knockout mouse, emphasiz-

ing the loss of lamellar structure and reduction in stromal

thickness at the posterior region and consequent reduced cor-

neal thickness. Though there is a 40% reduction in collagen

content within the posterior region of the stroma and an

increase in the interfibrillar space, there is no change in fibril

diameter. The conclusion is that the loss of dermatopontin

results in disruption of the long-range order of collagen fibrils.

Speaker’s Abstracts

Regulation of signal transduction byglycosylation

Robert S. HaltiwangerDepartment of Biochemistry and Cell Biology, Institute for Cell

and Developmental Biology, State University of New York at

Stony Brook, Stony Brook, NY, USA

The incredible diversity found in cell-surface glycoconjugate

structures led researchers over 30 years ago to propose that

complexity in carbohydrates must play a role in cellular com-

munication. Recent studies from a number of laboratories have

confirmed this hypothesis, demonstrating that cell-surface

glycoconjugates play significant roles in signal transduction

events. One striking example is the effect of O-fucose modifica-

tions on the Notch-signalling pathway. Notch is a cell-surface

receptor that is essential for proper development. The extracel-

lular domain of Notch contains up to 36-tandem epidermal

growth factor-like (EGF) repeats, many of which are predicted

to be modified at putative consensus sequences with O-fucose

and O-glucose saccharides. Genetic alterations (by knockout

or RNAi methodologies) in the enzyme responsible for the

addition of O-fucose to Notch, protein O-fucosyltransferase-

1, result in severe, embryonic lethal phenotypes resembling

Notch mutants. Thus, O-fucosylation appears to be essential

for proper Notch function. Elongation of the O-fucose mono-

saccharide by the b1,3-N-acetylglucosaminyltransferase, Fringe,

modulates Notch function, either increasing or decreasing

response from ligands depending on context. Although it is now

clear that O-fucose modifications affect Notch signalling, the

molecular mechanism by which this occurs is not known. As an

initial step in understanding how O-fucose glycans affect Notch

function, we are mapping O-fucose modifications to specific

sites on Notch. Already, we have demonstrated that O-fucose

modifies one of the EGF repeats involved in ligand binding,

suggesting that the sugars may play a role in Notch–ligand

interactions. Experiments to test the role of O-fucose modifica-

tions at specific sites are in progress. We have also found that



Fringe modifies O-fucose on some EGF repeats but not others.

Our initial analyses suggest that the basis of this specificity is

encoded within the sequences of the individual EGF repeats.

Site mapping has also confirmed the presence of O-glucose

saccharides on Notch. The evolutionarily conserved, predicted

O-glucose sites on Notch are as numerous as those for

O-fucose, suggesting that the O-glucose modifications will

play an equally important role in Notch biology. We have

recently identified an enzymatic activity capable of catalyzing

the addition of O-glucose to EGF repeats. Purification of the

protein O-glucosyltransferase is underway. These and other

results will be discussed.

Acknowledgement This work was supported by a grant from

NIH (GM61126).

Does the cellular glycome influence the bindingproperties and signalling functions of siglecs inthe immune system?

Tony Avril and Paul CrockerWellcome Trust Biocentre, School of Life Sciences, University of

Dundee, Dundee, UK

Siglecs are a family of up to 11 transmembrane sialic acid-

binding immunoglobulin superfamily. They are mostly

expressed on discrete cellular subsets of the immune system

where they are thought to be involved in both adhesive and

signalling functions. Each siglec has a distinct preference for

both the type of sialic acid as well as its linkage to other

sugars. As sialic acids are commonly found on glycoproteins

and glycolipids at the cell surface, siglecs can potentially inter-

act with these ligands both in cis (on the same cell) as well as in

trans (between cells). Cis interactions with sialic acids can

mask the sialic acid binding activity of siglecs and unmasking

can occur after sialidase treatment or in some cases by cellular

activation. We have focused on siglec-7 as a paradigm for a

new group of siglecs related to CD33 that includes siglecs-5, 6,

7, 8, 9, 10 and 11. Siglec-7 is expressed on mature human

natural killer (NK) cells and binds preferentially to glycans

containing a2,8-linked disialic acid. Similar to all CD33-

related siglecs, siglec-7 has two tyrosine-based motifs that are

predicted to mediate inhibitory signalling functions. When

expressed on NK cells, siglec-7 is masked by cis interactions

with sialic acids, but it can be unmasked following sialidase

treatment. P815 target cells were transfected with GD3

synthase to induce expression of GD3, a simple ganglioside

containing a2,8-linked disialic acid. Expression of GD3

increased their resistance to cytotoxicity mediated by sialidase-

treated NK cell killing but had little effect on killing

mediated by non sialidase-treated cells. To investigate whether

NK cells might express surface a2,8-linked disialic acids that

could explain the masked status of siglec-7, we have analysed

the binding of antibodies thought to bind specifically to this

structure. By FACS analysis, we observed preferential binding

to NK cells compared to other blood leucocytes including B

cells, T cells and monocytes. Real time PCR has been used to

investigate expression of all known a2,8-specific sialyltrans-

ferases by NK cells and other blood leucocytes. Taken

together, our findings indicate that NK cells express relatively

high levels of a2,8-linked disialic acids and that the binding

specificity of siglec-7 has become adapted to bind this struc-

ture, presumably to regulate NK-cell activation.

Conformations of cell surface oligosaccharidesand recognition by lectins from pathogens

Anne ImbertyCERMAV-CNRS, BP 53, Grenoble cedex 9, Grenoble, France

Pseudomonas aeruginosa is essentially a saprophytic bacter-

ium that can turn into an aggressive pathogen when in contact

with damaged and inflamed tissues such as burns and lungs of

cystic fibrosis (CF) patients. In all of these cases, the host cell

surfaces present altered glycosylations. The most studied

example is that of CF where point mutations in the cystic

fibrosis transmembrane conductance regulator (CFTR) gene,

results in altered ion movements and also influences the

N-glycosylation of the CFTR protein and other cell surface

glycoproteins. Increased fucosylation and decreased sialylation

with higher levels of Lewis x and Lewis a epitopes correlate

to P. aeruginosa-specific binding. In addition, both the mucus

covering the airway epithelia of CF patients and their salivary

mucins display higher levels of sialylated and sulfated Lewis

oligosaccharides that attract P. aeruginosa.

The galactose- and fucose-binding (PA-IL and PA-IIL)

lectins of P. aeruginosa contribute to the virulence of this

pathogenic bacterium, which is a major cause of morbidity

and mortality in CF patients. Database searching in newly

sequenced genomes indicates that the presence of these lectins

is characteristics of high environmental adaptability of some

opportunistic bacteria. The crystal structures of PA-IL and

PA-IIL lectins have been solved in complex with the corres-

ponding monosaccharides. Molecular modelling studies were

used in order to predict the binding mode of PA-IIL with

complex oligosaccharides. Combination of theoretical studies

with binding assays give clear indication that the histo-blood



group epitope Lewis a could be the biological ligand present in

the lung of CF patients.

References

Cioci G. et al. (2003) Structural basis of calcium and galactose

recognition by the lectin PA-IL of Pseudomonas aeruginosa.

FEBS Lett. 555, 297–301.

Imberty A. et al. (2004) Structures of the lectins from

Pseudomonas aeruginosa: insights into molecular basis for

host glycan recognition. Microb. Infect. 6, 221–228.

Mitchell E. et al. (2002) Structural basis for oligosaccharide-

mediated adhesion of Pseudomonas aeruginosa in the lungs of

cystic fibrosis patients. Nat. Struct. Biol. 9, 918–921.

MS strategies for high throughput glycomicsand glyco-proteomics

Anne Dell, Mark Sutton-Smith, Maria Panico,

Sara Chalabi, Nyet-Kui Wong, Paul Hitchen, Jihye

Jang-Lee, Simon North, Victoria Ledger, Simon

Parry, Stuart Haslam and Howard MorrisDepartment of Biological Sciences, Imperial College of Science,

Technology and Medicine, London, UK

Ultra-high sensitivity mass spectrometric strategies for defin-

ing the primary structures of highly complex mixtures of

glycopolymers are revolutionizing structural glycobiology in

the postgenomic era. MS strategies incorporating MALDI-MS

and ES-MS/MS enable very complex mixtures from biological

extracts and/or glycopolymer digests to be screened, thereby

revealing the types of glycans present and, importantly, pro-

viding clues to structures that are likely to be functionally

important.

This lecture will overview strategies employed in our labora-

tory which enable the glycome of cells, tissues and organs to be

examined and the glycoforms of individual glycoproteins to be

identified. The types of structural problems that can be solved

with current technology will be exemplified by some of our

recent research in the following areas:

. Development and application of glycomics screeningstrategies for examining glycosylation changes in cellsand tissues of knockout mice.

. Defining changes in glycosylation of murine T and B cellsupon cytokine activation.

. Characterization of the ovarian cancer antigen, CA125.

. Sequencing and site analysis of N- and O-glycans inmurine ZP3, the putative sperm receptor.

. Characterization of mucin glycosylation in cystic fibrosis-related research.

. Glyco-proteomic investigations of changes in O-glycosylationof CD8 during T-cell development.

. Glyco-proteomic analyses of novel glycosylation ofbacterial proteins.

Acknowledgements This work is supported by the BBSRC,

the Wellcome Trust and the NIH. Anne Dell is a BBSRC

Professorial Fellow.

Carbohydrate microarrays and the unravelling ofligands for effector proteins of the immunesystem

Ten FeiziThe Glycosciences Laboratory, Imperial College Department of

Medicine, Northwick Park and St Mark’s Hospital Campus,

Harrow, Middlesex, UK

Optimism (Feizi 1985) that the carbohydrate sequences of glyco-

proteins, glycolipids and proteoglycans are bearers of crucial

biological information is being well borne out (Lindahl et al.

1994; Feizi 2000; Crocker 2001). Knowledge that the human

genome encodes no more than 30 000–50 000 proteins has, in

the mean time, served to emphasize the importance of oligosac-

charide chains as modulators of the activities and functions of

the proteins in health and disease, through recognition processes

mediated by carbohydrate-recognizing receptors. The challenge

is to develop high throughput technologies to determine the

roles of specific oligosaccharide sequences as ligands for

carbohydrate-recognizing proteins. The neoglycolipid (NGL)

technology (Feizi et al. 1994) for generating lipid-linked oligo-

saccharide probes has many features that render it adaptable for

this challenge. This technology is now the basis of a carbohy-

drate array system that is applicable both to structurally

defined oligosaccharides and to those derived from biological

sources, glycoproteins, proteoglycans, cells and even whole

organs (Fukui et al. 2002). We observe that such repertoires

of NGLs are robust probes when presented on nitrocellulose

membranes, thus permitting sensitive and high throughput

detection of ligands for carbohydrate-binding proteins. We

show that carbohydrate-recognizing proteins single-out their

ligands, not only in the arrays of homogenous oligosacchar-

ides, but also in arrays of heterogeneous oligosaccharides.

The unique feature is that deconvolution strategies are

included with thin-layer chromatography (TLC) and mass

spectrometry for sequencing the ligand-positive components.

I shall discuss the applications in elucidating structures of

carbohydrate differentiation antigens and the ligands of car-

bohydrate-binding receptors of the immune system.



References

Crocker P.R. (2001) Mammalian Carbohydrate Recognition

Systems. Berlin, Germany: Springer-Verlag.

Feizi T. (1985) Demonstration by monoclonal antibodies that

carbohydrate structures of glycoproteins and glycolipids are

oncodevelopmental antigens. Nature 314, 53–57.

Feizi T. et al. (1994) Neoglycolipids – probes of oligosaccharide

structure, antigenicity, and function. Methods Enzymol. 230,

484–519.

Feizi T. (2000) Progress in deciphering the information content of

the ‘glycome’ – a crescendo in the closing years of the

millennium. Glycoconj. J. 17, 553–565.

Fukui S. et al. (2002) Oligosaccharide microarrays for high-

throughput detection and specificity assignments of carbohy-

drate–protein interactions. Nat. Biotechnol. 20, 1011–1017.

Lindahl U. et al. (1994) More to heparin than anticoagulation.

Thromb. Res. 75, 1–32.

Changes in mucin-type O-glycosylation in breastcancer: implications for the host immuneresponse

Joyce Taylor-Papadimitriou, Joy M. Burchell,

David Miles and Robert SewellCancer Research UK Breast Cancer Biology Group, GKT Medical

School, Thomas Guy House, Guy’s Hospital, London, UK

The major epithelial mucin, which is expressed by more than

90% of breast carcinomas and epithelial breast cancer cell lines,

is MUC1. The MUC1 membrane mucin has a relatively simple

structure, where a tandem repeat (TR) region carrying multiple

O-glycans forms the major part of the extracellular domain. The

gene shows a size polymorphism, due to variation in the number

of tandem repeats, each of which has five potential glycosylation

sites. MUC1 has been found to be a good model for studying

mucin-type O-glycosylation both in vitro and in vivo.

Changes in the pattern of glycosylation of MUC1 have been

observed in breast cancer (Burchell et al. 2001) and have led to

an interest in MUC1 as a possible target antigen for immuno-

therapeutic intervention (Taylor-Papadimitriou et al. 2000).

The mucin expressed at high levels in the normal lactating

mammary gland carries mainly core 2-based O-glycans (2–3

per tandem repeat). In breast cancers, the level of the ST3 Gal

1 enzyme is elevated (Burchell et al. 1999) and this is asso-

ciated with increased numbers of Sialyl T O-glycans being

added, as predicted from in vitro studies, showing competition

for the core 1 substrate between this sialyl transferase and

the relevant core 2 enzyme (Dalziel et al. 2001). Moreover,

the TR domain is more densely glycosylated. While the Sialyl

T O-glycan is commonly found on normal cells (e.g. resting T

cells), it is cancer associated in the mammary gland. However,

several lines of evidence indicate that this MUC1 glycoform, as

expressed on breast cancers, can be immunosuppressive,

maybe reflecting an inhibitory interaction with a lectin, pos-

sibly a siglec (Nath et al. 1999), on immune effector cells. In

25–30% of breast cancers, O-glycosylation is terminated very

early so that GalNAc (Tn) or NeuAc a2,6 GalNAc (Sialyl Tn)

glycans appear. Sialyl Tn is highly tumour-specific and is

detected immunohistochemically only when the enzyme

ST6GalNAcI is expressed. The Tn and Sialyl Tn will bind to

different lectins, including MGL and could stimulate the

immune response. Preliminary studies in mouse models sug-

gest that an immunization protocol which includes MUC1-

carrying Sialyl Tn O-glycans can protect against tumour

growth, while clinical studies in breast cancer patients using

immunogens based on the Sialyl Tn glycan are underway.

References

Burchell J. et al. (1999) An 2,3 sialytransferases (ST3Gal I) is

elevated in primary breast carcinoma. Glycobiology 9, 1307–

1311.

Burchell J.M. et al. (2001) O-linked glycosylation in the

mammary gland: changes that occur during malignancy.

J. Mammary Gland Biol. Neoplasia 6, 355–364.

Dalziel M. et al. (2001) The relative activities of the C2GnT1 and

ST3Gal-I glycosyltransferases determine O-glycan structure

and expression of a tumour-associated epitope on MUC1.

J. Biol. Chem. 276, 11007–11015.

Nath D. et al. (1999) Macrophage–tumour cell interactions:

identification of MUC1 on breast cancer cells as a potential

counter-receptor for the macrophage-restricted receptor,

sialoadhesin. Immunology 98, 213–219.

Taylor-Papadimitriou J. et al. (2000) MUC1 vaccines and breast

cancer. Cancer Vaccines and Immunotherapy. pp. 135–164

(eds. P.L. Stern, P.L.C. Beverley, M.W. Carroll). Cambridge, UK,

Cambridge University Press.

Hyaluronan: a simple polysaccharide withstructural plasticity and functional diversity

Anthony J. DayMRC Immunochemistry Unit, Department of Biochemistry,

University of Oxford, Oxford, UK

Hyaluronan (HA) is a high-molecular weight glycosaminogly-

can that is involved in an extracellular matrix (ECM) organ-

ization and cell adhesion, essential to a wide range of normal



physiological processes (e.g. development, immunology and

reproduction). Its diverse biological activities may seem sur-

prising for a simple linear polysaccharide composed solely of a

repeating disaccharide of glucuronic acid and N-acetylgluco-

samine. However, HA is likely to be able to take up many

different conformational states, which are transient and

rapidly interchanging in solution (Day & Sheehan 2001). It

has been suggested that the interaction of HA with different

HA-binding proteins (hyaladherins) may stabilize particular

conformations of the polysaccharide leading to HA-protein

complexes with distinct architectures and unique biological

properties (Day & Sheehan 2001). Consistent with this

hypothesis, recent studies on TSG-6 (Blundell et al. 2003)

and CD44 (Teriete et al. 2004) and molecular modelling of

other members of the Link module superfamily reveal signifi-

cant structural diversity in the HA-binding domains in these

related hyaladherins. Furthermore, there is evidence emerging

that the complexes formed between polymeric HA and pro-

teins can exhibit a wide range of higher order structures

depending on the hyaladherin(s) involved. In some cases,

these complexes may play mainly a structural role in ECM

(e.g. the link protein/aggrecan/HA aggregates that provide

load bearing function to cartilage), while in other cases they

act as cell binding/activation sites (e.g. HA cables formed

during inflammation). There is evidence that the function of

HA can be modulated by its interaction with proteins. For

example, preincubation of HA with TSG-6 enhances/induces

the binding of HA to cell surface CD44 on constitutive/indu-

cible lymphocyte backgrounds (Lesley et al.); these activities

are probably caused by the formation of cross-linked HA

fibrils that lead to receptor clustering. HA can also become

modified by the covalent attachment of heavy chains (HCs),

derived from the intera-inhibitor, where TSG-6 plays an essen-

tial role in this process (Mukhopadhyay et al.); this reaction is

critical for the formation of a HA-rich ECM required for

successful ovulation and fertilization. HC-HA complexes are

also formed during inflammation but their function is not yet

clear.

References

Blundell C.D. et al. (2003) The Link module from ovulation- and

inflammation-associated protein TSG-6 changes conformation

on hyaluronan binding. J. Biol. Chem. 278, 49261–49270.

Day A.J. & Sheehan J.K. (2001) Hyaluronan polysaccharide chaos

to protein organisation. Curr. Opin. Struct. Biol. 11, 617–622.

Lesley J. et al. (2004) TSG-6 modulates the interaction between

hyaluronan and cell surface CD44. J. Biol. Chem. 279, 25745–

25754.

Mukhopadhyay D. et al. (2004) Specificity of the tumor necrosis

factor-induced protein 6-mediated heavy chain transfer from

the intera-inhibitor to hyaluronan: Implications for the

assembly of the cumulus extracellular matrix. J. Biol. Chem.

270, 11119–11128.

Teriete P. et al. (2004) Structure of the regulatory hyaluronan

binding domain in the inflammatory leukocyte homing receptor

CD44. Mol. Cell. 13, 483–496.

Biosynthesis of heparan sulfate – how regulateddoes it need to be?

Ulf LindahlDepartment of Medical Biochemistry and Microbiology, Uppsala

University, Uppsala, Sweden

Heparan sulfate proteoglycans (HSPGs) are ubiquitous in ani-

mals. The functional significance of the structural variability

exhibited by the constituent HS chains remains a fundamental

question in cell and developmental biology. The polysacchar-

ides influence cell behaviour, by modulating the activities of

signalling proteins or through interactions in the extracellular

matrix. HS is synthesized as a linear polymer of alternating

glucuronate (GlcA) and N-acetylglucosamine (GlcNAc) units.

The resultant [GlcA-GlcNAc]n polymer is partially modified

through a series of N-deacetylation/N-sulfation, GlcA C5-

epimerization and O-sulfation reactions (Esko & Lindahl 2001)

and may be further modified by selective removal of O-sulfate

groups (Ai et al. 2003). Owing to the variable extent of these

reactions in different cells and tissues, and at different devel-

opmental stages, the mature HS products display different

structures, typically expressed through sulfated domains of

variable length and composition (Esko & Lindahl 2001;

Casu & Lindahl 2001). These domains provide binding sites

for protein ligands and thus constitute the structural basis for

the diverse functional roles of HS. The regulated diversity of

HS structure is demonstrated through compositional analysis

of HS from various organs as well as by immunohistochemical

recognition of HS epitopes (van Kuppevelt, T. H et al. 1998).

So far, a precise structure–function relation has been defined

only for the antithrombin-binding pentasaccharide sequence

(Casu & Lindahl 2001).

HS is essential throughout development, as mouse embryos

devoid of HS-polymerizing enzymes fail in gastrulation

(Gallagher 2001). By contrast, targeted disruption of genes

encoding polymer-modifying enzymes is associated with sur-

prisingly mild phenotypes. Embryos lacking N-deacetylase/

N-sulfotransferase-1, 2-O-sulfotransferase (Gallagher 2001)

or GlcA C5-epimerase (Li et al. 2003), thus show major

defects incompatible with postnatal life, but also apparently



normal organ systems known to rely on HS-binding morpho-

gens or growth factors for their development. Moreover,

transgenic mice overexpressing heparanase appear essentially

normal (Zcharia et al. 2004), in spite of an overall drastic

reduction in HS chain size. How stringent are structure/func-

tion relations in HS–protein interactions?

References

Ai X. et al. (2003) Qsulf1 remodels the 6-O sulfation states of cell

surface heparan sulfate proteoglycans to promote Wnt signal-

ing. J. Cell Biol. 162, 341–3513.

Casu B. & Lindahl U. (2001) Structure and biological interactions

of heparin and heparan sulfate. Adv. Carbohydr. Chem.

Biochem. 57, 159–206.

Esko J.D. & Lindahl U. (2001) Molecular diversity of heparan

sulfate. J. Clin. Invest. 108, 169–173.

Gallagher J.T. (2001) Heparan sulfate: growth control with a

restricted sequence menu. J. Clin. Invest. 108, 357–361.

Li J.P. et al. (2003) Targeted disruption of a murine glucuronyl

C5-epimerase gene results in heparan sulfate lacking 1-

iduronic acid and in neonatal lethality. J. Biol. Chem. 278,

28363–28366.

van Kuppevelt T.H. et al. (1998) Generation and application of

type-specific antiheparan sulfate antibodies using phage display

technology. Further evidence for heparan sulfate heterogeneity

in the kidney. J. Biol. Chem. 273, 12960–12966.

Zcharia E. et al. (2004) Transgenic expression of mammalian

heparanase uncovers physiological functions of heparan sulfate

in tissue morphogenesis, vascularization, and feeding behavior.

FASEB J. 18, 252–263.

Some new insights on the molecular structureand function of heparan sulfate

John GallagherCancer Research UK Department of Medical Oncology,

University of Manchester, Christie Hospital NHS Trust,

Manchester, UK

It is well established that the heparan sulfate (HS) polysacchar-

ide contains sulfated domains (S-domains) composed of

sequences of N- and O-sulfated disaccharides separated by

regions of low sulfation enriched in N-acetylated disaccharide

units. Recent work using the enzyme K5 lyase has shed new

light on the molecular organization of HS. K5 lyase is pro-

duced by bacteriophage K5A and it facilitates phage infectivity

by degrading the polysaccharide coat of Escherichia coli K5;

the K5 polysaccharide has an identical structure to the poly-

meric precursor of HS, named heparan, composed of GlcNAc

1-4 GlcA repeat units. We found that K5 lyase reduced the

average size of HS derived from mouse 3T3 cells from 45 kDa

to 7 kDa. Studies of its substrate specificity indicated a strong

preference for sequences of four or more N-acetylated disac-

charide units. N-sulfation, the initial step in the conversion of

heparan to HS, inhibited the action of the enzyme. One import-

ant consequence of this was that the regions of alternating

N-acetylated and N-sulfated disaccharides, the NA/NS domains

(GlcA 1-4 GlcNAc 1-4 GlcA 1-4 GlcNS-), that are common in

HS are not degraded by K5 lyase. On the basis of these and

other findings, we can refine our original model of HS to

define these domains as the predicted transition zones between

the highly sulfated S-domains and the N-acetylated sections

of the polymer chain. The close apposition of NA/NS- and

S-domains, with their distinctive sulfation patterns, creates

composite regions of hypervariable structure with an enhanced

potential for imparting specificity to HS–protein interactions.

The spacing of the K5 lyase cleavage sites is very similar to that

of the heparinase I sites (i.e. the S-domains) indicating near

maximal separation of the unmodified and highly modified

regions. The model is suggestive of a regulated mechanism of

HS biosynthesis and has implications for the proposed role of

HS as a template for the assembly of multiprotein signalling

complexes on the cell surface.

Phage display-derived antibodies: new tools tostudy heparan sulfate diversity

Toin van KuppeveltDepartment of Biochemistry, Nijmegen Centre for Molecular Life

Sciences, University Medical Centre, Nijmegen, The Netherlands

Heparan sulfates comprise a heterogeneous group of polysac-

charides involved in many basic cellular phenomena including

proliferation, migration and differentiation. Their biosynthesis

allows for the formation of a large number of different struc-

tures. Specific HS structures are likely involved in the binding

of HS to proteins, such as growth factors. Anti-HS antibodies,

obvious tools to study structural heterogeneity, are difficult to

obtain due to the poor immunogenicity of HS. To circumvent

this, we have adopted antibody phage display technology,

because this system allows for the generation of antibodies

against self-antigens.

A human semisynthetic antibody phage display library

(Nissim et al. 1994) and a semisynthetic library in which the

CDR 3 region of the antibodies contained an XBBXBX amino

acid sequence (a consensus heparin-binding site) were used to

select antibodies to HS using biopanning strategies.



A large number of antibodies were selected, sequenced and

characterized. A number of chemical groups in HS, important

for antibody recognition, were identified. Antibodies revealed

a large and regulated domain diversity of HS in a number of

tissues (Dennissen et al. 2002). The up/down-regulation of

specific HS structures in various pathologies including cancer

and nephropathies were identified (Jenniskens et al. 2003;

Smetsers et al. 2003). Transfection of cells with cDNAs encod-

ing the antibodies could be used to block HS in vitro and to

study its physiological relevance.

It is concluded that phage display-derived antibodies to HS

may provide new tools to study the role of HS in health and

disease.

References

Dennissen M.A. et al. (2002) Large, tissue-regulated domain

diversity of heparan sulfates demonstrated by phage display

antibodies. J. Biol. Chem. 277, 10982–10986.

Jenniskens G.J. et al. (2003) Phenotypic knock out of heparan

sulfates in myotubes impairs excitation-induced calcium spik-

ing. FASEB J. 17, 878–880.

Nissim A. et al. (1994) Antibody fragments from a ‘single pot’

phage display library as immunochemical reagents. EMBO J.

13, 692–698.

Smetsers T.F. et al. (2003) Localization and characterization of

melanoma-associated glycosaminoglycans: differential expres-

sion of chondroitin and heparan sulfate epitopes in melanoma.

Cancer Res. 63, 2965–2970.

The role of proteoglycans in regeneration andplasticity

James FawcettCambridge University Centre for Brain Repair, Robinson Way,

Cambridge, UK

Chondroitin sulfate proteoglycans (CSPGs) are up-regulated

in glial scar tissue and inhibit axon regeneration. Not only

are the protein cores up-regulated, but also there is more

glycosaminoglycan (GAG) attached to them. The final stage

of GAG synthesis is sulfation, which can occur in three posi-

tions. Both 6-sulfated GAG and the sulfotransferase that

sulfates N-acetylgalactosamine in the 6 position is specifi-

cally up-regulated in glial scar tissue, in inhibitory glial cells

and in astrocytes treated with tumour growth factor a (TGF-

a) and TGF-b. Removal of GAG chains by digestion with

chondroitinase or inhibition of GAG synthesis with chlorate

or b-D-xylosides, removes much of the inhibition from

CSPGs in vitro. We therefore tested to see whether GAG

digestion by chondroitinase would promote axon regenera-

tion in vivo. We first treated mechanical lesions of the nigros-

triatal tract and saw regeneration of about 4% of axons back

to their target. Next, dorsal column lesions of the spinal cord

at C4 were treated. Both sensory and corticospinal axons

regenerated in treated cords, and there was rapid return of

function in beam and grid walking tests. The return of func-

tion was so rapid that we hypothesized that some of it might

be due to enhanced plasticity. Many neuronal cell bodies and

dendrites are coated in thick perineuronal nets of inhibitory

CSPGs and tenascin-R, which would certainly be expected to

prevent the formation of new synapses. We therefore tested

the effects of chondroitinase treatment in a plasticity model:

ocular dominance shift in the visual cortex following mono-

cular deprivation. Monocular deprivation in adult animals

normally produces no ocular dominance shift. However, in

adult animals in which the cortex was treated with chondroi-

tinase, there was a large shift in response to monocular

deprivation.

Poster Abstracts

Modulation of heparan sulfate/chemokineinteractions using RNA interference

Hrant Arzoumanian, Terry Bennet, Zara Watson

and Noel CarterSunderland Pharmacy School, University of Sunderland,

Sunderland, UK

Introduction The glycosaminoglycan heparan sulfate (HS)

on the luminal surface of the vascular endothelium plays a

vital role in promoting leucocyte migration during inflamma-

tion via specific interaction chemokines. HS is modified by

sulfation and this appears to regulate chemokine binding. We

previously observed that under pro-inflammatory conditions

[tumour necrosis factor a (TNF-a) and interferon-g (IFN-g)],

the endothelial cell line HMEC-1 showed an increased

sulfation HS. This promoted a significant increase binding of

the chemokine RANTES and transendothelial migration of

leucocytes. TNF-a and IFN-g clearly modulated HS bio-

synthesis, in part by up-regulating the NDST 1 and 2 genes

responsible for N-sulfation of the glycan, resulting in struc-

tural changes that favour HS/chemokine interactions (Carter

et al. 2003). We have attempted to down-regulate the expression



of the gene NDST1 by RNA interference in order to investi-

gate the structural and functional effects of reduced HS

N-sulfation.

Materials and methods A double-stranded interference RNA

was designed and synthesized in vitro to target the sulfotrans-

ferase NDST-1 and used to tranfect the HMEC-1 cell line.

A number of assays measured its effect on HS structure and

function. Cell surface N-sulfation was measured by flow

cytometry using the antibody F58 10E4 (Seikagaku, Japan).

Cell surface binding of the chemokine MCP-1 was visualized

by immunohistochemistry. The functional effect of MCP-1

binding was measured using a trans-endothelial migration

assay.

Results Surprisingly, the transfected cells appeared to show a

small but marked increase in cell surface N-sulfation. This was

consistent with previous results transfecting a full-length

NDST-1 antisense construct into the cell line, A549 (unpub-

lished). The increase in N-sulfation observed was supported

functional date. Namely, transfected cells show an increased

binding of the chemokine MCP-1 and this promoted a statis-

tically significant increase in transendothelial migration of

leucocytes.

Discussion The results observed were contrary to expecta-

tions. It would appear that the interference RNA molecule is in

some way interfering in the regulation of the NDST-1 expres-

sion leading to an increase not decrease in gene expression.

This has yet to be verified but we are beginning real time

expression studies to validate this. We have hypothesized

two possible reasons for this observation; endothelial cells

may be able to sense a reduction in NDST activity and

up-regulate gene expression. Or the presence of the interference

RNA itself promotes NDST-1 expression. The latter is sup-

ported by growing evidence that short double-stranded RNAs

are thought to up-regulate the pro-inflammatory STAT tran-

scription factor family (Shuey et al. 2002).

References

Carter N.M. et al. (2003) Endothelial inflammation: the role of

differential expression of N-deacetylase/N-sulphotransferase

enzymes in alteration of the immunological properties of

heparan sulphate. J. Cell Sci. 116, 3591–3600.

Shuey D.J. et al. (2002) RNAi: gene-silencing in therapeutic

intervention. Drug Discov. Today 7, 1040–1046.

Direct and indirect manipulation of theMEK-ERK pathway regulates the formation of apericellular HA-dependent matrix by chickarticular surface cells without modifying CD44expresssion

Edward R. Bastow, Caroline P.D. Wheeler-Jones

and Andrew A. PitsillidesDepartment of Veterinary Basic Sciences, Royal Veterinary

College, Royal College Street, London, UK

Introduction Recent evidence suggests that hyaluronan (HA)

facilitates the mechano-dependent joint cavity-forming pro-

cess through the elaboration and retention of a HA-rich peri-

cellular matrix in the developing joint interzone (IZ). The

presumptive joint IZ phenotype shows a capacity to bind and

synthesize HA and also exhibits elevated activated ERK, prior

to synovial joint cavity formation (Lamb et al. 2001; Edwards

et al. 1994; Dowthwaite et al. 1998). We have found that

immobilization, which induces embryonic joint fusion with

loss of the joint IZ phenotype, also reduces ERK activity levels

in the IZ. As the signalling events regulating the synthesis and

binding of HA have yet to be determined, we hypothesize that

ERK activation plays a pivotal role in determining the pre-

sumptive joint IZ phenotype through HA synthetic and bind-

ing capacity.

Materials and methods Chick articular surface (AS) cells

were harvested from proximal tibiotarsal joints of embryos

by collagenase digestion. Pericellular coat formation was

assessed using the erythrocyte exclusion assay and cell-coat

area ratios determined. ERK activity was modulated by tran-

sient transfection of GFP constructs of constitutively active

(CA-) or dominant negative (DN-) forms of MEK, the direct

upstream regulator of ERK or by treatment with the MEK

inhibitor PD98059 (50 mm). ERK activation was monitored by

immunochemistry. CD44 expression and ERK activation in

PD98059-treated cells were monitored by immunoblotting

and medium HA concentrations by ELISA.

Results AS cells form large pericellular coats that are lost

following hyaluronidase treatment and thus dependent upon

HA for their construction. Treatment with PD98059 signifi-

cantly reduced pericellular coat formation after 6 h. In paral-

lel, we confirmed that PD98059 diminished active ERK

expression without modifying overall levels of ERK, suggest-

ing that the elaboration of large HA-pericellular coats is

dependent upon MEK’s activation of ERK. Western blot ana-

lysis of PD98059-treated cells showed that loss of pericellular

coats was not, however, associated with any decreased levels



of the cell surface HA receptor CD44. Although treatment

with PD98059 did not change medium HA concentration

after short times of exposure, at times (up to 6 h) during

which coat loss was evident, prolonged treatment over 24 h

significantly decreased medium HA concentration. Consistent

with a role for ERK in pericellular coat formation, transfection

with DN-MEK diminished, while CA-MEK increased, both

active ERK expression and coat formation efficiency. We

also found that, commensurate with this modification in coat

forming efficiency, cells expressing DN-MEK exhibited a sig-

nificant reduction in labelling of free HA on the cell surface.

Discussion These studies extend our recent work to indicate

that: (i) direct modulation of ERK activation by transfection

with its endogenous upstream regulator modifies cell surface-

associated HA (ii) PD98059-induced blockade of ERK activa-

tion restricts medium HA release and (iii) ERK-mediated

changes in pericellular coat elaboration are independent of

changes in cellular CD44 expression. These findings suggest

an intimate relationship between ERK activation and the

formation/retention of HA-rich pericellular matrices in vitro

and highlight the role for ERK activation in regulating joint

line-related differentiation.

References

Dowthwaite G.P. et al. (1998) An essential role for the interaction

between hyaluronan and hyaluronan binding proteins during

joint development. J. Histochem. Cytochem. 46, 641–651.

Edwards J.C.W. et al. (1994) The formation of human synovial

joint cavities – a possible role for hyaluronan and CD44 in

altered interzone cohesion. J. Anat. 185, 355–367.

Lamb K.J. et al. (2001) Active ERK-1/2 selectively colocalises

with polymerised actin at developing joint surfaces. Trans.

Orthop. Res. Soc. 27, 123.

Age-related changes in human meniscalglycosaminoglycans

Gareth Blackburn, Meneerah Al-Jafary, Tom

Huckerby and Robert LauderDepartment of Biological Sciences, Lancaster University,

Lancaster, UK

Introduction With an increased human lifespan, a major

challenge is now to ensure a concomitant increase in health-

span. Meniscal damage and degradation are common and are

strongly correlated with subsequent osteoarthritis. Indeed,

meniscal damage has been identified in about 60% of people

over 60. Markers of pathology will facilitate intervention but

first require normal age-related changes to be established.

Methods Undamaged vascular and avascular regions of med-

ial and lateral human menisci were comminuted and the tissue

extracted into 4-M GuHCl and subject to associative CsCl

density gradient centrifugation. Aggrecan and the small leucine

rich PGs (SLRPs) were isolated and their GAG profiles

examined by HPAEC fingerprinting, following enzyme depoly-

merization, and by an NMR spectroscopy.

Results and discussion Analysis of aggrecan and the SLRPs

show that there is a complex and dynamic pattern of KS, CS

and DS abundance and distribution within human menisci,

which changes with age.

The abundance of SLRPs is higher in the avascular than

vascular tissues, however, this is not reflected in the abun-

dance of aggrecan which is present at similar levels in both

tissue regions. The data show no other significant differences

between medial and lateral and between vascular and avascu-

lar tissue regions.

Analysis of the sulfation pattern of CS following digestion

by ACII lyase, shows that in both aggrecan and SLRPs the

4-sulfation level falls with age from 20 to 35% in young

tissues to 10–20% in older. Subsequent analyses following

ABC lyase depolymerization, to include DS, shows very

significant change with age from CS + DS 4-sulfation levels of

ca. 40–55% in young tissue to ca. 15–30% in older.

The difference between these datasets represents the contri-

bution made by 4-sulfated DS. Thus, analysis of the difference

suggests that DS makes a decreasing contribution to the CS/DS

profile with age.

Indeed, this is confirmed by an NMR analysis of these sam-

ples. Analysis of the resonances in the region 1.95–2.2 p.p.m.

(ref to TSP) allows the estimation of the contribution made by

DS, CS and KS. These data show that, in aggrecan, the con-

tribution made by DS chains falls from ca. 10% in younger

tissues to ca. 2–4% in older tissues.

NMR analysis also shows that KS levels fall with age

from ca. 15–20% in younger tissues to 5–10% in older

tissues. Analysis of the structure of the KS chains shows

chains with a structure similar to that of in articular carti-

lage but that at all ages there are very low levels of fuco-

sylation (ca. 1–5%).

Previous studies of age-related changes in CS/DS and KS

structures have shown significant changes in the first 17

years of life, with only modest nonpathological changes after

that time. These data from meniscal tissues do not show such a

dramatic halting of normal age-related changes. Indeed, the



data show gradual age-related changes in DS, CS and KS

abundance and structure throughout life.

These baseline age-related changes data will now allow the

analysis of pathology-related changes.

Molecular recognition and modulation ofhepatocyte growth factor activity by heparanand dermatan sulfates

Krista Catlow,* Jon A. Deakin,* Maryse

Delehedde,† David G. Fernig,† Mauro S.G.

Pavao,‡ John T. Gallagher* and Malcolm Lyon**Cancer Research UK Department of Medical Oncology,


Manchester, UK; †School of Biological Sciences, University of

Liverpool, Liverpool, UK; ‡Universidade Federal do Rio de

Janeiro, Rio de Janeiro, Brazil

Introduction Hepatocyte growth factor/scatter factor (HGF/

SF) is an unusual growth factor in that it binds both heparan

sulfate (HS) (Lyon et al. 1994) and dermatan sulfate (DS)

(Lyon et al. 1998) glycosaminoglycans (GAGs) with similar

high affinities. Both these GAGs act as co-receptors for HGF/

SF in the activation of the Met receptor (Lyon et al. 2002).

Our aim was to determine the sequences in HS and DS that

specifically interact with and modulate HGF/SF activity.

Materials and methods A structurally unique DS, which

possesses O-sulfation at carbon-6 of the hexosamine residue

(and not carbon-4 as in mammalian DS), was obtained from

the sea cucumber, Ascidia nigra. A variety of HS- and DS-like

structures were also generated using various chemical modifi-

cation procedures (specific desulfations and carboxyl reduc-

tions). The ability of these various GAG species to compete

with cell surface GAGs for HGF/SF binding was tested using

radiolabelled HGF/SF and MDCK cells. The modified GAG

structures and the A. nigra DS are currently being tested for

their ability to act as co-receptors for the interaction between

HGF/SF and Met by studying cell signalling and cellular

response assays, using the sulfated GAG-deficient CHO-745

cell line.

Results Unexpectedly, A. nigra DS was found to bind HGF/

SF strongly with a KD of around 1 nM. This interaction is

20-fold stronger than that of between HGF/SF and mammalian

DS, but similar to that of with HS. A. nigra DS also stimulated

HGF/SF-mediated Erk activation and migration in CHO-745

cells. Studies using the modified GAG species showed that, in

the case of HS, 6-O-sulfate and N-sulfate groups are most

important for HGF/SF binding. For HGF/SF binding to DS,

hexosamine O-sulfate is most important. HGF/SF was also

found to bind 6-O-sulfated GAGs more strongly than 4-O-

sulfated ones.

Discussion The data show that there is flexibility in the

structures recognized by HGF/SF, and this explains the ability

of the growth factor to bind both HS and DS. However, there

are still observable preferences in GAG structure, such as

6-O-sulfation over 4-O-sulfation. Information on HGF/SF-

binding GAG structures is valuable for the design of HGF/SF

antagonists that could be useful therapeutically in the treat-

ment of solid tumours where HGF/SF-Met activity is up-

regulated.

References

Lyon M. et al. (1994) Interaction of hepatocyte growth factor

with heparan sulfate. J. Biol. Chem. 269, 11216–11223.

Lyon M. et al. (1998) Hepatocyte growth factor/scatter factor

binds with high affinity to dermatan sulfate. J. Biol. Chem.

278, 271–278.

Lyon M. et al. (2002) The mode of action of heparan and

dermatan sulfates in the regulation of hepatocyte growth

factor/scatter factor. J. Biol. Chem. 277, 1040–1046.

Oligomeric collagen XVIII-derived endostatinrequires cell surface heparan sulfate in order toinduce morphological changes in endothelial andepithelial cell lines in vitro

A.R. Clamp,* F.H. Blackhall,* A. Henrioud,*

G.C. Jayson,* K. Javaherian,† J. Esko,‡

J.T. Gallagher* and C.L.R Merry**Cancer Research UK Department of Medical Oncology,


Manchester, UK; †Department of Surgery, Children’s Hospital,

Harvard Medical School, Boston, MA, USA; ‡Department of

Cellular and Molecular Medicine, University of California at San

Diego, La Jolla, CA, USA

Introduction Monomeric endostatin (HEM) has been exten-

sively investigated as an endogenous anti-angiogenic molecule.

In contrast, oligomeric endostatin (HED) induces a promigra-

tory phenotype in endothelial cells in vitro that can be inhibited

by an EM in a putative negative feedback loop (Kuo et al.

2001). Endostatin is heparin-binding but the role of cell-surface



glycosaminoglycans (GAG) in regulating the activity of HEM/

HED is unclear.

Methods and results Bovine aortic endothelial (BAE) cells

plated onto a Matrigel substratum spontaneously form

capillary-like tubules. Addition of 50 nM HED, 16 h after plating

induces the migration of BAE cells away from these tubules.

This pro-migratory action can be inhibited by coincubation

with HEM. We have demonstrated that exogenous GAGs can

also inhibit the actions of HED. This property is size depen-

dent, with heparin-derived oligosaccharides containing more

than 20 monosaccharide units being optimal inhibitors. HED

(50 nM) was also shown to induce marked morphological

changes in the Chinese hamster ovary (CHO) epithelial cell

line plated in Matrigel, i.e. cell elongation, lamellipodia for-

mation and intercellular bridging. This novel observation

allowed us to address the role of cell surface GAGs in HED

function using a panel of CHO mutants with defined defects in

GAG biosynthesis. CHO 745 cells, which lack all cell surface

sulfated GAGs, fail to respond to HED stimulation, while

CHO pgsD cells, which specifically lack cell surface heparan

sulfate (HS), displayed an attenuated phenotype on HED sti-

mulation with less cell elongation/intercellular bridging. Not-

ably, CHO pgsF17 cells, which lack 2-O-sulfation of HS,

respond normally to HED indicating that HS 2-O-sulfate

groups are not necessary for the bioactivity of HED.

To investigate the endostatin–GAG interaction further,

zero-length cross-linking was utilized. This indicated that mul-

tiple HEM subunits can simultaneously bind to a single oligo-

saccharide chain with up to 3 HEM molecules cross-linking to

a heparin dp22. HED cross-linking experiments indicate that

both endostatin subunits in this molecule may bind to the same

oligosaccharide chain. These results suggest that one potential

anti-angiogenic mechanism of HEM is to block the GAG-

binding sites for HED.

Discussion Cell surface GAGs are absolutely necessary for

the bioactivity of HED. HS is required for a wild-type

response, although 2-O-sulfation is not essential. The ability

to use a panel of GAG-mutant CHO cells provides a well-

understood system in which to investigate the role of HS in

HEM : HED regulation.

Reference

Kuo C.J. et al. (2001) Oligomerization-dependent regulation of

motility and morphogenesis by the collagen XVIII NC1/

endostatin domain. J. Cell Biol. 152, 1233–1246.

An ultrastructural investigation of dermato-pontin-knockout mouse corneas

L.J. Cooper,* A.J. Bentley,* I.A. Nieduszynski,*

N.J. Fullwood,* T.S. Ellis,* A. Thomson,*

A. Utani1,† H. Sinkai† and G.M. Brown**Department of Biological Sciences, Lancaster University,

Lancaster, UK; †Department of Dermatology, Chiba University

School of Medicine, Chuouku, Chiba, Japan

Introduction The corneal stroma is composed of a network

of heterotypic collagen fibrils, proteoglycans and matrix pro-

teins. Transparency of the tissue principally requires the uni-

formity of fibril diameters and interfibrillar distances and the

presence of a quasi-hexagonal lattice arrangement of parallel

fibrils. Keratan sulfate proteoglycans (KSPGs) have a crucial

role and the KS chains are clearly required for the maintenance

of transparency. Undersulfation of corneal KS results in tissue

opacity and the lumican (a KSPG) knockout mouse shows

corneal opacity and the disruption of collagen fibril diameters

and interfibrillar distances (Chakravarti et al. 1998). Biochem-

ical analysis has shown that dermatopontin is an abundant

component of the extracellular matrix and that it interacts

with KSPGs via the KS chains. This study aims to determine

whether dermatopontin has a direct role in corneal matrix

organization by investigating the corneal ultrastructure of

dermatopontin-null (dpt–/–) mouse corneas.

Materials and methods Conventional light microscopy was

used to compare the corneal thickness of dpt–/– mice (Takeda

et al. 2002) with that of the wild-type. Collagen fibril distribu-

tion was studied using transmission electron microscopy and the

datasets analysed using SIS-pro image analysis software to deter-

mine fibrillar volume, shape factor, fibril diameter and spacing.

Results Light microscopy demonstrated that dpt–/– corneas

show a 24% reduction in average stromal thickness compared

to wild-type (P < 0.001). The epithelium and Descemet’s mem-

brane appear normal. Examination of dpt–/– stroma by trans-

mission electron microscopy indicates a significant disruption

to lamellar organization in the posterior region while the

central and anterior regions appear largely unaffected com-

pared to wild-type. The collagen fibrils in dpt–/– stroma show a

pronounced increase in interfibrillar spacing as well as exhibit-

ing a lower fibril volume fraction. There is no apparent differ-

ence in fibril diameter between dpt–/– and wild-type mice.

Discussion Collectively, these data suggest that dermatopontin

plays a key role in collagen fibril organization and deposition.



Like the cornea from lumican-knockout mice (Chakravarti et al.

1998), the defects in collagen organization in dpt–/– cornea

appear to be most severe in the posterior stroma. It is likely

that dermatopontin interacts with the KS chains on lumican

and this interaction is involved in the maintenance of stromal

architecture.

References

Chakravarti S. et al. (1998) Lumican regulates collagen fibril

assembly: skin fragility and corneal opacity in the absence of

lumican. J. Cell Biol. 141, 1277–1286.

Takeda U. et al. (2002) Targeted disruption of dermatopontin

causes abnormal collagen fibrillogenesis. J. Invest. Dermatol.

119, 678–683.

New approaches for mapping the recognitionsites in protein–GAG interactions: a study of GAGbinding by hepatocyte growth factor/scatterfactor

Jon A. Deakin,* Daniel Lietha,† Ermanno

Gherardi,† John T. Gallagher* and Malcolm Lyon**Cancer Research UK Department of Medical Oncology, University

of Manchester, Christie Hospital NHS Trust, Manchester, UK;†Growth Factors Group, MRC Centre, Cambridge, UK

Introduction The minimal size and structure of GAG that

binds to a protein, as well as the site of GAG interaction on the

protein surface, are crucial recognition features for under-

standing, and potentially modulating, protein–GAG interac-

tions. Methods that are easily and generally applicable for

investigating such interactions, irrespective of the GAG species

involved, are very limited. We have been developing such

methodologies using the example of hepatocyte growth fac-

tor/scatter factor (HGF/SF). We have previously shown HGF/

SF to have an unusual GAG-binding specificity, in that it binds

with high affinity to, and is activated by, both heparan and

dermatan sulfates. There are two naturally occurring, highly

truncated variants of HGF/SF. These comprise only the

N-terminus and either the first one (NK1) or two (NK2) of

the four Kringle domains of the a-chain of HGF/SF, and

without a b-chain. Some controversy presently exists as to

whether GAG-binding in HGF/SF requires the N-terminus

alone, or additionally the second Kringle domain.

Materials and methods We have developed a protocol for

analysing protein–GAG interactions utilizing fluorescently

tagged GAG oligosaccharides run in a protein gel mobility

shift assay under native associative conditions. The activity

of oligosaccharides has been assessed by their ability to sti-

mulate HGF/SF-mediated activation of signals downstream

of the Met receptor in sulfated GAG-deficient CHO pgsA-

745 cells.

Results HS tetrasaccharides and DS hexasaccharides are

the minimal binding and activatory species for HGF/SF.

The basis for this size difference between HS and DS spe-

cies is not presently known. However, their ability to effi-

ciently compete with each other does indicate a shared

binding site. The three proteins HGF/SF, NK1 and NK2

all display identical GAG-binding specificities and minimal

oligosaccharide-binding sizes. This suggests that NK1, the

smallest variant, contains the entire minimal GAG-binding

functionality of full-length HGF/SF, and thus the second

Kringle domain is not required. Identification of NK1 and

tetrasaccharides as the minimal binding partners has

allowed us to directly target the binding site on NK1.

Fluorescently tagged tetrasaccharides have been crosslinked

into the binding site on NK1 using the zero-length cross-

linking methodology. After tryptic digestion, the specific

fluorescently tagged peptides can be recovered for subse-

quent sequencing to locate the GAG-binding site within the

known amino acid sequence of the protein.

Discussion Methods have been developed which can help to

identify the respective domains on both GAG and protein

involved in GAG-protein recognition. These are relatively

easy approaches, applicable to physiological solution condi-

tions and having the advantage of being widely applicable to

many protein interactions involving any uronate-containing

GAGs. In addition to HGF/SF, the gel mobility shift assay

has also been applied by us to a variety of other diverse

proteins.

Role of heparan sulfate in embryoid bodyformation

K.J Drummond, E.A. Yates, P.A. Murray and

J.E. TurnbullSchool of Biological Sciences, University of Liverpool, Crown

Street, Liverpool, UK

Introduction Embryoid bodies (Ebs) are layered, ordered

aggregates of cells which form when murine embryonic

stem (ES) cells are grown in suspension. Ebs are made up of



primitive endoderm cells overlaying an epithelium of

epiblast cells, separated by a basement membrane and sur-

rounding a central cavity. The generation of Ebs from undif-

ferentiated ES cells has been used as a model for early

development. The process of EB formation has been

shown to require signalling through fibroblast growth factor

(FGF) receptor 2, implicating heparan sulfate (HS) which

is a necessary coreceptor for the formation of a signalling

complex.

Heparan sulfate proteoglycans are found both on the surface

of cells and in the extracellular matrix. Within the cells, a newly

synthesized HS chain is partially modified by a family of

enzymes, many of which have multiple isoforms with differing

substrate preferences. These modifications include the addition

of sulfate groups at up to four positions on each disaccharide

unit. The pattern of modifications that a HS saccharide under-

goes alters its structure, affecting the ability of the HS to bind and

regulate different FGF-FGF receptor combinations, and so

regulate signalling outcomes.

It is hypothesized that the complement of modification

enzymes that a cell expresses determines the structure of the

HS that the cell produces, thereby altering the signalling response

of the cell to HS-binding factors such as FGFs. In this way, an

ES cell could alter its HS to allow signalling by, for example, a

particular FGF, setting in motion a chain of intracellular events

leading to differentiation.

Materials and methods Methods used in this study include

tissue culture, RT-PCR and immunocytochemistry.

Results We have found that undifferentiated ES cells express

HS as observed by cell staining with anti-HS antibodies. They

also display particular expression patterns of the different HS

modification enzyme isoforms as measured by RT-PCR. Stud-

ies on alterations in HS synthesis and structure during the

process of EB formation are underway. In addition, the effect

of the addition of exogenous heparin (a highly sulfated analo-

gue of HS) and HS saccharides with variant structures on EB

formation is being investigated.

Discussion Alterations in the profile of modification

enzymes expressed by ES cells as they differentiate may correl-

ate with changes in the structure of the HS they make. These

changes may allow the differentiating cells to control the way

in which they respond to their environment.

These studies will allow the dissection of the structural

requirements for a functional role of HS in embryoid body

formation and stem cell differentiation.

The use of SA-b-Gal to assess cell senescence inintervertebral disc cells

E.H. Evans,* D. Kletsas,† J. Urban,‡ J. Menage*

and S. Roberts**Centre for Spinal Studies, Robert Jones & Agnes Hunt

Orthopaedic Hospital, Oswestry, Shropshire, UK; †Laboratory of

Cell Proliferation & Ageing, Demokritas, Athens, Greece;‡Oxford University, Oxford, UK

Introduction The intervertebral disc is reported to age faster

than other connective tissues with significant degenerative

changes already seen in the second decade of life. In other

tissues, senescent cells have an altered phenotype, often with

a decreased synthetic ability and response to anabolic cyto-

kines. They appear to contribute to age-related pathologies

such as the degeneration of articular cartilage in osteoarthritis.

Little is known of cell senescence in the intervertebral disc. In

this study, we have investigated the production of senescence-

associated-ß-galactosidase (SA-ß-Gal), a ‘biomarker’ of cell

senescence, in intervertebral disc cells both in cultured popula-

tions in vitro, and in vivo, in pathological human discs.

Material and methods Intervertebral disc cells were

extracted from coccygeal bovine discs and cultured for

3 months. Confluent cell preparations were stained for SA-

b-Gal (Fenton et al. 2001) at passages 0, 1, 2, 3 and 4.

Preparations were fixed in 3% paraformaldehyde in phosphate-

buffered saline (PBS) for 5 min at room temperature and

incubated for 24 h at 37 �C in SA-ß-Gal solution containing

1 mg/ml 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside

(X-Gal, Sigma, Poole, UK), 5 mmol/l potassium ferrocyanide,

5 mmol/l potassium ferricyanide, 150 mmol/l NaCl, 2 mmol/l

MgCl2 and 40 mmol/l trisodium citrate, titrated with

NaH2PO4 to pH 6.0. Lysosomal (nonsenescent) ß-galactosi-

dase activity was detected using the same solution but titrated

to pH 4.0. After staining, preparations were rinsed in ice-cold

PBS, dehydrated and mounted. Pathological human disc from

patients with disc herniations or discogenic back pain were also

stained for SA-b-Gal (immersing the tissue in the stain solution

overnight) then cryosectioning (10mm thick) and fixing. Articular

cartilage was studied for comparison (Price et al. 2002).

Results Cultured intervertebral discs demonstrated some

staining for SA-ß-Gal at all passages investigated, but there

was little change in staining with passage number. Cells in

most pathological human discs demonstrated staining for SA-

ß-Gal. Positive cells were seen more commonly in herniated

discs [7/9 (78%); mean age: 466 13] than in those removed



from patients with discogenic back pain [2/6 (33%); mean

age: 326 5].

Discussion To our knowledge, this is the first study of cell

senescence in intervertebral disc cells. The greatest level was

seen in tissue from herniated discs where cell cluster formation

and cell proliferation are common (but the mean age of the

herniation group was slightly higher than the discogenic back

pain group). However, for a tissue demonstrating such signifi-

cant age-related degenerative changes, there is surprisingly

little expression of SA-ß-Gal in comparison with that found

in other pathological cartilages. These preliminary data sug-

gest that, unlike the situation in osteoarthritis, early cell senes-

cence is not a major contributing factor in the pathogenesis of

disc degeneration.

Acknowledgement This project was funded by ‘EURODISC’

(QLK6-CT-2002-02582).

References

Fenton M. et al. (2001) Cellular senescence after single and

repeated balloon catheter denudations of rabbit carotid

arteries. Arterioscler. Thromb. Vasc. Biol. 21, 220–226.

Price J.S. et al. (2002) The role of chondrocyte senescence in

osteoarthritis. Aging Cell 1, 57–65.

PVA combined with 0.8% alginate: a potential3-dimensional vehicle to deliver and retain cellsduring autologous repair of connective tissues?

B. Gargiulo,* J. Menage,* H. Evans,*

J.P.G. Urban,† B. Caterson,‡ C. Curtis,‡

S.M. Eisenstein* and S. Roberts**Centre for Spinal Studies, Robert Jones and Agnes Hunt

Orthopaedic Hospital, Oswestry, Shropshire, UK; †University of

Oxford, Oxford, UK; ‡Cardiff School of Biosciences, University

of Cardiff, Cardiff, UK

Introduction Autologous chondrocyte implantation (ACI) is

routinely used for the repair of articular cartilage defects. A

similar method may be employed to treat degenerate interver-

tebral discs or other connective tissues. A system in which cells

could not only be delivered, but also retained would offer

advantages compared to ACI. Such a vehicle would also

allow a homogeneous distribution of cells throughout the

defect and enhance nutrient penetration to the seeded cells.

Materials and methods Bovine nucleus cells were isolated via

enzyme digestion and expanded in number to passage 3. The

cells were resuspended in 0.8% alginate and loaded into poly-

vinyl alcohol (PVA) cubes. These constructs were placed into a

solution of calcium chloride to ‘gel’ the alginate. Constructs

were cultured in DMEM + 10% FBS within 15-ml conical

tubes rotated at 37 �C for up to 28 days. Cell distribution/

morphology and proliferation were assessed on H&E and Ki-

67 stained sections, respectively. The re-expression of a disc

cell phenotype was assessed using toluidine blue staining and

immunohistochemistry (with antibodies to collagen types I, II,

IIA, VI and X and to the glycosaminoglycans, chondroitin-4-

and -6-sulfate and keratan sulfate. RT-PCR was performed

using oligonucleotide primers to collagen types I, II and X,

aggrecan, link protein and small leucine-rich PGs.

Results H&E staining of 10-mm thick cryosections revealed

an even distribution of loaded cells throughout the scaffold at

day 1 being maintained through to day 28. Toluidine blue

staining showed the presence of GAGs, increasing with time.

Ki-67 staining indicated that approximately 5% of cells were

proliferating at all time points. Immunohistochemistry demon-

strated the production of collagen types I, II, IIA, VI and X,

and the glycosaminoglycans, chondroitin-4-, -6-sulfates and

keratan sulfate. RT-PCR results showed mRNA expression

of fibromodulin throughout the experiment, lumican at days

14, 21 and 28. Types II and X collagen were present at days 21

and 28.

Discussion Combining 0.8% alginate with PVA retained

100% of the seeded cells and allowed an even distribution of

cells throughout the scaffold. The immunohistochemistry and

RT-PCR demonstrated that the system allowed the bovine

nucleus cells to express phenotypic markers expressed by disc

cells in vivo. These preliminary results indicate that the PVA/

alginate system could act as a suitable delivery device for cells

during autologous repair of the intervertebral disc or other

connective tissues such as meniscus.

Highly diverse heparan sulfate analoguelibraries: a novel resource for bioactivityscreening of proteins

Scott E. Guimond, Susannah J. Patey, Edwin

A. Yates and Jeremy E. TurnbullSchool of Biological Sciences, University of Liverpool,

Liverpool, UK



Introduction Fibroblast growth factors (FGFs) are a multi-

potent family of growth factors that are important for many

biological processes, including development and wound heal-

ing. Normal, protease sensitive, prion protein (PrPC) can be

converted to the protease resistant, infectious, form (PrPSc)

believed to be associated with the pathogenesis of transmissi-

ble spongiform encephalopathies. FGFs signal through a

family of tyrosine kinase receptors, the FGF receptors

(FGFR) with the aid of heparan sulfate (HS), while the role

of HS in the biology of PrPC is currently unknown, although

depleting cells of HS can prevent production of PrPSc. HS, or

its more highly sulfated relation heparin, can exert both

positive and negative regulatory activities on a particular

FGF-FGFR combination. The nature of this regulation is deter-

mined by the structure of the HS that binds to the proteins. This

structure is at least partially determined by the presence of

particular sulfate groups along the sugar backbone. Identifica-

tion of specific sulfate groups that can regulate the activity of

proteins has been a long-term goal in the field. Previously,

heparins that had been completely lacking sulfates at specific

positions were used to determine the binding and activity

requirements for a particular protein. However, this may not

necessarily allow for a full examination of the regulatory pro-

perties of HS. Here, we present a heparan sulfate analogue

library produced by the partial, combinatorial desulfation of

heparin. This library was the used to examine the structural

properties of heparin required for FGF-1 signalling through

FGFR2c as well as the interactions of HS with PrPC.

Materials and methods Porcine intestinal mucosal heparin

was subjected to chemical desulfation and enzymatic cleavage.

Polysaccharides and oligosaccharides derived from gel filtra-

tion chromatography and ion exchange chromatography were

tested for their ability to activate FGF signalling through FGF

receptors using a BaF3 assay system. Optical biosensors and

cell assays were used to study the interaction of PrPC with

chemically modified heparin.

Results This library possessed vastly more heterogeneity than

tissue heparan sulfates, allowing for more systematic screening

to help identify those minimal structural features associated with

activity. This library was used to examine the different structural

features in heparin that support FGF-1 signalling through

FGFR2, showing that HS activity was not strictly dependant

on size or charge. In addition, small, low-sulfated heparins

were found to interfere with the PrPC–heparin interaction.

Discussion This supports the idea that overall structural fea-

tures of the HS, rather than just the presence or absence of

specific sulfate groups is important for the regulation of pro-

tein activity. Future efforts will be focused on further subfrac-

tionating the library and identifying specific structural features

in HS that support FGF-1 activity through FGFR2 and other

FGFRs as well as the role of HS in the normal function of

prion diseases, which may allow for the generation of novel,

heparin-based, therapeutics.

Utilization of cartilage graft technology for thetesting of novel chondro-therapeutic agents

Amanda Hall,*Anthony Hayes,*Liesbeth Brown,†

Ross Tubo† and Bruce Caterson**Connective Tissue Biology Laboratory, University of Cardiff,

Cardiff, UK; †Genzyme Corporation, Boston, MA, USA

Introduction The aims of the current study were to (i) tissue

engineer a cartilage graft with structural and biochemical

properties of native articular cartilage in vivo, with potential

for use in cartilage repair technologies and (ii) utilize this

model as a test system to evaluate the efficiency of novel

therapeutics for future research into cartilage metabolism in

health and disease.

Materials and methods Articular cartilage was harvested

from hock joints of (young) 7-day and (old) 18-month bovine

sources. Cells were isolated by enzymatic digestion and seeded

at a range of cell densities (2, 4, 6, 8, 10 and 12 · 106 cells/

insert) into type II collagen-coated Millipore filter inserts and

cultured as described previously (Kandel et al. 1995). In order

to mimic a catabolic effect on cartilage, some samples were

treated with IL-1a (10 ng/ml) for 24 h in the absence or pre-

sence of experimental drugs. Proteoglycan (PG) release, detect-

able in the medium, was analysed by colorimetric assay

(Farndale et al. 1986). At the end of the culture period, cartil-

age grafts were fixed, sectioned and stained with Alcian Blue

or immuno-fluorescently labelled with a panel of monoclonal

antibodies recognizing several components of the graft extra-

cellular matrix.

Results Full-depth chondrocytes from both young and old

bovine sources produced a stratified hyaline tissue with dis-

tinct zones after 2 weeks in culture. These zones approximated

to the surface, middle and deep zones that characterize native

articular cartilage in vivo. Increased culture time and seeding

density produced cartilage of an increased thickness and

cellularity, respectively. Grafts produced from young cartilage

contained approximately 3 times more sulfated PG than grafts



produced from an old cartilage, indicating an increased matrix

secretion in these cultures. Histologically, the old grafts were

also thinner and more weakly stained with Alcian Blue, indi-

cating a lower sulfated PG content. Addition of IL-1a to the

cultures resulted in a dramatic PG release from the cartilage

grafts, manifest histologically as a loss of Alcian Blue staining

in the upper third of the cartilage tissue. Immunofluorescent

staining identified subtle changes in matrix composition and in

the structure and catabolism of matrix proteoglycans in

response to both IL-1a and the experimental drugs tested.

Discussion The grafts produced had many structural and

biochemical similarities to articular cartilage in vivo. These

grafts may better integrate with the host cartilage in cartilage

repair procedure. This culture system also provides ideal con-

ditions to analyse the response of engineered grafts to cata-

bolic factors that occur in the arthritic joint, along with ideal

conditions for research into drug therapies. Advantages of this

culture system, in comparison with an explant system, are that

effects can be analysed within a 24-h period. Future work will

include applying fatty acids, modified glucosamine and some

Asian herbal remedies to this culture system and analysing

their potential chondroprotective effects.

References

Kandel R.A. et al. (1995) Transplantation of cartilagenous tissue

generated in vitro into articular joint defects. Artif. Cells Blood

Substit. Immobil. Biotechnol 23, 565–577.

Farndale R.W. et al. (1986) Improved quantitation and discrimin-

ation of sulphated glycosaminoglycans by use of dimethyl-

methylene blue. Biochim. Biophys. Acta. 883, 173–177.

An interaction between opticin and heparansulfate may provide the molecular basis forvitreoretinal adhesion

V. John Hindson,* John T. Gallagher† and

Paul N. Bishop**Wellcome Trust Centre for Cell-Matrix Research, School of

Biological Sciences, University of Manchester, Manchester, UK;†Cancer Research UK Department of Medical Oncology,


Manchester, UK

Introduction Opticin is a member of the extracellular matrix

small leucine-rich repeat (SLRP) proteoglycan/protein family,

which was originally identified in the eye associated with the

collagen fibrils of the vitreous humour. A putative heparin/

heparan sulfate (HS) binding motif (RKERKRR) was identi-

fied at the N-terminus of human opticin, but this is absent in

the bovine form. Furthermore, the strength of attachment

between the vitreous and the retina was observed to be spe-

cies-dependent and related to the presence or absence of this

motif. We hypothesized that opticin cross-links the collagen

fibrils of the vitreous to HS proteoglycans in the inner limiting

lamina (a basement membrane on the inner surface of the

retina), contributing towards vitreoretinal adhesion.

Materials and methods Recombinant human and bovine

opticin were expressed in 293-EBNA cells and purified to

apparent homogeneity. Solid phase assays and surface plas-

mon resonance studies were used to characterize interactions

between immobilized heparin/HS and opticin.

Results Solid phase and BIAcore data revealed that human

opticin binds heparin/HS and binds to heparin with a dissocia-

tion constant of approximately 20 nM. By contrast bovine

opticin, which lacks the basic cluster, bound severalfold less

tightly. Competition studies with heparin oligosaccharides

indicated that the heparin/HS binding site is greater than 6

monosaccharides in length. Heparin, HS, chondroitin sulfate

A (CS-A), dermatan sulfate and hyaluronan all competed with

heparin for binding to human opticin but CS-C did not.

Discussion Work to date suggests that the N-terminal

sequence RKERKRR contributes significantly to the binding

of opticin to heparin/HS. Vitreoretinal adhesion plays a key

role in a number of eye diseases and inhibitors of the opticin–

HS interaction could be of therapeutic value.

The molecular evolution of the collagen X(C1q/TNFa) supergene family

Julie Huxley-Jones,* David L. Robertson† and

Raymond P. Boot-Handford**Wellcome Trust Centre for Cell-Matrix Research, University of

Manchester, Manchester, UK; †School of Biological Sciences,

University of Manchester, Manchester, UK

Introduction Collagen X is a nonfibrillar short-chain col-

lagen that contains a globular domain at the C-terminus

(NC1 domain) responsible for trimerization of the protein

(Marks et al. 1999). Homologous NC1 domains are found in

an extensive family of trimeric proteins (often referred to as



the C1q/TNF super-family) including C1q and TNF (Shapiro

& Scherer 1998). This family may have radiated during verte-

brate evolution (Kishore et al. 2002), but how and when the

family first evolved has not been determined. We report here

that Ciona intestinalis, a urochordate that represents one of

the closest invertebrate relatives of vertebrates, has two genes

containing the C1q/TNFa NC1 domain. Using these sequences

to root a detailed phylogenetic analysis of the supergene family

members found in mammals allows us to describe not only the

relationships between family members but also the temporal

order with which family members evolved in vertebrates.

Materials and methods A variety of human NC1 domain

amino acid sequences (e.g. type X collagen, TNFa, Clq)

were used to BLAST the Ciona genome. Two sequences

(Ciona 247 & 377) were identified. Sequences with homology

to the NC1 domains of the Ciona proteins, and human type X

collagen and TNFa were identified by BLAST using the non-

redundant protein and human genome databases. The NC1

sequences were aligned using ClustalX and trees produced by

three independent methods: Neighbor Joining, Bayesian and

Maximum Likelihood. Information on protein expression was

obtained from the scientific literature and EST databases.

Results Extensive BLAST analyses of the Ciona genome

identified two genes that contained domains with significant

sequence homology to the trimerizing NC1 domain of the

C1q/TNFa supergene family. The human genome contained

27 genes with such a domain and in addition, three chipmunk-

specific hibernation proteins were identified using the non-

redundant protein database. A striking feature of identified

genes is that most (22 out of 32), including one of the Ciona

genes (377), encode a collagenous domain just N-terminal to

the NC1 domain. Phylogenetic analyses demonstrated that

specific subgroupings or clades have evolved within this highly

divergent family including the TNF/emilin clade, the collagen

X/VIII clade, the C1q clade and the hibernation protein clade.

Comparisons of the domain structures of the encoded pro-

teins, and the exon structures encoding the NC1 domains,

strongly supports the majority of the phylogenetic groupings

of genes even where statistical support is not high. The major-

ity of the vertebrate genes encoding C1q/TNF NC1 domains

have lost the intron apparent in both Ciona genes although a

subset of vertebrate genes, including precerebellins, that phy-

logenetically cluster appear to have conserved this intron.

Discussion We have identified, for the first time, invertebrate

sequences from which the C1q/TNF supergene family of pro-

teins, including types VIII and X collagen have evolved and

demonstrated that this super-gene family has radiated exten-

sively during vertebrate evolution. The precise clustering of

genes, based on phylogenetic analyses and supported by the

overall gene and domain structures of the encoded proteins,

provides new insights into the evolution of the vertebrate

extracellular matrix, the versatility of the collagen triple hel-

ical domain and potential functions of novel genes

References

Kishore U. et al. (2002) Recent progress in the understanding of

the structure-function relationships of the globular head

regions of C1q. Immunobiology 205, 355–364.

Marks D.S. et al. (1999) Metaphyseal chondrodysplasia type

Schmid mutations are predicted to occur in two distinct three-

dimensional clusters within type X collagen NC1 domains

that retain the ability to trimerize. J. Biol. Chem. 274,

3632–3641.

Shapiro L. & Scherer P.E. (1998) The crystal structure of a

complement-1q family protein suggests an evolutionary link to

tumor necrosis factor. Curr. Biol. 8, 335–338.

The differentiation of ES cells into neuroecto-dermal precursors is associated with an increasein the levels and sulfation of heparan sulfateproteoglycans

Claire E. Johnson,* Marios P. Stavridis,† Brett E.

Crawford,‡ Valerie A. Wilson,† Jeffrey D. Esko,‡

Austin G. Smith,† John T. Gallagher* and

Catherine L.R. Merry**Cancer Research UK Department of Medical Oncology,


Wilmslow Road, Manchester, UK; †Institute for Stem Cell

Research, University of Edinburgh, King’s Buildings, Edinburgh,

UK; ‡Department of Cellular and Molecular Medicine,

Glycobiology Research and Training Center, University of

California at San Diego, La Jolla, CA, USA

Introduction Heparan sulfate (HS) is involved in determin-

ing a cell’s response to its local environment; helping to define

cell fate by acting as co-receptor for a variety of growth

factors/cytokines and modulating tissue patterning by binding

morphogens. The complex, tissue-specific structure of the HS

polysaccharide is thought to be tightly regulated by differential

expression of its biosynthetic pathway components.

Materials and methods We use directed differentiation

of embryonic stem cells (ESC) to investigate changes in HS



during neural lineage progression, a process dependent on

HS-dependent factors, such as FGF-4.

Results Using an HS-specific antibody, FACS analysis has

revealed a significant increase in HS as cells commit to a neural

lineage, whilst ICC analyses suggest that HS redistributes from

plaques on the cell surface. RT-PCR analysis of an extensive

range of proteoglycan biosynthetic enzymes/core proteins

revealed significant changes with differentiation, e.g. up-

regulation of syndecan 4. Confirmation of these data is being

sought by other methods, e.g. FACS. We also performed a

structural comparison of an ESC-derived HS with HS from a

differentiating culture. ESC-derived HS is poorly sulfated com-

pared with previously characterized HS from adult tissues.

Increases were seen in N- and O-sulfation patterns of HS

from differentiated cells.

Discussion This is the first investigation into the role of HS

in ESCs, and in neural differentiation in vitro. Differences

uncovered will be correlated with alterations in the respon-

siveness of cells to inductive factors, e.g. FGF4.

The effects of human intervertebral disc aggrecanon neuronal and endothelial cell growth

W.E.B. Johnson,* B. Caterson,† S.M. Eisenstein,*

D.M. Snow‡ and S. Roberts**Centre for Spinal Studies, Robert Jones and Agnes Hunt

Orthopaedic Hospital, Oswestry, UK; †University of Cardiff,

Cardiff, UK; ‡University of Kentucky, Lexington, KY, USA

Introduction The development of ‘discogenic’ low back pain

has been associated with increased nerve growth into degen-

erated intervertebral discs, particularly in neovascularized

areas. A marked loss of proteoglycans, particularly from the

disc’s inner regions, is also a marked feature of the degenera-

tive process. As proteoglycans from other tissues can have

inhibitory effects on nerve growth, we have hypothesized

that the alterations in proteoglycan content seen in disc degen-

eration may affect the growth of nerves and blood vessels into

the disc. To test our hypothesis, we have established cell

culture assays to determine the effects of human intervertebral

disc aggrecan, which forms the major proteoglyan found in the

disc, on neuronal and endothelial cell growth.

Materials and methods Aggrecan (A1D1 preparations) iso-

lated from the outer (anulus fibrosus, AF) and inner (nucleus

pulposus, NP) regions of human lumbar intervertebral discs

was incorporated into culture substrata, using methods pre-

viously described (Snow et al. 1990). Chick dorsal root ganglia

(DRG) and the cell line SH-SY5Y were used as models of nerve

growth. HMEC-1 and Eahy-926 cell lines were used as models

of endothelial cell growth.

Results Human disc aggrecan inhibited SHSY-5Y cell attach-

ment, SHSY5Y neurite outgrowth and induced sensory DRG

neurite growth cone turning in a concentration-dependent

manner. Sensory neurites emanating from DRG across permis-

sive substrates (collagen/laminin) were repelled by high (1 mg/

ml), but not low (0.01 mg/ml) concentrations of disc aggrecan

and became aligned to the aggrecan border. Disc aggrecan

similarly inhibited endothelial cell adhesion, cell spreading

and migration. HMEC-1 and Eahy926 cells migrated over

collagen substrates (type I) until they encountered disc aggre-

can, where they either stopped migrating or, more commonly,

changed their direction of movement and aligned to the aggre-

can border. In general, aggrecan isolated from the AF was

more inhibitory than that isolated from the NP. The inhibitory

effects were partially abrogated following enzymic deglycosy-

lation of the aggrecan, or if aggrecan was added in solution

(i.e. was nonsubstrate bound).

Conclusions This study provides evidence that disc aggrecan

inhibits neuronal and endothelial growth and migration and there-

fore supports a hypothesis that a loss of aggrecan from degenerated

discs predispose the tissue to vascular and neuronal invasion.

Reference

Snow D.M. et al. (1990) Sulfated proteoglycans in astroglial

barriers inhibit nerve growth in vitro. Exp. Neurol. 109,

111–130.

ADAMTS-4 activity on a proteoglycan vs. apolypeptide is controlled by the ancillarydomains

Gavin C. Jones,* Mireille N. Vankemmelbeke,†

Jan H. Verheijen,‡ Claudia DeDood,‡ Anthony

J. Day§ and David J. Buttle¶

*Rheumatology Research Unit, Addenbrooke’s Hospital, Hills

Road, Cambridge, UK; †Centre for Biomolecular Sciences,

University of Nottingham, Nottingham, UK; ‡Gaubius

Laboratory, Department of Biomedical Research, TNO

Prevention and Health, Leiden, The Netherlands; §MRC

Immunochemistry Unit, Department of Biochemistry, University

of Oxford, Oxford, UK; ¶Division of Genomic Medicine,

University of Sheffield, Children’s Hospital, Sheffield, UK



Introduction In combination with the catalytic domain, the

ancillary domains of the ADAMTS’ are proposed to regulate

activity via interactions with sulfated GAGs, the extracellular

matrix (ECM) and cell surface. Interactions with both GAGs

and the ECM have been attributed to the thrombospondin

(TSP) type 1 motifs and spacer region (Kuno and Matsushima

1998; Flannery et al. 2002). ADAMTS-1, -4 and -5, all undergo

cleavage within their respective spacer regions (Flannery et al.

2002;Rodriguez-Manzaneque et al. 2000; Georgiadis et al. 2002),

an event which has been reported to increase activity towards

the interglobular domain of aggrecan and decrease the heparin

affinity of ADAMTS-4 (Flannery et al. 2002; Gao et al. 2002).

Materials and methods V5- and His-tagged recombinant

human ADAMTS-4 constructs terminating after the catalytic

(nDTS), disintegrin-like (nTS), TSP (nS) or spacer region

(Full) were expressed in High-Five cells. Proteoglycanase activ-

ities of the resultant proteins were assayed with solution

digests of aggrecan and a polyacrylamide-entrapped aggrecan

particle assay. Proteolytic activity was measured using a novel,

nonglycosylated, reporter substrate assay.

Results All forms of ADAMTS-4 were active to varying

degrees in the reporter substrate assay. Digestion of aggrecan in

solution digests was apparent in all proteins with the exception

of the catalytic domain in isolation (nDTS). Activity towards

aggrecan decreased with increasing truncation of the protein.

Discussion Removal of the cysteine-rich-spacer domain and

further C-terminal truncations decrease the activity of

ADAMTS-4 towards aggrecan, whilst the proteolytic activity

remains intact. Cleavages releasing the ancillary domains of

ADAMTS’ may therefore alter the catalytic activity of these

enzymes against proteoglycans and also nonglycosylated poly-

peptides. More information is required about potential sub-

strates for the processed forms of ADAMTS-4.

References

Kuno K. & Matsushima K. (1998) ADAMTS-1 protein anchors at

the extracellular matrix through the thrombospondin type I

motifs and its spacing region. J. Biol. Chem. 273, 13912–

13917.

Flannery C.R. et al. (2002) Autocatalytic cleavage of ADAMTS-4

(Aggrecanase-1) reveals multiple glycosaminoglycan-binding

sites. J. Biol. Chem. 277, 42775–42780.

Rodriguez-Manzaneque J.C. et al. (2000) Characterization of

METH-1/ADAMTS1 processing reveals two distinct active

forms. J. Biol. Chem. 275, 33471–33479.

Georgiadis K. et al. (2002) ADAMTS-5 is autocatalytic at a

E753-G754 site in the spacer domain. Orthop. Res. Soc. Trans.

27, 167.

Gao G. et al. (2002) Activation of the proteolytic activity of

ADAMTS4 (aggrecanase-1) by C- terminal truncation. J. Biol.

Chem. 277, 11034–11041.

A novel keratanase-generated keratan sulfateantibody and its application to structuralanalysis of skeletal and corneal keratan sulfate

B.C. Kerr, C.E. Hughes and B. CatersonConnective Tissue Biology Laboratories, Department of

Bioscience, University of Cardiff, Cardiff, Wales, UK

Introduction The objective of this study was to make mono-

clonal antibodies specific for keratanase-generated neoepi-

topes in keratan sulfate (KS) and to use them along with

existing KS monoclonal antibodies (e.g. 5D4, IB4) to investi-

gate KS sulfation pattern motifs in connective tissue proteo-

glycans during development, ageing and disease.

Methods Bovine nasal cartilage aggrecan (BNC A1D1) was

trypsin digested, generating a range of GAG-peptide frag-

ments. The sample was then subjected to anion-exchange

and size exclusion chromatography to separate KS peptides

from CS attachment domain fragments. Fractions were ana-

lysed by Western blotting for positive immunoreactivity for

KS, then pooled and keratanase digested to generate ‘KS stub’

antigens. Immunization and fusions were carried out as pre-

viously described (Caterson et al. 1983; Hughes et al. 1992).

Screenings involved the use of a range of antigens; including

keratanase vs. keratanase II-digested bovine cartilage aggrecan

and bovine corneal KS-PGs. A new monoclonal antibody,

BKS-I, was identified that specifically recognized a keratanase-

generated neoepitope on both skeletal and corneal KS. This

novel monoclonal antibody was used along with existing KS

monoclonal antibodies 5D4 and 1B4 to investigate KS structure.

Results and discussion Bovine trypsin-generated aggrecan

KS-peptides were chondroitinase ABC treated and either

keratanase or keratanase II treated. The digests were run on

SDS-PAGE and immunolocated with monoclonal antibody

5D4 (that recognizes linear disulfated N-acetyl lactosamine

disaccharide-containing segments in KS) and the new ‘KS-stub’

monoclonal antibody BKS-I. Our results indicated that there

was reduced monoclonal antibody 5D4 immunostaining after

keratanase pretreatment. However, keratanase II digestion

completely removed all 5D4 structural epitopes. In contrast,

BKS-I showed no immunostaining on the untreated KS-peptides



but strong staining on keratanase treated samples and no staining

after keratanase II digestion. Similar patterns of immunoreactivity

were observed with Western blot analysis of untreated, keratanase

treated and keratanase II treated corneal KS-PGs.

Conclusion These data indicate that monoclonal antibody

BKS-I recognizes a nonreducing terminal neoepitope-containing

sulfated N-acetylglucosamine adjacent to a nonsulfated

lactosamine disaccharide. We also conclude that skeletal KS

must have a structure with four possible variations opposed to

the generic structures, proposed as being made of disulfated

disaccharides at the nonreducing end, followed by a series of

monosulfated disaccharides at the middle and nonsulfated

disaccharides nearer the linkage region. 5D4 staining,

observed after keratanase digestion, indicates that there must

be a minimum structure of a pentasulfated hexasaccharide

remaining on the KS chain ‘stubs’ near the linkage region of

skeletal and corneal KS. The BKS-I monoclonal antibody can

be used to demonstrate differential substitution of KS GAG

chains in the CS attachment region of cartilage aggrecan with

ageing. It has also proven useful for immunohistochemical

analyses identifying the sites of KS–PG association with col-

lagen lamellae of cornea.

References

Brown G.M. et al. (1998) Human aggrecan KS undergoes

structural changes during adolescent development. J. Biol.

Chem. 273, 26408–26414.

Caterson B. et al. (1983) Identification of a mAb that specifically

recognises corneal and skeletal KS. J. Biol. Chem. 258,

8848–8854.

Hughes C.E. et al. (1992) mAbs recognising protease-generated

neoepitopes from cartilage PG degradation. Applications to

studies of human link protein cleavage by stromelysin. J. Biol.

Chem. 267, 16011–16014.

Conformation of glycosaminoglycans by ionmobility mass spectrometry

Lan Jin,* Perdita E. Barran,* Malcolm Lyon† and

Dusan Uhrın**University of Edinburgh, School of Chemistry, Joseph Black

Building, Edinburgh, UK; †Cancer Research UK Department of

Medical Oncology, University of Manchester, Christie Hospital

NHS Trust, Manchester, UK

Introduction Glycosaminogycans (GAGs) are polysaccharides

found on most animal cell surfaces and extracellular matrices.

They constitute an important class of biologically active macro-

molecules that are implicated in the biological activity of mor-

phogens, growth factors, cytokines, chemokines and enzymes.

Intervention in signalling pathways they are involved in, repre-

sents a valuable therapeutical opportunity for cancer treatment,

wound healing and specific organ regeneration. GAGs are linear

polysaccharides with alternating uronic acid and hexosamine

residues. Their polysaccharide precursors are extensively mod-

ified (epimerization, N-deacetylation N-, O-sulfation) creating a

wide heterogeneity to their structures. The binding specificity of

GAGs is encoded in their primary structures but ultimately

depends on how this basis set of functional groups is presented

to a protein in three-dimensional space.

Methods As a part of our efforts to characterize the confor-

mation of GAGs, we present here our preliminary results of

conformational studies of heparin-derived oligosaccharides by

ion mobility mass spectrometry. This MS technique is

designed to study conformation of mass-selected gas-phase

ions. When ions drift through an inert gas under the influence

of a weak electric field, their arrival time depends on their

collisional cross sections. From this data, the experimental

cross sectional areas are easily derived. These are then com-

pared with those obtained from candidate geometries gener-

ated from molecular modelling, and hence conformation(s) of

studied compounds can be determined.

Results and discussion We have measured the cross sectional

areas of four heparin oligosaccharides: one disaccharide and three

tetrasaccharides. Amongst these are two fully sulfated species and

also two tetrasaccharides each with one sulfate group missing.

Our samples were prepared by enzymatic digestion of heparin

and contain a nonreducing terminal unsaturated glycosyluronic

acid ring. We employ the GLYCAM 2000 force field (Woods)

and the AMBER (Case et al. 2002) suite of programs to model the

gas-phase conformations of the oligosaccharides. We have gener-

ated AMBER parameters for sulfate groups using ab initio

calculations and are in the process of generating structures for

which we calculate the cross sectional areas using a projection

approximation (Wyttenbach et al. 1997). We present a compar-

ison of experimental cross sections with those of calculated

models. We also compare them with cross sections calculated

using oligosaccharide fragments extracted from X-ray and

NMR structures of heparin oligosaccharides – protein complexes.

References

Case D.A. et al. (2002) AMBER 7. San Francisco, CA: University

of California.

Woods R.J. [retrieved from http://glycam.ccrc.uga.edu].



Wyttenbach T. et al. (1997) Effect of the long-range potential

on ion mobility measurements. J. Am. Soc. Mass Spectr. 8,

275–282.

Identification of major heparin-binding proteinsin plasma using electrophoresis and massspectrometry

Ruth Killeen,* Robin Wait,† Shajna Begum,†

Elaine Gray* and Barbara Mulloy**National Institute of Biological Standards and Control, South

Mimms, Potters Bar, Herts., UK; †Kennedy Institute of

Rheumatology Division, Faculty of Medicine, Imperial College

London, London, UK

Introduction The anticoagulant glycosaminoglycan heparin

binds to the plasma protein antithrombin, potentiating its

activity and so inhibiting the coagulation cascade. Among

other therapeutic applications, it is used to prevent and treat

deep vein thrombosis. As heparin binding is an attribute

shared by many proteins other than antithrombin, sequestra-

tion by plasma proteins could potentially reduce the available

concentration of therapeutically administered heparin, and

thus modulates its biological activity. Although the heparin-

binding ability of some abundant plasma proteins is well

characterized, the binding potential of the vast majority of

plasma constituents is unknown. We have therefore initiated

a systematic study of the heparin-binding plasma proteome,

using affinity capture on immobilized heparin, followed by

salt elution and protein identification by mass spectrometry.

Materials and methods Five platelet-depleted human plasma

pools were fractionated by affinity chromatography on

HiTrap heparin columns. Bound material was eluted stepwise

with 0.5 M, 0.7 M, 1.2 M and 2.2 M NaCl. The approximate

protein concentration of each plasma pool and fraction was

determined by optical density reading at 280 nm. The fractions

were separated by SDS-PAGE and protein bands were visual-

ized by staining with Coomassie blue or silver nitrate. Stained

bands were excised, digested in gel with trypsin, and the

liberated peptides were analysed by tandem electrospray

mass spectrometry using a Micromass Q-Tof. Proteins were

identified by correlation of the uninterpreted tandem mass

spectra to entries in the UniProt database.

Results Over 50 proteins were identified in whole plasma.

No systematic differences in protein composition were

observed between the different plasma pools. Heparin-binding

proteins recovered in salt washes of 0.7 M or above included

fibronectin, histidine-rich glycoprotein, apolipoproteins, com-

plement factors C3 and C4b, and antithrombin III. Antithrom-

bin was the major protein eluting at 1.2 M NaCl or above.

Discussion The rationale of this study was to investigate

plasma for the presence of proteins potentially able to compete

with antithrombin for therapeutically administered heparin.

Of over 50 different plasma proteins identified in this prelim-

inary study, only antithrombin remained bound to heparin

above 1.2 M NaCl. We anticipate that use of larger volumes

of albumin- and immunoglobulin-depleted plasma will reveal

low concentrations of additional high-affinity binding pro-

teins, but these are unlikely to bind significant amounts of

heparin. However, in the present study, we detected several

abundant proteins, which bound to immobilized heparin at

0.7 M NaCl, and these may reduce the bioavailability of

heparin by nonspecific binding.

Heparan sulfate is essential for neuronmigration and axon outgrowth in Caenorhabditiselegans

Tarja Kinnunen, Joanne Townsend and Jeremy

TurnbullSchool of Biological Sciences, University of Liverpool, Liverpool,

UK

Introduction The importance of heparan sulfate proteogly-

cans (HSPGs) has been highlighted by a number of human

genetic disorders associated with mutations in genes encoding

for HSPG protein cores or biosynthetic enzymes required for

heparan sulfate assembly.

Materials and methods We have studied the role of HS

and HSPGs in the nervous system development using the nema-

tode Caenorhabditis elegans as a model organism. Our focus

has been on the single C. elegans homologues of the vertebrate

syndecans, sdn-1 and heparan 2-O-sulfotransferase, hst-2.

Results and discussion C. elegans syndecan, sdn-1 and

heparan 2-O-sulfotransferase, hst-2, expressions coincide

with the start of morphogenesis in mid-embryonic stages

after most cell divisions have occurred. Sdn-1 is expressed

primarily in neuronal cells, whereas hst-2 is more widely

expressed in neurons, hypodermis (epidermis), pharynx and

the gonad leader cells (DTC). A deletion mutant of sdn-1

which lacks the putative heparan sulfate (HS) attachment



sites has specific neuron migration and axon pathfinding

defects. Hst-2 mutants have partially overlapping yet diverse

neuronal migration defects. In both sdn-1 and hst-2 mutants,

the serotonergic hermaphrodite-specific neurons (HSNs) fail

to migrate to their correct position in the vicinity of the vulva

and hence fail to innervate the vulval muscles causing sdn-1

and hst-2 mutants to become egg-laying defective (egl) and

accumulate embryos within the mother. Canal-associated neu-

rons (CANs) that, like the HSNs, have a stereotyped long-

distance postembryonic migration pattern also fail to migrate

to their correct position in sdn-1 mutants, whereas in hst-2

mutants the CANs migrate normally. These results suggest

that specific 2-O-sulfation of sdn-1 HS is essential for HSN

migration, whereas it is dispensable for CAN migration.

Hst-2 mutants have additional cell migration defects that

are not detected in sdn-1 mutants suggesting that other HSPGs

such as perlecan/unc-52 and glypican/gpn-1 may be respon-

sible for these defects.

A method for fluorescent HPLC analysis of CS/DSglycosaminoglycans

Robert LauderDepartment of Biological Sciences, Lancaster University,

Lancaster, UK

Introduction Chondroitin and dermatan sulfate (CS/DS) show

considerable species, tissue, age and pathology-related structural

heterogeneity. In addition within chains their sulfation patterns

are not random. To elucidate their structure/function relation-

ships, methods for complete characterization are required.

A method for keratan sulfate (KS) fingerprinting (Whitham

et al. 1999) has been extended for the linkage, repeat and chain

cap regions of CS/DS, including the acquisition of CS/DS ratios.

Methods Chains were depolymerized by 1 U/100 mg of

chondroitin ABC endolyase or ACII lyase at 50 mg/ml in

0.1 M ammonium acetate, pH 8 and for 15 h at 37 �C. Alter-

natively, chains were de-N-acetylated by hydrazinolysis at

98 �C for 24 h at 10 mg/1 ml in anhydrous hydrazine with

100 mg/ml hydrazine sulfate. Then, they were depolymerized

by 3.9 M sodium nitrite/0.28 M acetic acid at 0 �C for 4 h.

Unreduced chains were released from their protein core in

0.5 M LiOH at 4 �C for 12 h.

Materials were fluorescently labelled with 2-AA as pre-

viously described (Whitham et al. 1999) and characterized by

HPAEC using a Dionex AS4-SC column at 50 �C and 2 ml/min

with constant 15% 1 M NaOH. A 5-min isocratic period of

85% H2O/0% 2 M NaCl was followed by a linear gradient of

0–30% 2 M NaCl over 60 min. The oligosaccharides were

monitored using a lex of 315 nm and a lem of 400 nm.

Results and discussion This method resolves repeat region

di-, tri- and tetrasaccharides, capping oligosaccharides and

linkage regions and can be used to profile known and

unknown oligosaccharides. Unsulfated oligosaccharides elute

between 2 and 10 min, monosulfated between 7 and 30 min,

disulfated between 25 and 40 min and trisulfated between 49

and 54 min. Allied with data on size, oligosaccharide identifi-

cation is facilitated.

Hydrazinolysis/nitrous acid depolymerization of CS/DS

chains results in disaccharides from CS with 4- or 6-sulfation

and from DS with 4-sulfation which retain IdoA and GlcA

structures and which can be distinguished chromatograph-

ically. The methodology was used to examine CS/DS from

shark, whale, bovine and human tracheal, articular and menis-

cal cartilage and cornea.

Tracheal cartilages show predominantly 4-sulfation with

porcine sources being more highly 4-sulfated (ca. 75%) than

bovine (ca. 65%). Articular cartilage comprises mainly

6-sulfated GalNAc (ca. 95% in the adult), while adult meniscal

cartilage shows only ca. 85%. Tracheal and articular cartilage

aggrecan showed no IdoA; however, it represented ca. 20% of

the uronic acids of bovine meniscal aggrecan, showing the

presence of DS.

Corneal CS/DS has a very low level of 6-sulfation

(<ca. 5%) but shows an equal abundance of unsulfated and

4-sulfated residues and contains high levels, ca. 50%, of IdoA

residues.

Shark cartilage shows ca. 75% 6-sulfation with signifi-

cant levels of uronic acid 2-sulfation found only between a

4-sulfated residue and a 6-sulfated residue, reflecting sulfo-

transferase specificity. Shark cartilage contains modest (ca.

1–5%) levels of DS that may be contaminants of preliminary

isolation.

This method extends a previous method to now allow the

complete examination of KS, CS and DS chains by a single

rapid chromatographic method.

Reference

Whitham K.M. et al. (1999) An improved method for the

structural profiling of keratan sulfates: analysis of keratan

sulfates from brain and ovarian tumors. Glycobiology 9,

285–291.



New insights into the alternating sequences ofheparan sulfate

Kevin J. Murphy,*‡ Malcolm Lyon,* Catherine

L. R. Merry,* James E. Thompson,†

Ian S. Roberts† and John T. Gallagher**Cancer Research UK Department of Medical Oncology,


Manchester, UK; †School of Biological Sciences, University of

Manchester, Manchester, UK

Introduction The sulfated domains (S-domains) within HS

are thought to be the primary binding site for protein ligands

(Gallagher and Lyon, 2000). Studies on the binding of multi-

meric chemokines to HS have suggested that N-acetylated

sequences (NAc-domains) act to space the S-domains correctly

so binding can occur at more than one HS binding site within a

protein (Lortat-Jacob et al. 1995; Stringer et al. 2002). So far,

little functional significance has been ascribed to either alter-

nating N-sulfate/NAc or lone N-sulfate types of sequence,

although alternating sequences are thought to occur at the

borders between S-domains and NAc-domains, forming transi-

tion zones between the two. Existing scission techniques used to

excise oligosacchrides from the intact chain for structural and

functional analysis disrupt the transition zones (heparinase III),

or the S-domains (heparinase I) or both (low pH nitrous acid).

Materials and methods Radiolabelled HS was isolated from

cultured 3T3 fibroblasts HS. Its patterns of degradation using

bacteriophage K5 lyase as well as conventional heparinases

I/III and nitrous acid were compared. K5 lyase-resistant frag-

ments were characterized by size exclusion chromatography

and strong-anion exchange HPLC.

Results The work presented demonstrates how the enzyme

K5-lyase specifically cleaves only within NAc-domains requir-

ing sequences of at least five consecutive N-acetylated dis-

accharides within the HS chain. The enzyme leaves intact

S-domains together with their flanking transition zones in situ.

Discussion The characterization of oligosaccharides gener-

ated by K5 lyase cleavage of the intact HS chain provides new

insights into the domain structure of HS.

References

Gallagher J.T. & Lyon M. (2000) Molecular structure of heparan

sulphate and interactions with growth factors and morphogens.

In: Proteoglycans: Structure, Biology and Molecular Interac-

tions. pp. 27–59 (ed. R.V Iozzo) New York: Marcel Dekker

Inc.

Lortat-Jacob H. et al. (1995) Molecular organization of the

interferon ~aa-binding domain in heparan sulphate. Biochem. J.

310, 497–505.

Stringer S.E. et al. (2002) Characterization of the binding site on

heparan sulphate for macrophage inflammatory protein 1a.

Blood 100, 1543–1550.

Site-specific N-linked glycosylation analysis onthe human salivary mucin MUC5B using PrecursorIon Discovery on the CAPLC Q-TOF system

Mark Ritchie,* David Knight,† Sara Kirkham†

and David Thornton†

*Waters MS Technology Centre, Manchester, UK; †Wellcome

Trust Centre for Cell-Matrix Research, University of Manchester,

Manchester, UK

Introduction Mucins are large highly O-glycosylated oligo-

meric glycoproteins that form the polymer matrix of mucus,

which forms part of the innate immune system. MUC5B, the

predominant gel-forming mucin in saliva, is around 600 kDa

in mass before glycosylation. O-linked glycosylation is abun-

dant in defined repeated domains and may account for 80% of

the observed mass of the secreted. Not much is known about

the N-linked glycosylation, although there are at least 34

potential sites. Despite little being known about the structure,

N-glycosylation has been shown to be important but not

essential for mucin synthesis (Perez-Vilar & Hill 1999).

Here, we aim to study the N-linked glycosylation site specifi-

cally using mass spectrometry

Materials and methods A sample of purified human MUC5B

mucin was digested with trypsin and separated by size exclu-

sion chromatography (Thornton et al. 1999). The previously

uncharacterized fractions were analysed by online LC Q-Tof

MS in order to characterize any N-glycosylated peptides pre-

sent. Glycopeptide discovery was steered by the Precursor Ion

Discovery function, which detects potential glycopeptides by

looking for sugar oxonium ions during intermittent high-

collision energy surveys. Candidate ions are then selected for

MSMS, which reveals both the carbohydrate content and the

glycosylation site location.

Results Glycopeptides were located and site specifically ana-

lysed. Data suggests N-linked occupancy of numerous sites,

and microheterogeneity was observed.

‡Present address: School of Applied Science, University of Huddersfield,

Huddersfield, UK.



Discussion This is the first study to investigate the N-glycans

on oligomeric gel-forming mucins. The N-glycans are localized

in the N- and C-terminal portions of the MUC5B polypeptide

in areas which have sparse O-glycosylation. These regions of

the molecule are known to be involved in the disulfide bond-

mediated oligomerization of the mucin monomers. Further-

more, they may be potential sites for interaction with other

components of saliva that are important for biological func-

tion. It may be that they have a role in stabilizing these

important regions of the molecule, or they may be sites of

interaction with other salivary components.

References

Perez-Vilar J. & Hill R.L. (1999) The structure and assembly of

secreted mucins. J. Biol. Chem. 274, 31751–31754.

Thornton D.J. et al. (1999) Salivary mucin MG1 is comprised

almost entirely of different glycosylated forms of the MUC5B

gene product. Glycobiology 9, 293–302.

Studying the role of heparin in the formation ofFGF1-FGFR2 complexes using gel chromatography

Christopher J. Robinson,* Nicholas J. Harmer,†

Tom L. Blundell† and John T. Gallagher**Cancer Research UK Department of Medical Oncology,


Manchester, UK; †Department of Biochemistry, University of

Cambridge, Cambridge, UK

Introduction The fibroblast growth factors (FGFs) are a

family of at least 24 proteins which have diverse roles in

regulating cell proliferation, migration and differentiation dur-

ing embryogenesis, angiogenesis and tissue repair. Four FGF

receptor tyrosine kinases (FGFRs) have been identified, each

of which binds a specific subset of the FGF family. FGF bind-

ing induces dimerization of the FGFRs leading to their activa-

tion through transphosphorylation. Heparin or heparan

sulfate (HS) is essential for the activity of the FGFs, because

they form an integral part of the signalling complex on the cell

surface. Our group has been using gel filtration FPLC to study

the role that heparin plays in the formation of FGF1-FGFR2

complexes.

Methods A Superdex 200 HR 10/30 FPLC column was cali-

brated using protein standards and used to estimate the size of

protein complexes which form in the presence of heparin. The

column was run in 150 mM NaCl, 50 mM HEPES, pH 7.2

buffer and complexes could be visualized by protein UV absor-

bance at 280 nm.

Results In agreement with published data (Pellegrini et al.

2000), we found that 2 : 2 : 1 complexes formed spontaneously

in solution between FGF1, FGFR2IIIc and heparin decasac-

charides (dp10). Although heparin dp6–8 fragments could

bind FGF1 and FGFR2 simultaneously, only dp10 and larger

fragments efficiently formed 2 : 2 : 1 complexes. These com-

plexes appear to be relatively stable, as they remain associated

following prolonged incubation with heparinase. FGF1 was

able to bind heparin saccharides strongly even in the absence

of receptor. In contrast, FGFR2 had very little affinity for either

FGF1 or heparin dp12 separately, but bound efficiently to the

FGF1-heparin complex. Dimerization of FGF1 appears to be

essential for subsequent dimerization of FGFR2. Selectively

desulfated heparin saccharides could only bind FGF1 singly

and formed 1 : 1 : 1 complexes with FGFR2, which did not

then associate with one another.

Discussion Our data supports the idea that heparin plays a

dual role in the formation of FGF1 signalling complexes, firstly

by mediating interactions between FGF1 and FGFR2 and sec-

ondly by acting as a template for the dimerization of FGF1-

FGFR2 subunits. This technique could be easily adapted to

study other heparin/HS-mediated ligand–receptor interactions

and could provide new insights into the mechanisms of forma-

tion of multiprotein signalling systems on the cell surface.

Reference

Pellegrini L. et al. (2000) Crystal structure of fibroblast growth

factor receptor ectodomain bound to ligand and heparin.

Nature 407, 1029–1034.

The analysis of the fibroblast growth factorligand-receptor complex using a quartz crystalmicrobalance-dissipation

Tim Rudd,*,† Laurence Duchesne,*

David G. Fernig* and Richard J. Nichols†

*School of Biological Sciences, University of Liverpool, Liverpool,

UK; †Department of Chemistry, University of Liverpool,

Liverpool, UK

Introduction The quartz crystal microbalance-dissipation

(QCM-D) is a device that measures both the mass of adsorb-

ates upon a surface and the energy dissipated by the surface.



The latter quantity, known as dissipation (D), gives infor-

mation about the viscoelastic properties of species adsorbed

to the surface leading to insights into their shape and

conformation.

Methods The fibroblast growth factor (FGF) receptor-ligand

signalling complex is simulated within the QCM-D by the

immobilization of heparin-derived oligosaccharides to a

gold-sputtered QCM-D crystal surface, and then the surface

is interrogated with various growth factors and a recombinant

receptor. Fibroblast growth factor receptor 1 (FGFR1) extra-

cellular domain (immunoglobulin loops II and III and the acid

box of the FGFR1IIIc), FGF-1, FGF-2, FGF-7 and hepatocyte

growth factor/scatter factor (HGF/SF) all bind to immobilized

heparin-derived oligosaccharides with different changes in D

relative to mass for the same surface.

Results For example, complexes of FGF-1 and octasaccharide

are, on a mass basis, far more dissipative than complexes of

HGF/SF. One interpretation of these results is that octasacchar-

ides are relatively flexible, or at least contain hinge regions and

that different FGFs have a preference for binding above the

hinge (so more dissipative) or on the hinge (so more rigid).

Binding of FGFR1 produced complexes that were less dissipa-

tive than the FGF-1 complexes. Intriguingly, deglycosylation of

the FGFR1 increased the amount of binding to heparin tetra-

saccharide, and this complex on a mass basis is more dissipative.

Conclusion By using the QCM-D, we are able to gain insights

into novel aspects of these protein-glycosaminoglycan complexes.

Purification and characterization of endocan(endothelial cell-specific molecule-1), acirculating proteoglycan involved in tumourprogression and inflammatory diseases

Stephane Sarrazin,* David Bechard,* Philippe

Lassalle,† Malcolm Lyon,‡ Arnaud Scherpereel,†

Hugues Lortat-Jacob,§ Taoufik Ouatas* and

Maryse Delehedde**ENDOTIS Pharma, Therapeutic Glycomic Department, Parc

Eurasante, Loos, France; †INSERM U416, Institut Pasteur de

Lille, Lille, France; ‡Cancer Research UK Department of Medical

Oncology, University of Manchester, Christie Hospital NHS

Trust, Manchester, UK; §Institut de Biologie Structurale,

CEA-CNRS UMR 5075, Grenoble, France

Introduction By virtue of the multiplicity of their protein-

binding partners (e.g. growth factors, cytokines/chemokines),

proteoglycans have been shown to be involved in the regulation

of a large number of pathophysiological processes including

cancer and inflammatory diseases. We have studied and char-

acterized endocan, also called endothelial cell-specific molecule-1

(ESM-1), which represents a new group of circulating pro-

teoglycans. Endocan is mainly expressed by endothelial cells but

also by epithelial cells from lung, gut and kidney. Structurally,

endocan is constituted of a mature polypeptide of 165 amino

acids with a single glycosaminoglycan chain covalently linked

to the serine at position 137 (Bechard et al. 2001).

Methods and results We showed that human umbilical vein

endothelial cells expressed endocan specifically with a single

chain of dermatan sulfate (DS) as glycosaminoglycan moiety.

As shown by surface plasmon resonance, the DS chain directly

interacts with cytokines and growth factors including hepatocyte

growth factor/scatter factor and could be responsible for endo-

can’s biological activities. Human embryonic kidney 293 cells,

which have been genetically engineered to overexpress endocan,

induce tumour growth when injected subcutaneously in SCID

mice. Moreover, inflammatory cytokines such as TNF-a and

IL-1 have been shown to increase the synthesis and the secretion

of endocan from human umbilical vein endothelial cells.

Conclusion These results suggest that circulating levels of

endocan may represent a novel marker for cancer and inflam-

matory diseases. Further studies on its GAG structure could

help us to better understand the biological activities of endo-

can and to design future glycomic-based therapies.

Reference

Bechard D. et al. (2001) Endocan is a novel chondroitin sulfate/

dermatan sulphate proteoglycan that promotes hepatocyte

growth factor/scatter factor mitogenic activity. J. Biol. Chem.

276, 48341–48349.

Investigating the role of heparin sulfateproteoglycans in hereditary multiple exostoses(HME) tumourigenesis

Z.M. Scholefield,* J.r Hassell,† L. West,†

P. Govindraj,† J.T. Hecht,‡ G. Rushton,*

R. Oldershaw,§ T. Hardingham,§ C.L.R. Merry*

and J.T. Gallagher**Cancer Research UK Department of Medical Oncology,


Manchester, UK; †Shriners Hospital for Children, Tampa, FL,

USA; ‡Department of Pediatrics, University of Texas-Houston

Medical School, Houston, TX, USA; §School of Biological

Sciences, University of Manchester, Manchester, UK



Introduction Heparin sulfate (HS) has long been implicated

in the bone deformity hereditary multiple exostoses (HME),

and it is now clear that HME is associated with mutations in

the HS biosynthetic genes EXT1 and EXT2. Interestingly,

HME is also associated with an increased risk of chondro-

and osteo-sarcomas.

Methods and results Preliminary analysis of GAG samples

purified from fibroblasts of both HME and isolated non-HME

exostoses patients reveal a dramatic shift in the ratio of

CS : HS, with the HME and isolated cases having a much

higher proportion of CS relative to normal controls. This is

true in the case of both shed and cell surface material but is far

more extreme in the latter, with the HS reducing from

approximately 45% in the controls to less than 10% in

HME patients. Initial analysis also reveals shortened chain

length within these samples; indeed they often have two popu-

lations of chains present. Simple analysis of the total disac-

charide composition of these samples demonstrates no

significant differences against controls. However, detailed

analysis of the subpopulations of chains (as determined by

chain length) within these samples as well as cartilaginous

samples from exostoses patients may provide further insight

into the changes that occur within the biosynthetic pathway

following disrupted EXT function.

We are also carrying out immunocytochemistry with a vari-

ety of HS-specific antibodies with the aim to further investi-

gate normal HS structure and localization. This is being

carried out on human primary chondrocytes isolated from

normal patients and also adult mesenchymal stem cells as

they undergo differentiation into chondrocytes. HS has been

identified in both these cell types, and it is hoped that the

manipulation of these cells through RNAi of different enzymes

of the HS biosynthetic pathway will provide a suitable model

for studying what changes may occur in cellular HS structures

over the initial differentiation process in the growth plate.

Discussion Together, these investigations should provide a

good model to allow us to determine the role of HS in chon-

drocyte differentiation and maturation in both normal and

diseased states.

Structural and functional studies onhyaluronan-protein aggregates produced in vitro

Nicholas T. Seyfried,* Gillian F. McVey,* Jayesh

Dudhia† and Anthony J. Day**MRC Immunochemistry Unit, Department of

Biochemistry, University of Oxford, Oxford, UK;

†Department of Veterinary Basic Sciences, Royal Veterinary

College, London, UK

Introduction The proteoglycan aggrecan forms link protein-

stabilized complexes with hyaluronan (HA) that provide cartilage

with its load bearing properties. Aggrecan binding to both HA

and cartilage link protein are mediated through its N-terminal

G1-domain, which is composed of an immunoglobulin fold and

two contiguous Link modules. Similar aggregates (potentially

containing members of the new link protein family) in which

the proteoglycan versican substitutes for aggrecan are likely to

contribute to the structural integrity of many other tissues.

Materials and methods Human cartilage link protein (LP1)

and the G1-domains of human aggrecan and versican were

cloned and expressed in Drosophila S2 cells. The recombinant

proteins were purified from media using a combination of ion-

exchange chromatography and reverse-phase HPLC. A micro-

titre plate assay was used to determine the minimum length of

HA necessary for high affinity binding; the interaction of

biotinylatd-LP1 to immobilized G1-domains was also deter-

mined. The ability of the recombinant proteins to form com-

plexes in solution was assessed with HA oligosaccharides of

defined length (i.e. HA10-HA40) using a combination of gel

filtration and protein cross-linking experiments. Cross-linking

products were identified by in-gel digestion with trypsin fol-

lowed by MALDI-TOF mass spectrometry.

Results SDS-PAGE showed the three recombinant proteins

to be highly pure and that they all contained N-linked glyco-

sylation; amino acid sequencing revealed that aggrecan has

undergone some differential processing at its N-terminus.

LP1, G1-aggrecan and G1-versican were all functionally active

showing HA-binding properties that are similar to those

described for the native proteins; e.g. HA10 was the minimum

length that could compete effectively for polymeric HA bind-

ing. Gel filtration and protein cross-linking studies reveal that

LP1 and G1-aggrecan interact in both the absence and pre-

sence of HA. Interestingly, LP1 and G1-versican did not inter-

act in the absence of HA yet could be cross-linked in the

presence of HA24 but not HA20 or shorter oligosaccharides.

However, when G1-versican was immobilized on a microtitre

plate, it was found to interact with biotinylated-LP1, which is

consistent with recent reports in the literature. Treatment of

G1-aggrecan or G1-versican with PNGase F (which removes

most of the N-linked glycans from these proteins) did not

affect the formation of aggregates in the presence of HA,

indicating that glycosylation is not required for structural

integrity or functional activity. Surprisingly, for G1-aggrecan



HA of approximately 40 sugar units appears to be the min-

imum size that can fully accommodate two protein molecules,

whereas in the case of versican this occurs with HA between

HA20 and HA24.

Conclusion Aggrecan and versican appear to have distinct

properties both with regard to their interaction with LP1 and

HA, consistent with the hypothesis that different hyaladherins

bind and capture distinct conformations of HA (Day and

Sheehan 2001). Work is now in progress to produce aggrecan/

LP1 and versican/LP1 ternary complexes for crystal studies.

Reference

Day A.J. & Sheehan J.K. (2001) Hyaluronan polysaccharide chaos

to protein organisation. Curr. Opin. Struct. Biol. 11, 617–622.

Anti-inflammatory actions of green teacatechins and ligands of peroxisomeproliferator-activated receptors

M. Trekli, D. Buttle and F. GuesdonDivision of Genomic Medicine, University of Sheffield, Sheffield,

UK

Introduction Green tea catechins and peroxisome prolifera-

tor-activated receptor (PPAR) agonists have been shown to

reduce inflammation associated with collagen-induced arthri-

tis in mice (Haqqi et al. 1999; Cuzzocrea et al. 2003) and to

inhibit other markers of inflammation (Vankemmelbeke et al.

2003; Sabatini et al. 2002; Singh et al. 2003; Jiang et al. 1998).

We are investigating the mechanisms by which catechins and

PPAR agonists interfere with the signalling pathways of the

pro-inflammatory cytokines, IL-1 and TNF.

Materials and methods Epigallocatechin gallate (EGCG),

epicatechin gallate (ECG), epigallocatechin (EGC), epicatechin

(EC) and fenofibrate were from Sigma Aldrich, Poole, UK.

Rosiglitazone was from Cayman Chemical, Ann Arbor, MI,

USA. Cytokines and IL-8 ELISA were from NIBSC. IL-6 ELISA

from eBiosciences (San Diego, CA, USA). Cells used were

primary fibroblasts from inflamed periodontal tissue or osteoar-

thritic chondrocytes from discarded tissue following surgery.

Results We show the significant suppression of cytokine-

induced IL-6 and IL-8 release by EGCG, which is dose- and

cell-type dependent. The other three catechins have a much

lesser effect. Preliminary results show that PPAR agonists also

interfere with TNF-induced IL-8 production.

Discussion Green tea has a long history of human consump-

tion, and epidemiological studies have shown that it can reduce

inflammatory diseases (Greenwald et al. 2002). Our results

confirm that EGCG has the strongest anti-inflammatory effect,

compared with the other three catechins. They also indicate that

the mechanism of action of EGCG is cell-type dependent. This

could be because the compound targets components of pro-

inflammatory signalling pathways whose expression is

restricted to certain differentiated cell types. Alternatively, the

different sensitivities of cells to EGCG could reflect differences

in their ability to metabolize the compound. Further experi-

ments are needed to discriminate between these hypotheses.

References

Cuzzocrea S. et al. (2003) Reduction in the evolution of murine

type II collagen-induced arthritis by treatment with rosiglita-

zone, a ligand of the peroxisome proliferator-activated

receptor. Arthritis Rheum. 48, 3544–3556.

Greenwald P. et al. (2002) Micronutrients in cancer chemopre-

vention. Cancer Metastatis Rev. 21, 217–230.

Haqqi T.M. et al. (1999) Prevention of collagen-induced arthritis

in mice by a polyphenolic fraction from green tea. Proc. Natl.

Acad. Sci. U.S.A 96, 4524–4529.

Jiang C. et al. (1998) PPAR-gamma agonists inhibit production of

monocyte inflammatory cytokines. Nature 391, 82–86.

Sabatini M. et al. (2002) Effects of agonists of peroxisome

proliferator-activated receptor gamma on proteoglycan degrad-

ation and matrix metalloproteinase production in rat cartilage

in vitro. Osteoarthritis Cartilage 10, 673–679.

Singh R. et al. (2003) Epigallocatechin-3-gallate selectively

inhibits interleukin-1-induced activation of mitogen activated

protein kinase subgroup c-Jun N-terminal kinase in human

osteoarthritis chondrocytes. J. Orthop. Res. 21, 102–109.

Vankemmelbeke M.N. et al. (2003) Selective inhibition of

ADAMTS-1, -4 and -5 by catechin gallate esters. Eur. J.

Biochem. 270, 2394–2403.

Epitope mapping of proteins for heparin bindingsites

Romain R. Vives,* Elodie Crublet,* Jean-Pierre

Andrieu,* Jean Gagnon† and Hugues

Lortat-Jacob**Institut de Biologie Structurale, CNRS-CEA-UJF, Grenoble,

France; †Laboratoire d’Immunologie, Hospital SUD, Grenoble

cedex 9, France

Introduction Heparan sulfate (HS) is a cell surface and

basement membrane, sulfated polysaccharide involved in a



huge array of biological processes, such as cell prolifera-

tion, chemo-attraction, inflammation, matrix assembly,

embryo development or viral attachment. These multiple

functions stem from HS ability to bind and modulate the

activity of a large number of proteins. The interaction of

HS with its ligands is primarily, although not exclusively,

of an electrostatic nature and involves the recognition of

basic domains on the protein by specific motifs of the

polysaccharide. Importantly, subtle differences in HS struc-

ture have been shown to dramatically affect the polysac-

charide activity, as observed for HS regulation of bFGF

signalling. Therefore, the identification of both protein

and saccharide structural determinants implicated in HS/

protein binding is critical for understanding the biological

role of the interaction. In this study, we have developed a

simple technique enabling the identification of HS-binding

sites on proteins.

Materials and methods Briefly, proteins were immobilized

on heparin beads, using an adapted protocol of a zero-length

two-step cross-linking method and submitted to proteolytic

digestion. Peptides retained on beads, i.e. containing amino

acids involved in heparin binding, were then identified by solid

phase N-terminus protein sequencing performed directly on

the heparin beads.

Results This technique was used to analyse a number of HS

protein ligands, including the envelope protein gC of a herpes

virus (pseudorabies) and the chemokine RANTES. Results

obtained pinpointed residues known to be critical for HS

binding and thus validate the method.

Conclusion This technique constitutes a novel tool that

should greatly facilitate HS binding site cartography of the

ever-expanding family of heparin-binding proteins and pro-

vides an attractive alternative to more complex approaches

such as site-directed mutagenesis.

Increased turnover of proteoglycan aggregatesin tendon vs. cartilage

A.D. Waggett, S.G. Rees and B. CatersonConnective Tissue Biology Labratories, Cardiff University School

of Biosciences, Museum Avenue, Cardiff, UK

Introduction Although the function of proteoglycans (PGs)

within the tendon extracellular matrix are not fully under-

stood, changes in their turnover have been associated with

tendinopathies (Riley et al. 1994). In contrast to cartilage,

aggrecanases are constitutively expressed and active in tendon

(Rees et al. 2000), indicative of a high rate of aggrecan turn-

over. Clinical trials investigating the use of active site MMP

inhibitors have been confounded by side effects, which involve

tendonitis and ‘musculoskeletal syndrome’. Such side effects

may relate to nonspecific inhibition of tendon aggrecanases

required to maintain normal metabolic homeostasis. The pur-

pose of this study, therefore, was to compare the rate of turn-

over of tendon and cartilage PGs derived from the same joint

and to determine the effect of MMP inhibitors (actinonin and

marimastat) on aggrecan catabolism.

Materials and methods Deep digital flexor tendon explants

from compressed and tensional regions were dissected from

young and mature bovine. Explants were precultured and

then cultured for a further 4 days with or without marimastat

(0–2 mM) or actinonin (0–200 mM). PG and lactate quantifica-

tion, Western blot analysis of degradation products and RT-

PCR analyses were performed. In a separate experiment for

measurement of PG turnover, explants were set up as

described above and then pulse chase labelled with [35S]

sulfate. The rate of turnover of 35S-labelled PGs from the

matrix of tendon (and articular cartilage obtained from the

same animal) was subsequently calculated from the amount

of 35S-labelled macromolecules appearing in the medium

each day and that remaining in the matrix of explants at the

termination of culture.

Results PG turnover (presumably predominantly aggrecan)

was markedly higher in tendon vs. cartilage. This difference was

apparent in tendons from all regions and ages. Both marimastat

and actinonin inhibited aggrecanase-mediated PG catabolism in

both tendon and cartilage explants. As expected, mRNA expres-

sion for the aggrecanases, MMPs and TIMPs was unaffected by

the addition of these inhibitors to the culture medium.

Discussion Aggrecan turnover in tendon is higher than that

of articular cartilage, which may be attributed to distinct

physiological properties of this PG in tendon. Importantly,

immunohistochemical staining for aggrecan in tendon indi-

cates its presence in between collagen fibres and fibril bundles

(Vogel et al. 1999), and thus aggrecan aggregates may dissi-

pate resultant compressive loads by resisting the flow of

water in these locations. In addition, aggrecan may facilitate

the sliding of fibrils during the small amount of elongation

of the tendon whilst under tension. Thus, the half-life of

tendon aggrecan is significantly reduced because it constantly



participates in repeated resistance to compression. Our data

also demonstrates that both marimastat and actinonin can

inhibit aggrecanase-mediated PG catabolism in tendon cultures.

This suggests that the occurrence of ‘musculoskeletal syndrome’

in clinical trial patients may be due to the fact that these

inhibitors affect the activity of aggrecanases in tendon, thus

preventing them from playing their normal role in tendon

aggrecan turnover and consequently perturbing normal physio-

logical function.

References

Rees S.G. et al. (2000) Catabolism of aggrecan, decorin and

biglycan in tendon. Biochem. J. 350, 181–188.

Riley G.P. et al. (1994) Glycosaminoglycans of human rotator

cuff tendons–changes with age and in chronic rotator cuff

tendonitis. Ann. Rheum. Dis. 53, 367–376.

Vogel K.G. et al. (1999) Biology of the Synovial Joint. Harwood

Academic Publishers, pp. 301–313.

Analysis of CD44 hyaluronan-binding domainmutants by NMR

A.J. Wright,*† S. Banerji,‡ M. Noble,†

D.G. Jackson,‡ I.D.Campbell† and A.J. Day†

*MRC Immunochemistry Unit, University of Oxford, Oxford,

UK; †Department of Biochemistry, University of Oxford, Oxford,

UK; ‡MRC Human Immunology Unit, University of Oxford,

Oxford, UK

Introduction Hyaluronan (HA) is a ubiquitous high molecu-

lar mass glycosaminoglycan composed of a repeating disac-

charide. CD44, the major cell surface receptor for HA, has a

HA-binding domain (CD44_HABD) at the N-terminus of the

protein, the 3D structure of which has been determined by

both NMR and X-ray crystallography (Teriete et al. 20004);

NMR spectra collected on the protein in complex with HA

oligosaccharides has allowed us to predict how they may

thread across the interaction surface. Amino acids previously

implicated in HA binding include R41, Y42, R78 and Y79

(Peach et al. 1993; Bajorath et al. 1998), which form a cluster

on the surface of the Link module-like region (Teriete et al.

2004), as well as residues in the C-terminal extension (R150,

R154, K158 and R162) (Peach et al. 1993). The position of the

putative-binding residues in the C-terminal segment, and

NMR data, led to the hypothesis of two modes of HA

binding (Teriete et al. 2004). Here, this hypothesis is tested

by NMR studies of single-site mutants in the context of the

CD44_HABD construct.

Materials and methods Four mutants of CD44_HABD were

made, each with a single residue substitution (R150A, R154A,

K158A and R162A). These constructs were expressed as 15N-

labelled proteins in Escherichia coli, refolded and purified to

homogeneity. 1H–15N HSQC spectra were acquired on the

mutants in the presence of varying concentrations of HA

hexasaccharide (HA6) and compared to the wild-type con-

struct to determine changes of protein fold and ligand binding.

Results The mutants R150A, R154A and K158A have simi-

lar HSQC spectra to wild-type CD44_HABD except for local

chemical shift perturbations around the altered residue. Con-

versely, the R162A mutant has widespread chemical shift

differences compared to wild-type indicating that this muta-

tion disrupts the fold. On binding HA6, the R150A, R154A

and K158A mutants all experience shift perturbations similar

to that seen with the wild-type protein.

Discussion The interaction of HA6 with wild-type

CD44_HABD has been found to cause a significant conforma-

tional change in the protein (Teriete et al. 2004). The results here

indicate that this ligand-induced rearrangement can also occur in

the R150A, R154A and R158A mutants. Therefore, these muta-

tions do not seem to affect the binding of CD44 to HA6. It has

been shown previously that the R162A mutation has reduced

affinity for HA compared to wild-type protein (Peach et al.

1993). This loss of function may be due to the perturbation of

the protein fold, and it is possible therefore that R162 does

not participate directly in binding. Work is in progress to test

the functional activity of these CD44_HABD mutants using

ELISA-like assays and to investigate their chemical shift

perturbation in the presence of longer oligosaccharides.

References

Bajorath J. et al. (1998) Identification of CD44 residues

important for hyaluronan binding and delineation of the

binding site. J. Biol. Chem. 273, 338–343.

Peach R.J. et al. (1993) Identification of hyaluronic acid binding

sites in the extracellular domain of CD44. J. Cell Biol. 122,

257–264.

Teriete P. et al. (2004) Structure of the regulatory hyaluronan

binding domain in the inflammatory leukocyte homing receptor

CD44. Mol. Cell. 13, 483–496.



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