Annals of the Rheumatic Diseases 1993; 52: S22-S29

Role of protein glycosylation in immune regulation

Elizabeth F Hounsell, Michael J Davies

Most cell surface and secreted proteins areglycosylated - that is, they have one or moreoligosaccharide chains covalently attached tospecific amino acids. These oligosaccharidesusually make up a significant amount of thehydrodynamic mass of the molecules and alsohave effects on protein conformation, surfaceexpression, stability, circulating half life,activity, and antigenicity. In addition, oligo-saccharide sequences are themselvesrecognised as antigens and as ligands forcarbohydrate binding proteins (lectins). Theimmune system exemplifies the diversity ofoligosaccharide structure and function, whichthis review will aim to communicate. Inparticular, the relevance of oligosaccharideheterogeneity, antigenicity, and immuneregulatory activity in autoimmunity andmicrobial pathogenesis will be considered.Firstly, a general description of the structureand three dimensional arrangement of proteinglycosylation will be given. Then, we describethe structure of the major glycoprotein effectormolecules involved in immune regulation andconcentrate on the role of T cell -y 8 cells intuberculosis and rheumatoid arthritis (RA).

General description ofproteinglycosylation (also see Glossary)Protein glycosylation is of two major typescalled N- or 0-linked depending on the linkageof the oligosaccharide chain throughasparagine (NH2) or serine/threonine (OH),respectively. N-linked chains typically have apentasaccharide core of mannose (Man) andN-acetylglucosamine (GlcNAc) residues withadditional mannose residues (high mannosechains) or backbone galactose-N-acetyl-glucosamine sequences with and without chainterminating sialic acid residues (complexchains). Hybrid chains are defined as thosehaving some high mannose branches and somecomplex-type sequences. The core andbackbone residues of complex chains arevariously decorated with blood group andrelated antigenic sequences involving substitu-tion with fucose, (Fuc), N-acetylgalactosamine(GalNAc), and galactose (Gal).' 2 Thepresence of the different chains can bedistinguished not only by chemicalcomposition but also by their susceptibility toenzyme digestion - all are released by peptide-N-glycosidase F, whereas endoglycosidase Honly releases high mannose chains.

0-linked glycosylation discussed in thisarticle is characterised by core regions based onN-acetylgalactosamine linked to protein, withgalactose, N-acetylglucosamine, or N-acetyl-galactosamine attached. 1- Extension of thechains is by substitution patterns similar to

those of the outer branches of N-linkedglycosylation (although there are differences).In both chains the diversity of oligosaccharidesequences is manifest in the large number ofways the monosaccharides can be linkedtogether (that is, through 1-4 hydroxyl groups,with and without branching, in (x or 3configuration, etc). Each linkage has aparticular set of allowed conformations insolution which provide specific orientations offunctional groups for molecular recognition.Such epitopes usually extend over a tri- toheptasaccharide sequence, but topographicalepitopes caused by folding back of longcarbohydrate chains onto themselves or ontoprotein are possible. In addition, the oligo-saccharide cores of 0-linked chains are in closeassociation with the protein backbone, whichstrongly influences protein conformation andoligosaccharide-protein antigenicity.' Oftenmultiple 0-glycosylation sites are clustered inone region of the protein to accentuate theseconformational effects (for example, CD8discussed below).

N-linked chains are in general lessstereochemically restricted around the protein-oligosaccharide linkage than their 0-linkedcounterparts, and, in addition, haveconsiderable flexibility at branch points withinthe chain. The oligosaccharide moieties,therefore, sample a large amount ofconformational space, but in some cases theymay be restrained by interaction with theprotein backbone or adjacent oligosaccharidesattached to the same protein. The paradigm forprotein-to-N-glycosylation interaction is theFc region of IgG where electron density foroligosaccharides has been shown by x raycrystallography which can be interpreted asconstraints by the protein to impose a singleconformational state.' In other glycoproteinsthat have been characterised by x raycrystallography the oligosaccharide conform-ation could not be discerned at high resolution- for example, studies of the major histocom-patibility complex (MHC) glycoproteinmolecule HLA-A2.6 For still furtherglycoproteins, crystals of sufficiently highquality for x ray crystallography are not formedunless some of the glycosylation is modified.This can be for the reasons already mentioned- that is, large hydrodynamic volume (inparticular when multiple glycosylation ispresent); effects on protein conformation; or,inherent oligosaccharide flexibility. Anadditional factor, discussed next, is themicroheterogeneity of oligosaccharide struc-ture at each glycosylation site within a protein.Because of the difficulties of their visualisationby x ray studies the characterisation of protein

Clinical ResearchCentre, Watford Road,Harrow, MiddlesexHAl 3UJ, UnitedKingdomE F HounsellM J DaviesCorrespondence to:Dr Hounsell.

S22

on Septem

ber 8, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.52.S

uppl_1.S22 on 1 M

arch 1993. Dow

nloaded from

Role of protein glycosylation in immune regulation

glycosylation has largely relied on enzymatic,gel, and chromatographic methods of analysiswith, when possible, high resolution physi-cochemical techniques (in particular, massspectrometry and nuclear magnetic resonancespectroscopy).24 7

Microheterogeneity of oligosaccharidesequences of glycoproteinsAll glycoproteins exhibit heterogeneity suchthat they exist as a population having aspectrum of oligosaccharide sequences (glyco-forms). Variation occurs in the number andlength of branches (or antennae) leading fromthe cores, together with alterations in sequenceand peripheral substitutions. Of relevance tothe present article is the documentation of theglycoforms of IgG where a large number ofdifferent biantennary N-linked chains were

found, including some having short chainsexposing GlcNAcp residues to which Galresidues and additional substitutions are

normally linked.8 The presence of these chains,termed Galo is suggested to be a marker of RAthat may be functional by reducing theintramolecular oligosaccharide-to-proteininteraction discussed above or by effectingbinding to GlcNAc, specific endogenouslectins, or both. This binding would have to bespecific to the GlcNAcP1-6/4/2Man sequenceas GlcNAc, 1-3Gal and GlcNAccx1-4Gal are

found quite commonly as chain terminatinggroups in mucms.-The studies quoted in reference 8 were

carried out on IgG pooled from human serum.From other studies it is known thatglycoproteins from single donors can also showconsiderable heterogeneity - for example,more than 250 different glycoforms from theTamm-Horsfall proteins of the urine of one

donor.9 There is also considerable variation incell-cell glycosylation - for example, inglycoproteins from single cell lines.'0 Thestructural changes in IgG are also mirrored incell studies from patients with RA" or from thelpr autoimmune mouse model.'2 These studiesprovide support for the contention thatparticular clones of B cells can be stimulatedto express immunoglobulins of a restrictedphenotype.'3 Since the original observations ofGalo in rheumatoid patients8 several otherimmune disorders, granulomatous-typediseases such as tuberculosis, and age matchedcontrols have shown the prevalence of this typeof IgG glycosylation."1 Although, in general,there is a large variability in the glycosylationof glycoproteins in normal and diseased states,the Galo phenotype remains relativelyrestricted to IgG. The relevance of thisobservation within the context of thecomplexity of autoimmunity remains obscure.This review explores other areas where protein

glycosylation may play a part in immuneregulation.

Direct role ofprotein glycosylationIn addition to the heterogeneity in the types ofglycoprotein chains discussed above, there are

further glycosylation patterns found in, forexample, parasite and mammalian glycophos-phatidylinositol lipid anchors'4 and bacterialglycoconjugates, including cell wall peptido-glycans, capsular polysaccharides, and0-antigen-type specific lipopolysaccha-rides.'5 16 These molecules are highlyantigenic, but in the case of lipopoly-saccharides and peptidoglycan are more likelyto exert their arthritogenic affect by mechan-isms involving direct mitogenicity.'5 Themammalian proteoglycans are an additionaltype of glycosylation which needs to bediscussed in this context. One of the initialresponses in the rheumatoid joint is breakdownof the proteoglycans which, together withcollagen, make up cartilage. Hyperimmun-isation with host proteoglycans will lead toexperimental arthritis. ' Proteoglycans areclassically very high molecular weightmolecules of different uronic acid-hexosaminerepeating disaccharide sequences havingmultiple variable sulphation.'8 Relatively shortregions of proteoglycan sequences, however,can also be found on membrane-typeglycoproteins - for example, the invariantchain of class II MHC'9 and lymphocyteantigen CD44.2" Therefore besides directdamage to the joint, the enzymes induced inarthritis which degrade proteoglycans may alsodirectly affect T cell function. Proteoglycansare also common constituents of secretorygranules of a variety of haemopoietic cells.2' Inmast cells evidence is accumulating for theirfunction during degranulation in controllingthe release of mast cell proteases,22 which arethe mediators of allergic inflammatoryreactions. In addition, proteoglycans atendothelial cell membranes are necessary foran intact microvasculature, the breakdown ofwhich is an essential component of therheumatoid process.23

Other glycoproteins are being increasinglyimplicated in the inflammatory process - inparticular, the recruiting of neutrophils to areasof endothelial cell damage. Studies have shownthat particular oligosaccharide structures on asubset of neutrophil glycoproteins arerecognised by the selectin ELAM- 1 induced onactivated endothelial cells.24 The majoroligosaccharide structure involved, called sialylLewis X, belongs to a family of previouslycharacterised oligosaccharide differentiationantigens25 which have now found a role incellular interactions. These interactions areinfluenced by cytokines such as tumournecrosis factor.26 Other cytokines, theinterleukins IL-1 and IL-2, have been shownto have lectin properties and respectively bindto Tamm-Horsfall glycoprotein27 (also calleduromodulin when found in the urine ofpregnant females with more glycosylation thanthe Tamm-Horsfall glycoprotein discussedabove) and high mannose oligosaccharides.28This may explain the well known immuno-suppressive effect of the oligosaccharides ofTamm-Horsfall glycoprotein/uromodulin andalso of the high mannose chains of yeast. Asecond mechanism for the immunosuppressiveeffects of polysaccharides of microbial origin is

S23

on Septem

ber 8, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.52.S

uppl_1.S22 on 1 M

arch 1993. Dow

nloaded from

Hounsell, Davies

their uptake by macrophages and disruption ofthe normal paths of antigen presentation.29Mention should also be made here of (a) thelectin properties of IgD30; (b) the stronghomologies between a rat IgE binding proteinand galactose binding proteins such as CBP35and Mac-2 macrophage antigen31; and (c) theglycosylation inhibiting factor, which alters theglycosylation of IgE binding proteins causingselective suppression of IgE synthesis.32

Effect of glycosylation on immune proteinfunctionIn addition to the specific functions ofoligosaccharides in immune regulation, glyco-sylation has several regulatory effects onprotein function and recognition. Theoligosaccharide chains of glycoproteins tend tocover a large surface area inhibiting immunerecognition and processing of the underlyingprotein. Many of the effector molecules inimmune regulation, including the immunoglo-bulins mentioned above, are glycosylated. Thecytokine receptors so far characterised areglycoproteins, as are some of the cytokinesthemselves.33 Oligosaccharides are known toinfluence the conformation of the protein to

Antigen bindingpocket -4

which they are attached and this is particularlytrue of the multiple 0-glycosylation found, forexample, on the receptor for IL-234 and onCD8. The first evidence for the importance of0-glycosylation in lymphocyte antigens wasreported for CD45.35 For CD8 it has beenshown36 that 0-glycosylation must be removedbefore crystallisation can take place for x raycrystallography studies. Interestingly, therelated molecule CD4, which has a roleequivalent to that of CD8 in their respectivebinding to class I and class II MHCmolecules,37 has a very different glycosylationpattern with no reported 0-glycosylation buthaving four possible N-linked sites, theoccupancy of which is a prerequisite for correctfolding and membrane expression.38 MHCmolecules are themselves glycosylated and thiscan influence antigen presentation.39 In fig 1we have chosen to illustrate the relative sizesand orientation of a single oligosaccharidechain as present on the human class I MHCmolecule HLA-A2. It depicts the minimumsized N-linked complex chain which in thenative molecule could have additionalbranches and peripheral substitution. Asdiscussed, most glycoproteins have more thanone glycosylation site. Figure 2 presents a

Oligosaccharide

Figure 1 A computergraphics molecular model ofMHC class I HLA-A2 ar ta3 domains (270 amino acids) takenfromBrookhaven files ofcrystallographic studies carried out by Bjorkman et al.6 The protein carbon backbone is accentuated witha ribbon. The a, and a2 region a helices surround the antigen binding pocket. A disialyated biantennary nonasaccharideis attached at the consensus N-linked glycosylation site, Asn 86 (top right), at the end of the a, a helix. This represents one

ofmany possible solution conformers of the oligosaccharide chain which will explore space within a dome of the dimensionsshown. Modelling was carried out using Biosym software on a silicon graphics IRIS workstation.

S24

on Septem

ber 8, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.52.S

uppl_1.S22 on 1 M

arch 1993. Dow

nloaded from

S25Role ofprotein glycosylation in immune regulation

CD4 CD8Of

28 1

117-1180119@

126*130-

133*137 *

-SH-SH

TCRaf

75 P

4 80

145 >41194120

124179 >

41274128

190 N4141

226 P

415

4125

4161

Figure 2 The glycosylation patterns of the human T cell glycoproteins CD44, CD4, CD8 (ao8 heterodimer), and the T cellreceptor (HPB-MLT a chain, HPB-ALL ,B chain). Consensus N-glycosylation sites are denoted by *, characterised0-glycosylation sites by *, aind potential proteoglycan attachment sites by U. SH represents free cysteines capable offorming interchain disulphide bridges and -S-S represents interchain disulphide bridges.

synopsis of the oligosaccharide chains ofCD44, CD4, CD8 aot, and the T cell receptor(TCR) a,B chains showing their variousglycosylation patterns taken from references20, 36, 38, and 40-43.

Type 1 T1

A more complicated pattern of cell specificglycosylation of T cell yb receptors hasemerged (fig 3), which we discuss here in fullto illustrate the type of diversity encountered,together with the evidence implicating this T

ype 2abc Type 2bc

* 132

179 t

4195

249 ,

265 I271 1

280 N

I-SH-SH--SHSH

106

4132

140

4195

193 N

-SH 209l215k224k

Y

Figure 3 The diversity of yb T cell receptor structures; glycosylation sites are classified as infig 2. 111 represents the Cyl C2

exon; M represents the C-y2 C2 exon copy a; 3 represents the Cy2 C2 exon copy b; * represents the C-y2 C2 exon copy c.

CD44

425

457

410041104 120

273 l

300Q

4254

180 U190 U231 U

257 U

Lull-

202 >

257>26310

272k

Liz

111

4132

4195

- SH

SH-SHSH-SH

r/

8

-SH-SH--SH-SH

__

'Y

on Septem

ber 8, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.52.S

uppl_1.S22 on 1 M

arch 1993. Dow

nloaded from

Hounsell, Davies

cell subset in the aetiology of RA andtuberculosis.

T cell -y receptor structureThe TCR 8 chain is a polypeptide of 40kilodalton size with two N-linked glycosylationsites (one high mannose oligosaccharide andone complex oligosaccharide, as determined byendoglycosidase H and peptide N-glycanase Fdigests).44 In addition, there are two intra-molecular disulphide bridges and potential forone intermolecular disulphide bridge.44 4 In Tcell lines y chains exhibit much more structuraldiversity, being expressed as one of threeforms. All three forms have two intramoleculardisulphide bridges and at least two potentialN-glycosylation sites. The type 1 receptor wasfirst isolated from a peripheral bloodlymphocyte cell line.45 This form expresses twoglycoforms of the y chains, a 40 and a 36kilodalton form, both forms containing fourN-linked glycosylation sites.46 Treatment withendoglycosidase H reduced both glycoforms toa core peptide of 31 kilodaltons.45 Thisindicates differential glycosylation of the twopeptides, notably in the distribution of highmannose and hybrid oligosaccharides. Thetype 1 receptor uses the Cy1 gene. Within this,one copy of the C2 exon is expressed,containing two consensus N-glycosylation sitesand a cysteine residue. This cysteine is able tocrosslink with the cysteine of the 8 chainforming a disulphide bridge.44 It is this featurewhich is the main distinguishing characteristicof the type 1 receptor.

In addition to the type 1 form, two non-intermolecular disulphide linked forms of thereceptor have been described.44 45 The 8 chainsare similar to those expressed by the type 1receptor, but the -y chains show significantdiversity in their constant regions.44 Both formsuse the C-y2 gene for their constant region. The2abc form expresses three copies of the C2exon (copies a, b, and c). The resultingpolypeptide has a size of 55-60 kilodaltons,which reduces to 40 kilodaltons on endogly-cosidase H treatment, again showing differ-ential glycosylation and the probable use of allfive consensus N-glycosylation sites.The second non-disulphide linked form of

the receptor (type 2bc) expresses two copies ofthe C2 exon (copies b and C).44 This results ina glycosylated peptide of 40 kilodaltons, whichreduces to 35 kilodaltons, on endoglycosidaseH treatment. The amino acid sequence givesa theoretical non-glycosylated size of 34-8kilodaltons, which together with tunicamycindata (an inhibitor of N-linked glycosylation)shows the glycosylation to be essentially allN-linked.44 The 5 kilodalton loss in molecularweight indicates that unlike the type 2abcreceptor only two of the six possibleglycosylation sites are used. The differences in

the number of glycans added can be explainedby the fact that the protein encoded by the C2aexon copy has an altered secondary or tertiarystructure making more of the 2abc y chainconsensus N-glycosylation sites accessible.47

Expression of the type 1 receptor ispredominant in peripheral blood lymphocytes,

although if the type 2 receptors are expressed,types 1 and 2 are usually found in equalproportions.47 This diversity ofT cell receptorsis unique to y8 T cells and as yet no distinctfunctional roles have been attributed to eitherTCR type, though a report has suggesteddifferent cytoskeletal organisation andmorphology between the Cy 1 and Cy2forms.49

Like o43 TCRs, ry8 TCRs are associated withthe CD3 glycoprotein to facilitate cellsignalling and activation. The CD3 'y8Epeptide cores are the same for both T cellsubsets (c43 and y8).f0 However, glycosylationof the CD38 subunit is different. In ot3 T cellsthe CD38 subunit carries one high mannoseand one complex oligosaccharide,5' whereas iny6 T cells both oligosaccharides are fullyprocessed to the complex form.50 y6 CD3seems to increase intracellular calcium ontriggering and is a substrate for protein kinaseC activation.52 53 Thus if the differentglycosylation is important it may function inthe regulation of T cell signalling via the -yTCR/CD3 pathway.

Role ofTCR y8 cells in antigen andsuperantigen recognitionHeat shock proteins are the major antigensdescribed to be recognised by -y8 T cells. Inboth a live and heat killed Mycobacteriumtuberculosis infection model, -yb T cellsproduced interferon -y, granulocyte-monocytecolony stimulating factor, IL-3, and tumournecrosis factor Ol.5 The nature of the dominantantigen or antigens recognised remains to becharacterised fully as up to 400 differentcomponents may be involved,55 but themycobacterial 65 kilodalton heat shock protein(hsp65) is a major contender56 57 and, inparticular, a peptide corresponding to residues181-195.58 59 Heat shock proteins are a highlyconserved set of proteins with both bacterialand mammalian homologues. The 60kilodalton family (including M tuberculosishsp65) is thought to influence the folding andtrafficking of mitochondrial proteins.606'The 65 kilodalton heat shock protein has

been described as a possible antigen in severalautoimmune disorders. It has been suggestedto be a putative yb T cell ligand on Daudicells67 and a ligand on B cells in lupusnephritis.63 Rheumatoid arthritic synovial fluid-y8 T cells reactive to M tuberculosis were firstisolated in 1989,64 with one clone reacting tothe hsp65 of M bovis-BCG. An earlier ratmodel of adjuvant arthritis had shown theantigenicity of hsp65 peptide 180-188,65though this epitope is only present in thebacterial protein and not its mammalianhomologue. More recently, both -yb and ot3 Tcell clones have been isolated, which arestimulated by both mycobacterial and humanhsp65, supporting the possibility of hsp65involvement in autoimmune disorders.66 67Data for hsp65 as a major antigen of RA arenot entirely conclusive.68 A recent review hasshown that recombinant hsp65 fromEscherichia coli also contained small quantitiesof E coli derived antigens.69 Thus stimulation

S26

on Septem

ber 8, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.52.S

uppl_1.S22 on 1 M

arch 1993. Dow

nloaded from

Role of protein glycosylation in immune regulation

of rheumatoid T cells by the recombinanthsp65 may in fact be by contaminating E coliantigens. Coupled with this, a second grouphas found that highly purified M bovis-BCGhsp65 is poorly stimulatory, leading theauthors to conclude that hsp65 is not a majorantigen for rheumatoid synovial fluid T cells.70The role of hsp65 in RA is thus not clear cutand it may be that hsp65 continues thedegenerative disease process after stimulationby a different antigen.7' It is of interest herethat heat shock protein may mimic the MHCin a similar manner to that proposed for HIVgp 1 20.72 73

No alternative mycobacterial antigens ofpossible relevance for human RA have yet beencharacterised. Mycoplasmal mitogens havebeen proposed in murine models.74 7Superantigen stimulation has been used toexplain the increased usage ofVP 14 ot3 T cellsin RA.76 In addition, the superantigenstaphylococcal enterotoxin D has been shownto stimulate CD4 V16 (o3 T cells to producerheumatoid factors.77 There is also evidencethat superantigens may stimulate -y6 T cells inRA: Vy982 T cell clones from rheumatoidarthritic synovial fluid have been shown torecognise a short tetanus toxin peptide in thecontext of MHC class II78; this Vy982 subsetmay be stimulated by tne acetone precipitablefraction of M tuberculosis.78 79 There is alsoevidence for clonal expansion of V-y9 T cells inthe synovial fluid of patients with RA.80

Similarly, in M tuberculosis infection there isincreasing evidence that hsp65 is not a major-y T cell antigen, and indeed there is someevidence that it may be a poor M tuberculosisantigen in general.82 The major subset of y5 Tcells which expands in response to Mtuberculosis is Vy982, which predominantlyexpress the C-y1 form of the TCR discussedabove.83 84 The same V y9 subset has beenshown to be stimulated by the superantigen,staphylococcal enterotoxin A,85 which requiresthe presence of the MHC class II. This isdirectly analogous to the activation of ot3 cellsby superantigens.86 A second VWy9-seekingsuperantigen has been isolated from Mtuberculosis, 6 87 again requiring MHC class II.The antigen seems to be a low molecularweight, protease resistant ligand which loses itsantigenicity on incubation with the plantlectins UEA1, SBA, and DBA. Thus it hasbeen postulated that the active components areglycosylated determinants having chainterminating ot-linked fucose and GalNAca(1-3)GalNAc for which these lectins are specific.87This is important not only from the point ofview ofM tuberculosis infection but is the firsttime a possible non-protein superantigen hasbeen described.The mechanism by which antigens are

presented to -yi T cells remains largelyunresolved. Conflicting reports exist on therequirement for P2 microglobulin and henceclass I restriction of -y8 T cells,88 89 though therequirement may depend on the T cell typestudied. Restriction by non-classical class Imolecules including Qa-1, has been shown.90The initial discovery of -yb T cells in

rheumatoid synovial fluid noted therequirement of antigen presenting cells forstimulation by mycobacterial antigens, but thiswas suggested not to be class I or class IIrestricted.64 Later a class II restricted V-y982 Tcell response was characterised against atetanus toxin peptide.78 However, only a smallpercentage of yyb T cells are CD4+; themajority express either CD8&x chains only, orare CD4-CD8-, and it has been suggested thatCD4+ y8 T cells are more likely to be a fetalT cell subset which has persisted postnatally.9"In general, y8 and (o3 T cells produce a similarrange of cytokines,92 but the proposed minorsubset of CD4+ -y8 T cells shows productionof IL-2 and granulocyte-monocyte colonystimlating factor and low levels of cytotoxicactivity, whereas the CD4- y8 T cells exhibitlower levels of cytokine production and ahigher cytotoxic activity.93 Based on this yi Tcells can be subdivided into a CD4+ 'helper'subset and a CD4- 'cytotoxic' subset.

ConclusionStudy of the possible role of superantigens inautoimmune disorders has recently become thevogue. Care should be exerted in interpretationof the data, however, owing to the complexityof the interactions of the effector moleculesinvolved in immune regulation. An importantaspect of these interactions is the role ofglycosylation in regulating glycoproteinactivity, antigenicity, and recognition.

Protein glycosylation may be involved inseveral stages in the aetiology of RA: (a)initiating antigen; (b) control of T cell inter-actions; (c) control of antibody function; (d)microvasculature breakdown; (e) breakdown ofsynovial gel/water structure; and () specificsignals to inflammatory cells.Of crucial importance is the role that

proteoglycan sequences have in maintainingthe endothelial cell surface barrier, thephysicochemical properties of the synovium,and cartilage structure. The release of enzymeswhich breakdown these proteoglycans couldalso directly affect lymphocyte immunefunction by altering cell surface molecules andintracellular trafficking.

GlossaryCD: Clustered Determinant lymphocyte antigens.

Consensus N-glycosylation site: The nitrogen (N) of theAsn amino acid in the sequence Asn-X-Ser/Thr hasthe potential to be glycosylated, where X is any aminoacid except proline.

N-linked protein chains: high mannosecomplexhybrid

0-linked glycoprotein chains: The hydroxyl group ofany Ser or Thr amino acid usually in high Ser/Thr/Pro- containing regions is glycosylated.

Peptidoglycan: Repeating bacterial cell wall polymer ofamino acids, muramic acid, and N-acetylglucos-amine.

Polysaccharides: In addition to plant (e.g. cellulose) and

S27

on Septem

ber 8, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.52.S

uppl_1.S22 on 1 M

arch 1993. Dow

nloaded from

Hounsell, Davies

mammalian (e.g. glycogen) polymers, bacteria havediverse long chain molecules of often complexrepeats defined as: (a) capsular antigens (b)0-antigen lipopolysaccharide (LPS).

Proteoglycan: Components of mammalian extracellular

matrix, membrane cell surface molecules, secretorygranules, and cartilage.

Selectin: Mammalian carbohydrate binding protein also

called leccams (lectin cell adhesion molecules).

1 Hounsell E F, Feizi T. Gastrointestinal mucins. Structures

and antigenicities of their carbohydrate chains in health

and disease. Med Biol 1982; 60: 227-36.

2 Hounsell E F, Davies M J, Renouf D V. Studies of

oligosaccharide and glycoprotein conformation. Biochemi

Soc Trans 1992; 20: 259-63.

3 Hounsell E F, Lawson A M, Stoll M S, et al.

Characterisation by mass spectrometry and 500-MHz

proton nuclear magnetic resonance spectroscopy of

penta- and hexasaccharide chains of human foetal

gastrointestinal mucins (meconium glycoproteins). Eur.7Biochem 1989; 186: 597-610.

4 Chai W, Hounsell E F, Cashmore G C, et al. Neutral

oligosaccharides of bovine submaxillary mucin. A

combined mass spectrometry and 'H-NMR study. Eur_

Biochem 1992; 203: 257-68.

5 Rudd P M, Leatherbarrow R J, Rademacher T W, Dwek

R A. Diversification of the IgG molecule byoligosaccharides. Mol Immiunol 1991; 28: 1369-78.

6 Bjorkman P J, Saper M A, Samraoui B, Bennett W S,Strominger J L, Wiley D C. Structure of the human class

I histocompatibility antigen, HLA-A2. Nature 1987; 329:

506-12.7 Hounsell E F. Structural and conformational

characterization of carbohydrate differentiation antigens.Chem Soc Rev 1987; 16: 161-85.

8 Sutton D L, Fernandes A, Leung D, et al. Primary sequenceof the N-linked oligosaccharides associated with IgG.Nature 1985; 316: 452-7.

9 Hard K, van Zadelhoff G, Moonen P, Kamerling J P,

Vliegenthart J F G. The Asn-linked carbohydrate chains

of human Tamm-Horsfall glycoprotein. Novel sulfated

and novel GalNAc-containing N-linked carbohydratechains. Eur_7Biochem. 1992; 209: 895-915.

10 Hard K, Damm J B L, Spruiit M P N, et al. The

carbohydrate chains of the ,Bsubunit of human chorionic

gonadotropin produced by the choriocarcinomas cell line

BeWo. Novel 0-linked and novel bisecting-GlcNAc-containing N-linked carbohydrates. Eur 7 Biochem 1992;

205: 785-98.11 Bodman K B, Sumar N, MacKenzie L E, et al.

Lymphocytes from patients with rheumatoid arthritis

produce agalactosylated IgG in vitro. Clin Exp Irnniunol

1992; 88: 420-3.12 Mizuochi T, Hamako J, Nose M, Titani K. Structural

changes in the oligosaccharide chains of IgG in

autoimmune MRIJMp-lpr/lpr mice. _7 Immunol 1990;

145: 1794-8.13 Haynes G A, Maini R N, Malcolm A D B. Immunoglobulin

V genes expression and mRNA sequencing in rheumatoid

arthritis. Ann Rheum Dis 1990; 49: 460-8.

14 Ferguson M A J. Lipid anchors on membrane proteins.Current Opinions in Structural Biology 1991; 1: 522-9.

15 Stimpson S A, Esser R E, Carter P B, Balfour Sartor R,Cromartie W J, Schwab J H. Lipopolysaccharide induces

recurrence of arthritis in rat joints previously injured bypeptidoglycan-polysaccharide. _7 Exp Med 1987; 165:

1688-702.16 Jennings H. Capsular polysaccharides as human vaccines.

Adv Carbohydr Chenm Biochem 1983; 41: 155-208.

17 Leroux J-Y, Poole A R, Webber C, et al. Characterization

of proteoglycan-reactive T cell lines and hybridomas from

mice with proteoglycan-induced arthritis. _7 Immunol

1992; 148: 2090-6.

18 Poole A R. Proteoglycans in health and disease: structures

and functions. Biochem37 1986; 236: 1-14.

19 Sant A J, Cullen S E, Giacoletto K S, Schwartz B D.

Invariant chain is the core protein of the Ia-associated

chondroitin sulfate proteoglycan. .7 Exp Med 1985; 162:

1916-34.20 Stamenkovic I, Amiot M, Pesando J M, Seed B. A

lymphocyte molecule implicated in lymph node homing

is a member of the cartilage link protein family. Cell 1989;

56: 1057-62.

21 Steward W P, Christmas S E, Lyon M, Gallagher J T.

synthesis of proteoglycans by human T lymphocytes.

Biochim Biophys Acta 1990; 1052: 416-25.

22 Le Trong H, Newland G F J, Miller H R P, Charbonneau

H, Neurath H, Woodbury R G. Amino acid sequence of

a mouse mucosal mast cell protease. Biochemistry

28: 391-5.

23 Lopes-Virella M F, Virella G. Immunological

microbiological factors in the pathogenesis

atherosclerosis. Clin Immunol Immunopathol 1985;

377-86.

24 Lowe J B, Stoolman L M, Nair R P, Larsen R D, Berhend

T L, Marks R M. ELAM-1-dependent cell adhesion to

vascular endothelium determined by a transfected human

fucosyltransferase cDNA. Cell 1990; 63: 475-84.

25 Feizi T. Cell-cell adhesion and membrane glycosylation.Current Opinlionis in Structuiral Biology 1991; 1: 766-70.

26 Gamble J R, Smith W B, Vadas M A. TNF modulation of

endothelial and neutrophil adhesion. In: Beutler B, ed.

Ttionour necrosisfactors: the miiolecules and their cpnergilig role

i?nmedicine New York: Raven Press, 1992: 65-86.

27 Muchmore A V, Decker J M. Evidence that recombinant

ILl A exhibits lectin-like specificity and binds to

homogeneous uromodulin via N-linked oligosaccharides.Immunol 1987; 138: 2541-6.

28 Sherblom A P, Sathyamoorthy N, Decker J M, Muchmore

A V. IL-2, a lectin with specificity for high mannose

glycoproteins. JlmnImunol 1989; 143: 939-44.

29 Harding C V, Roof R W, Allen P M, Unanue E R. Effects

of pH and polysaccharides on peptide binding to class II

major histocompatibility complex molecules. Proc Natl

Acad Sci USA 1991; 88: 2740-4.

30 Amin A R, Lakshmi Tamma S M, Oppenheim J D, et al.

Specificity of the murine IGD receptor on T cells is for

N-linked glycans on IgD molecules. Proc NatlAcad Sci

USA 1991; 88: 9238-42.

31 Cherayil B J, Weiner S J, Pillai S. The mac-2 antigen is a

galactose-specific lectin that binds IgE. _7 Exp Med 1989;

170: 1959-72.

32 Katamura K, Iwata M, Mori A, Ishizaka K. Biochemical

identification of glycosylation inhibiting factor. Proc Natl

AcadSci USA 1990; 87: 1903-7.

33 Arai K-I, Lee F, Miyajima A, Miyatake S, Arai N, Yokota

T. Cytokines: coordinators of immune and inflammatorvresponses. Annu Rev Biochemn 1990; 59: 783-836.

34 Nikaido T, Shimizu A, Ishida N, et al. IL2 receptorstructure. Nature 1989; 311: 631-5.

35 Childs R A, Dalchau R, Scudder P, Hounsell E F, Fabre

J W, Feizi T. Evidence for the occurrence of

0-glycosidically linked oligosaccharides of poly-N-acetyllactosamine type on the human leucocytecommon antigen. Biochemn Biophys Res ComnipiunI 1983;110: 424-31.

36 Leahy D J, Axel R, Hendrickson W A. Crystal structure ofa soluble form of the human T cell coreceptor CD8 at 2.6

A resolution. Cell 1992; 68: 1145-62.

37 Parham P. The box and the rod. News and views CD8 and

CD4. Nature 1992; 357: 538.

38 Tifft C J, Proia R L, Camerini-Otero R D. The folding and

cell surface expression of CD4 requires glycosylation. _7Biol Chem 1992; 267: 3268-73.

39 Neefjes J J, De Bruijn M L H, Boog C J P. et al. N-linked

glycan modification on antigen-presenting cells restores

an allospecific cytotoxic T cell response. N Engl Med

1990; 10: 533-88.

40 Pascale M C, Malagolin N, Serafini-Cessi F, Migliaccio G,Leone A, Bonatti S. Biosynthesis and oligosaccharidestructure of human CD8 glvcoprotein expressed in a rat

epithelial cell line. YBiol Chemi 1992; 267: 9940-7.41 Norment A M, Littman D R. A second subunit of CD8 is

expressed in human T cells. EMBO371988; 7: 3433-9.42 Saito H, Kranz D M, Takagaki Y, Hayday A C, Eisen H

N, Tonegawa S. Complete primary structure of a

heterodimeric T-cell receptor deduced from cDNA

sequences. Nature 1984; 309: 757-62.

43 Yoshikai Y, Anatoniou D, Clark S P, et al. Sequence and

expression of transcripts of the human T-cell receptor3-chain genes. Nature 1984; 312: 521-4.

44 Hochstenbach F, Parker C, McLean J, et al.

Characterization of a third form of the human T cell

receptory/8. YExp Med 1988; 168: 761-76.45 Brenner M B, McLean J, Scheft H, et al. Two forms of the

T-cell receptor y protein found on peripheral blood

cytotoxic T lymphocytes. Nature 1987; 325: 689-94.

46 Krangel M S, Band H, Hata S, McLean J, Brenner M B.

Structurally divergent human T cell receptor y proteinsencoded by distinct C-y genes. Science 1987; 237: 64-7.

47 Band H, Hochstenbach F, Parker C M, McLean J, KrangelM S, Brenner M B. Expression of human T cell receptor-½y structural forms.YIJmmnunol 1989; 142: 3627-33.

48 Littman D R, Newton M, Crommie D, et al.

Characterization of an expressed CD3 associated Ti

-y-chain reveals a C-y domain polymorphism. Natuire 1987;

326: 85-8.

49 Grossi C E, Ciccone E, Migone N, ct al. Human T cells

expressing the -y/8 T-cell receptor (TcR- 1): Cyl -andC-y2-encoded forms of the receptor correlate with

distinctive morphology, cytoskeletal organization, and

growth characteristics. Proc Natl Acad Sci USA 1989; 86:

1619-23.

50 Krangel M S, Bierer B E, Devlin P, et al. T3 glycoproteinis functional although structurally distinct on human

T-cell receptor y T lymphocytes. Proc Natl Acad Sci USA

1987; 84: 3817-21.51 Borst J, Alexander S, Elder J, Terhorst C. The T3 complex

on human T lymphocytes involves four structurally

distinct glycoproteins. _7 Biol Chem 1983; 258: 5135-4 1.

52 Oettgen H C, Terhorst C, Cantley L C, Roseoff P H.

Stimulation of the T3-T cell receptor complex induces a

membrane-potential-sensitive calcium influx. Cell 1985;

40: 583-90.53 Wange R L, Tony Kong A-N, Samelson L E. A tyrosine-

phosphorylated 70-kDa protein binds a photoaffinityanalogue ofATP and associates with both the C chain and

CD3 components of the activated T cell antigen receptor.

7 Biol Cheii 1992; 267: 11685-8.

S28

on Septem

ber 8, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.52.S

uppl_1.S22 on 1 M

arch 1993. Dow

nloaded from

Role of protein glycosylation in immune regulation

54 Bames P F, Grisso C L, Abrams J S, Band H, Rea T H,Modlin R L. yb T lymphocytes in human tuberculosis.J3InfectDis 1992; 165: 506-12.

55 Schoel B, Gulle H, Kaufmann S H E. Heterogeneity of therepertoire of T cells of tuberculosis patients and healthycontacts to Mycobacterium tuberculosis antigensseparated by high-resolution techniques. Infect Immun1992; 60: 1717-20.

56 Haregewoin A, Soman G, Hom R C, Finberg R W. Humany5' T cells respond to mycobacterial heat-shock protein.Nature 1989; 340: 309-11.

57 O'Brien R L, Happ M P, Dallas A, Palmer E, Kubo R, BornW K. Stimulating of a major subset of lymphocytesexpressing T cell receptor -y by an antigen derived frommycobacterium tuberculosis. Cell 1989; 57: 667-74.

58 O'Brien R L, Happ M P, Dallas A, et al. Recognition of asingle hsp-60 epitope by an entire subset of yS Tlymphocytes. Immunol Rev 199 1; 121: 155-70.

59 Born W, Hall L, Dallas A, et al. Recognition of a peptideantigen by heat shock-reactive y8 T lymphocytes. Science1990; 249: 67-9.

60 Langer T, Neupert W. Heat shock proteins hsp60 andhsp70: their roles in folding, assembly and membranetranslocation of proteins. Curr Top Microbiol Immunol1991; 167:3-30.

61 Koll H, Guiard B, Rassow J, et al. Antifolding activity ofhsp60 couples protein import into the mitochondrialmatrix with export to the intermembrane space. Cell 1992;68: 1163-75.

62 Fisch P, Malkovsky M, Kovats S, et al. Recognition byhuman V-yN82 T cells of a GroEL homolog on DaudiBurkitt's lymphoma cells. Science 1990; 250: 1269-73.

63 Rajagopalan S, Mao C, Datta S K. Pathogenicautoantibody-inducing y/8 T helper cells from patientswith lupus nephritis express unusual T cell receptors. ClinImmunol Immunopathol 1992; 62: 344-50.

64 Holoshitz J, Koning F, Coligan J E, De Bruyn J, StroberS. Isolation of CD4- CD8- mycobacteria-reactive Tlymphocyte clones from rheumatoid arthritis synovialfluid. Nature 1989; 339: 226-9.

65 van Eden W, Thole J E R, van der Zee R, et al. Cloningof the mycobacterial epitope recognized by T lymphocytesin adjuvant arthritis. Nature 1988; 331: 171-3.

66 Haregewoin A, Singh B, Gupta R S, Finberg R W. Amycobacterial heat-shock protein-responsive y8 T cellclone also responds to the homologous human heat-shockprotein: a possible link between infection andautoimmunity. _lInfect Dis 1991; 163: 156-60.

67 Quayle A J, Black Wilson K, Le S G, et al. Peptiderecognition, T cell receptor usage and HLA restrictionelements of human heat-shock protein (hsp) 60 andmycobacterial 65-kDa hsp-reactive T cell clones fromrheumatoid synovial fluid. Eur J Immunol 1992; 22:1315-22.

68 Crick F D, Gatenby P A. Limiting-dilution analysis of Tcell reactivity to mycobacterial antigens in peripheralblood and synovium from rheumatoid arthritis patients.Clin Exp Immunol 1992; 88: 424-9.

69 Life P F, Bassey E 0 E, Hill Gaston J S. T-cell recognitionof bacterial heat-shock proteins in inflammatory arthritis.Immunol Rev 1991; 121: 113-35.

70 Res P C M, Telgt D, van Laar J M, Pool M 0, BreedveldF C, de Vries R R P. High antigen reactivity inmononuclear cells from sites of chronic inflammation.Lancet 1990; 336: 1406-8.

71 Kaufmann S H E. Heat shock proteins and autoimmunity:fact or fiction? The evidence from animal modelssuggesting the involvement of heat shock protein Iautoimmune disease is supported by some, but not all,data from autoimmune disease patients. Current Biology1991; 1: 359-61.

72 Rippmann F, Taylor N R, Rothbard J B, Green N M. Ahypothetical model for the peptide binding domain of hsp70 based on the peptide binding domain ofHLA. EMBOJ 1991; 10: 1053-9.

73 Hounsell E F, Renouf D V, Liney D, Dalgleish A G,Habeshaw J. A proposed molecular model for the

carboxyl terminus of HIV-1 gpl20 showing structuralfeatures consistent with the presence of a T-cellalloepitope. MolAspects Med 1991; 12: 283-96.

74 Kirchner H, Brehm G, Nicklas W, Beck R, Herbst F.Biochemical characterisation of the T-cell mitogenderived from Mycoplasma arthritidis. Scand J Immunol1986; 24: 245-9.

75 Atkin C L, Cole B C, Sullivan G J, Washburn L R, WileyB B. Stimulation of mouse lymphocytes by a mitogenderived from Mycoplasma Arthritidis. 7 Immunol 1986;137: 1581-9.

76 Paliard X, West S G, Lafferty J A, et al. Evidence for theeffects of a superantigen in rheumatoid arthritis. Science1991; 253: 325-9.

77 He X, Goronzy J, Weyand C. Selective induction ofrheumatoid factors by superantigens and human helper Tcells. J Clin Invest 1992; 89: 673-80.

78 Holoshitz J, Vila L M, Keroack B J, McKinley D R, BayneN K. Dual antigenic recognition by cloned human y8 Tcells. J Clin Invest 1992; 89: 308-14.

79 Olive C, Gatenby P A, Serjeantson S W. Variable geneusage of T cell receptor y- and b-chain transcriptsexpressed in synovia and peripheral blood of patients withrheumatoid arthritis. Clin Exp Immunol 1992; 87: 172-7.

80 Doherty P J, Yang S X, Laxer R M, Silverman E D, InmanR, Pan S. Evidence for clonal expansion ofT cell receptorV-yII+ T cells in the synovial fluid of patients with arthritis.JlImmunol 1992; 149: 295-9.

81 Kabelitz D, Bender A, Schondelmaier S, Schoel B,Kaufmann S H E. A large fraction of human peripheralblood -y/8 T cells is activated by Mycobacteriumtuberculosis but not by its 65-kD heat shock protein. JExp Med 1990; 171: 667-79.

82 Orme I M, Miller E S, Roberts A D, et al. T lymphocytesmediating protection and cellular cytolysis during thecourse of Mycobacterium tuberculosis infection.Evidence for different kinetics and recognition of a widespectrum of protein antigens. J Immunol 1992; 148:189-96.

83 Kabelitz D, Bender A, Prospero T, Wesselborg S, Janssen0, Pechhold K. The primary response of human y/8' Tcells to Mycobacterium tuberculosis is restricted toVy9-bearing cells. J ExpMed 1991; 173: 1331-8.

84 Casorati G, de Libero G, Lanzavecchia A, Migone N.Molecular analysis of human y/8 clones from thymus andperipheral blood. J Exp Med 1989; 170: 1521-30.

85 Rust C J J, Verreck F, Vietor H, Koning F. Specificrecognition of staphylococcal enterotoxin A by human Tcells bearing receptors with the Vy9 region. Nature 1990;346: 572-4.

86 Johnson H M, Russell J K, Pontzer C H. Staphylococcalenterotoxin microbial superantigens. FASEB J 1991; 5:2706-12.

87 Pfeffer K, Schoel B, Plesnila N, et al. A lectin-binding,protease-resistant mycobacterial ligand specificallyactivates Vy9+ human -yb T cells. J Immunol 1992; 148:575-83.

88 Correa I, Bix M, Liao N-S, Zijlstra M, Jaenisch R, RauletD. Most -y8 T cells develop normally in 132-microglobulin-deficient mice. Proc NatlAcad Sci USA 1992; 89: 653-7.

89 Pereira P, Zijlstra M, McMaster J, Loring J M, Jaenisch R,Tonegawa S. Blockade of transgenic lyS T celldevelopment in j32-microglobulin deficient mice. EMBOJ 1992; 11: 25-31.

90 Vidovic D, Roglic M, McKune K, Guerder S, MacKay C,Dembic Z. Qa-I restricted recognition of foreign antigenby a y8 T-cell hybridoma. Nature 1989; 340: 646-50.

91 Bucy R P, Chen C-L H, Cooper M D. Tissue localizationand CD8 accessory molecule expression of Ty8 cells inhumans.JIImmunol 1989; 142: 3045-9.

92 Christmas S E. Cytokine production by T lymphocytesbearing the gamma-delta T cell antigen receptor. ChemImmunol 1992; 53: 32-46.

93 Morita C T, Verma S, Aparicio P, Martinez-A C, Spits H,Brenner M B. Functionally distinct subsets of human -y/8T cells. EurJtImmunol 1991; 21: 2999-3007.

S29

on Septem

ber 8, 2020 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.52.S

uppl_1.S22 on 1 M

arch 1993. Dow

nloaded from