Glycobiolog). vol. 2 no. I pp. 5-23, 1992

MINI REVIEW

Polysialylation: from bacteria to brains

Frederic A.Troy, II

Department of Biological Chemistry, University of California School of Medicine, Davis, CA 95616-8365, USA

Key words: dolichol recognition sequenceslE.coli K1 neural pathogenicitylneural cell adhesion molecules/polysialic acid/ sialyltransferases

Introduction and perspective

Polysialic acid (polysia) is an oncodevelopmental antigen in human kidney and brain, and may enhance the metastatic potential of Wilms tumour cells (Roth et al., 1988b) and neuro- blastomas (Livingston et al., 1988). These novel carbohydrate chains also appear to have a regulatory role in cell growth, differentiation, fertilization and neuronal pathogenicity. PolySia chains are linear homopolymers of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneurarninic acid (NeuSGc) residues joined internally by a-2,8-, a-2,9- or a-2,8/a-2,9-ketosidic linkages (Table I, reviewed in Troy, 1979). The degree of polymerization (DP) can extend beyond 200 sialic acid (Sia) residues (Rohr and Troy, 1980). Oligomers of deaminated Sia (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid), desig- nated KDN, have also been described (Nadano et al., 1986). PolySia chains constitute a structurally unique group of carbo- hydrate residues that covalently modify surface glycoconjugates on cells that range in evolutionary diversity from bacteria to human brains (Troy, 1990).

Structural variation in the oligo- and polySia moieties on membrane polysialoglycoproteins (PSGPs) is considerable, due chiefly to variation in acetylation at the 4, 7, 8 and 9 positions of NeuSAc, Neu5Gc and KDN (Higa and Varki, 1988; Iwasaki et al., 1990). While Sia residues can also be substituted with lactyl and phosphate groups at C9, and methyl and sulphate groups at C8 (Manzi et al., 1990), these substitutes have yet to be reported in polySia. The structural diversity of the Sia residues, and the ability of cells to modulate surface expression of the polySia epitope, probably account for the remarkable functionally diverse processes that these molecules appear to mediate.

This review will focus on recent developments that have emerged regarding the occurrence, structure, function, and bio- synthesis of polysialosyl carbohydrate residues in sources as disparate as bacterial capsules, fish eggs, nerve tissue and human tumours. Prokaryotic-derived reagents that allow the de- tection and analyses of polySia residues will also be reviewed.

Polysialylated bacterial capsular polysaccharides

Occurrence and structure

As shown in Table I, the polySia capsular antigens of neuro- invasive Escherichia coli K1 and Neisseria meningitidis

Table I. Summary of the occurrence and structure of polysialic acid-containing glycoconjugates

Organism Reported structure

Bacrerial capsular polysaccharides

Escherichia coli K 1 Escherichia coli K92 (Bas-12 and N-67)

Neisseria meningiridis Group B Neisseria meningiridis Group C

Pasreurella haemolyrica A2 Morarella nonliquefaciens

Fish egg polysialoglycoproreir~s

Lake trout eggs (Salvelinus nammycush )

Rainbow trout eggs (Salrno gairdneri)

Vitelline envelope

Conical vesicles

Ovarian fluid

Kokanee salmon eggs (Oncorhynchus nerka adonis)

Embryonic neural membranes

Sialogangliosides Neural cell adhesion molecules

Non-neural rissues

Postnatal rat kidney, heart and muscle

Elecrrophorus elecrricus (Electroplax sodium channel)

Turnours (see Table 111)

(-8Neu5Aca2-),,; 7-0-Ac. 9-0-Ac- Alternating (-8Neu5Aca2.) and (-9Neu5Aca2.) linkages

(-8NeuSAca2-),, (-9NeuSAca2-),,

(-8Neu5Aca2-),, (-8Neu5Aca2-),,

(-8Neu5Aca2-),, KDNa2-(8NeuSGca2-),, (-8KDNa2-),, KDNa2-3Gal- : KDNa2- 8KDN- KDNa2 -6GalNAc-; 9-OAcKDNa2 - 8KDN

KDNa2- 8Neu5Ac-; KDNa2-3GalNAc- KDNa2- 8Neu5Gc-; KDNa2 - 6GalNAc- 9-OAcKDNa2 - 8Neu5Gc-

poly(KDN)-gp (same as VE)

(-8Neu5Gca2-),,; 4-0-Ac, 7-0-Ac-, and 9-0-Ac KDNa2-(8NeuSGca2-),,; 9-0-Ac-KDN-

(-8Neu5Aca2-),,; expressed on [EIN-CAM

(-8Neu5Aca2-),

serogroup B strains are composed of poly(-8Neu5Aca2-), residues. The capsule surrounds the bacterial cells and represents the outermost surface structure. The E.coli K1 and N.meningitidis Group B capsules are structurally and irnrnuno- logically identical, and are virulence determinants associated with neonatal meningitis in humans (Robbins et al., 1974). The capsular antigen of N.meningitidis serogroup C is also a homo- polymer of Sia, but the residues are joined internally through a-2,9 linkages (Bhattacharjee et al. , 1976). E. coli K92 and Bos-12 strains contain a polySia capsule that is antigenically related to the serogroup C capsule, except that it consists of Neu5Ac residues alternately linked through a-2,8 and a-2,9-ketosidic linkages (Egan et al., 1977). All of the above- mentioned strains are primary pathogens isolated from humans.

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Some substrains of E.coli Kl and N.meningitidis can modifytheir Neu5Ac residues by Oacetylation at C7-C9 (Higa andVarki, 1988). O-Acetylation increases the immunogenicity ofthe poorly immunogenic E.coli Kl polySia capsule anddecreases its neuroinvasive pathogenicity (Higa and Varki,1988). Poly(-8Neu5Aca2-)n capsules have also been reportedon Pasteurella haemolytica A2 and Moraxella nonliquefaciens,both gram-negative microorganisms aetiologically associatedwith infections (Adlam etai, 1987). No oligo-polySia chainscontaining Neu5Gc or KDN residues have yet been describedin bacteria, although KDN has been reported as a constituent ofa heteropolysaccharide capsule of Klebsiella ozaenae K4(Knirel etai, 1989). Sia has been reported as a serologicaldeterminant in Salmonella ngozi and Salmonella dahlea(Kedzierska et al., 1968), and is present in capsules of Type IIIStreptococcus (Baker and Kasper, 1976; Glode etai., 1977)and Bacteroides fragilis (Kasper, 1976). The occurrence ofsialyl multimers in these strains, however, has not beenreported. In A', meningitidis Group B, the reducing terminuson approximately one-third of the polySia chains is reported tobe covalently attached to 1,2-diacylglycerol through a phos-phodiester linkage (Gotschlich etai., 1981). Capping of thereducing terminus of several E.coli type II capsular poly-saccharides with phosphatidic acid has also been reported(Schmidt and Jann, 1982). It has been proposed that the lipidmay anchor the E.coli K5 capsule to the outer membrane. It isnot understood why only a fraction of the polymers are sub-stituted with phosphatidic acid, nor how lipidation is controlled.

The attachment of polySia to membrane proteins in E. colt K1was first shown in 1979 (Troy and McCloskey, 1979), anobservation that was later corroborated by Rodriguez-Aparicioetai. (1988). Evidence confirming the attachment of polySiachains to a 20 kDa membrane protein was subsequentlypublished (Weisgerber and Troy, 1990). The 20 kDa proteinwas implicated in the initiation of polySia synthesis and/or inthe transmembrane translocation of polySia chains across theinner membrane (Weisgerber and Troy, 1990).

Function of bacterial polySia capsules

Expression of the polySia capsule on the surface of neuro-invasive E.coli Kl and N.meningitidis serogroup B and C cellsis important in pathogenesis, since it appears to facilitatebacterial invasion and colonization of the meninges in neonates(Table II). Little is known about how the capsule can influencethe pathogenic character of these strains, except that itsexpression may represent an elaborate survival mechanism thatevolved to trick the human immune system by masking thesomatic O-antigen moieties of lipopolysaccharide (reviewed inTroy, 1979; Robbins et al., 1980; Silver and Vimr, 1990). Thiscould enhance virulence by facilitating evasion of the humoraldefence mechanisms. Further, the polyanionic capsule providesa barrier against phagocytosis, while concealing surface struc-tures that can activate complement (Bortolussi etai., 1979;Horowitz and Silverstein, 1980). Poly(-8Neu5Aca2-)n, but notpoly(-9Neu5Aca2-)n, is poorly immunogenic, leading to theconcept of 'antigenic mimicry'. The central idea is that ofimmune tolerance because the bacterial poly(-8Neu5Aca2-)chains are structurally identical to the polySia epitope on theembryonic form of vertebrate neural cell adhesion molecules(N-CAMs) that are expressed during embryonic development(Finne etai., 1983b; Whitfield etai., 1984a). Antigenicmimicry helps explain how E.coli Kl and N.meningitidis Group

Table II. Possible functions of polysialosyl carbohydrate residues

I Bacterial polysialic acid capsules

Virulence determinant, polyanioruc shield that masks O-antigenand renders cells resistant to phagocytosis

Facilitates bacteria] invasion and colonization of neonatal brainsReceptor for binding bacteriophages

II Fish egg and ovarian fluid polysialoglycoproteins (PSGPs)

Implicated in fertilizationMay serve as recognition markers for sperm and initiate gamete

interaction by mediating sperm bindingIn vitelline envelope and ovarian fluid, may protect eggs from

artificial activation, bacterial invasion and mechanicaldestruction

O-Acetylation and KDNcosylation renders polySia chains resistantto depolymenzation by exo- and endo-siahdasei

Maintenance of net negative charge may be important in cell-to-cell recognition processes and interactions

III Embryonic neural membranes (N-CAMs)

Embryonic form of N-CAM implicated in early embryonicdevelopment and mediates cell adhesive interactions including,

neunte fasciculationneuromuscular interactionscell migration

The amount of polySia on N-CAM is critical for normalmorphogenesis and neural development

IV Expression on tumours

In developing human kidney, and perhaps in human brain, polySiais an oncodevelopmental antigen

On some human tumours (e.g. neuroblastomas), polySiaexpression may enhance the metastatic potential of tumour cells

V Voltage-sensitive sodium channel from eel electroplax

Function of polySia unknown. By analogy with bacterial polySiacapsules, postulated that polyanioruc surface charge over thechannel may maintain a solute reservoir and shield channelfrom toxins

B may escape immune surveillance, and also why it has beendifficult to develop an effective vaccine using Kl or Group Bcapsular polysaccharides as antigens (Wyle etai., 1972).Finally, the capsule in E.coli Kl strains is a receptor for polySiaspecific bacteriophages (e.g. K1F) that require expression ofthe capsule for infectivity (Vimr et al., 1984).

Regulation of polySia capsule expression in E.coli Kl:genetic organization of the kps gene cluster.

A surprisingly complicated pathway at both the genetic andbiochemical level is involved in regulating surface expressionof the polySia capsule. At the genetic level, this can beappreciated in Figure 1, which summarizes the genetic organ-ization of the kps gene cluster in E.coli Kl. kps is a designationfor 'capsular polysaccharide'. The kps gene complex has beencloned and is encoded in ~ 17—19 kb of DNA that consists ofthree coordinately regulated regions (Silver etai, 1981;Boulnois et al., 1987; Silver and Vimr, 1990). The multigeniccluster encodes for at least 12 proteins that are required for thesynthesis, activation and polymerization of Sia (region 2),energetics and translocation (region 3), and export of polySiachains to the cell surface (region 1). The gene complex maps

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GENETIC ORGANIZATION OF THE kps GENE CLUSTER IN E. coll K1

Region 3 Region 2

(1.6 kb) kb)

kptU I *P*T I neuB II nauA ntuC

Energetic* /trenslocatlonacross IM

ntuE\ ntuS

Sla synthesis, activation and polymerization

kptS kptC |

Region 1(10-12 kb)

I IkptD I kptE I kptF | | kpaB

Polyslallc acid chain export

Fig. 1. Genomic organization of the kps gene complex encoding the polySia capsule in E coli Kl The function of proteins encoded by the different genesimplicated in synthesis of the capsule (region 2). translocatlon across the inner membrane (region 3) and export to the cell surface (region 1) is described inthe text and references cited therein. A single line between kpsM and kpsT. neuE and neuS indicates that the genes are overlapped

at 64 map units on the E. coli chromosome (0rskov et al.,1976; Vimr, 1991) and is absent from common laboratoryisolates of E.coli. It has been suggested that encapsulated E.colistrains may have acquired the kps gene block by ?. trans-positional event (Vimr, 1991).

The molecular organization of region 1 and 3 gene loci thatflank the region 2 cluster is architecturally similar in all E.coligroup II capsular polysaccharides. These loci share commoncomponents and appear to be functionally interchangeable(Roberts et al., 1988). This gene organization is also commonto serogroups of N.meningitidis Group B and Haemophilusinfluenzae, leading to the suggestion that capsule expression ingram-negative bacteria evolved from a common molecularorigin (Frosch et al., 1991). In contrast, region 2 genes arespecific for each type II capsular polysaccharide, and code forthose enzymes required for sugar synthesis, activation and poly-merization. Thus, the size of the region 2 cluster reflects thestructural complexity of the polysaccharide. For example, theregion 2 cluster for polySia expression in Kl is contained in-5 .8 kb of DNA, while the synthetic region for E.coli K5 is~ 7 kb and the K4 polysaccharide is - 21 kb of DNA (Boulnoisand Jann, 1989). K5 is a heteropolysaccharide consisting of(-4-/SG)cUA-l,4-aGlcNAc-l-) residues, while the K4 capsuleis a structurally more complicated fructosylated chondroitin.

Region 2 gene cluster. In E. coli K1, the central region 2 genes(5.8 kb) code for two soluble enzymes, Sia synthase (NeuB)and CMP-Sia synthetase (NeuA), a 50 kDa protein that hasbeen purified and sequenced (Vann etai, 1987). A thirdsoluble enzyme, GlcNAc/ManNAc epimerase (NeuC) is alsocoded by region 2. The neuC gene product is a 45 kDa proteinthat is essential for ManNAc production, the sugar precursorfor Sia. At least two membrane-associated proteins, NeuE andNeuS, are implicated in the initiation, polymerization andpossibly translocation of polySia chains across the innermembrane (Weisgerber and Troy, 1990; Steenbergen andVimr, 1991; Weisgerber et al., 1991). None of the enzymatic

Table m. Expression of polysialic acid on human tumours

Tumours expressing poly(-8Neu5Aa2-)n residues

Neuroblastomas (CHP-134: CHP-126A; CHP-212. CHP^KD: IMR-32/5;NB-BM, SK-NAS)

Nephroblastomas (Wilms tumour)MedulloblastomasPhaeochromocytomasMedullary carcinomas of the thyroidSmall cell lung cancersPituitary adenomas

Human tumour expression (-9Neu5Aca2-)2 residues

Ovarian teratocarcinoma (PA1 embryonal carcinoma cells)

Tumours tested but found not to express polysialic acid

Clear cell sarcomas of kidney, cystic nephromas, renal cell carcinomas,transitional cell carcinomas and papillomas of renal pelvis, ureter orunnary bladder

Ewing sarcomasHepatoblastomasRhabdomyosarcomasCarcinomas of the stomach, colon, exocnne pancreas, lung and

oesophagusPancreatic endocrine tumoursMelanomas

See the text for details and references to primary publications

activities of the membrane-associated polysialyltransferasecomplex have been solubilized and purified.

The complete nucleotide and deduced protein sequence forNeuS, a CMP-Neu5Ac:poly-a-2,8 sialosyl sialyltransferasefrom E.coli Kl, was recently published by Weisgerber et al.(1991). The gene codes for a 47 kDa protein that has poly-sialyltransferase activity, based on its ability to elongateexogenous acceptors. A 47 kDa protein was also expressed inminicells, suggesting that the enzyme is not processed aftertranslation (Weisgerber et al., 1991). These results correct anearlier report by Steenbergen and Vimr (1990) that implicated

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of KpsS, and the remaining proteins in this cassette, exceptKpsD, remain unknown. Silver et al. (1987) have shown thatKpsD is a periplasmic protein that is required in some unknownway to export polySia and other type II capsules to the cellsurface. Jann and colleagues have shown that E.coli K5 withmutations in region 1, in contrast to region 3, synthesizes full-length lipidated chains (Kroncke etai, 1990). An importantgoal for future studies will be to devise experimental strategiesto determine the biochemical function of these proteins that con-stitute the presumed export apparatus.

Biosynthesis of polySia in E.coli Kl

The biosynthetic pathway leading to the formation of poly-sialosyl carbohydrate chains has been most thoroughly studied,and is best characterized in E.coli Kl (Troy et al., 1975; Vijayand Troy, 1975; Troy and McCloskey, 1979; Rohr and Troy,1980; Whitfield et al., 1984a; Whitfield and Troy, 1984; Weis-gerber and Troy, 1990). Because information derived from thebacterial system transcends to the eukaryotic cell, and hasalready proven a good model for developing methods to identifypolysialyltransferases in unfertilized fish embryos (Kitajimaet al., 1988a) and embryonic rat brains (Vimr etai, 1984;Troy et al., 1987), the E.coli Kl pathway will be reviewed insome detail.

The major steps in the synthesis of Neu5Ac, CMP-Neu5Acand polySia in E.coli Kl are summarized in reactions 1—5.

Enz. Neu5Ac + Pi(1) ManNAc + PEP _

(2) Neu5Ac + CTP Enz. 2 ^ CMP-Neu5Ac + PPi

(3) CMP-Neu5Ac + P-C55 P ° l y S T ^ Neu5Ac-P-C;, + CMP

(4) n(Neu5Ac)P-C5J poiyST ^ (Neu5Ac)n-P-C5S + n(P-C,3)

(5) (Neu5Ac),,-P-C,5 poiyST ^ (Neu5Ac),-acceptor+«(P-C55)

+ endogenous acceptor

where Enz. 1 is Neu5Ac synthase (NeuB), Enz. 2 is CMP-Neu5Ac synthetase (NeuA), poiyST is CMP-Neu5Ac:poly-a-2,8-sialosyl sialyltransferase complex (NeuE/NeuS), PEP isphosphoenolpyruvate and P-C55 is undecaprenylphosphate.Reactions 1 and 2 are catalysed by soluble enzymes, whilereactions 3-5 are catalysed by enzymes of the membrane-bound polysialyltransferase complex.

Genes for the enzymes catalysing these reactions are encodedin region 2 of the kps gene complex, as described above.

In E.coli Kl, Sia synthase (Enz. 1) is a cold-sensitive enzymethat is synthesized at 15°C, but is reversibly inactivated at lowtemperatures (Merker and Troy, 1990). This explains in partwhy these cells are acapsular when grown at 15°C, anobservation first reported by Troy and McCloskey (1979) andconfirmed by Bortolussi et al. (1983) and 0rskov et al. (1984).CMP-Sia synthetase (Enz. 2) is also cold sensitive and it islikely that the polysialyltransferase is too (Merker and Troy,1990). Thus, growth of E.coli Kl at temperatures below~20°C has pleiotropic effects on polySia expression. Siasynthase has not been purified from E. coli K1. The enzyme islabile even to dialysis and is difficult to assay in soluble extractsat protein concentrations of <20 mg/ml (Merker and Troy,1990). In cell-free homogenates, the E.coli Kl Sia synthase is

dependent on the addition of ManNAc and PEP, suggesting thatthe enzyme may not require ManNAc-6-P, as the mammalianenzymes do (Merker and Troy, 1990). The E.coli Kl enzymeis distinct from the meningococcal enzyme (Masson andHolbein, 1985), a conclusion consistent with the report that nogenetic homology exists between cloned kps genes from thesetwo genera (Echarti et al., 1983). In contrast to the paucity ofinformation about Sia synthase, CMP-Sia synthetase (Enz. 2)from E. coli has been cloned and sequenced by Vann et al.(1987).

The exact number of enzymes in the polysialyltransferasecomplex required for the initiation and polymerization of Sia(reaction 3-5) is not known. Chain synthesis is initiated (re-action 3) by the transfer of Sia from CMP-Sia to undecaprenyl-phosphate (P-C55), which functions as an intermediate carrierof sialyl residues (Troy et al., 1975). The addition of each Siaresidue is coupled to the energetically favourable hydrolysis ofCMP-Sia and the subsequent hydrolysis of PPi. The extent towhich Sia residues are polymerized on undecaprenylphosphateis not known, but similar oligomeric sialyl undecaprenylphos-phate intermediates have been described for polySia synthesisin N.meningitidis Group B (Masson and Holbein, 1985).

PolySia chain growth occurs by the addition of Sia to thenon-reducing terminus of the growing chain (tail growth) inwhich the activated linkage in CMP-Sia is used for its ownaddition (Kundig et al, 1971; Rohr and Troy, 1980). Neitherthe sialyltransferase activity responsible for initiating polySiasynthesis (an 'initiase') or for catalysing chain polymerization(a polymerase) have been isolated.

In spite of arguments concluding that polySia chain initiationand elongation are catalysed by one sialyltransferase (NeuS)(Steenbergen and Vimr, 1990), it seems likely that at least twoenzymes are involved. Given the extraordinarily high degree ofspecificity that characterizes sialyltransferases in general, itseems unlikely that a single active site would catalyse both thetransfer of one Sia residue to the phosphate residue of undeca-prenylphosphate, and a second Sia unit to the OH group at Cg

on Sia. While one transferase could possess two active sites,identification of an undecaprenol dolichol recognition sequencein NeuE adds further support to the hypothesis that separatesialyltransferases are required to initiate polySia synthesis andpolymerize the chains. As discussed above, our analysis ofthe deduced protein sequence of NeuE and NeuS indicates thatNeuE may be an initiase responsible for starting polySia chainsynthesis (Ye and Troy, 1991). It is also possible that Siaresidues are fully polymerized on undecaprenylphosphateby the sequential addition of monomers (n = ~ 200 in reaction4). Such a pre-assembled 'activated' polysialyl lipid may betranslocated across the inner membrane, while linked to un-decaprenylphosphate, presumably catalysed by a translocase.Alternatively, a polysialylated reactive phosphoryl intermediatecould be transferred en bloc to a translocater protein,designated 'endogenous acceptor' in reaction 5, which thenshuttles the polySia chains across the membrane. A 20 kDasialylated membrane protein that may initiate its own sialylationand/or facilitate polySia chain translocation, has been identifiedin E.coli Kl (Weisgerber and Troy, 1990), but the relationshipbetween sialylation and translocation is not clear. The func-tional domain of the E.coli Kl polysialyltransferase is locatedon the cytoplasmic surface of the inner membrane (Janas andTroy, 1989; Troy et al., 1990a, c), meaning that a mechanismfor the vectorial translocation of polySia chains across the innermembrane must exist (see below). Our recent studies have

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identified long chains of an activated form of polySia on thecytoplasmic surface of sealed inside-out vesicles (IOV) pre-pared from a translocation-defective mutant of E.coli Kl (Choand Troy, 1991). The reactive nature of these polySia chainscould be verified by their en bloc transfer to exogenousacceptors or, in the presence of CMP-[l4C]Neu5Ac, furtherelongated. The polySia chains are presumably activated byattachment to undecaprenylphosphate. Irrespective of the natureof the activated linkage, this unexpected finding provides newinsight into the molecular mechanisms underlying polySia chainsynthesis and translocation. Of importance, it also underscoresthe need for substantiating claims of polysialyltransferaseactivity in translocation-defective mutants when the in vitroassay measures the apparent formation of [l4C]polySia aftermembranes containing pre-existing polySia chains are incu-bated with CMP-[14C]Neu5Ac. In such cases, the transfer ofonly a single Sia residue could be interpreted, incorrectly, aspolymer synthesis. Similarly, conclusions regarding the mech-anism of polymerization that are based on studies where shortsialyl oligomers are used as exogenous acceptors, and where nopolymerization occurs (Steenbergen and Vimr, 1990), are alsoof dubious significance.

PolySia chain initiation requires protein synthesis

Several new features of polySia chain synthesis emerged fromstudies to elucidate the temperature-induced alteration incapsule synthesis, including: (i) activation of polySia chainsynthesis in membranes prepared from cells grown at 15°Coccurs in low-density vesicles (LDV; Q = 1.11 g/cm3)(Whitfield etal., 1984a); (ii) LDVs also catalyse proteinsynthesis, and protein synthesis is a prerequisite for theactivation of polySia synthesis in vivo (Whitfield et al., 1984b)and in vitro (Whitfield and Troy, 1984); (iii) protein synthesisis not required, however, for the subsequent elongation of poly-Sia chains (Whitfield etal., 1984b); (iv) synthesis of ~ 12membrane proteins is temporarily correlated with surfaceexpression of the capsule (Whitfield and Troy, 1984; Whitfieldet al., 1985). These studies were carried out before the geneticorganization of the kps gene cluster and predicted sizes of thecorresponding proteins were known. Interestingly, of the 12proteins shown to be linked to polySia expression by thetemperature-upshift experiments, at least half have molecularmasses now ascribed to the kps gene cluster, including KpsM,KpsT, NeuE, and KpsC, D, and E (Whitfield et al., 1985). Adiminution or absence of proteins with Mr similar to that ofKpsM, KpsT, NeuE and NeuS was observed when cells weregrown at 15°C (Troy and McCloskey, 1979; Whitfield andTroy, 1984; Whitfield etal., 1985), suggesting that theexpression of these genes may, in part, be regulated by Sia,since Sia is not synthesized when cells are grown at 15°C(Merker and Troy, 1990).

Other genes involved in regulating Sia metabolism

An added level of complexity emerged in the regulation of Siametabolism in E.coli with the discovery of two new genes con-trolling an inducible catabolic system for Sia (Vimr and Troy,1985a). nariT codes for a Sia-specific permease and nan A foryV-acylneuraminate pyruvate lyase (Sia aldolase; E.C. 4.1.3,3),which degrades Sia to ManNAc and pyruvate. These geneswere mapped by transduction to near glnF at 69 units on the

E.coli K12 genetic map, clearly establishing that the catabolicgenes are genetically distinct from the biosynthetic genes (Vimrand Troy, 1985a). nan A has been cloned (Kawakami etal.,1986) and sequenced. Regulation of Sia aldolase, induced bySia, is necessary for dissimilating Sia and for modulating thelevel of metabolic intermediates in the Sia pathway (Vimr andTroy, 1985b).

Topology of the polysialyltransferase complex

In spite of considerable biochemical and genetic information onthe regulation of surface expression of the polySia capsule inE.coli Kl, there was, until recently, nothing known about thetopology of the polysialyltransferase complex. To determine thetransmembrane organization of this complex, membranevesicles of defined orientation were used to assay poly-sialyltransferase activity. Sealed right-side-out vesicles (ROV)have the same topology as intact cells, whereas IOV, preparedby disrupting the cells in a French press, have the oppositeorientation (Owen and Kaback, 1978). Thus, sealed IOV havea unique orientation since enzymes normally located on theinner surface of the cytoplasmic (inner) membrane appear onthe exterior side of the vesicles. Enzymes showing such atopological orientation can be assessed using impermeablesubstrates (e.g. CMP-Neu5Ac) and enzymes (e.g. trypsin). Thesideness and impermeability of ROV and IOV were verified us-ing voltage-sensitive fluorescent probes (Scherman and Henry,1980; Cabrini and Verkman, 1986). This strategy was used toshow that there was no polysialyltransferase activity in ROVabove a background level attributed to vesicles that wereinverted during preparation (Janas and Troy, 1989; Troy et al.,1990a). In contrast, IOV prepared by French press showed a5-fold increase in polysialyltransferase. Further, there was asubstantial increase in polysialyltransferase activity in ROVafter inversion by French press or sonication, or by permeabil-ization with Triton X-100 or toluene. These data show that thefunctional domain of the polysialyltransferase complex islocated on the inner face of the inner membrane. Confirmationof this conclusion was provided by showing that there was onlya slight decrease in polysialyltransferase activity when ROVwere treated with trypsin and then inverted. In contrast, > 90%of the transferase activity was lost when ROV were invertedbefore trypsin treatment (Troy et al., 1990c). This new infor-mation regarding the topology of the polysialyltransferasecomplex requires that polySia chains must somehow traversethe inner membrane before being exported and assembled in theouter leaflet of the outer membrane. Presumably, as describedearlier, KpsM, KpsT and perhaps region 2 gene products areinvolved in the initial translocation step, while proteins encodedin region 1 participate in the subsequent export pathway.

A second important feature to emerge from these studies wasthe finding that full-length polySia chains were present in thecytoplasm (Troy et al., 1990b). This finding means that at leastfor the polySia capsule, polymerization precedes translocationof the chains across the inner membrane and, therefore, poly-merization does not need to be coupled to transport. This is incontrast to the report of Boulnois and Jann (1989), who havepostulated that synthesis of the K5 polysaccharide is coupled toexport. It is difficult to envisage a model in accord with thispostulation because the K5 polysaccharide also grows by theaddition of new sugar residues to the non-reducing terminus(tail growth).

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Energy dependence of the E. coli Kl polysialyltransferase

The importance of a membrane potential for activation of poly-Sia in E.coli Kl membranes was first reported by Whitfieldet al. in 1984. The initial finding was that energy uncouplers,e.g. carbonyl cyanide m-chlorophenylhydrazone (CCCP),inhibited in vitro activation of polySia. While the exact energy-requiring processes were not defined, it was proposed that themembrane potential may be required for maintenance of amembrane conformation necessary for structural coupling ofprotein synthesis and translocation of the polySia chains(Whitfield etal., 1984a). In related studies, Osborn andcolleagues have shown that the formation of the first lipid-linked intermediate in O-antigen synthesis in Salmonellatyphimurium, galactosylpyrophosphoryl undecaprenol, is de-pendent on maintenance of the proton motive force (Marinoetal., 1991). Studies to further delineate the energy require-ment for polysialyltransferase activity in E. coli K1 were carriedout recently in sealed IOV (Troy et al., 1990a). The effects ofenergy-rich compounds, ATPase inhibitors and uncouplers ofthe proton electrochemical potential gradient (A/tH+) werestudied. Both ATP hydrolysis and NADH and lactate oxidationcaused an increase in polysialyltransferase activity, while thenon-hydrolysable ATP analogue, AMP-PNP, reduced thisactivity. Polysialyltransferase activity was markedly inhibitedby treatment of IOV with the photoaffinity probe, 8-azido-ATP.A partial restoration of polysialyltransferase activity wasobserved after the addition of NADH. These changes in enzymeactivity were found to correlate with changes in A/tH+, asmeasured by quenching of 9-amino-6-chloro-2-methoxyacri-dine fluorescence. The quantitative results of these experimentsconfirmed that the polysialyltransferase activity in IOV ismodulated mainly by AjtH + , and that the high-energy phos-phoryl potential of ATP is involved in polySia synthesis. Itis proposed that the hydrolysis of ATP could be used in thechain-elongation reaction to drive the polymerization reactionforward. Since polySia synthesis in IOV does not involve trans-location of these polymers across the inner membrane, therequirement of energized membranes for full activity of themembranous polysialyltransferase complex is established. How-ever, as described below, both A/tH+ and the transmembraneelectrical potential gradient (A^) are required to translocatepolySia chains across the inner membrane.

Translocation of polySia chains across the inner membrane

The functional domain of the polysialyltransferase is localizedon the cytoplasmic surface of the inner membrane and polySiachains are fully polymerized inside the cell before being trans-located (Troy et al., 1991). To study the molecular mechanismof translocation, an in vivo labelling system using sphaeroplastsprepared from K1 cells that are unable to degrade Sia becauseof a defect in Sia aldolase (nanA mutation) was developed(Troy etal., 1991). PolySia chains that have been trans-located across the inner membrane were differentiated fromthose chains remaining inside by their accessibility to depoly-merization by endo-yV-acylneuraminidase (Endo-N). Afterpulse-labelling sphaeroplasts with [MC]Neu5Ac, synthesis andtranslocation were followed kinetically and the theory of com-partmental analysis was applied to determine polySia chain dis-tribution among different compartments. The effects of CCCPas a modulator of A^H + , and valinomycin as a modulator ofA¥, on translocation rates were assessed. The results of these

studies revealed that both the A/*H+ and the A* were requiredfor the vectorial translocation of polySia chains across the innermembrane. Further, if the A/xH+ was collapsed with CCCP,fully polymerized polySia chains remained inside sphaero-plasts, demonstrating that chain polymerization was not coupledto chain translocation (Troy et al., 1991). While the molecularmechanism linking the A/*H + and A* to chain translocation isnot known, these studies suggest that energized membranesmay be required to integrate conformational changes and cross-talk among various components of the polysialyltransferase-polySia translocation apparatus (Troy et al., 1990b). The ATP-binding domain of KpsT and the consensus dolichol bindingdomain in KpsM (Ye and Troy, 1991) further implicate thesetwo proteins of region 3 in the energy-dependent synthesis andtranslocation of polySia chains across the inner membrane. Akey challenge now is to further elucidate the components andmechanism of the membrane machinery that moves capsularpolysaccharide chains across the membrane, and to define theenergetics more precisely. In this regard, we have postulatedthat the relatively large A*, up to -150 mV (negative inside),that is generated across the E.coli inner membrane may facili-tate the movement of these polyanions across the membrane(Troy etal., 1990b).

Polysialylated glycoproteins in fish eggs

Occurrence and structure

A common misconception is that the N-CAM was the firsteukaryotic glycoprotein reported to contain polySia residues.The first was, in fact, a novel PSGP isolated from unfertilizedeggs of rainbow trout (RT) by Inoue and Iwasaki in 1978.These PSGPs are the major glycoprotein components in corticalvesicles (alveoli) of a number of Salmonidae fish eggs (Iwasakiand Inoue, 1985; Iwasaki et al., 1985). The RT PSGP containsoligo/poly(-8Neu5Gca2-) residues (Table I) that are attached toO-linked oligosaccharide chains. Neu5Gc accounts for -60%(w/w) of the mass of these glycoproteins. In a series of elegantstructural studies, the Inoues and colleagues have elucidated thecomplete structure of both the oligosaccharide chains and theapoprotein moieties of the PSGPs (Nomoto etal., 1982;Iwasaki and Inoue, 1985; Kitajima et al., 1986; Iwasaki et al.,1990). What has emerged is an extraordinary array of struc-turally diverse glycoconjugates whose presence appears to beubiquitous in a number of Salmonid fish. The PSGP from RT

eggs, for example, is composed exclusively of Neu5Gc residues(Inoue and Iwasaki, 1980; Iwasaki etal., 1984). In contrast,lake trout (Lj-; Salvelinus namaycush) egg PSGP contains twodistinct types of oligo/polysialyl chains, i.e. (-8Neu5Gca2-)n

and (-8Neu5Aca2-)n (Iwasaki et al., 1990). No hybrid struc-tures having both Neu5Ac and Neu5Gc residues have beenreported. There is considerable oligo-polydispersity in thelength of the polySia chains in these PSGPs. While the averageDP of the sialyl units is ~ 6 , some chains contain up to ~24sialyl residues (Inoue and Iwasaki, 1980; Nomoto et al., 1982).The PSGPs are polydisperse with a weight average Mr of2X1O5 (Inoue and Inoue, 1986; Kitajima etal., 1986). Thestructures of the asialo-oligosaccharide moieties from fourspecies of Salmonidae eggs have been reviewed (Iwasaki andInoue, 1985). The complete amino acid sequence of theapoprotein moieties from two genera of Salmonid fish eggs hasalso been published (Kitajima et al., 1988b). The proteinbackbone of PSGPs is composed of tandem repeats of - 25

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tridecapeptide units (Inoue and Inoue, 1986; Kitajima etal.,1986).

An even more bewildering structural complexity emerged inSalmonid fish when Inoue et al. (1988) described for the firsttime a naturally occurring KDN-containing glycoprotein (gp) inthe vitelline envelope (VE) of RT eggs. KDN occurred at thenon-reducing terminus and was the only ulosonic acid found inthis glycoprotein. When deaminated Sia residues are present asterminal, non-reducing sugars, they can be linked a2-*3 to Gal,or a2-*6 to GalNAc (Table I). A similar, yet slightly lowermolecular weight deaminated neuraminic acid-rich glycoproteinwas found in the ovarian fluid (OF) of ovulating RT (Kanamoriet al., 1989). Further structural analyses of the Sia-containingglycoproteins of RT eggs showed another unique KDN-con-taining sialyloligosaccharide in which KDN capped thenon-reducing termini of oligo (-8Neu5Gca2-) chains, formingKDNa[8Neu5Gca2-]m where n = 1-7 residues (Nadanoetal., 1986). The structure of this novel poly(KDN) oligo-saccharide is shown below.

[-8KDNa2-]nN6

GalNAcal Thr/(Ser)3

KDNa2 - 3Gal/31 - 3GalNAca 1

where n is - 6-7 residues.Recently, oligo/poly KDN chains have been isolated as

poly(KDN)-gps from both VE and OF from Salmonid fish(Inoue et al., 1991). Poly(KDN)-gps isolated from the VE andOF have identical O-linked oligosaccharides containinga-2,8-linked oligo(KDN) chains. The amino acid compositionof the apoprotein moiety of KDN-gp and Sia-gp isolated fromdifferent species of Salmonid fish are alike, suggesting thatthese PSGPs may share similar functions. In the VE and OF ofsome species of Salmonid, poly(Neu5Gc)-gp replaces the poly-(KDN)-gp (Inoue et al., 1991). In this case, the poly(Neu5Gc)-gp accounts for —50% of the mass, with only small amountsof KDN present. The trisaccharide structure for the poly(Neu-5Gc)-gp is identical to that described above for poly(KDN)-gp(Inoue et al., 1991).

Recently, a new sialic acid analogue (9-O-acetyl-KDN) anda-2,8-linked O-acetylated poly(Neu5Ac) chains were describedin a PSGP from the unfertilized eggs of the Kohanee salmon(Oncorhynchus nerka adonis) (Inoue etal., 1988). Thesepoly(Neu5Gc) chains were shown by 'H NMR to contain 4-0-Ac-, 7-O-Ac- and 9-0-Ac esters of Neu5Gc. 9-0-Ac-KDNresidues can also cap the non-reducing termini of the Oacetyl-ated poly(Neu5Gc) chains in PSGPs from the Kohanee salmon.The discovery of 9-0-Ac-KDN extends the family of naturallyoccurring sialic acids, while the discovery of O-Ac-poly(Neu-5Gc) extends the range of structural diversity in polySia-con-taining glycoconjugates. As summarized in Table I, KDN in theVE of Salmonid fish can be linked a2-"3 to Gal, a2-*6 toGalNAc, a2—8 to KDN and, as the 9-OAcKDN derivative,a2—8 to KDN. In cortical vesicles, KDN can be linked eithera2-"3 or a2-*6 to GalNAc, a2-»8 to Neu5Ac or Neu5Gc and,as 9-OAc-KDN, a2-*8 to Neu5Gc. In contrast to neural glyco-proteins, e.g. N-CAM, no oligo-polySia chains have beenreported in fish eggs that are attached to N-linked oligo-saccharides. Finally, the first example of the natural occurrenceof a KDN-containing ganglioside (KDN-GM3; KDNa2,3Gal-

/31,4Glc/31-ceramide) in trout sperm has been reported by Songet al. (1991). Neither KDN, poly(KDN) nor poly(Sia) have yetbeen reported in mammalian gametes.

Function of polySia residues in fish

While the precise function of polySia in fish PSGPs has notbeen determined, elucidation of the structural features of theoligosaccharide chains has revealed an extraordinary variationin chemical structures (Table I). This diversity in structure pre-dicts a probable diversity in function and the details of some ofthese functions are beginning to emerge (Table II).

As Inoue et al. (1991) have noted, all vertebrate eggs areenveloped by a carbohydrate-enriched capsule, designated theVE, zona pellucida or chorion. These extracellular coatsusually function in species-specific cell—cell recognition eventsduring fertilization and early embryogenesis, and in providingthe egg with a protective barrier (Inoue etal., 1991). As aclass, the Salmonidae fish egg PSGPs are present in the VE andas major components in the cortical vesicles that localize in thecortex of unfertilized eggs (Table I). After fertilization or eggactivation, cortical vesicle exocytosis occurs and the highmolecular weight PSGPs undergo a rapid proteolysis at theperivitelline space to their repeating unit (Kitajima and Inoue,1988; Song et al., 1990). The polySia moieties are not depoly-merized, but their presence in the VE may serve initially as arecognition marker for sperm binding. The extreme polyanioniccharacter of the PSGPs also suggests that they could be con-formational determinants important in maintaining the correctthree-dimensional structure of the protein. Results in support ofthe sperm recognition hypothesis were reported by Kanamoriet al. (1991) who showed that the KDN-containing glyco-protein of the VE of RT eggs was localized in the second layerof the envelope, and has strong sperm-agglutinating activity.The O-linked KDN-glycoprotein oligosaccharide structureshown above inhibits sperm agglutination, leading to the sug-gestion that KDN-glycoproteins specifically recognize spermand thus may mediate egg—sperm interaction (Inoue, 1991). Insupport of this possibility, Inoue and colleagues have evidencethat the oligo(KDN) glycan chains interact specifically with thehomologous sperm. This species-specific interaction can bedemonstrated by light microscope when sperm are treated withfluorescein isothiocyanate (FITC)-labelled KDN-gp moleculesisolated from the egg surface (Inoue, 1991). This is an excitingnew development in KDNology because it shows thatoligo(KDN) glycan units may mediate egg-sperm interaction.KDN-ganglioside [(KDN)GM3] molecules that are located onthe sperm cell surface could also act as a ligand in species-specific sperm-egg interactions, although no direct evidenceexists to support this possibility.

The polySia-gps in the vitelline envelope have also beenpostulated to protect eggs from bacterial invasion (Table II).Similarly, because of the high viscosity and polyanionic natureof ovarian fluid KDN(Sia)-gps, these molecules may protecteggs from artificial activation, mechanical destruction andbacteria] invasion (Kanamori etal., 1989).

Capping of the non-reducing termini of oligo-polySia chainswith KDN protects these chains from degradation byexosialidases, and this may be important in egg activation(Nadano etal., 1986). O-Acetylation of the poly(Neu5Gc)chains also renders them resistant to depolymerization bybacterial exosialidases and a bacteriophage-derived endo-jV-acylneuraminidase (Endo-N) that is specific for cleaving

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a-2,8-linkages in either poly(Neu5Ac) or poly(Neu5Gc) chains(Nadano et al., 1986) (see below). While the significance ofthis resistance to sialidases is not known, maintenance of the netnegative surface charge again suggests that polySia mayfunction as a conformational determinant to regulate cell-to-cellrecognition processes and interactions.

interesting findings will be important in future studies tocharacterize the sialyltransferase activities and clone theenzymes. The potential relationship between sialyltransferase(s)catalysing Neu5Ac/Neu5Gc sialylation and KDNosylationawaits further studies.

Biosynthesis of polySia chains in unfertilized fish eggs

Detection of CMP-KDN synthetase and CMP-Sia.poly-a-2,8-sialyltransferase (polyST) in unfertilized rainbowtrout eggs

Despite the extensive structural information on the polySiamoieties of fish egg PSGPs (Table I), and cloning and sequenc-ing of cDNAs coding for the apoPSGPs of RT eggs (Sorimachiet al., 1988), relatively little is known regarding how the poly-Sia chains are synthesized. As a first step in elucidating the bio-synthesis of polySia in unfertilized RT eggs, Ito et al. (1991)recently reported the detection of CMP-KDN synthetase andpolyST activities in embryonic RT eggs. Using soluble andmembrane fractions prepared by French press disruption ofimmature oocytes obtained from RT, these initial studiesrevealed two important findings. First, CMP-KDN synthetaseactivity was detected in the supernatant fraction, based on theconversion of [I4C]KDN to CMP-[I4C]KDN in the presence ofCTP. Concomitant with a loss of radioactivity in KDN was theformation of a new nucleotide-containing component with theproperties expected for CMP-[I4C]KDN. [14C]KDN was syn-thesized from [l4C]Man and pyruvate in 89% yield using animmobilized Sia aldolase (Auge and Gautheron, 1987). CMP-KDN appears to be more labile than CMP-Neu5Ac (F.Ito andF.A.Troy, unpublished). Since CMP-KDN synthetase has notbeen purified, it remains to be determined if separate condens-ing enzymes synthesize CMP-KDN, CMP-Neu5Ac and CMP-Neu5Gc. Second, polysialyltransferase activity was detected inboth the membrane and supernatant fractions using CMP-[l4C]Neu5Ac as substrate. A nearly 2-fold increase in poly-a-2,8-sialyltransferase activity occurred when PSGPs from Rjwere added as exogenous acceptors. PolySia was confirmed asthe product of the sialyltransferase reaction by its sensitivity todepolymerization by purified Endo-N. Further informationabout the enzymology of sialylation was provided recently byKitazume et al. (1991), who used asialo-PSGP and PSGP toidentify two distinct sialyltransferases that are differentially ex-pressed during oogenesis. A cytosolic CMP-Neu5Ac (or Neu-5Gc):a-N-acetylgalactoside a-2,6-sialyltransferase (a-2,6-ST)catalysed the initial transfer of Sia onto the C6 proximalGalNAc residues on asialo PSGP. Expression of this enzymepreceded expression of a cortical alveolar-derived CMP-Neu-5Ac (or Neu5Gc):a-2,8-sialosyl sialyltransferase (a-2,8-ST)activity that was responsible for poly-or-2,8-sialylation. Akinetic study of the maturation of PSGP during oogenesiscorroborated the temporal expression of the sialyltransferases,and revealed that synthesis of the asialo core oligosaccharideswas completed before sialylation. Interestingly, Kitazume et al.(1991) have shown that both a-2,6- and a-2,8-ST activities aresecreted from the Golgi to cortical vesicles (or cortical alveoli),and that extensive sialylation occurs in these vesicles. Further,activity of the cortical vesicle-derived a-2,8-ST was dramat-ically reduced by freeze-thawing and could be resuscitated bythe addition of Tween 80 (Kitazume etai, 1991). These

Polysialylated glycoproteins in the nervous system

N-CAMs are vertebrate cell surface sialoglycoproteins thatfunction in embryonic development and mediate a variety ofcell—cell adhesive interactions, including neurite fasciculation,neuromuscuiar interactions and cell migration [reviewed inEdelman (1985); Cunningham et al. (1987) and Rutishauseret al. (1988)]. In the vertebrate embryo, N-CAMs areexpressed on neuroepithelia, neurons, glial and muscle cells. Aunique structural feature of N-CAM is the presence of longchains of poly(-8Neu5Aca2-) residues (DP>55) that areattached as outer branches to presumed tri- and tetraantennarychains of N-linked oligosaccharides (Finne, 1982; Hoffmanet al., 1982; Finne et al., 1983a; Margolis and Margolis, 1983;McCoy and Troy, 1986). No c*2,9 linkages, KDN or Neu5Gcresidues have been reported attached to N-CAM.

N-CAM has been extensively studied and its gene has beencloned (Murray et al., 1984; Goridis et al., 1985; Cunninghamet al., 1987; Owens et al., 1987). Surface expression of thepolySia epitope is developmentally regulated in the central andperipheral nervous systems. The embryonic form of N-CAM([E]N-CAM) contains a high Sia content that undergoes apost-natal conversion to the adult form ([A]N-CAM) with a lowSia content (Hoffman etal., 1982). The reduction in Sia ispostulated to increase homophilic adhesive interactions betweencells, the [A]-N-CAM being most adhesive and presumablycontaining shorter oligomers of polySia. [A]N-CAM containsonly one-third as much polySia as the embryonic form, yet theirpolypeptides are identical (Finne, 1982; Hoffman et al., 1982;Finne et al., 1983a). PolySia can exist in some adult tissues,e.g. olfactory (Chuong and Edelman, 1984), and in adult frogretina (R.D.McCoy and F.A.Troy, unpublished). While poly-sialylated N-CAM is most abundant on nervous tissue, it isnot confined to the nervous system. Transient expression ofN-CAM has been reported in kidney (Roth etal., 1987),skeletal muscle, mesenchymal tissue, adrenal and testis, and insome of these cases the molecule is polysialylated (Finne et al.,1987). The variation in Sia content accounts for the poly-dispersity in apparent molecular mass of N-CAM moleculesobserved in SDS-PAGE (Hoffman et al., 1982). Western blotanalyses with anti-polySia antibodies show N-CAM immuno-reactivity with molecular masses extending from — 180 kDa to> 250-300 kDa (Vimr et al., 1984; Roth et al., 1987, 1988d).There are three major N-CAM polypeptides with molecularmasses of 120, 140 and 180 kDa (Hoffman et al., 1982). Threeof the seven potential asparagine-1 inked attachment sites in[E]N-CAM are polysialylated, and these sites are located in thecentral region of the polypeptide chains (Crossin et al., 1984;Hemperly et al., 1986). Synthesis of N-CAM in the presenceof tunicamycin prevents N-glycosylation and, as a consequence,polysialylation (Hemperly et al., 1986). It has been estimatedthat, on average, each N-CAM polypeptide should contain-130-150 sialyl residues (Livingston etal., 1988). Theearlier reports of the DP of Sia on [E]N-CAM estimated thatthese chains contained —8—16 sialyl residues (Finne, 1982;Rothbard etal., 1982). These original estimates were low,

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Structure, function and synthesis of polysialk acids

probably because of the lability of these polymers to evenmildly acidic conditions, temperature (Troy and McCloskey,1979) or contaminating sialidases.

The development and application of the prokaryotic-derivedprobes to detect polySia chains in eukaryotes provided directproof that had been lacking for the existence of long-chainpoly(-8Neu5Aca2-) residues on [E]N-CAM (Vimr et al.,1984). The presence of such chains in N-CAM was firstinferred from sugar compositional analysis by Hoffman et al.(1982) who reported a Sia:Gal ratio of - 16 in [EJ-N-CAM and~3 in [A]-N-CAM. The first direct proof that internally linkedSia was present in N-CAM was provided by Finne (1982), whoshowed internally linked a-2,8-Sia residues by mass fragmen-tography. Development of the prokaryotic reagents describedbelow made possible the following studies: (i) a determinationof the developmental^ regulated changes in surface expressionof polySia in neural membranes and sprouting neurons; (ii)identification of a polysialyltransferase that catalysed poly-sialylation of N-CAM in neonatal neuronal tissue; (iii) in vivostudies that provided evidence for the role of polySia inmodulating cell adhesion between living cells; (iv) elucidationof the length of polySia chains that are expressed on humanneuroblastoma cells, and which may enhance the metastaticpotential of these tumours. The essential features of thesestudies, and other corollary studies, will be summarized.

PolySia expression on fetal rat brain membranes and sprout-ing neurons

The temporal expression of polySia residues on fetal rat brainmembrane (Vimr et al., 1984) and sprouting neurons (Rosen-berg et al., 1986) was studied using the prokaryotic-derivedreagents described below. H.46 was used in Western blotexperiments to detect in neonatal neural tissue, but not adultbrain tissue, polySia immunoreactivity with Mr expected for[E]N-CAM (180-240 000) (Finne et al., 1983b; Vimr et al.,1984). PolySia could also be detected with H.46 after rocketimmunoelectrophoresis of samples solubilized with TritonX-100. Treatment of brain with Endo-N abolished the immuno-reactivity. The Endo-N-solubilized material was isolated,reduced with NaB3H4 and shown by HPLC analysis to contain(-8Neu5Aca2-)3_6. Treatment of the 3H-labelled oligoSia withexosialidase quantitatively converted the radioactivity tosialitol, establishing that the Endo-N-derived (-8Neu5Aca2-)oligomers were composed solely of Sia. The pre-existence ofpolySia chains in rat brain membranes was confirmed by show-ing that these membranes functioned as an exogenous acceptorof [14C]Neu5Ac residues from CMP-[l4C]Neu5Ac in theE.coli EV11 polysialyltransferase assay (Vimr etal., 1984).These data thus provided structural proof that antibodiesspecific for detecting the long-chain (DP > 200) poly(-8Neu-5Aca2-) capsule in E.coli Kl strains also detected similarantigenic species in N-CAM, verifying the presence of inter-nally linked (-8Neu5Aca2-) residues on [E]N-CAM. Thesimplicity of using anti-polySia antibodies and phage-derivedEndo-N in combination to identify and study the developmen-tally regulated expression of polySia residues on N-CAM wasconfirmed by Roth et al. (1987) and Finne et al. (1987).

The 5B4 antigen expressed on sprouting neurons from fetal,but not from adult rat brain, was also shown to containa-2,8-linked polySia using H.46 and Endo-N (Rosenbergetal., 1986). The 5B4 antigen was known to be a develop-

mentally regulated membrane glycoprotein whose expressionon neurons was coincident with neuronal sprouting (Ellis et al.,1985; Wallis etal., 1985). A monoclonal antibody (mAb) to5B4 recognized a diffuse antigen of - 185-255 000 Mr onfetal (17-day gestation) rat brain membranes. In contrast, mAb5B4 recognized an antigen of — 140 000 Da in adult rat brain,and the biochemical relationship of the fetal 5B4 antigen to thatin adult brain was not known. Because no brain proteins otherthan N-CAM were known to be polysialylated, these studiessuggested that the 5B4 antigen was a member of the N-CAMfamily, and exemplified the powerful advantage of using Endo-N in conjunction with anti-polySia antibodies to detect and con-firm the presence of polySia residues in nervous tissue.

Biosynthesis of polySia in neural tissue

Identification of a CMP-Neu5Ac:poly-a-2,8-sialosyl sialyltrans-ferase in fetal rat brain. The initial identification of aCMP-Neu5Ac:poly(-8Neu5Aca2-)sialosyl sialyltransferase (poly-sialyltransferase) in a eukaryotic organism was reported in 1985(McCoy etal., 1985). The enzyme was first described andshown to be differentially expressed in developing neural tissue.A Golgi-enriched fraction from 20-day-old fetal rat brain con-tained a membrane-associated polysialyltransferase activity thatcatalysed the incorporation of [l4C]Neu5Ac from CMP-[l4C]Neu5Ac into endogenous acceptor molecules. Endo-Nwas used to show that at least one-third of the [l4C]Siaresidues were incorporated in polySia, a conclusion confirmedby structural studies (McCoy etal., 1985). In SDS-poly-acrylamide gels, the major l4C-labelled species migrated witha mobility expected for [E]N-CAM. The l4C-labelled peakalso showed immunoreactivity to H.46 that was abolished bypretreatment with Endo-N. The addition of N-CAM to theGolgi polysialyltransferase stimulated [14C]Sia incorporation3-fold. The product of this reaction was also sensitive to Endo-N and contained poly(-8Neu5Aca2-) chains, thus showing thatN-CAM can serve as an exogenous acceptor for polysialylationin vitro. [l4C]Sia incorporated into adult rat brain membraneswas resistant to Endo-N, showing that expression of the poly-sialyltransferase activity is apparently restricted to an earlystage in development. This conclusion was confirmed by Breenet al. (1987) who subsequently reported that a Golgi-enrichedfraction from postnatal (12-day), but not adult rats, catalysedthe sialylation of N-CAM. More recent studies led to thesuggestion that N-CAM sialylation may be controlled byisoforms of the Golgi sialyltransferases (Breen and Regan,1988). The validity of this supposition could be tested directlyif the sensitivity of the various sialylated products to Endo-Nwas determined. In the absence of such a control, it is notpossible to know how much of the [l4C]Sia was incorporatedinto polySia in comparison to monosialylated species.

Based on results obtained from immunolabelling experi-ments, Zuber and Roth (1990) have raised the interestingpossibility that polysialylation of N-CAM in Wilms tumourmay occur partially or exclusively at the cell surface, sincepolySia was only detected at this site. Given the complexity ofthe pathway for the synthesis, transmembrane translocation andexport of the polySia capsular chains in E.coli Kl (summarizedabove), it will be surprising if the pathway regulating thesynthesis and surface expressions of polysialosyl carbohydrateunits in eukaryotic systems is less complicated (Janas andTroy, 1989).

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Polysialic acid as a regulator of N-CAM-mediatedadhesion

The role of Sia in N-CAM-mediated adhesion was first shownby Cunningham et al. (1983), and by Hoffman and Edelman(1983). These studies showed that removal of Sia by exosiali-dase treatment of [E]N-CAM increased the apparent bindingaffinity of the molecule. These studies predated informationabout the existence of polySia in neural tissue, and an under-standing of the use of Endo-N to study the structure andfunction of polySia residues. When it became evident that thesialic acid-rich form of N-CAM was less adhesive and thatEndo-N could selectively remove the polySia moiety atphysiological pH, the functional importance of polySia inneuronal morphogenesis could be tested directly. The initialstudies were carried out in collaboration with Rutishauser andcolleagues, who injected purified Endo-N into the eyes of3.5 day old chick embryos (Rutishauser et al., 1985). Sagittalsections of the eyes were examined by light microscopy andcompared to a control eye that was injected with buffer alone.Distribution of N-CAM in the retina was visualized by im-munoperoxidase staining of frozen sections in the more dorsalpart of the eye. The results of these experiments showed thatinjection of Endo-N produced a striking array of abnormalitiesin those regions of the neural retina that contained the highestconcentrations of N-CAM. These perturbations included adramatic thickening of the neural epithelium in the posterioreye, a failure of cells in this eye to elongate radially, formationof an ectopic optic fiber layer and an incomplete association ofthe pigmented epithelium with the neural retina. These resultsthus provided the first direct evidence that the polySia onN-CAM had a regulatory effect on adhesion between livingcells, and that the amount of polySia was critical for normalneural development.

Rutishauser and colleagues have continued to exploit Endo-Nin studies that have confirmed the importance of polysialylatedN-CAM in development [summarized in Rutishauser et al.(1988) and Rutishauser (1989)]. They have reported thatincreased levels of polySia on N-CAM are associated with moreplasticity in cell-to-cell interactions during cell migration andaxon outgrowth (Sunshine et al., 1987), yet may decrease theactivity of choline acetyltransferase in chick sympatheticganglion cells in culture (Acheson and Rutishauser, 1988).Based on immunoelectron microscopic studies using anti-poly-Sia antibodies and Endo-N, they have proposed that polySiaon N-CAM can control contact-dependent cell interactions,perhaps by influencing membrane—membrane apposition(Rutishauser et al., 1988). Results showing that removal ofpolySia with Endo-N from a neuroblastoma/sensory neuron cellhybrid also increased cell—substrate interactions, provide furtherevidence for the importance of polySia in mediating adhesiveprocesses (Rutishauser, 1989; Acheson et al., 1991). Land-messer et al. (1990) have suggested that N-CAM polySia mayalso regulate intramuscular nerve branching during embryonicdevelopment. Again, Endo-N was used to specifically removepolySia chains from the axon surface during innervation ofchick muscle. Depolymerization of polySia increased axonfasciculation and decreased nerve branching. Hall et al. (1990)reported the curious finding that differences in polySia residueson NP-40 detergent-solubilized N-CAM do not directly affectthe homophilic binding sites. This result seems contradictory tothe in vivo results showing that [A]N-CAM containing shortchains of polySia are more adhesive than [E]N-CAMs that con-tain longer chains of polySia.

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That polySia may modulate N-CAM-dependent axonalgrowth during development was suggested by Doherty et al.(1990), based on results showing that embryonic chick retinalganglion cells (RGCs) extended longer neurites when grown onhuman N-CAM transfected 3T3 cells than non-transfected cells.Neurite outgrowth from RGCs was inhibited by removing poly-Sia from neuronal N-CAM or adding anti-polySia antibodies.

It thus seems likely that expression of the polySia epitopeon N-CAMs is critical for regulating a variety of events inthe multifactorial pathways of neuronal development. Whilehypotheses abound, the challenge now is to develop newstrategies and approaches to determine the molecular mech-anisms underlying the interesting phenomenology that has beendeveloped. In this regard, a clever set of experiments have beencarried out by Livingston et al. (1990) who prevented the de-velopmentally regulated polysialylation of N-CAM in Xenopusembryos by microinjecting mRNA coding for expression of/3-galactoside:a-2,6-sialyltransferase (a2,6-ST). Presumably,expression of the a2,6-ST blocked polysialylation by alteringthe Siaa2,3Gal linkages of N-CAM. Polymerization of Siaresidue on N-linked oligosaccharides in N-CAM is reported tooccur by extending Siaa2,3-Gal- and not Siaa2,6-Gal-residues(Finne, 1982). The altered pattern of polySia expressionappeared to correlate with abnormal neural development,although clear defects in motor function were not evident. Thislatter observation may reflect the plasticity of neural develop-ment and the involvement of multiple factors (Livingston et al.,1990).

Polysialic acid expression in extraneural tissues

Roth and associates (Bitter-Suermann and Roth, 1987; Rothet al., 1987, 1988d) and Finne et al. (1987) were the first toreport the expression of polysialylated N-CAM outside neuraltissue. They described the temporal expression of [E]N-CAMin the postnatal developing rat kidney. Expression was develop-mentally regulated since polySia immunoreactivity disappearedconcomitantly with postnatal kidney development. PolySia wasalso re-expressed in Wilms tumour (see below). The develop-mentally related decrease in rat kidney polySia was similar tothat reported on N-CAM during neural development, leadingRoth and others to suggest that the kidney polySia, like brain,may also function in regulating cell—cell contacts during kidneydifferentiation and development (Roth et al., 1987). mAbs havealso shown the presence of polySia in newborn heart andmuscle, where it appears to be associated with N-CAM (Finneetal, 1987).

With the exception of the bacterial and fish egg PSGPs, theonly protein other than N-CAM reported to contain polySiais the voltage-sensitive sodium channel from Electrophoruselectricus electroplax (James and Agnew, 1987, 1989). Again,the use of anti-polySia antibodies and Endo-N enabled Jamesand Agnew to confirm the presence of polySia. The sodiumchannel appears to be the only protein in electroplax mem-branes to be polysialylated, implying the presence of a specificpolysialyltransferase committed to polysialylating this protein(James and Agnew, 1989). No information is available regard-ing this enzyme, the length of the polySia chains or theirfunction.

Polysialic acid expression on human tumours

Polysialylated N-CAM was first reported on human tumours in1987 when Lipinski et al. (1987) and Livingston et al. (1987)

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reported its expression on human neuroblastomas, and Bitter-Suermann and Roth (1987) demonstrated its presence on Wilmstumours. Immunoreactivity of N-CAM to anti-polySia anti-bodies and sensitivity to Endo-N digestion were key criteria forproof of structure (Livingston etal., 1988). These initialreports generated a number of subsequent studies that con-firmed the presence of polysialylated N-CAM on human neuro-blastomas and Wilms tumour (Bitter-Suermann and Roth, 1987;Roth etal., 1988b, c, d; Livingston etal., 1989; Moolenaaretal., 1990; Glick etal., 1991). Glick etal. (1991) showedthat polySia was not expressed on FG-Met-2 human pancreaticcancer cells, but was expressed on a number of humanneuroblastoma cells (Table UT).

The most comprehensive study of polySia in human tumourswas a comparative study on urinary tract tumours and non-neuroendocrine tumours by Roth etal. (1988c). Again, ananti-polySia mAb detected polySia on Wilms tumors, but not onclear cell sarcomas of kidney, cystic nephromas, renal cellcarcinomas, transitional cell carcinomas or papillomas of therenal pelvis, ureter or urinary bladder (Table III). Ewing sar-comas, hepatoblastomas, rhabdomyosarcomas, and carcinomasof the stomach, colon, exocrine pancreas, lung and oesophaguswere also immunohistochemically negative for polySia. Sub-sequent studies by Heitz et al. (1990) demonstrated polySia ina number of neuroendocrine tumours, including phaeochromo-cytomas, neuroblastomas, medullary carcinomas of the thyroid,small cell cancer of the lung and pituitary adenomas (Table 111).Carcinoids of various organs, pancreatic endocrine tumours andmelanomas did not express polySia, leading to the suggestionthat polysialylated N-CAM expression may be a useful criterionfor the differential diagnosis of neuroendocrine tumours (Heitzet al., 1990). Anti-polySia mAbs were also used by Moolenaaretal. (1990) to detect polySia expression in small cell lungcancers, a report recently confirmed by Komminoth et al.(1991). In this study, small cell lung carcinomas were positivefor polySia, irrespective of their histological type. Becausemature and atypical bronchial and gastrointestinal carcinoidslacked polySia, Komminoth et al. concluded that polySiaexpression can be used to distinguish neuroendocrine tumoursof the lung.

An extensive screening of human neuroectodermal tumoursfor polySia using an anti-polySia antibody in Western blotanalyses by Figarella-Branger et al. (1990) showed variation inthe polysialylated N-CAM molecules expressed. The highlysialylated N-CAM isoforms characteristic of [E]N-CAM wereexpressed in all four medulloblastomas and four of sevenneuroblastomas examined. The low sialylated N-CAM (140kDa), more characteristic of [A]N-CAM, was present inependynomas and astrocytomas. This observation led Figarella-Branger etal. (1990) to suggest that the decrease in poly-sialylation might reflect a change critical for the conversion ofhuman neuroblastomas into benign ganglioneuromas. Such achange in polysialylation might thus be useful to differentiatebetween neuroblastomas and other human neuroectodermaltumours. Interestingly, Figarella-Branger also reported thatpolySia could be detected in the cerebrospinal fluid frompatients with meningeal spread of medulloblastomas, and thatthese assays may help to assess the metastatic spread of poly-sialylated N-CAM-positive tumours. This is a promising obser-vation that will be important to follow up. Weisgerber et al.(1990) also reported the presence of polysialylated N-CAM(120 kDa) in the cerebrospinal fluid of young children andshowed that its expression decreases during the first year.

Roth et al. (1988d) first reported that polySia was temporar-ily expressed in developing human kidney, but not in adultkidney, and was re-expressed in Wilms tumour. This findingled to the important conclusion that polySia is an oncodevelop-mental antigen in human kidney. The observation that longchains of polySia are expressed on human neuroblastomas,some of which are metastatic to the bone marrow (Livingstonet al., 1988; Glick et al., 1991), suggests that it may also bean oncodevelopmental antigen in brain, since the embryonicform of N-CAM is infrequently expressed in human braintissue.

Based on an analogy to the function of the polySia capsule inneuroinvasive E.coli Kl and N.meningitidis Group B cells,which allows these pathogens to migrate into the brain, weoriginally proposed that surface expression of the polySiaepitope on neuroblastomas may provide these cells withenhanced metastatic potential (Livingston et al., 1987, 1988).Our supposition was based on the assumption that the presenceof such a large polyanionic shield on the surface of the cellmight mimic the bacterial capsular polysaccharides (Troy,1979) and thereby alter normal cell-to-cell interaction andprocesses. Others have also recently suggested that poly-sialylated N-CAM may raise the invasive potential of tumours(Roth et al, 1988b; Moolenaar et al, 1990; Roth and Zuber,1990).

Reference is too frequently made in the literature to the 'long-chain' or 'highly polysialylated' forms of N-CAM, based onimmunoreactivity with anti-polySia antibodies. It is importantto note that this methodology does not provide any informationabout the length of the polySia chains, other than to suggest thatthey probably contain at least 10 Sia residues. It is a formidabletask to accurately determine the DP of polySia chains contain-ing ;> 100 Sia residues (Rohr and Troy, 1980), and there is onlyone study that has examined this aspect of the polysialylation ofN-CAM (Livingston et al, 1988). In this study, partial Endo-Ndigestion of [3H]glycopeptides from [3H]GlcN-labelled humanneuroblastoma cells released a series of [3H]sialyl oligomerswith lengths exceeding 55 Sia residues. This number begins toapproximate the 130-150 Sia residues that have been estimatedto be present on [E]N-CAM chains (Livingston et al, 1988).

All of the poly(-8Neu5Aca2-)n chains expressed on humantumours (Table III) are believed to be associated with N-CAMand, in many of the studies cited above, this has been directlyconfirmed. The work of Fukuda et al. (1985), describing thepresence of a novel a-2,9-linked disialosyl structure (9-Neu-5Aca2-)2 on human embryonal carcinoma cells, has interestingimplications for future studies. a-2,9-Linked sialic acid chainshave not been previously reported in eukaryotes, their onlyprevious citing being restricted to N.meningitidis Group C andE.coli Bosl2 and K92 (Table I). Thus, the studies of Fukudaet al., showing that polylactosamine chains on surface glyco-proteins of Mr 80-120 000 are capped at their non-reducingtermini with a-2,9 disialosyl residues, raise two importantpossibilities. First, that the a-2,9-sialosyl structure will not berestricted to a single ovarian teratocarcinoma but, rather, maybe a decisive epitope on other glycoconjugates implicated innormal development, and that is re-expressed in other tumours.Second, that polyor2,9 sialosyl chains can be expected to befound and that these chains may play an important part in themetastatic phenotype, analogous to their polya-2,8 counterpart.What are needed at this time to follow up Fukuda's intriguingfindings are facile, sensitive and specific reagents to detect andanalyse polya2,9-sialyl residues.

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Prokaryotic-derived reagents for the detection and analysisof polySia residues

In 1984, the development of prokaryotic-derived reagents fordetecting poly(-8Neu5Aca2) chains on nervous tissue glyco-conjugates (and bacteria) was reported as an original findingfrom our laboratory (Vimr et al., 1984). Two of these probeswere bacteria-derived enzymes, while the third was an equine-derived antibody. These reagents are highly specific and sen-sitive for either recognizing, synthesizing or depolymerizingpolySia residues. The three reagents are: (i) a bacteriophage-induced poly(-8Neu5Aca2-)Endo-A'-acylneuraminidase (Endo-N),produced by infection of E.coli Kl with a lytic bacteriophage;(ii) the polysialyltransferase complex from E. coli K1 describedabove that can transfer activated Sia residues to exogenousacceptors containing oligo- or polySia; (iii) an equine poly-clonal IgM antibody, designated H.46, prepared by immunizinga horse with N.meningitidis serogroup B (Sarff et al., 1975).H.46 was made and generously provided by Dr John Robbins(National Institutes of Health).

The facility with which these reagents could be used to dis-cover and confirm the presence of poly(-8Neu5Aca2-) residuesin extracts of neural tissue, tissue slices or whole cells spawneda great interest in, and provided a way to study, polysialylationin eukaryotic systems that had not been possible previously.The strategy and details for the use of these reagents have beenpublished previously (Vimr etal., 1984; McCoy and Troy,1986); therefore, only a brief summary of the rationale of theiruse for identifying polySia is summarized.

Endo-N

Endo-N can serve as a specific molecular probe to detect andselectively modify polySia chains. The enzyme catalyses thedepolymerization of polySia chains according to the followingreaction:

(-8Neu5Aca2-),-X- (-8Neu5Aca2-)2-X

The soluble form of the enzyme was purified to homogeneityand characterized (Hallenbeck et al., 1987a, b; Troy etal.,1987). The enzyme requires at least five Neu5Ac or Neu5Gcresidues for activity. The products of limit digestion from thepolySia capsule of E. coli K1 or the embryonic form of N-CAMare mainly DP4, with some DP3 and DPI ,2 (Hallenbeck et al.,1987a). The purified Endo-N derived from bacteriophage K1Finfection of E.coli K12/K1 hybrids also effectively cleaves thealternating (-8Neu5Aca2-) and (-9Neu5Aca2-) polymers, butnot poly(-9Neu5Aca2-) chains (Hallenbeck etal., 1987a).Similar phage-induced enzymes have been described byKwiatkowski etal. (1982), Finne and Makela (1985) andPelkonen et al. (1989). Endo-N has been used to detect polySiaresidues in bacteria (Vimr etal., 1984), mammalian tissues(Vimr etal., 1984; McCoy etal., 1985; Finne etal., 1987;Lipinski etal., 1987; Roth etal., 1987; Acheson and Rutis-hauser, 1988; Livingston et al., 1988; Moolenaar et al., 1990),fish egg PSGP (Kitajima etal., 1988a) and the eel electricorgan (James and Agnew, 1987, 1989).

E.coli Kl CMP-Neu5Ac:poly-a-2,8-sialosyl sialvltransferase(polysialyltransferase)

The E.coli Kl polysialyltransferase complex catalyses the trans-fer of [l4C]Neu5Ac from CMP-[14C]Neu5Ac to endogenous

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and exogenous acceptors containing pre-existing a-2,8-linkedpolySia residues (Troy et al., 1975; Vijay and Troy, 1975;Troy and McCloskey, 1979; Whitfield et al., 1984a; Whitfieldand Troy, 1984; Weisgerber and Troy, 1990). The enzyme canbe used to attach [14C]Neu5Ac to exogenous acceptors contain-ing at least three a-2,8-linked sialyl residues. E.coli EV11, amutant derived from a hybrid of E.coli K12 and a Kl poly-Sia-expressing strain, is defective in vitro in catalysing theendogenous synthesis of polySia. IOV from EV11 can, how-ever, transfer Neu5Ac from CMP-Neu5Ac to exogenousacceptors (Vimr et al., 1984). Thus, restoration of [l4C]poly-Sia synthesis in E.coli EV11 membranes by the addition ofexogenous acceptors is a sensitive indicator for determining thepresence of pre-existing oligo/poly(-8Neu5Aca2-) residues(Vimr etal., 1984; Livingston et al., 1988).

Anti-polysialosyl antibody (H. 46)

The presence of poly(-8Neu5Aca2-) associated with eitherbacteria, fish egg PSGPs or other eukaryotes can be detected byWestern blot, rocket immunoelectrophoresis or immunohisto-chemical detection methods using the H.46 antibody (Finneet al., 1983b; Vimr et al., 1984; McCoy et al., 1985; Living-ston etal., 1988). The presence of polySia is confirmed ifpre-treatment with Endo-N abolishes H.46 immunoreactivityand the solubilized material is shown to contain short sialyloligomers (Vimr et al., 1984; McCoy et al., 1985; Livingstonet al., 1988). This control is important when using antibodiesto detect polySia because an IgM has been reported that cross-reacts with polynucleotides, including DNA (Kabat et al.,1986). H.46 and the mAb are specific, however, for polySia.

The preparation of mouse monoclonal IgG antibodies (mAb)that recognize the a-2,8-linked polySia epitope with highaffinity (Frosch etal., 1985), and their subsequent use toidentify these residues in a variety of normal and pathologicalstates (Bitter-Suermann and Roth, 1987; Finne et al., 1987;Lipinski etal., 1987; Roth etai, 1987, 1988a; Aaron andChesselet, 1989; Moolenaar et al., 1990), has highlighted thesimplicity and the potential of this method. The shortesta-2,8-linked sialyl oligomer required to inhibit or bind theseantibodies is - D P 8-10 (Finne and Makela, 1985; Jenningsetal., 1985). Jennings and colleagues have shown that theunusual length is required in order for the antibody to recognizea conformational epitope that is contained within the internal sixsialyl residues (Michon etal., 1987). This conclusion issupported by the recent studies of Hayrinen et al. (1989).

Immunochemical probes for detecting KDN and oligo-polyKDN structures in glycoconjugates have recently been reportedby Kanamori et al. (1991). An IgG raised against the KDN-gpisolated from the vitelline envelope recognizes this epitope, andbinding is inhibited by oligo (KDN). This antibody also reactswith KDN-ganglioside (KDN)GM3, and thus will be animportant reagent to search for oligoKDN chains in othersources.

Summary and future perspectives

Studies to understand polysialylation of surface glycoconjugatesin sources as distinct as neuroinvasive bacteria and human brainhave emerged as an exciting new aspect of molecular, cell anddevelopmental biology, and of microbiology, tumour-, neuro-and glycobiology. Studies in these areas will probably becomeincreasingly more important as we seek to determine the

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function and consequences of polysialylation, and its molecularinvolvement in bacterial pathogenesis, fertilization, celladhesive interactions, neural development, cancer metastasisand as oncodevelopmental antigens in some human tumours.The challenge now for future studies is to determine thefunction of these molecules and how their synthesis and surfaceexpression are regulated. This will also probably require agreater understanding of the three-dimensional conformation ofthe polysialosyl carbohydrate chains.

In prokaryotes, these studies will have to elucidate how themultienzyme kps complex and undecaprenyl phosphate areorganized to catalyse the initiation and polymerization of poly-Sia chains, and the mechanism whereby the translocation andexport machinery transports these polyanions across the innermembrane and to the outer membrane. It will also be imperativeto determine how the high-energy phosphoryl potential of ATPis linked to synthesis, and how both A/*H+ and A* are in-volved in the transmembrane translocation of polySia chains.Finally, the significance of the intriguing new findings thatseveral proteins of the kps cluster contain polyisoprenyl (doli-chol) recognition sequences, and that the deduced proteinsequence of both NeuE and NeuS are evolutionarily related toDNA polymerases and other DNA binding proteins, must bedetermined.

The immediate challenge in the eukaryotic systems will be toclone the polysialyltransferase(s) responsible for catalysing thepolysialylation of fish egg PSGPs, N-CAMs, and possibly otherproteins and glycolipids. A key problem will be to discoverhow these enzymes are regulated, since the amount of polySiaappears to be critical for mediating key events in fertilization,normal morphogenesis of nerve tissue and, perhaps, in influ-encing the metastatic potential of human cancers in a prog-nostically important way. It will also be essential to determinewhere polysialylation occurs, whether oligomerization of Siaon dolichylphosphate occurs, what signals determine whichN-linked oligosaccharide chains are polysialylated and themechanism for trafficking these molecules to the cell surface,if synthesized elsewhere. Whether any of the eukaryoticproteins are evolutionarily related to the proteins encoded bythe kps cluster in E.coli Kl will also be important to determine.Given the complexity of polySia synthesis, translocation, exportand assembly in prokaryotes, it does not appear likely that thepathway regulating the surface expression of polySia ineukaryotes will be any simpler.

As the reagents described here to detect and analyse polySiaresidues become more readily available, and newer methods aredeveloped, it is anticipated that the occurrence and function ofpolysialylated glycoconjugates will continue to expand. It isalso likely that these studies will reveal an even greater role ofpolySia in biological processes, as well as alteration in expres-sion of these molecules that will be associated with an increas-ing number of pathophysiological states, touching perhaps allorgan systems. A great deal remains to be accomplished in thisnew, exciting and vibrant area of sialobiology.

Acknowledgements

The paUence and expert secretarial assistance of Becky Greer is gratefullyacknowledged. I also express my most sincere appreciation to Dr Jean Ye forher expertise in the sequence analysis of the kps gene cluster, for her criticalreading of the manuscript and for her tireless efforts in assembling thereferences. I am also indebted to Mr Jin-Won Cho for his valuable contributionsto our computer analysis of kps and Figure 2. This work was supported byresearch grant AI-09352 from the National Institutes of Health.

Abbreviations

CCCP, carbonyl cyanide m-chlorophenylhydrazone: DP, degree of polymer-ization; Endo-N. poly(-8Neu5Aca2-)endo-A'-acylneuraminidase; F1TC.fluorescein isothiocyanate; IOV, inside-out vesicles; KDN, 2-keto-3-deoxy-r>glycero-r>galacto-nonulosonic acid (trivial name, deaminated neuramirucacid); LDV, low-density vesicles; Lj, lake trout; mAb, monoclonal antibodyN-CAM, neural cell adhesion molecules, [A] is adult and [E] is embryo;Neu5Ac, W-acetylneuraminic acid (sialic acid); Neu5Gc, A'-glycolylneuraminicacid; ORF, open reading frame, PEP, phosphoenolpyruvate; polySia. poly-a-2,8-linked polysialic acid; PSGPs, polysialoglycoproteins; RGC. retinalganglion cell; ROV, nght-side-out vesicles; RT, rainbow trout; Sia. sialic acid;a-2,6-ST, CMP-Neu5Ac (or Neu5Gc):a-A'-acefylgalactoside a-2.6 sialyltrans-ferase; a-2,8-ST, CMP-Neu5Ac (or Neu5Gc):a2,8-sialosyl sialyltransferase;VE, vitelline envelope.

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Troy.F.A., Vijay.I.K. and Tesche.N. (1975) Role of undecaprenyl phosphatein synthesis of polymers containing sialic acid in Escherichia coli J Biol.Chem., 250, 156-163.

Troy.F A , Hallenbeck.P.C, McCoy,R.D. and Vimr.E R (1987) Detection ofpolysialosyl-containing glycoproteins in brain using prokaryotic-denvedprobes. Methods Enzymol., 138, 169-185

Troy.F.A., Janas.T. and Merker.R.I. (1990a) Topology of the poly-a2,8-sialyltransferase in E.coli Kl and energetics of polysialic acid chaintranslocation across the inner membrane Glycoconjugate J., 7, 383.

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Structure, function and synthesis of polysialic acids

Whitfield.C, Vimr.E.R., Costerton,J.W. and Troy,F.A. (1985) Membraneproteins correlated with expression of the polysialic acid capsule inEschenchia coli Kl. J. Baaerioi, 161, 743-749.

Wyle.F.A., Artenstein.M.S., Brandt,B.L., Tramont.E.C, Kasper.D.L..Altieri,P.L., Berman.S.L. and Lowenthal.J.P. (1972) Immunologicresponses of man to group B meningococca] polysaccharide vaccines.J. Infect. Dis., 126, 514-522.

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cell adhesion molecule N-CAM in Wilms tumor and their subcellulardistributions. Eur J. Cell Bioi, 51, 313-321.

Received on October 29, 1991, accepted on Nowmber 5, 1991

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