structural diversity in the a24blinked polysialic acid ...mild acid hydrolysisltlc method-each...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 31, Issue of November 5, pp. 23675-23684, lYYB 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Structural Diversity in the a24bLinked Polysialic Acid Chains in Salmonid Fish Egg Glycoproteins OCCURRENCE OF POLY(Neu5Ac), POLY(Neu5Gc), POLY(Neu5Ac, Neu5Gc), POLY(KDN), AND THEIR PARTIALLY ACETYLATED FORMS*

(Received for publication, June 21, 1993)

Chihiro Satot, Ken Kitajimat, Ichiro TazawaS, Yasuo Inouets, Sadako Inouell, and Frederic A. Troy, 1111 From the $Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo-7, Tokyo 113, Japan, the Wchool of Pharmaceutical Sciences, Showa University, Hatanodai-1, Tokyo 142, Japan, and the IIDepartment of Biological Chemistry, Uniuersity of California, School of Medicine, Davis, California 95616-8365

a243-Linked polysialic acid (polySia) chains termi- nate 0-linked oligosaccharide chains on Salmonidae fish egg polysialoglycoproteins (PSGPs). Expression of these surface PSGPs are developmentally regulated and the polySia epitope is functionally implicated in a number of distinct species-specific cell-cell recognition events during fertilization and early embryogenesis. To better understand the functional diversity of these PSGPs, structural studies of the polySia chains isolated from three genera and eight species of Salmonidae fish eggs were carried out by chemical, immunochemical, enzymatic, and ’H NMR methods. A remarkable degree of structural diversity was found, including differences in the N-acyl groups, i.e. N-acetylneuraminic acid (NeuBAc) or N-glycolylneuraminic acid (NeuBGc), and in the presence of either 0-acetyl substitution at C4, C,, or C9 or 0-lactyl substitution at CS. The presence of heteropolymers containing both Neu5Ac and Neu5Gc residues was also an unexpected finding. Ac- cordingly, the different forms of a243-linked homo- and heteropolymers of these polySia structures include: poly(NeuSAc), poly(NeuBGc), poly(Neu5,xAcz), poly(NeuSGcxAc), poly(NeuBAc, NeuBGc), poly- (NeuBAc, Neu5,scAcz), poly(NeuBAc, NeuBGcxAc), poly(Neu5Gc,Neu5,xAcZ), and poly(NeuBGc, Neu5- GcxAc), where x represents the site of acetylation at carbon atom 4, 7, or 9. The significance of this new structural information, together with our recent finding of a24b l inked polydeaminoneuraminic acid, poly(KDN), in the rainbow trout egg vitelline envelope, is that it demonstrates the natural occurrence of multiple forms of a24b l inked polySia chains in Salmon- idae fish glycoproteins that have not been previously described. The results also predict that a remarkable array of polysialylated glycoconjugates is yet to be discovered in animals other than teleost fishes.

The first eukaryotic glycoprotein shown to contain a 2 4 -

* This research was supported in part by Grants-in-Aid for General Scientific Research Grant 04453160, International Scientific Pro- gram-Joint Research Grant 04044055 from the Ministry of Education, Science, and Culture of Japan, and National Institutes of Health Grant AI-09352. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J TO whom correspondence should be addressed. Fax: 81-3-5684- 2394.

linked oligo/polysialic acid (polySia)’ chains was isolated from the unfertilized eggs of rainbow trout (Oncorhynchus mykiss) by Inoue and Iwasaki in 1978 (1). Prior to this discovery, polySia structures had only been known in some neuroinvasive Escherichia coli K1 and Neisseria meningitidis Gp B strains (2, 3). Following the development of highly specific and sensitive methods using anti-polySia antibodies and endo-N-acylneuraminidase (Endo-N) to detect a 2 4 - l i n k e d poly(Neu5Ac) chains (4-6), glycoproteins bearing poly- (NeuSAc) chains have been identified in a wide variety of animal species ranging from insect to human (7-13). Expres- sion of polySia chains on the embryonic form of vertebrate neural cell adhesion molecules (N-CAM) and on sodium channel proteins are two examples of polysialylated glycoproteins that have been structurally characterized (10, 11, 14,15). The functional involvement of polysialylated N-CAM in mediating a variety of cell-cell adhesive interactions, including neurite fasciculation, neuromuscular interactions, and cell migration, is a field that is still emerging.

The polySia chains from rainbow trout egg polysialoglycoproteins (PSGPs) were found to contain only N-glycolylneuraminic acid (Neu5Gc) residues (1). In contrast, vertebrate N- CAM (5, 16) and the a-subunit of the adult rat brain Na’

The abbreviations used are: polySia, polysialic acid; KDN, 2-keto- 3-deoxy-D-glycero-D-gahcto-nononic acid or naturally occurring deaminoneuraminic acid; Sia, sialic acid; PSGP, polysialoglycopro- tein; H-PSGP, high molecular weight PSGP isolated from the unfertilized eggs of Salmonidae fish eggs; L-PSGP, low molecular weight PSGP isolated from the fertilized eggs of Salmonidae fish eggs; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; poly(Neu5Ac), a2-%linked homopolymer of Neu5Ac or (- 8Neu5Aca2+),; poly(Neu5Gc), a 2 4 - l i n k e d poly(Neu5Gc) or (+ 8Neu5Gca2+),; poly(Neu5,xAcn), a 2 4 - l i n k e d homopolymer of N - acetyl-x-O-acetylneuraminic acid, where x is carbon atom 4, 7, or 9; poly(Neu5GcxAc), a 2 4 - l i n k e d homopolymer of x-0-acetyl-N-glycolylneuraminic acid poly(Neu5Ac, Neu5Gc), a 2 4 - l i n k e d hetero- polymer made up of Neu5Ac and Neu5Gc; poly(Neu5Ac, Neu5,xAcz), poly(Neu5Ac, Neu5GcxAc), poly(Neu5Gc, Neu5,xAc2), and poly(NeuSGc, Neu5GcxAc), a2-8-linked heteropolymers made up of two different component sialic acids, where x represents the site of acylation a t C4, C,, or C9; poly(KDN), a 2 4 - l i n k e d polydeaminoneuraminic acid; N-CAM, neural cell adhesion molecule; KDN-gp, KDN-rich glycoprotein isolated from vitelline envelope and/or female ovarian fluid of rainbow trout; mAb.735, monoclonal antibody specific t o a24 - l inked poly(Neu5Ac); H.46, polyclonal antibody specific to a 2 4 - l i n k e d poly(Neu5Ac); MM-GLC, mild methanolysis-GLC; MH/MM-GLC, mild acid-mild methanolysis-GLC; Endo-N, hacte- riophage K1F-derived endo-N-acylneuraminidase; GLC, gas liquid chromatography; TLC, thin-layer chromatography; ’H NMR, proton nuclear magnetic resonance spectroscopy; dansyl, 5-dimethylamino- naphthalene-1-sulfonyl.

23675

23676 Diversity in a 2 4 - L i n k e d Polysialic Acid Structure

channel (10) contain only Neu5Ac residues. A second unique structural feature of the rainbow trout PSGP is that the poly(Neu5Gc) chains are capped at their nonreducing termini by deaminated neuraminic acid (2-keto-3-deoxy-D-glycero-~- galacto-nononic acid; KDN) residues (17). Recently, a structurally new type of glycoprotein, KDN-rich glycoprotein (KDN-gp) from the vitelline envelope of rainbow trout, was described (18). KDN-gp is a large mucin-type glycoprotein which contains a number of 0-linked glycan units each having an a24"nked oligo/poly (KDN) chain (19). While the exact molecular involvement of the polySia chains on PSGPs in cellular processes continues to be elucidated, what has emerged is that these carbohydrate chains appear to function in a remarkably diverse number of cellular processes, including species-specific cell-cell recognition events during fertilization and early embryogenesis (20,21). Furthermore, KDN- gp may mediate egg-sperm interaction (21,22). To determine if this functional diversity may be related to structural diversity in the polySia chains, we initiate studies to determine the structures of the polySia chains on Salmonidae fish egg PSGPs isolated from the eggs of eight different fish species from three genera, Oncorhynchus, Salmo, and Saluelinus. In this paper we describe a wide range of diversity in polySia structure including differences in the relative proportion of monomeric units (Neu5Ac, Neu5Gc, and KDN), the presence of hybrid structures, and the extent of 0-acylation (acetyl and lactyl groups) in the PSGPs. Additional structural studies also revealed the occurrence of three distinct types of PSGP molecules. Two were homopolymers containing only poly- (Neu5Ac) or poly(Neu5Gc) chains. The third type was a hybrid polySia structure that contained both Neu5Ac and Neu5Gc residues in the same chain. The significance of these studies also underscores the importance of using chemical and physical methods to identify polySia residues. For example, any search for polysia-containing glycoconjugates using the presently available immunoprobes such as monoclonal mAb.735 (6) and polyclonal H.46 (2, 23), which are specific for identifying only a 2 4 - l i n k e d poly(Neu5Ac) chains, would not detect large number of the new structures reported here. Our findings predict that the structural diversity in the polySia chains in mammalian glycoproteins may be considerable, and that the different structures revealed here will not be unique to fish.

EXPERIMENTAL PROCEDURES

Salmonidae Fish Eggs-Unfertilized and fertilized (30-60 min postfertilization) eggs were supplied by courtesy of the following institutions. Lake trout (Saluelinus namaycush), brook trout (Saluel- inus fontinalis), kokanee salmon (Oncorhynchus nerka adonis), and brown trout (Salmo trutta fario), the National Research Institute of Aquaculture, Nikko Branch; rainbow trout (0. mykiss) and Japanese common char (Saluelinus leucomaenis pluuius), the Gunma Prefec- tural Fisheries Experimental Station at Kawaba; rainbow trout (0. mykiss), the Okutama Fish Farm, Department of fishery, Tokyo Metropolitan Government; chum salmon (Oncorhynchus keta), the Wokkaido Salmon Hatchery, Chitose Branch; land-locked cherry salmon (Oncorhynchus masou ishikawai), the Gunma Prefectural Fisheries Experimental Station at Hakoshima.

PSGPs and Other Materials-H- and L-PSGPs from the unfertilized and fertilized eggs of each of the above Salrnonidae fish species were prepared essentially by the methods previously described (24, 25). PSGPs isolated from the various salmonid fish eggs were designated as follows: lake trout (S . numaycush), PSGP(Sn); brook trout (S. fontinalis), PSGP(Sf); Japanese common char (S. leucomaenis pluuius), PSGP(Slp); rainbow trout (0. mykiss), PSGP(0rn); chum salmon (0. keta), PSGP(0k); land-locked cherry salmon (0. masou ishikawai), PSGP(0mi); kokanee salmon (0. nerku adonis), PSGP(0n); brown trout (S. trutta fario), PSGP(Stf). PSGPs oh- tained from eggs partially activated, either by insemination or im- mersion in water, were found to have the molecular size between H-

PSGP and L-PSGP. PSGP(0n) was kindly provided by Yu Song of our laboratory.

Neu5Ac and colominic acid (Na+ form; (+8Neu5Aca2+), where n = 30) were purchased from Sigma. Antiserum containing polyclonal IgM antibodies (H.46) specific to a-2,b-linked poly(Neu5Ac) was kindly provided by Dr. J. B. Robbins, (National Institutes of Health, Bethesda). Exosialidase (Arthrobacter ureafaciens) was purchased from Nacalai.

Chemical Analyses-Sialic acids were quantified or monitored by the thiobarbituric acid (26, 27) and the resorcinol methods (28). The hexose content was estimated by the phenol-sulfuric acid method (29). The molar ratios of component monosaccharides of glycopep- tides and glycoproteins were determined by GLC as reported previously (30). Amino acids and hexosamines were determined quantita- tively by the methods described previously (31,32). NH2- and COOH- terminal analyses were carried out by using the dansyl (5-dimeth- ylaminonaphthalene-1-sulfonyl) chloride procedure (31, 33, 34). Au- tomated amino acid sequence analysis was carried out as previously described (31,32).

Quantitation of Neu5Ac and Neu5Gc in PSGP-The individual sialic acid components in PSGPs were quantitated by the mild acid hydrolysis/subsequent mild methanolysis-GLC (MH/MM-GLC) proo- cedure as previously described (35). To a dry sample containing 10 pg of myo-inositol as an internal standard, was added 300 p1 of 0.1 M trifluoroacetic acid. The resulting solution was heated at 80 "C for 3 h. The aqueous acid was removed under a stream of dry NZ and then dried in a vacuum desiccator for 30 min. The residue was treated with 0.5 ml of 0.05 N methanolic HCl at 80 "C for 1 h (36). The resulting methyl ester methyl ketosides were trimethylsilylated and analyzed by GLC on a glass column (3 mm X 1 m) of 1.5% OV-17 on Chromosorb W, operated at temperatures from 128 to 230 "C (4 "C/ min). These analyses were carried out on a Shimadzu GC-14A gas chromatograph connected to a Shimadzu C-R6A chromatopac.

Mild Acid HydrolysislTLC Method-Each sample of PSGPs (400 pg as Neu5Ac) and colominic acid was partially hydrolyzed in 200 ~1 of 50 mM sodium acetate buffer (pH 4.8) a t 37 "C for 48 h, after mild alkaline treatment (0.1 M NaOH, 25 "C, 60 min) and neutralization with 1 M HC1. Aliquots were subjected to TLC analysis as previously described (37, 38).

Thin Layer Chromatography-Approximately 1-10 pg of sample were spotted on a TLC sheet (0.2-mm thick silica gel plastic sheet; Kiesel Gel 60, Merck). The TLC plate was developed with 1-propanol, 25% NHaOH, water (6k2.5) (38, 39). The sialic acids were detected by spraying the TLC plate with resorcinol reagent and heating at 80 "C.

Separation of Oligosialic Acids Released from PSGP(S1p) and Frac- tionation According to C h i n Length-The partial acid hydrolysate (50 mM sodium acetate buffer, pH 4.8, 37 "C, 3 days) of PSGP(S1p) was chromatographed on Sephadex G-75 (1.3 X 83 cm; eluted with water). The fraction containing oligosialic acids was subjected to DEAE-Sephadex A-25 chromatography (1 X 13 cm) after desalting on a Sephadex G-25 column (1.8 x 115 cm; eluted with 5% aqueous ethanol). The oligomers were eluted with a linear gradient of NaCl (0-0.4 M) in 10 mM Tris-HC1 (pH 7.8). Each oligomer fraction was desalted and analyzed by TLC. About 1.0 mg of dimer fraction was subjected to preparative silica gel TLC (Kiesel Gel 60, Merck; solvent, 1-propanol, 25% NH40H, water = 6:1:2.5 (v/v), developed for 11.5 h). A lane from the TLC plate was cut off for monitoring by visuali- zation with the resorcinol reagent. Each sialyl oligomer was eluted from the silica gel plate with 20% ethanol, and quantitated by the resorcinol method.

Identification of Each Sialyl Dimer by GLC-Twenty to 100 pg of each sialyl dimer were evaporated to dryness, and reduced in 0.25 ml of 1 M NaBHl at 25 "C for 3 h. After destroying excess NaBH4 with 0.3 ml of 1 M acetic acid, each dimer fraction was subjected to Dowex 5O-X2 (H+ form; 1 X 5 cm) and eluted with 4 column volumes of water. The eluate was dried after addition of 15 ml of methanol, which was repeated twice. Sialitol and the nonreducing terminal residue of each dimer were analyzed by the MM-GLC method (35) on a Shimadzu GC-14A gas chromatograph, equipped with a glass column of 2% OV-101 on Gas-chrom Q (3 mm x 2 m) or 1.5% OV- 17 on Chromosorb W (3 mm X 1 m). The column was run from 180 to 230 "C (2 'C/min) using NP as the carrier gas and a flame ionization detector.

Digestion of PSGPs with Endo-N-PSGP was first treated with mild alkali (0.1 M NaOH, 25 "C, 1 h), dialyzed after neutralization with HC1 and lyophilized. PSGP (100 pg as Neu5Ac) was dissolved in 100 p1 of 20 mM Tris-HC1 buffer (pH 7.5) and treated with 5.0 pg

Diversity in cu243-Linked Polysialic Acid Structure 23677

(about 10 milliunits) of bacteriophage K1F-derived endo-N-acylneuraminidase (Endo-N) (40) a t 37 "C for 20 h (37). The digests were analyzed by TLC.

Ouchterlony Immunodiffusion Experiments with Anti-poly (Neu5Ac) Polyclonal Antibody, H.46-Immunoreactivity of the antiserum H.46 with various salmonid fish egg PSGPs was tested by Ouchterlony double diffusion method (41). Immunodiffusion was carried out using 0.9% agar in 0.15 M NaCl, 0.01 M sodium phosphate buffer (pH 7.2), and 0.1% NaN3. The center well was filled with 20 11 of H.46 (50 mg/ml) and outer wells were filled with 10 1 1 of each PSGP (37).

Immunoprecipitation Experiment-An H.46 IgM fraction was used. The IgM fraction was obtained by gel filtration on a Sephacryl S-300 column (2.9 x 45 cm; equilibrated and eluted with phosphate-buffered saline) of 50% saturated ammonium sulfate precipitate of a horse antiserum (H.46) against group B N . meningitidis (23). PSGP(Sn) and PSGP(0m) were incubated with 560 pg of H.46 antibody in 1.5 ml of phosphate-buffered saline (10 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCI) at 25 "C for 1 h and further incubated at 4 "C for 72 h. After centrifugation at 8000 X g for 30 min, the precipitate was washed twice with phosphate-buffered saline and once with water (42, 43), and analyzed for sialic acid by the MH/MM- GLC method (35).

Exosialidase Digestion of H-PSGPfSn) and H-PSGP(Sf)-Intact PSGP(Sn) and PSGP(Sf) (each 100 pg as Neu5Ac) were exhaustively digested with 12.5 milliunits of A. ureafaciens exosialidase in 200 pl of 50 mM sodium acetate buffer (pH 5.5) before and after mild alkaline treatment (0.1 M NaOH, 25 "C, 60 min) followed by neutralization with 1 N HCl (44). Release of free sialic acid was monitored by the thiobarbituric acid assay.

Preparation of Sialidase-resistant Sialic Acid and Characterization by 'H NMR Spectroscopy-L-PSGP(Sn) containing only Neu5Ac residues (5.0 mg as Neu5Ac) was exhaustively digested with 0.625 units of A. ureafaciens exosialidase in 50 mM sodium acetate buffer (pH 5.5) (44), and the digest was subjected to Sephadex G-50 column (1.6 X 149 cm) chromatography, eluted with 0.1 M NaCl to eliminate free sialic acid released. The sialidase-resistant sialic acid-containing material was desalted on a Sephadex G-25 column (1.8 X 115 cm), lyophilized, and then subjected to mild acid hydrolysis with 0.01 N trifluoroacetic acid at 70 "C for 30 min. The hydrolysate was chromatographed on a Sephadex G-50 column (1.6 X 149 cm) after neutralization and eluted with 0.1 M NaCl. Free sialidase-resistant sialic acid fraction was subjected to preparative TLC on a Silica Gel 60 plate and developed in 1-butanol, 1-propanol, water (5:10:3, v/v) to separate the modified sialic acids from their parent unsubstituted sialic acid(s). The modified sialic acids were further purified by passage through a Sephadex G-25 column (1.8 X 115 cm) and then subjected to 400-MHz 'H NMR spectral measurement according to the method previously described (44).

RESULTS

Compositional Analyses of PSGPs from Eight Different Species of Salmonidae Fishes

T h e objective of this experiment was to determine if the structures of the PSGP molecules present in the eggs of different salmonid fish orders and/or species were similar, or if in fact order- and/or species-specific differences existed. A second objective was to determine how many different forms of the oligo/polySia chains were present in these PSGP molecules. Since it was demonstrated that PSGPs from Saluelinm fish eggs contained both Neu5Ac and Neu5Gc (39), while those from rainbow trout (0. mykiss), Pacific salmon (0. keta), and land-locked cherry salmon (0. m o u ishikawai) contained exclusively Neu5Gc residues (1, 39, 45), this study focused on the oligo/polysialyl chains of the PSGPs isolated from Salvelinus fish eggs.

The PSGP(Sn) isolated from the unfertilized and fertilized eggs of three individual lake trout varied widely in molecular size. Therefore, each lake trout PSGP(Sn) from a single fish was first purified by chromatography on a DEAE-Sephadex A-25 anion-exchange column. Each PSGP gave rise to four fractions which eluted at NaCl concentrations of 0.18-0.27,

0.27-0.32, 0.32-0.34, and 0.34-0.40 hi. These fractions were pooled and chromatographed separately on a Sephacryl S-200 column. Subfractions thus obtained were pooled and designated PSGP(Sn)-I, PSGP(Sn)-11, PSGP(Sn)-111, PSGP(Sn)-IV, and PSGP(Sn)-V, in accord with their increasing Neu5Gc content. The relative molar ratios of NeuSAc and Neu5Gc in these PSGP(Sn) are shown in Table I, together with those of the other two species of Salvelinus, four Oncorhynchus species, and one Salmo fish. The carbohydrate and amino acid compositions of Salmonidae (3 genera, 8 species) H-PSGPs are shown in Tables I and 11.

Amino Acid Sequence Analysis of PSGPs Isolated from Salmonidae Fish Eggs

T h e NHZ-terminal amino acid of L-PSGP(Sn) and L- PSGP(Sf) was determined by the method of Gray (33). Asp was the only amino acid detected. In contrast, their COOH- terminal amino acids were Asp and Ser (Scheme I). L- PSGP(Sn) and L-PSGP(Sf) were then subjected to auto- mated amino acid sequence analysis. The results indicated that both L-PSGP(Sn) and PSGP(Sf) contained glycododeca- and glycotridecapeptide. The molar ratios of the dodeca- and tridecapeptide were estimated to be approximately 70:30 for PSGP(Sn) and 75:25 for PSGP(Sf) by the method described previously (31,46). At cycles where glycosylated amino acid residues were present, no phenylthiohydantoin-deriva-

TABLE I Carbohydrate composition of PSGPs isolated from Salmonidae fish

eggs Values are molar ratios relative to GalNAc taken as 1.0. Sialic acid

content (Sia) was determined by the resorcinol method. Neu5Ac and Neu5Gc contents were determined by the MH/MM-GLC method and are expressed in mol %.

PSGP Fuc Gal GalNAc KDN Sia Neu5Ac Neu5Gc

PSGP(Sn)-I 0.27 1.1 1.0 0.039 3.4 100 0 PSGP(Sn)-I1 0.14 0.93 1.0 0.093 1.6 94 6.0 PSGP(Sn)-111 0.23 1.0 1.0 0.074 3.6 77 23 PSGP(Sn)-IV 0.10 1.1 1.0 0.13 3.2 30 70 PSGP(Sn)-V 0.24 1.1 1.0 0.11 2.3 13 87 PSGP(Sf)-I 0.089 1.3 1.0 0.19 2.7 IO 90 PSGP(Sf)-I1 0.16 1.1 1.0 0.031 3.4 6.2 94 PSGP(S$)" 0.087 1.3 1.0 0.052 2.6 10 90 PSGP(Om)b 0.098 1.1 1.0 0.32 3.6 0 100 PSGP(0rni)" 0.21 1.1 1.0 0.047 3.6 0 100 PSGP(0k) 0.46 1.5 1.0 0.005 4.7 0 100 PSGP(0n)-I" 0.28 1.5 1.0 0.16 4.6 0 100 PSGP(0n)-II' 0.26 1.6 1.0 0.16 4.3 10 90 PSGP(Stf) 0.19 1.1 1.0 0.09 5.4 2.6 97

Data taken from Ref. 39. * Data taken from Ref. 31. E Data taken from Ref. 46.

TABLE I1 Amino acid composition of PSGPs isoluted from Salmonidae fish eggs

Values are molar ratios relative to Thr taken as 2.0. PSGP Species Asp Thr Ser Glu Pro Gly Ala

PSGP(Sn)-I S. namuycush 1.8 2.0 1.8 0.93 1.1 0.95 2.7 PSGP(Sf)-I S. fontinalis 2.1 2.0 2.0 1.2 1.1 1.1 3.2 PSGP(Slp)" S. leucomaenis 1.8 2.0 1.7 0.95 1.1 1.1 3.3

PSGP(0k) 0. keta 2.4 2.0 3.0 1.1 1.2 1.4 3.1 PSGP(0mi) 0. masou ishikawai 1.9 2.0 2.5 1.0 1.2 1.0 3.0 PSGP(Om)* 0. mykiss 2.1 2.0 2.1 1.2 1.2 2.3 3.4 PSGP(On)-II' 0. nerka adonis 2.8 2.0 1.7 0 1.2 1.8 2.8 PSGP(Stf) S. truttafariu 2.2 2.0 2.2 1.1 1.2 1.1 3.2

pluviw

Data taken from Ref. 39. Data taken from Ref. 31. Data taken from Ref. 46.

23678 Diversity i n a 2 4 - L i n k e d Polysialic Acid Structure SCHEME I

Amino acid sequences of L-PSGPs isolated from fertilized eggs of Salmonidae fish species c" indicates 0-glycosylation site) These peptide sequences also represent the smallest repeating units of H-PSGPs and values in brackets show molar ratios of L-PSGPs

when two molecular forms are present in the same fish species. Lake trout (S . namaycwh) L-PSGP(Sn)

Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp [701 Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser [301

Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp [751 Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser ~ 5 1

Asp-Ala -Thr*-Ser*-Glu-Ala -Ala -Thr . -Gly -Pro-Ser -Asp [ W

Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp ~ 5 1 Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser [351

Chum salmon (Oncorhynchus keta) L-PSGP(0k)" Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser [1001

Land-locked cherry salmon (0. musou ishikawai) L-PSGP(0mi)" Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser [loo1 Rainbow trout (0. mykiss) L-PSGP(Om)b Asp-Asp-Ala -Thr*-Ser ."Ala -Ala -Ala -Thr*-Gly-Pro-Ser -Gly [1001 Kokanee salmon (0. nerka adonis) L-PSGP(OnT

Asp-Ala-Thr*-Ser*-Asp-Ala-Ala-Thr*-Gly-Pro-Ser-Asp [501 Asp-Asp-Ala-Thr*-Ser*-Asp-Ala-Ala-Thr*-Gly-Pro-Ser-Gly [501

Brook trout (S . fontinalis) L-PSGP(Sf)

Japanese common char (S. leucomaenis pluuius) L-PSGP(S1p)"

Brown trout (S . trutta fario) L-PSGP(Stf):

' Taken from Ref. 48. *Taken from Ref. 20.

Taken from Ref. 46.

tives were identified. These Thr/Ser residues were assigned, as shown below, based on the amino acid composition and accumulated data indicating that the tri- or tetrapeptide sequence around the glycosylation sites in PSGP was strictly conserved among Salmonidae fishes (20, 31, 46-48). These data, taken together with previous studies (20, 31, 46-48), show that the structures of H-PSGP(Sn) and H-PSGP(Sf) were expressed as a mixture of the following two tandem repeats of glycododeca- and glycotridecapeptides in the molar ratios described above (* denotes the site of 0-glycosylation): (-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp)", (-Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser- Ser),.

The brown trout L-PSGP(Stf) was also found to be a 65:35 mixture of the glycododeca- and glycotridecapeptides as found for L-PSGP(Sn) and L-PSGP(Sf). The amino acid sequences of L-PSGPs for salmonid fish species are summarized in Scheme I (20,46-48).

Poly(o1igo)sialic Acid Structures in Salvelinus Fish Egg PSGP

Initial carbohydrate analysis revealed the presence of both Neu5Ac and Neu5Gc residues in the lake trout PSGP. Expres- sion of both Sia residues was in contrast to the expression of only Neu5Gc residues in four Oncorhynchus species (Table I). These data suggested an apparent order difference and prompted further examination of other Salvelinus fish egg PSGPs. The data in Table I shows that the three Salvelinus PSGPs (designated Sn, Sf, and Slp) all expressed both Neu5Ac and Neu5Gc residues, although the ratio of Neu5Ac to Neu5Gc ranged from 6 to loo%, depending on the subfraction analyzed.

Identification of Three Distinct PolySia Forms in the Salvelinus Fish Egg PSGPs

The finding of both Neu5Ac and Neu5Gc in Salvelinus PSGPs raised further questions about the molecular organization of these Sia residues in the oligo/polysialyl chains. To determine this organization, the molecular characteristics of lake trout PSGP(Sn), brook trout PSGP(Sf), and Iwana PSGP(S1p) was studied by: (a) structural analysis of sialyl

4

1 2 3 4 5 6 7 8 FIG. 1. Thin layer chromatographic analysis of oligosialic

acids derived from Salvelinus fish egg PSGPs after pH 4.8- catalyzed hydrolysis at 37 "C for 2 days. As standards, the partial acid hydrolysates of PSGP(0m; polyNeu5Gc) and colominic acid (polyNeu5Ac), after addition of Neu5Gc and Neu5Ac, respectively, were run in lanes 1 and 2, respectively. The numbers by the spots in lanes 1 and 2 represent the degree of polymerization of oligo

PSGPs: lane 3, PSGP(Sn)-I (100% Neu5Ac); lane 4 , PSGP(Sn)-I1 (Neu5Gc) and oligo(Neu5Ac), respectively. Lane 3-8, hydrolysates of

(Neu5Ac/NeuSGc, 94:6); lane 5, PSGP(Sn)-111 (Neu5Ac/Neu5Gc, 77:23); lane 6, PSGP(Sf)-I1 (Neu5Ac/Neu5Gc, 6:94); lane 7, PSGP(Slp) (Neu5Ac/Neu5Gc, 10:90); lane 8, PSGP(0mi) (100% Neu5Gc). Arrow indicates the origin.

oligomers released by mild acid hydrolysis and Endo-N digestion; and ( b ) using the anti-polySia antibody H.46, which specifically reacts with a 2 4 - l i n k e d poly(Neu5Ac), but not with a24bl inked poly(Neu5Gc) chains.

TLC Analysis of Mild Acid Hydrolysates of PSGPs-TO investigate the nature of the polySia chains in subfractions I through I11 of lake trout PSGP(Sn), PSGP(Sf1, and PSGP(Slp), these PSGPs, together with PSGP(Omi1, were first treated with mild alkali to remove 0-acyl groups (see below). Following neutralization they were then hydrolyzed with mild acid in sodium acetate buffer (pH 4.8) at 37 "C for 2 days. The sialyl oligomers released were examined by TLC (Fig. 1). As controls, sialyl oligomers released by the controlled acid hydrolysis of rainbow trout PSGP(0m) and colominic acid were chromatographed as authentic standards of (Neu5Gc), (Fig. 1, lane 1 ) and of (Neu5Ac), (Fig. 1, lane 2).

Diversity i n a 2 4 - L i n k e d Polysialic Acid Structure 23679

Carbohydrate analysis of a subfraction of lake trout PSGP, PSGP(Sn)-I, showed the absence of Neu5Gc (Table I). Thus, the structures of oligo/polySia chains present in this fraction corresponded to oligoSia containing only Neu5Ac (Fig. 1, lane 3). Lake trout PSGP(Sn)-I is therefore unlike all of the other PSGPs so far examined (1,20, 31,44-48). In contrast to this structure, PSGP(0mi) contained only Neu5Gc residues. Ac- cordingly, the oligoSia residues released by partial acid hydrolysis of land-locked cherry salmon PSGP(0mi) (Fig. 1, lane 8) migrated identically with reference oligo(Neu5Gc) (Fig. 1, lane 1 ). Carbohydrate analysis of lake trout PSGP(Sn) subfractions I1 through V showed that this PSGP contained both Neu5Ac and Neu5Gc residues (Table I). Upon comparison with reference homooligomers of Neu5Gc and Neu5Ac (Fig. 1, lanes 1 and 2) it can be seen that the oligoSia chains in PSGP(Sn)-I11 consisted of a mixture of oligo(Neu5Ac) and oligo(Neu5Gc) (Fig. 1, lane 5 ) . The occurrence of heterosialyl oligomers consisting of both Neu5Ac and Neu5Gc residues was not observed in PSGP(Sn). Rather, PSGP(Sn) appeared to carry both oligo/poly(Neu5Ac) and oligo/poly(Neu5Gc) chains, although the relative proportions of Neu5Ac and Neu5Gc residues in the five subfractions varied markedly from subfraction to subfraction, as shown in Table I.

Sia analysis revealed that two other Salvelinus PSGPs, brook trout PSGP(Sf) and Iwana PSGP(Slp), also contained both Neu5Ac and Neu5Gc residues (Table I). However, the TLC profiles for the partial acid hydrolysates from both of these PSGPs were found to differ from those for lake trout PSGPs. As shown in Fig. 1, lanes 6 and 7, both PSGP(Sf) and PSGP(S1p) exhibited additional bands with intermediate migration rates compared to the homooligosialic acids consisting of only Neu5Gc (lane 1 ) or Neu5Ac (lane 2) residues. Thus, these data suggested that both brook trout and Iwana PSGPs contained heteropolymeric polySia chains containing both Neu5Ac and Neu5Gc residues in the same chain.

Identification of Sialyl Dimers Obtained from Mild Acid Hydrolysis of PSGP(S1p)"The above analysis revealed for the first time distinctive differences in the polySia chains of lake trout PSGP(Sn) and Iwana PSGP(Slp), suggesting the presence of heteropolysia chains in PSGP(S1p). To confirm this structural differences, a detailed analysis of the disialic acid fraction obtained from partial acid hydrolysis of PSGP(S1p) (9.5 mg as Neu5Ac) was carried out. The partial acid hydrolysate of PSGP(S1p) (50 mM acetate buffer, pH 4.8, 37 "C, 3 days) was first subjected to DEAE-Sephadex A-25 column chromatography to fractionate the oligoSia mixture according to chain length (Fig. 2 A ) . The di-, tri-, and tetraSia fractions, separated according to size, were pooled and subfractionated by TLC (Fig. 2B). From a closer examination of the chro- matogram, the presence of a series of heterooligomers made up of Neu5Ac and Neu5Gc was seen in each of fraction. The dimer fraction (Fig. 2B) revealed, for example, four possible disialic acids derived from Neu5Ac and Neu5Gc. The homo- geneity of each of bands was verified by TLC (Fig. 2C), and the sequences of the disialic acids were determined (a) from their relative migration rates on TLC when compared with those for the reference oligo(Neu5Ac) and oligo(Neu5Gc) and (b) alkaline borohydride treatment followed by the MM-GLC analysis. As summarized in Table I11 the disialic acid sequences identified in PSGP(S1p) were: Neu5Aca2+8Neu5Ac, Neu5Aca2+8NeuSGc, Neu5Gca2+8Neu5Ac, and Neu5 Gc(r24Neu5Gc.

Quuntitation of Sialyl Dimers Obtained from Mild Acid Hydrolysis of Salvelinus PSGPs-In order to further charac- terize the hybrid structures, the disialic acid fractions obtained from the mild acid hydrolysates of three Salvelinus

(A)

0.12 - dimer

0 20 40 60 80 Fraction number

1

2

1 3

2 r* 4

3 5 6 ,

5 4 7

1

2

1

3 2 Q, *

4

3 5

4 6 7

5

0 '

FIG. 2. Chromatographic separation and identification of the sialic acid oligomers produced on mild acid hydrolysis of PSGP(Slp). A , fractionation of partial acid hydrolysates of PSGP(S1p) according to chain length on a column (1 X 10 cm) of DEAE-Sephadex A-25 (Cl- form) at pH 7.8, 10 mM Tris-HCI buffer. The eluent was a linear gradient of NaCl from 0 to 0.4 M as shown (broken line), and the effluent was assayed by the resorcinol method and collected in 3-ml fractions. Yield monomer, 140 pg; dimer, 2,500 pg; trimer, 1,200 pg; tetramer, 730 pg; pentamer, 510 pg. B, comparative TLC of each of the di-, tri-, and tetrasialic acid mixtures obtained from PSGP(S1p) as shown in A. Di, tri, and tetra correspond to the dimer, trimer, and tetramer fraction in A, respectively. (Gc), and (Ac), represent the oligosialyl standards of Neu5Gc and Neu5Ac as shown in Fig. 1. C, purity of each of the sialic acid dimers obtained by preparative TLC. Lanes a-d correspond to Neu5Aca2+8Neu5Ac, Neu5Aca2+8Neu5Gc, Neu5Gca24Neu5Ac, and Neu5Gca2+ 8Neu5Gc, respectively. Arrow indicates the origin.

TABLE 111 Identification of four different sinlic acid dimers obtained from mild

acid hydrolysis of PSGP(S1p) by MM-GLC analysis after alkaline borohvdride treatment

Neu5Ac Neu5Acol Neu5Gc NeuBGcol Disialic acid sequence

a + + - - Neu5Aca24Neu5Ac b + - - + Neu5Aca24Neu5Gc

+ + - Neu5Gca24Neu5Ac d + + Neu5Gca24Neu5Gc C -

- - a a-d correspond to the bands on TLC in Fig. 2C.

PSGPs, i.e. PSGP(Sn), PSGP(Sf), and PSGP(Slp), were isolated by anion-exchange chromatography on Sephadex A- 25. Each dimer was further purified by preparative TLC chromatography, and each of the four disialic acids was ex-

23680 Diversity in a 2 4 - L i n k e d Polysialic Acid Structure

tracted and quantitated. As shown by the results summarized in Table IV, approximately 15 mol % of the Sia residues in PSGP(S1p) and PSGP(Sf) existed as hybrid oligo/polysialyl chains containing both Neu5Ac and Neu5Gc residues. No such hybrid structure was detected in PSGP(Sn). The variation of the relative yields of the disialic acids should be taken as an indication of the appreciable variation in (a) molecular organization of the component sialic acids (Neu5Ac and Neu5Gc) in oligo/polySia chains and (b) the rate constants of the acid-catalyzed hydrolysis of a24-s ia ly l bonds linking the 2 sialic acids in the four possible combinations. The former possibility was considered to be a major factor. Upon comparison with the molar proportions of Neu5Ac+Neu5 Ac: Neu5Ac+Neu5Gc+Neu5Gc+Neu5Ac: Neu5Gc+ Neu5Gc, calculated on the assumption that the sequence of polySia was random in both PSGP(Sf)-I and PSGP(S1p) with Neu5Ac/Neu5Gc = 10:90, these data strongly suggested that although hybrid structures of Neu5Ac and Neu5Gc occurred in both PSGP(Sf) and PSGP(Slp), the observed de- viation from the theoretical values was much larger in the former than in the latter (Table IV). The simplest explanation for this observation is that (a) both PSGP(Sf) and PSGP(Slp) contained hybrid oligo/poly(Neu5Ac, Neu5Gc) structures, but that they are not random copolymers and (b) the Neu5Ac residues tend to exist as clustered forms or long stretches of oligo/poly(Neu5Ac) chains in PSGP(Sf), compared to PSGP(S1p). Further evidence for this possibility was provided by immunoreactivity of PSGP(Sf) and PSGP(SZp) with H.46 anti-(4Neu5Aca2+), antibodies (see below).

Endo-N Digestion of Salvelinus PSGPs-We have previously reported that Endo-N catalyzes the hydrolysis of both cy24-linked poly(Neu5Ac) and poly(Neu5Gc) unless these chains are 0-acetylated (37, 44). We examined the susceptibility of the hybrid polySia chains having both Neu5Ac and Neu5Gc residues to Endo-N using PSGP(S1p) and PSGP(Sf). As shown in Fig. 3, Endo-N cleaved both PSGP(Sf) ( l a n e 4) and PSGP(S1p) ( l a n e 5 ) to produce heterotrimer (minor) in addition to Neu5Gc-trimer (major) and Neu5Gc-tetramer (major) and Neu5Gc- and Neu5Ac-dimers (minor). An Endo- N hydrolytic map of PSGP(0mi) was obtained and compared with that from the PSGP(Om), and the two maps were superimposable, confirming that PSGP( Omi) carried only poly(Neu5Gc) structure (data not shown).

Immunoreactivity of Salvelinus PSGPs with Anti- poly(Neu5Ac) IgM Antibody, H.46-To corroborate the exist- ence of poly(Neu5Ac) clusters on PSGP(Sf) but not on

TABLE IV Sialic acid dimers isolated from the mild acid hydrolysates of PSGPs

from three Salvelinus species Disialic acid” PSGP(Sn)-IV PSGP(Sn-I PSGP(S$)

a Neu5Ac+Neu5Ac 29 (30)b 5.0 (1)’ 2.7 (1)‘ b Neu5Ac+Neu5Gc 0 11 12 c Neu5Gc+Neu5Ac 0 2.8 3.5 d Neu5Gc+Neu5Gc 71 (70) 82 (81) 79 (81) ’ Each dimer was isolated by preparative TLC of the dimer frac-

tions and quantitated by the resorcinol method, as described under “Experimental Procedures.”

* Values in parentheses for PSGP(Sn) show mol % of the dimers estimated on the assumption that PSGP(Sn) molecules are composed of the homopolymeric chains of oligo/poly(Neu5Ac) and oligo/ poly(Neu5Gc) in the observed ratio of Neu5Ac:Neu5Gc = 30:70 (see text).

Values in parentheses for PSGP(Sf) and PSGP(S1p) are calculated based on the observed data Neu5Ac:Neu5Gc = 1090 for both PSGPs, and on the assumption that the sialic acid sequence of the heteropolymeric chains of oligo/poly(Neu5Ac,Neu5Gc) are random.

m o l %

1 2 3 4 5 FIG. 3. TLC Analysis of oligoSia released from Salvelinue

PSGP after depolymerization by Endo-N. Lanes 1 and 2 are standard sialyloligomers of (Neu5Gc). and (Neu5Ac)., respectively, as described in the legend to Fig. 1. Lane 3, PSGP(Sn); lane 4, PSGP(Sf); lane 5, PSGP(S1p). Arrow indicates the origin. Asterisks indicate heterotrimerb).

TABLE V Immunoreactivity of Salvelinus fish egg PSGPs to equine anti-poly(a24Neu5Ac) antibodies, H.46, as determined

by the Ouchterlony immunodiffusion method For comparison, the data for PSGP(Stf) and PSGP(0m) are also

included. ~~~~ ~ ~

PSGP fraction ’ ratio (Neu5Ac’ Immunoreactivity (Neu5Ac + Neu5Gc)

PSGP(Sn)-IV 30

PSGP(S1p) 10 PSGP(Sf)-I1 6.2

PSGP(Stf) PSGP(0m)

2.6 0

++O

+ - + -

++, strongly reactive; +, reactive; -, unreactive.

TABLE VI Sialic acid (Neu5Ac, Neu5Gc, and KDN) contents in both intact and

H.46 immunoprecipitated PSGPs Values are given in mol %.

PSGP(Sn)-IV PSGP(Sn)-V PSGP(0m)

Intact precipitate Intact Precipitate Intact Precipitate

Neu5Ac 29 91 12 95 0 0 Neu5Gc 67 0 84 0 94 0 KDN 4.2 9.1 4.5 4.7 6.3 0

~~

PSGP(Slp), immunoreactivity of Salvelinus PSGP(Sn), PSGP(Sf), and PSGP(SZp) with H.46 anti-(43Neu5Aca2+). antibody (n > 10) was examined by Ouchterlony immunodiffusion. H.46 IgM antibody was previously shown to specifically recognize poly(Neu5Ac) chains (37, 42). As shown in Table V, lake trout PSGP(Sn)-IV containing 71% Neu5Gc+ Neu5Gc and 29% Neu5Ac+Neu5Ac (Table IV) was the most immunoreactive fraction tested. The hybrid PSGP(Sf)-I1 was also immunoreactive, while the Iwana PSGP(S1p) was inac- tive (>lo p ~ ) . The PSGP(Stf) was also probed with the polyclonal antibody H.46 because this PSGP was found to contain Neu5Ac (Table I) and it too was immunoreactive.

Immunoprecipitation of Ldke Trout PSGP(Sn) with H.46 Antibody-H-PSGP (Mr about 200,000) contains approximately 75 a24- l inked oligo/polysialylglycan chains in a single molecule (31). I t is possible that in a significant part of the PSGP both poly(Neu5Ac) and poly(Neu5Gc) chains co- exist. To establish whether the poly(Neu5Ac) and poly(Neu5Gc) chains are present on a single PSGP molecule

Diversity in a24-Linked Polysialic Acid Structure 23681

such as those molecules present in PSGP(Sn)-IV and PSGP(Sn)-V, further structural evidence was provided by MH/MM-GLC analysis of the H-PSGP(Sn)-H.46 IgM com- plexes (Table VI). The H.46 antibody specifically recognized and was able to precipitate the poly(Neu5Ac)-PSGP present in the incubation with PSGP(Sn)-IV and -V. These results strongly indicate that the lake trout PSGP(Sn) is made up of the discrete two molecular species, one having only poly(Neu5Ac) the other having exclusively poly(Neu5Gc), in their varying proportion. These data, together with our previous results (37, 39, 44, 45) are in accordance with the presence of three distinct polySia forms in salmonid fish egg PSGPs, namely poly(Neu5Ac), poly(Neu5Gc), and poly(Neu5Ac, Neu5Gc).

Identification of 0-Acetyl- and 0-Lmtyl Sialic Acid Residues in PolySia Chains of PSGPs

To investigate further the nature of the polySia chains in Salvelinus PSGPs, these polymers were treated with A. ureafaciens exosialidase. The sialyl residues of PSGP(Sn) and PSGP(Sf) were only partially hydrolyzed by the exosialidase under the same condition in which the sialyl residues of rainbow trout PSGP( Om) were nearly completely hydrolyzed. The sialyl residues of PSGP(Sn) and PSGP(S/) were suscep- tible to the exosialidase upon removal of 0-acyl groups by treatment with 0.1 N NaOH at 25 "C for 60 min (Fig. 4). The resistance of the residual sialic acid residues in these PSGPs to the action of the exosialidase, even after treatment with mild alkali, is due to the presence of the sialidase-resistant Sia residues (49, 50), GalNAcpl+4(Siaa2+3)GalNAc/31+ 3Gal~14Gal~l+3GalNAcal+Thr/Ser, and the KDN- capped oligo/polySia chains (17, 51) (Fig. 4). In order to identify the nature and location of the acyl substitution on the Sia residues, PSGP(Sn) was first exhaustively digested with the exosialidase, and then the digest was subjected to hydrolysis of the ketosidic bonds of Sia under mild conditions (0.01 N trifluoroacetic acid at 70 "C for 30 min) (52). An aliquot of the released sialic acids was examined by 'H NMR spectroscopy, and the assignments of the chemical shifts to specific protons were facilitated by comparing the observed data with the literature values published for the relevant Sia

0- 0- 0 5 10 15 2 0 0 5 10 1s 2 0

(A) PSGP(Sn)-I and ( E ) PSGP(Sf)-I1 before and after mild FIG. 4. Kinetics of A. ureafaciens exosialidase digestion of

alkali treatment. Sialic acids liberated by exosialidase treatment were monitored by the thiobarbituric acid method before (0) and after (0) alkali treatment, as described under "Experimental Proce- dures."

Time, h Time, h

(53,54). Consequently, the released Sia were found to consist of a mixture of Neu4,5Acz (CH3 of 4-0Ac, 6 2.08 ppm), Neu5,7Ac2 (CH3 of 7-0Ac, 6 2.15 ppm), and Neu5,9Ac2 (CH, of 9-OAc, 6 2.12 ppm). In addition, the 'H NMR spectrum exhibited the chemical shifts of the substituted Neu5Ac which was tentatively identified as 9-0-lactyl Neu5Ac (H-3,,, 1.80 ppm; H-3,,, 2.22 ppm; CH3-CHOH-, 1.34 ppm ( J = 6.9 HZ); CH3-CHOH-, 4.10 ppm). Previously, we showed the presence of a mixture of Neu5Gc and its 4-, 7 - , and 9-0-acetyl derivatives as well as KDN- and 9-O-acetyl-KDN-cappedpoly(a2- 8NeuSGc; 44/7/9-0Ac) in kokanee salmon PSGP(0n) (44).

DISCUSSION

When PSGPs were first discovered in the eggs of rainbow trout and characterized, it appeared initially that all cortical alveolar-derived major glycoproteins might belong to this class of a 2 4 - l i n k e d poly(Neu5Gc)-containingglycoproteins. The PSGP(0m) associated with eggs in rainbow trout, for example, contained poly/oligosialyl chains consisting of only Neu5Gc residues (1). In general, Neu5Ac was thought to be completely absent in most Oncorhynchus PSGPs except, as reported herein, for PSGP(0n)-11, which contains 10 mol % Neu5Ac residues. Brook trout PSGP(S/) also contained mainly Neu5Gc (90~94%) and only small amounts of Neu5Ac, although the ratio of Neu5Ac to Neu5Gc was found to vary somewhat in individual fish (see Table I comparing PSGP(S/)-I and PSGP(Sf)-11). The present study reveals, however, an even more remarkable diversity in the polySia structures of the salmonid fish egg PSGPs than heretofore recognized. Subfractionation of lake trout PSGP(Sn), for example, revealed that some molecular species contained exclusively Neu5Ac residues, PSGP(Sn)-I, while others contained as little as 13 mol % Neu5Ac and 87% Neu5Gc (Table I). At least 15% of the Sia residues in this PSGP were 0- acetylated and a minor fraction (about 1.8%) of the Sia residues contained 0-lactyl groups. Other subfractions of PSGP(Sn) contained intermediate amounts of Neu5Ac and Neu5Gc residues. Fraction PSGP(Sn)-I1 contained 94% Neu5Ac and 6.0% Neu5Gc, while subfraction PSGP(Sn)-I11 was composed of 77% Neu5Ac and 23% Neu5Gc residues (Table I). The polySia chains in brook trout PSGP(Sf), the Japanese common char fish (Iwana) PSGP(Slp), kokanee salmon PSGP(0n)-11, and brown trout PSGP(St/) were also shown to contain varying amounts of Neu5Ac residues, ranging from as little as 2.6 mol % to 10 mol % (Table I).

The finding of both Neu5Ac and Neu5Gc in PSGP was unexpected, and raised further questions about the molecular organization of these polySia chains. Our results demonstrate that there are distinctive differences in the polySia structure between lake trout PSGP(Sn), brook trout PSGP(S/), and Iwana PSGP(S1p). The PSGP(Sn) species containing both Neu5Ac and Neu5Gc residues (PSGP(Sn)-11, 111, IV, and V) were shown to be a mixture of two dlscrete types of PSGP molecules, each of which contained homopolymers consisting solely of poly(Neu5Ac) or poly(Neu5Gc) chains in the same molecule (Table IV). By contrast, structural analysis of PSGP(Slp) and PSGP(Sf) revealed that these are heteropolymers containing both Neu5Ac and Neu5Gc residues in the same chains, namely poly(Neu5Ac, Neu5Gc), although the hybrid structure is not a random copolymer (Table IV). PSGP(S/) appeared to be particularly so, since PSGP(S/)-I1 was more immunoprecipitable with H.46 IgM antibody than PSGP(S1p) in spite of the fact that the percentage of Neu5Ac in the former even was lower than in PSGP(S1p) (Table I). Thus, three different Saluelinus species (lake trout, brook trout, and Iwana) were found to contain Neu5Ac, NeuSGc,

23682 Diversity i n a 2 4 - L i n k e d Polysialic Acid Structure

and Neu5,nAc2 in varying proportions related to the species. The absolute and relative amounts of Neu5Ac and Neu5Gc were variable in the individual preparations of lake trout PSGP(Sn). The position of 0-acetyl substitution at the Neu5Ac residues was determined to be C4, C7, and C9, whereas that of 0-lactyl substitution has not been conclusively determined. Previous studies with kokanee salmon PSGP(0n) established the presence of 0-acetylated Neu5Gc and O-acetylated KDN (44). We cannot exclude the possibility that part of the acetyl and lactyl groups in PSGPs may have been lost during purification procedures.

Based on these results we conclude that at least four different types of sialic acids are present in fish egg PSGPs: Neu5Ac, Neu5Gc, Neu5,xAcz, and NeuBGcxAc, where x is Cq, C,, or C9. This leads to the occurrence of diverged forms of a24-l inked homo- and heteropolymers of these polySia structures including poly(Neu5Ac), poly(Neu5Gc), poly(Neu5,xAc2), poly(Neu5GcrAc), poly(Neu5Ac, Neu5Gc), poly(Neu5Ac, Neu5,rAcp), poly(NeuSAc, Neu5GcxAc), poly(NeuSGc, Neu5,xAcz), and poly(Neu5Gc, Neu5GcnAc). The presence of 9-0-lactyl Neu5Ac residue was also suggested for PSGP(Sn)-I. Interestingly, in kokanee salmon PSGP(0n) 4-0-acetylNeu5Gc occurred almost exclusively (44). Th' IS new structural information, together with our finding of poly(a2- 8KDN) in the rainbow trout egg vitelline envelope (19), is significant because it demonstrates the natural occurrence of a wide variety of a24-l inked polySia structures in salmonid fish egg glycoproteins that have not been previously described. These structures are summarized in Table VII. These results predict that an equally remarkable array of polysialoglycoconjugates is yet to be discovered in animals other than teleost fishes.

The a24-linked poly(Neu5Gc) chains found in the rainbow trout PSGP showed a characteristic structure not yet reported in bacterial and mammalian polySia chains. A well studied example of polySia is the homopolymer of a 2 4 - linked Neu5Ac occurring in neuroinvasive E. coli K1 and N. rneningitidis Gp B strains (2-4, 55) . Monoclonal and polyclonal antibodies against this (4Neu5Aca2+), chain, uiz. mAb.735 and H.46, have been used to prove that the same structural and immunological epitope exists in various animal cells and tissues (4-13). Immunofluorescence of lake trout cortical alveoli demonstrated a specific localization of (-. 8Neu5Aca2+), chains in these Golgi-derived secretory vesi- cles (37). The H.46 antibody did not cross-react, however, with Iwana PSGP(Slp) which is composed of about 90 mol % Neu5Gc residues (Table V). These immunochemical proper- ties further support our conclusion that marked structural differences in polySia chains exist between PSGPs. H.46 antibody reacts preferentially with (4Neu5Aca2+), with n

greater than 10 (42) and, as such, has allowed the study of polySia structures in sources as disparate as bacteria and brains (3). To date, however, only limited immunochemical and enzymatic probes are available for the identification of polySia chains, and these are restricted in use to poly(8Neu5Aca2+) structures (4). An important aspect of this study was the necessity to develop sensitive methods to detect various forms of polySia chains containing Neu5Gc, KDN, and their acylated derivatives.

There is increasing evidence that surface expression of polySia chains may modulate a number of key biological processes including species-specific cell-cell recognition events during fertilization and embryogenesis (3,7,10, 13, 15, 21, 56). Examples of animal sialoglycoproteins found to contain polysialyl groups include the fish egg PSGPs (l), embryonic N-CAM of avians (14, 15), amphibians (12), and mammals (5, 16), voltage-sensitive Na+ channel from electroplax (11) and rat brain (lo), cell-surface glycoproteins on a number of human tumors (7-9), and an embryonal cell-surface glycoprotein of Drosophila rnelanogaster (13). Only poly(Neu5Ac) chains have been reported in all these cases, except for the fish egg PSGPs. However, it has been well known that both Neu5Ac and Neu5Gc occur in a wide variety of sialoglycoproteins and gangliosides obtained from various animal tissues and cells (57), although Neu5Ac is the principal Sia expressed in most human sialoglycoconjugates. In general, while human tissues usually yield only Neu5Ac, other mammals, especially pig, cow, sheep, rabbit, rat, and goat have significant levels of Neu5Gc. Neu5Ac and Neu5Gc do not appear to be biologically equivalent. The Neu5Gc residues in glycolipids, for example, are antigenic in humans (58) and the Neu5Gc residues are, in general, more resistant to hydrolysis by some exosialidases than Neu5Ac residues. Likewise 0-acetylated Sia residues are expected to be functionally different from their parent unsubstituted Sia (59). Indeed, 0-acetylation increases the immu- nogenicity of the E. coli K1 polySia capsule and decreases its neuroinvasive pathogenicity (60). Also, changes in the relative amounts of Neu5Ac and Neu5Gc are known to be associated with differentiation in rat intestine (61) and with differences in allotransplantability in mouse mammary tumor cells (62, 63). A major challenge for the future of polySia-glycoconjugate research is to determine if a similar structural diversity exists in polysialoglycoconjugates other than fish egg PSGPs. The failure to find poly(Neu5Gc) or poly(Neu5Ac, Neu5Gc) in polysialylated glycoconjugates such as N-CAMS may be due to limitations of the current methodologies available (see Table VII). Because of the lack of antibodies reactive with poly(Neu5Gc), for example, we have been unable to determine if poly(Neu5Gc)-containing N-CAM is expressed in any mammalian cells or tissues. When immunochemical probes which

TABLE VI1 Occurrence and reactiuities toward Endo-N and anti-(cu24-linked polysialic acid) in Salmonidae fish egg glycoproteins

PSA structure Substitution Glycoprotein Endo-N H.46

susceptibility reactivity Probes available

Poly(Neu5Gc) +O-Acetyl PSGP(Om), PSGP(Omi), += - No PSGP(OL), PSGP(Sn)

Poly(Neu5Ac) kO-Acetyl PSGP(Sn) +" + H.46'

Poly(Neu5Ac,Neu5Gc) +O-Acetyl PSGP(Sf), PSGP(Slp), +u + o r -d No PSGP(Stf), PSGP(0n)

Poly(KDN) +O-Acetyl KDN-gp(0m) - - mAb.kdn8kdn'

+O-Lactyl mAb.735'

a Endo-N is only reactive for polySia having no 0-acyl substitution. See Ref. 2. See Ref. 6. H.46 is reactive with PSGP(Sfl and PSGP(Stf) but not reactive with PSGP(S$) and PSGP(0n).

e See Ref. 4.

Diversity in a24-Linked Polysialic Acid Structure 23683

specifically recognize poly(Neu5Gc), or any other variety of polySia structures become available, it is likely that the number of examples of different polySia structures will likely expand to include various types of polysialylated glycoconjugates other than fish egg PSGPs. In support of this prediction are our recent studies that have been successful in developing a sensitive immunoprobe for a 2 4 - l i n k e d oligo/poly(KDN), and which has allowed us to detect poly(KDN) in sea urchin embryos (see Ref. 4).’

No experimental evidence is available to date to prove the exclusive presence of Neu5,xAcz and/or Neu5GczAc residues in the Salmonidae PSGPs. Taking into account the lability of 0-acetyl groups in polysaccharides to even very mild basic conditions, it is not surprising that we always find small amounts of unacetylated Neu5Ac and Neu5Gc, together with Neu5,rAcz and Neu5GcxAc. If Neu5,9Ac2 was the only Sia residue in a PSGP, the acetyl group would have been removed under the experimental conditions used initially to isolate and purify these molecules (64). The presence of a polysialyl 0-acetyltransferase activity, the enzyme responsible for 0- acetylation of the E. coli K1 capsular polySia chains, was reported to have preferential substrate specificity toward longer polySia chains (60). Acetyl groups were shown to be transferred to both the 7- and 9-positions of the Sia residues, and no acetylation was apparent in poly(Neu5Ac) of degree of polymerization < 14 (60). If a similar oligo/polysialyl 0- acetyltransferase is involved in the in vivo synthesis of salmonid PSGPs, then it appears that such long chain substrates as required for 0-acetylation of the E. coli K1 polySia may not be essential for fish egg PSGPs because these polysialyl chains are usually shorter (30, 31, 39, 44, 45).

A comparative study of the detailed structures of the core protein and polySia groups in PSGP from Salvelinus eggs may provide new evidence on the molecular basis of the phylogenetic relationship among different genera of Salmon- idae fishes. Our present results already raise a question about the classification of “brown trout” into Salmo genus because apo-L-PSGP(Stf) was found to be a 65:35 mixture of dodeca- and tridecapeptides, as found for apo-L-PSGP of Saluelinus fish species. Furthermore, PSGP(Stf) was also found to contain Neu5Ac residues (Table I). These data indicate that brown trout, presently classified in the genus Salmo, is more closely related to the Salvelinus species.

In summary, the significance of our new findings is %fold. First, a new class of structurally &verse polySia chains is reported that have not been previously described in other polysialylated glycoconjugates. Second, elucidation of the natural occurrence of multiple forms of Lu24-linked polySia chains may now account, in part, for the myriad functional activities that these structures appear to be involved in. While the major biological significance of polysialylation is poorly understood, even in the nervous system, it now seems clear that with such great structural differences in the polySia epitope alone, that a detailed understanding of their biological activity will be a challenging pathway to unravel. A further challenge that our collaborative efforts are directed towards is to understand the biochemical and genetic mechanisms that regulate surface expression of polysialylated glycoconjugates.

Acknowledgment-We thank Dr. Yutaka Muto, University of Tokyo, fo r obtaining the 400-MHz ‘H NMR spectra.

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structural diversity in the a24blinked polysialic acid ...mild acid hydrolysisltlc method-each...

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