The Human Blood Fluke Schistosoma mansoni Synthesizes Glycoproteins Containing the Lewis X Antigen*

THE JOURNAL OF BIOLOGICAL CHEMISTRY 1992 by The American Society for Biochemistry and Molecular Biolopy, Inc. Vol. 267, No. 28, lrrsue of October 5, pp. 2196-223, 1992 Printed in U.S.A. The Human Blood Fluke Schistosoma mansoni Synthesizes Glycoproteins Containing the Lewis X Antigen* Jayanthi Srivatsan, David F. Smith, and Richard D. Cummingst From the University of Georgia, Department of Biochemistry, Athens, Georgia 362 (Received for publication, May 6, 1992) Infection of vertebrates with the parasitic blood unusual oligosaccharides (Makaaru et al., 1992; Levery et al., fluke Schistosoma mansoni induces a variety of host 1992). immuneresponses,which are directed against both Most studies in recent years have been concerned with the protein and carbohydrate antigens. In this report, we general chemical structure of glycoprotein and glycolipid olidescribe our studies on the structures of antigenic oli- gosaccharides synthesized by schistosomula and adult schisgosaccharides derived from glycoproteins synthesized tosomes (Nyame et al., 1987, 1988a, 1988b, 1989; Makaaru et by S. mansoni. Immobilized antibodies derived from al., 1992). The unusual structural features of the complexthe sera of infected hamsters and mice bind to a family type sugar chains in the schistosome-derived glycoproteins of high molecular weight Asn-linked oligosaccharides in glycoproteins from the adult parasite. Structural suggested that these may be antigenic in infected animals. analysis of the major antigenic oligosaccharides re- Our present studies are focused on identifying the nature of vealed that they have high amounts of fucose-linked the antigenic carbohydrate determinants in glycoproteins syna1,3 to N-acetylglucosamine residues within the linear thesized by schistosomes. In this report, we present our findrepeating disaccharide (3Gal@l-4GlcNAcBl),, a poly- ings that s. mansoni adults can synthesize glycoproteins N-acetyllactosamine sequence containing the Lewis X containing the Lewis X antigen (Le ) Galbl-4(Fucculantigenic blood group. The remarkable ability of S. 3)GlcNAc within the repeating N-acetyllactosamine sequence mansoni to synthesize these vertebrate-type oligosac- (SGalpl-4GlcNAc/31),. charides may have implications in both the mechanisms of host-parasite interactions and on the development EXPERIMENTAL PROCEDURES of vaccines to prevent this disease in humans. Materials-Concanavalin A (ConA) -Sepharose was procured from Pharmacia LKB Biotechnology Inc. CNBr-activated Sepharose 4B, Protein A-Sepharose CLGB, Sephadex G-25-8, anti-mouse IgM alkaline phosphatase conjugate, galactose, lactose, fucose, a-methylglucoside, a-methylmannoside, Tetragonolobuspurpureas, Triton X-1, Schistosoma mansoni is one of the human blood flukes that causes the chronic disease, schistosomiasis, and is second only to malaria in generating morbidity and suffering in tropical zones worldwide (Capron et al., 1987). Schistosomes synthesize numerous antigenic glycoproteins in infected animals and humans, and many of these in both schistosomula and adult worms have been identified by monoclonal antibodies, lectin binding studies, and antibodies from the sera of infected individuals (Bennett and Seed, 1977; Simpson and Smithers, 198; Linder and Huldt, 1982; Weiss and Strand, 1985; MacGregor et al., 1985; Dissous et al., 1986; Hayunga and Sumner, 1986a, 1986b; Omer-Ali et al., 1986, 1991; Linder et ai., 1991). The antigenic determinants of many of these glycoproteins have been shown to reside in their carbohydrate moieties (Weiss and Strand, 1985; Weiss et al., 1986; Dissous et al., 1986; Gryzch et al., 1987; KO et al., 199). Recent studies have shown that there are antigenic determinants in schistosome-derived glycoprotein oligosaccharides, and some of the cross-reactive antigens are found in glycolipids (Weiss et al., 1986); glycolipids in schistosomes are now known to possess * This work was supported by National Institutes of Health Grant A126725 (to R. D. C.) and National Science Foundation Research Grant DMB-881164 (to D. F. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed Dept. of Biochemistry and Molecular Biology, Oklahoma Center for Molecular Medicine, University of Oklahoma Health Science Center, 94 S. L. Young Blvd., Oklahoma City, OK 7314. Tel.: 45-271-2546; Fax: 45-271- 391. N-acetylgalactosamine, N-acetylglucosamine, MOPS, Tris-base, Amberlite MB-3, anti-mouse alkaline phosphatase-conjugated IgM, neuraminidase, and P-D-galactosidase from jack bean were purchased from Sigma. a-1,3/1,4-fucosidase from Streptomyces sp. was obtained from Takara Biochemicals Inc. (Berkeley, CA). Affi-Gel 1 was obtained from Bio-Rad. a-l-fucosidase from bovine kidney, X-phosphate (BCIP), and nitro blue tetrazolium were procured from Boehringer Mannheim. Endo-@-galactosidase was purchased from V-labs (Covington, CA), and Pronase was obtained from Calbiochem (San Diego, CA). Anti-LeuM1 (CD15) monoclonal antibody was purchased from Becton Dickenson Immunocytometry Systems (San Jose, CA). /3-N- Acetylhexosaminidase was purified from jack bean meal (Li and Li, 1972). The radiolabeled sugars [6-3H]glucosamine (4 Ci/mmol), [2-3H]mannose (21 Ci/mmol) and [6-3H]galactose (25 Ci/mmol) were purchased from ICN Biochemical Co (Irvine, CA). Dulbecco s minimum essential medium was obtained from GIBCO and normal baboon serum from the animal facility, Department of Zoology, University of Georgia. Standard partially methylated, alditolacetate derivatives of mannose and glucosamine were chemically prepared by Ali Shilatifard, Department of Biochemistry, University of Georgia. The standards were separated using Hewlett-Packard mode 5892 gas chromatography system fitted with 3-m SP233 or 3-m DB-1 columns for neutral and amino sugars, respectively. Metabolic Radiolabeling of Adult Schistosome Wormpairs with Ra- dioactive Precursor Sugars-Eight-week-old schistosome wormpairs (KEB strain, Kenyan, baboon-passaged) were obtained under sterile conditions from the portal veins of BALB/cJ mice or hamsters as The abbreviations used are: ConA, concanavalin A; RCA-I, Ricinus communis agglutinin I; TPA, T. purpureas agglutinin; CHO, Chinese hamster ovary; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high pressure liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; INF, infected; UNINF, uninfected; BCIP, 5-bromo-4-chloro-3-indolyl phosphate. 2196

described previously (Nyame et al., 1987). At least 12 wormpairs were radiolabeled in oitro with 2 mci/ml of either [6-3H]glucosamine, [6- "Hlgalactose, or [2-3H]mannose in Dulbecco's modified Eagle's medium containing 5% normal baboon serum and 1% penicillin-streptomycin solution (1, units of penicillin G/ml and 1 mg of streptomycin/ml in.9% NaCI) in a final volume of.5 ml for 24 h. Incubations were conducted at 37 "C in a humidified incubator containing 5% CO,. At the end of the incubation, the worms were motile and viable, and the culture was devoid of bacterial and fungal contamination. The worms were washed three times with phosphate-buffered saline and stored at -8 "C till further use. Preparation of Radiolabeled Glycopeptides from Adult Schistosome Wormpairs-The radiolabeled adult schistosome wormpairs were sonicated in lysis buffer (.1 M Tris-base, ph 8., containing 1 mm CaCl,). Lipids were extracted with 2 volumes of chloroform/methanol/water (1:1:3) followed by chloroform/methanol (21). The protein extracts were dried under a stream of nitrogen and digested with 1 mg/ml Pronase in lysis buffer at 6 "C in toluene atmosphere for 24 b. After incubation, the samples were boiled for 5 min and desalted on a Sephadex G-25 (1 X 5-cm) column in water containing 7% n- propyl alcohol. The radiolabeled glycopeptides were recovered in the void fractions, pooled, and dried in shaker water bath under reduced pressure. Antibody Affinity Column Chromatography of fhlgalactose-, ("HI Mannose-, and fh]glucosamine-labeled Glycopeptides-Sera from 8- week infected hamsters (INF) and normal hamsters (UNINF) were heat inactivated at 56 "C for 3 min and centrifuged at 3 X g for 3 min to remove any precipitate. Two 1.5-ml (.7 X 4-cm) Protein A-Sepharose columns equilibrated with Tris-buffered saline, TBS-1 (1 mm Tris-base, ph 8., containing 1 mm EDTA,.15 M NaCl, and.2% NaN3), were saturated with 1. ml of sera diluted with TBS-1. The radiolabeled glycopeptides (1 X lo5 cpm) were applied to the columns and the columns were washed with TBS-1 and fractions of 1. ml were collected. The bound glycopeptides were eluted with.1 M sodium acetate, ph 2.8, containing.15 M NaC1. The fractions were neutralized by collecting in test tubes containing.1 ml of 1. M Tris-base, ph 8.. Aliquots of fractions (-5 ml) were mixed with Scintiverse BD (Fisher) for determining radioactivity in the scintillation counter. Coupling of Immune Serum from Infected Hamsters to CNBr- Sepharose 4B-The heat-inactivated immune serum from 8-week infected hamsters was dialyzed against 3 volumes of coupling buffer (.1 M NaHC3,.15 M NaCI, ph 8.3) overnight. The serum was then directly coupled to CNBr-Sepharose 4B at a concentration of 15 mg/ ml in the coupling buffer overnight at 4 "C as described previously (Harlow and Lane, 1988). The coupled gel was equilibrated with TBS- 1. A column of 25 ml was prepared in TBS-1, ph 8.. The radiolabeled glycopeptides were applied to the column and fractions of 3. ml were collected and the bound glycopeptides were eluted as described above. The bound glycopeptides were desalted by chromatography on a column of Sephadex G-25 (2.5 X 45-cm) in.1 M pyridine-acetate buffer, ph 5.4. The bound glycopeptides recovered in the void fractions were pooled and evaporated to dryness under reduced pressure. Lectin Affinity Chromatography of Radiolabeled Antigenic Glycopeptides-Immobilized column of T. purpureas lectin (TPA) in Affi-Gel 1 was prepared according to the manufacturer's instructions. Briefly, Affi-Gel (1-1.5 ml) is washed with 1 volumes of deionized water. Lectin solution (1. ml, 15-2 mg lectin/ml in.1 M MOPS buffer, ph 7.5, containing 4 mg/ml fucose) was mixed with Affi-Gel at 4 "C overnight. The uncoupled lectin was drained from the gel and mixed with 1. ml of.2 M ethanolamine for at least 2 h at 4 "C. The column was equilibrated with Tris-buffered saline-2, TBS-2 (1 mm Trisbase, ph 8., containing 1 mm CaC12, 1 mm MgCI2,.15 M NaCl, and.2% NaN3). The radiolabeled antigenic glycopeptides were fractionated on 2.- ml columns (.7 X 5-cm) of ConA-Sepharose equilibrated with TBS- 2 at room temperature (Cummings and Kornfeld, 1982; Merkle and Cummings, 1987). Glycopeptides bound to the columns were first eluted with 1 mm a-methylglucoside followed by 1 mm a-methylmannoside. Fractions of 2..ml were collected. The pooled glycopeptide fractions were dried, desalted, and were further fractionated on a 1.-ml column (.3 X 14-cm) of TPA equilibrated with TBS-2. The unbound glycopeptides were eluted with TBS-2, and the bound glycopeptides were eluted with TBS-2 containing.4% fucose. Fractions of.5 ml were collected. RCA-I agarose affinity chromatography was performed on a 2-ml column (.8 X 4-cm) at room temperature as described previously (Merkle and Cummings, 1987), and 1.-ml fractions were collected. The bound material was eluted with.1 M Oligosaccharides Antigenic S. mansoni 2197 lactose. Glycopeptides were desalted and separated from monosaccharides by chromatography on a column of Sephadex G-25 (1 X 5- cm) in 7% n-propyl alcohol. Analysis of Sugar Composition of Antigenic Glycopeptides-Strong acid hydrolysis of 13H]mannose- and [3H]galactose-labeled glycopeptides was performed in 2 N HCl at 1 "C for 4 h, and the hydrolysates were dried by evaporation under reduced pressure, resuspended in 2 p1 of water, and analyzed by descending paper chromatography on Whatman No.1 paper in solvent I, ethylacetate/pyridine/water (8:2:1) for 24 and 48 h, respectively. [3H]Glucosamine-labeled glycopeptides were hydrolyzed in 4 N HCI at 1 "C for 4 h. The released sugars were re-n-acetylated and analyzed by descending paper chromatography in solvent 11, n-butyl alcohol/pyridine/water (6:4:3) as described previously (Cummings and Kornfeld, 1982). The distribution of radioactivity on the paper chromatograms was determined by measuring the radioactivity of 1- cm segments of the strips in a liquid scintillation counter. Glycosidase Treatment-The radiolabeled total and antigenic glycopeptides were digested with exoglycosidases in 4 pl of 5 mm citrate buffer, ph 4.6, at 37 "C in toluene atmosphere for 48-96 h depending on the exoglycosidases used (Cummings and Kornfeld, 1982). Glycopeptides incubated at 37 "C without any exoglycosidases were used as control for these experiments. Neuraminidase, a-~fucosidase, P-N-acetylhexosaminidase, and P-D-galactosidase were used at a concentration of.25 unit/ml. The samples after digestion were boiled for 5 min and are subjected either to INF-Protein A- Sepharose affinity chromatography or analyzed by descending paper chromatography in solvent systems I and I1 for [3H]mannose- and [3H]glucosamine-labeled glycopeptides, respectively. [3H]Galactose-labeled antigenic glycopeptides were digested before and after a-fucosidase treatment with 4 milliunits of endo-p-galactosidase in 5 g1 of.1 M sodium acetate buffer, ph 5.6, at 37 "C in toluene atmosphere for 48 h (Cummings and Kornfeld, 1984). The native and defucosylated glycopeptides incubated at 37 "C without any enzyme were used as control for these experiments. After the treatment, the glycopeptides were boiled for 5 min and analyzed by descending paper chromatography in sovent system 111, ethylacetate/ pyridine/acetic acid/water (5:5:1:3) for 24 h. The paper chromatograms were scanned as described above. Methylation Analysis of Antigenic Glycopeptides-The [3H]glucosamine-, [3H]galactose-, and [3H]mannose-labeled antigenic glycopeptides were permethylated using a modification (Clark et al., 1991) of the iodomethane-naoh-dimethyl sulfoxide procedure (Guannarson, 1987; Cincanu and Kerek, 1984). The permethylated derivatives were hydrolyzed in 2 N trifluoroacetic acid for 2 h at 121 "C. The methylated sugars after hydrolysis were analyzed by thin layer chromatography on Silica Gel-G plates (2.5 X 16.5 cm) using acetone/ water/ammonium hydroxide (25:3:1.5) solvent system to separate methylated galactose species (Stoffyn et al., 1971). The [3H]galactoselabeled methylated standards, 2,4,6-tri-O-methylgalactose and 2,3,4,6-tetra--methylgalactose were prepared from [3H]galactoselabeled glycopeptides from Chinese hamster ovary cells as described previously (Cummings and Kornfeld, 1984). The sample lanes were marked traversely into.5-cm sections and scraped into scintillation vials and mixed with Scintiverse BD (Fisher) for the determination of radioactivity in the scintillation counter. The -methylmannitols and -methylfucitols obtained after hydrolysis and reduction (Lindberg, 1972) of the [3H]mannose-labeled methylated species were analyzed by HPLC as described earlier (Szilagyi et al., 1985) using a C-18 Zorbax reverse-phase column with slight modifications. The sample (2 pl) was injected and the eluate collected every 12 s directly into a 5.-ml scintillation vial at a flow rate of 1.8 ml/min for 15 min. The eluate was mixed with Scintiverse BD (Fisher) for the determination of radioactivity. The -methylalditolacetates of amino sugars were prepared after hydrolysis, reduction, and reacetylation of the [3H]glucosamine-labeled methylated derivatives, and these were analyzed by a Hewlett- Packard 571-A gas chromatography fitted with a DB-1 column (J & W Scientific, Folson, CA). The column was calibrated with unlabeled partially permethylated alditolacetate standards using flame ionization detector. The chromatographic analyses were carried out using the following program: initial temperature was maintained at 17 "C for 2 min, then raised to 25 "C at a rate of 2 "C/min, and was held at 25 "c for 2 min. Injector temperature was at 25 "C, and the detector temperature was also maintained at 25 "C with the flame ionization detector on. The combustion of radioactive derivatives produces tritiated water which was collected by inverting a precooled vial (-8 "C) for periods of 1-15 s/fraction between 1 and 25 min

2198 Antigenic S. mansoni after each injection. The condensate was mixed with 2. ml of Scintiverse BD and.2 ml of water to determine the radioactivity in the scintillation counter. SDS-PAGE and Western Blotting of Schistosome Glycoproteins- The schistosome wormpairs were solubilized (three times volume) with TBS-2 containing.1% Triton X-1 and the protease inhibitors, aprotinin, pepstatin, and dichlorocoumarin (1 pg/ml), leupeptin (1 pg/ml), trypsin inhibitor (.1%), and EDTA (1 mm) by sonication for 3 X 1 s, and the worm extract was centrifuged at 15, X g for 3 min followed by 1, X g for 1 h. The supernatant was applied to a column of TPA (.3 X 2-cm) equilibrated with the above buffer, and 1.5-ml fractions were collected. The column was washed overnight with the equilibration buffer to remove the unbound proteins and eluted with the buffer containing.4% fucose. The fractions containing the bound proteins were pooled and the BCA assay from Bio-Rad was carried out to determine the protein. The total, the TPA-unbound, and TPA-bound fractions were subjected to electrophoresis on 9% SDS-polyacrylamide gel under reducing conditions. The proteins were visualized by Coomassie staining (5% MeOH, 2% acetic acid,.1% Coomassie Blue) or silver staining. Proteins were electrophoretically transferred to nitrocellulose according to the manufacturer's instructions. Anti-mouse sialyl Le' monoclonal antibody and anti-mouse Le" monoclonal antibody were used as primary antibodies and anti-mouse alkaline phosphataseconjugated IgM was used as the secondary antibody and the reactive proteins were visualized by reaction with BCIP-nitro blue tetrazolium substrates. RESULTS Metabolic Radiolabeling of Adult Schistosome Wormpairs with Radioactive Precursor Sugars-Schistosome wormpairs were used to carry out the experiments on the antigenicity of the glycopeptides derived from the newly synthesized glycoproteins. The adult schistosome wormpairs were metabolically radiolabeled with tritiated precursor sugars, and the glycopeptides prepared by Pronase digestion of these glycoproteins were subjected to antibody affinity chromatography followed by serial lectin affinity chromatography. Metabolic radiolabeling is a useful tool in studying the structure of oligosaccharides in glycoproteins in minute quantities and avoids contamination of host glycoproteins during analyses (Cummings et al., 1989). The Adult Schistosome Glycopeptides Bind to the Antibodies Present in the Serum of Infected Hamsters-Experiments were carried out to determine if the radiolabeled glycopeptides prepared from schistosome glycoproteins have an affinity for immunoglobulins from the sera of infected hamsters. At least 25% of [3H]glucosamine-labeled glycopeptides bound to the INF affinity column (designated B glycopeptides), whereas there was negligible binding to the UNINF affinity column (Fig. 1A). A considerable amount of [3H]mannose- and [3H] galactose-labeled glycopeptides also bound to INF affinity column, as shown in Fig. 1, B and C, respectively. As with the ["H]glucosamine-labeled glycopeptides, we found that less than.4% of the [3H]mannose- and [3H]galactose-labeled glycopeptides bound to the UNINF affinity column (data not shown). N-Glycanase treatment of [3H]glucosamine-labeled glycoproteins released oligosaccharides that bound to the INF affinity column, but not to the UNINF affinity column, demonstrating that the antigenic glycopeptides contain com- plex-type N-linked oligosaccharides (Tarentino and Plummer, 1987). Similar studies with the sera from uninfected and infected mice showed that 13% of the [3H]glucosamine-labeled glycopeptides preferentially bound to the affinity column containing infected mouse serum (data not shown). These results demonstrate that schistosomes synthesize glycoprotein oligosaccharides that generate immune responses in infected hamsters and mice. We sought to characterize the structures of these antigenic oligosaccharides. The serum immunoglobulins from infected Oligosaccharides hamsters were covalently coupled to CNBr-Sepharose 4B and the antigenic [3H]glucosamine-, [3H]mannose-, and [3H]galactose-labeled glycopeptides were isolated as described above. The desalted, antigenic glycopeptides were subjected to serial lectin affinity chromatography to separate them on the basis of their structures. Fractionation of Antigenic Glycopeptides by Serial Lectin Affinity Chromatography-The antigenic glycopeptides were further fractionated by affinity chromatography on immobilized lectins to separate the glycopeptides and subject them to chemical analyses. The [3H]glucosamine-, [3H]mannose-, and [3H]galactose-labeled antigenic glycopeptides (designated the B glycopeptides) were applied to ConA-Sepharose affinity columns, and their fractionation profiles are shown in Figs. 2A, 3A, and 4A, respectively. ConA-Sepharose does not bind to highly branched tri- and tetraantennary and bisected biantennary N-linked oligosaccharides but binds to complex-type biantennary N-linked oligosaccharides and can be eluted with 1 mm a-methylglucoside (Ogata et al., 1975; Krusius et al., 1976; Cummings and Kornfeld, 1982; Merkle and Cummings, 1987). It binds with very high affinity to high mannose-type and hybrid-type N- linked oligosaccharides which require 1 mm a-methylmannoside for elution. A major portion of [3H]glucosamine- and ['HH]mannose-labeled B glycopeptides and less than 5% of ['H]galactose-labeled glycopeptides did not bind to the ConA column and were designated as B-I. The glycopeptides that were eluted with 1 mm a-methylglucoside and with 1 mm a-methylmannoside were designated as B-I1 and B-111, respectively. The [3H]glucosamine-, [3H]mannose-, and [3H]galactoselabeled B-I and B-I1 glycopeptides were further subjected to affinity chromatography on a column of TPA. TPA is a fucose-binding lectin and binds with high affinity to oligosac- charides containing fucose linked a1,3 to N-acetylglucosamine in the outer chain of N-linked oligosaccharides (Goldstein and Hayes, 1978). The glycopeptides bound by TPA (B- Ib and B-IIb) were eluted with.4% fucose. Most of the B-I glycopeptides bound to the affinity column (Figs. 2B, 3B, and 4B) and some of the [3H]mannose- and [3H]glucosaminelabeled B-IT glycopeptides also bound to the column (Figs. 2C and 3C). The B-I1 glycopeptides metabolically radiolabeled with [3H]galactose were not tested for their binding to TPA- Sepharose. We found that the radioactivity contained in these glycopeptides was mostly in glucose and not in galactose (Table I). Subsequent studies on the B-I1 glycopeptides have revealed that the N-linked oligosaccharides in this material lack galactose and have highly unusual structures which will be reported in another paper. The [3H]galactose-labeled B-Ib glycopeptides were further fractionated by affinity chromatography on column a of RCA- I-agarose. RCA-I-agarose binds with high affinity to complextype bi, tri-, and tetraantennary N-linked oligosaccharides that contain terminal galactose residues linked,b-1,4 to N- acetylglucosamine residues (Baenziger and Fiete, 1979; Merkle and Cummings, 1987). A significant portion (6%) of the B-Ib glycopeptides bound to the column (Fig. 4C) suggesting that complex-type N-linked chains in B-Ib glycopeptides contain terminal galactose residues. This suggestion was confirmed as described below by sequential exoglycosidase digestion and methylation analyses of [3H]galacto~e-labeled B-Ib glycopeptides. Analysis of Sugar Composition of Antigenic Glycopeptides- The [3H]glucosamine-labeled antigenic glycopeptides were hydrolyzed in strong acid, and the released monosaccharides were reacetylated and analyzed by descending paper chro-

S. mansoni Antigenic Oligosaccharides 2199 FIG. 1. Antibody affinity column chromatography of radiolabeled glycopeptides. The adult schistosomes were incubated with [6-3H]glucosamine, [2-: H]mannose, and [6-3H]galactose, and the derived radiolabeled glycopeptides were applied to Protein A-Sepharose affinity columns containing normal (UNINF) and immune (INF) sera from hamsters as described under Experimental Procedures. Panel A, [3H]glucosamine-labeled glycopeptides; panel B, [ Hlmannose-labeled glycopeptides; panel C, [ Hlgalactose-labeled glycopeptides. B B I a I ti I r I 1 2 3 4 1 1 2\ 3 4 5 B 3. a TPA C FIG. 2. Serial lectin affinity chromatography of [SH]glucosamine-labeled antigenic glycopeptides. [3H] Glucosamine-labeled antigenic glycopeptides were applied to columns of immobilized lectins as described under Experimental Procedures. The indicated fractions from the ConA-Sepharose column were pooled, dried, desalted, and applied to a column of TPA. 1. 1,.,.,.,.,., 1 2 3 4 5 1 2 3 4 5 matography (Table I). Thirty-two % of the radioactivity recovered from the B fraction was present in GalNAc and the remainder was present as GlcNAc. The B-I fraction contained mostly GlcNAc whereas B-I1 fraction contained both GlcNAc and GalNAc. [ HIMannose-labeled antigenic glycopeptides after strong acid hydrolysis were subjected to descending paper chromatography (Table I). The bound fraction contained 77% fucose and the remainder being mannose while B-I and B-I1 fraction contained mainly fucose. Only 16% of the radioactivity recovered from the B fraction was present as galactose and the rest of the radioactivity as glucose in the case of [3H]galactoselabeled antigenic glycopeptides (Table I). More than 5% of the radioactivity was recovered as galactose in B-I fraction whereas mainly glucose was present in B-I1 and B-I11 fractions, and all the radioactivity was present as galactose in B- Ib fraction. These data demonstrate that the antigenic glycopeptides are highly fucosylated and contain mainly mannose, galactose, and GlcNAc in addition to fucose. Because of their abundance, further structural analyses were focused on the B-Ib antigenic glycopeptides.

22 Oligosaccharides Antigenic S. mansoni t 4, t 1 2 3 FIG. 3. Serial lectin affinity chromatography of [3H]mannose-labeled antigenic glycopeptides. [3H]Mannose-labeled antigenic glycopeptides were applied to columns of immobilized lectins as described under Experimental Procedures. The indicated fractions from the ConA-Sepharose column were pooled, dried, desalted, and applied to a column of TPA. TABLE I Compositional analyses of radiolabeled antigenic glycopeptides 6 :m 12 1 3 4 5 L W gwo 2 4 2 a 2 4 6 8 FIG. 4. Serial lectin affinity chromatography of [3H]galactose-labeled antibody-bound glycopeptides. [3H]Galactose-labeled antigenic glycopeptides were applied to columns of immobilized lectins as described under Experimental Procedures. The indicated fraction from the ConA-Sepharose column waspooled, dried, desalted, and applied to a column of TPA. The glycopeptides bound to the TPA column werepooled, dried, desalted, and a portion was applied to a column of RCA-I- agarose (RCA). Glycopeptides L3H1 % radioactivity released as Mannose Man Fuc GalNAc GlcNAc Gal Glc B 23 77 32 68 16 85 B-I 6 94 6 94 6 4 B-I1 12 88 49 51 3 97 B-Ib ND ND ND ND 1 - Percent radioactivity was determined after strong hydrolysis and descending paper chromatography of aliquots of the radiolabeled glycopeptides as described under Experimental Procedures: Percent radioactivity refers to the percent of total radioactivity in the glycopeptide pool recovered in each of the constituent monosaccharides. ND, not determined. -, indicates that no radioactivity was present in that monosaccharide. 1 n 1 2 3 MIGRATION, CM FIG. 5. Endo-@-galactosidase treatment of [3H]galactose-labeled B-Ib glycopeptides. Glycopeptides were incubated with bovine kidney a-l-fucosidase for 96 h. The native and defucosylated glycopeptides (approximately 5 cpm) were digested with 4 milliunits of endo-@-galactosidase, and the treated material was analyzed by descending paper chromatography as described under Experimental Procedures. The migrations of standard oligosaccharides are shown. Antigenic Glycopeptides (B-Ib) Contain Lactosamine Chains-The high molecular weight (eluted in the void volume in Sephadex G-5 size exclusion column), the high content of fucose in these glycopeptides and the presence of idase. The native and the defucosylated glycopeptides were terminal galactose linked @-1,4 to N-acetylglucosamine as shown by affinity for immobilized RCA-I suggested that they digested with the enzyme as described under Experimental Procedures. The released fragments were analyzed by paper might contain polyfucosylated poly-n-acetyllactosamine se- chromatography, and the results are shown in Fig.5. The quences composed of the repeating disaccharide (3Gal@1-4GlcNAcP-1). To evaluate this possibility, the [3H]galactoselabeled B-Ib glycopeptides were treated with endo-p-galactosidase, an enzyme known to cleave poly-n-acetyllactosamine sequences at internal @1,4-linked galactosyl residues (Fukuda et al., 1978). Since fucosyl residues on the poly-n-acetyllactosamine chain can block the action of endo-@-galactosidase, a portion of the glycopeptides was pretreated with cy-l-fucosnative glycopeptides were resistant to endo-@-galactosidase, whereas the defucosylated B-Ib glycopeptides were highly susceptible to this endoglycosidase; 8% of the radiolabel was released as low molecular weight fragments. Endo-@-galactosidase typically releases both a disaccharide, GlcNAc@l-3Gal and a trisaccharide, Gal@l-4GlcNAc@l-3Gal which were recovered in the digestion of the schistosome antigenic oligosaccharides in the ratio of 41, respectively (Fig. 5). The slowly

Oligosaccharides Antigenic S. mansoni 221 migrating, higher molecular weight oligosaccharide (19%) is CHO cells indicating that fucose was present in a1,3 linkage probably a penta- or a hexasaccharide-containing fucose in B-I glycopeptides (data not shown). The release was not which can arise because of the inefficiency of the a-l-fucosi- quantitative since the enzyme is available with low specific dase and the inability of endo-p-galactosidase to cleave inter- activity and hence has to be used in excess to observe complete nally fucosylated poly-n-acetyllactosamine sequences (Fu- release of fucose. The fucose is present in a1,3 linkage in the kuda et al., 1984). These results demonstrate that the anti- outer chains of the glycopeptides and not the inner core since genic glycopeptides contain poly-n-acetyllactosamine chains TPA does not bind glycopeptides obtained from wild type with several GalPl-4GlcNAc repeating units that are fucosy- CHO cells that contain a1,g-linked fucose in the inner core lated. but appears to have high affinity for fucosylated polylactosa- Linkage Analyses Demonstrated That B-Ib Glycopeptides mine chains. Contain Terminal Fucose, and It Is Linked a1,3 to N-Acetyl- Methylation of the [3H]galactose-labeled B-Ib glycopepglucosamine with Terminal Galactose-The linkage patterns tides resulted in the recovery of mostly 2,4,6-tri-O-methylgaof mannose, galactose, and N-acetylglucosamine residues in lactose, indicating that most of the galactose residues are B-Ib glycopeptides were next examined which would clearly monosubstituted at the C-3 position (Table 11). This is the confirm the structure of the antigenic oligosaccharides. The typical linkage pattern seen for galactosyl residues in po1y-n- -methylmannitols of [3H]mannose-labeled B-Ib glycopep- acetyllactosamine sequences. This conforms with our obsertides were prepared and analyzed by HPLC as described under vation above showing the affinity of the B-Ib glycopeptides "Experimental Procedures." The ratios of various methylated for RCA-I and their susceptibility to endo-p-galactosidase residues to 2,4-di-O-methylmannitol, which was set to 1. and, therefore, the presence of galactose substituted at C-3 residue, are shown in Table 11. A mixture of radioactive 3,4- position in /3 linkage to N-acetylglucosamine. The methyladi-, 2,4-di-, 3,4,6-tri-O-, and 2,3,4,6-tetra-O-methylmanni- tion analysis of the defucosylated B-Ib glycopeptides gave a tols and 2,3,4-tri-O-methylfucitol were used as standards for pattern similar to the native glycopeptides (data not shown) HPLC. The results of methylating the [3H]mannose-labeled indicating that galactose is not fucosylated. Only a small B-Ib glycopeptides indicate that the glycopeptides are a mix- amount of galactose was found in the terminal position by ture of tri- and tetraantennary N-linked oligosaccharides methylation, and this is consistent with the observation that (Nyame et al., 1989), which are core structures commonly exoglycosidase treatment of the [3H]galactose-labeled B-Ib found in vertebrate glycoproteins. Radioactive fucose follow- glycopeptides with P-galactosidase alone released only 9% of ing methylation was recovered as 2,3,4-tri-O-methylfucitol, the galactose (data not shown). indicating that fucosyl residues are in terminal unsubstituted Methylation of the [3H]glucosamine-labeled B-Ib glycopeppositions. tides, the compositional analysis of which indicated that the The presence of fucose in a1,3 linkage was shown by the radioactivity was primarily in N-acetylglucosamine, showed digestion of [3H]mannose-labeled B-I glycopeptides with that 81% of the radioactivity was recovered as 6--methylal,3/1,4-fucosidase from Streptomyces sp. (Sano et al., 1992), N-acetylglucosamine (Table 11). This derivative arises from and the amount of fucose released was analyzed by paper N-acetylglucosamine residues that are substituted at both chromatography as described earlier. [3H]Mannose-labeled positions C-3 and C-4, as shown in Fig. 6. The 3,6-di-- ConA-I fraction from wild type Chinese hamster ovary (CHO) methyl-n-acetylglucosamine may likely represent the core N- cells was used as negative control and that from NeoLewis acetyglucosamine present in N-linked sugar chains substituted at C-4 position core to mannose in,6 linkage. The results cells (CHO cell line transfected with al,3-fucosyltransferase) (Zhou et al., 1991) as a positive control. Fucose was released from B-I and Neolewis glycopeptides but not from those of TABLE I1 Methylation analyses of radiolabeled B-Ib glycopeptides Methylated derivatives derived from B-lb glycopeptides % radioactivity" Ratiob ["HlMannitols from ['HH]mannose-labeled glycopeptides 3,4-Di-O-methyl 3 1. 3,6-Di-O-methyl 18.6 2,4-Di-O-methyl 3 1. 3,4,6-Tri-O-methyl 22.7 ["HIGalactose from ['H]galactose-labeled glycopeptides 2,4,6-Tri-O-methyl 91 1. 2,3,4,6-Tetra-O-methyl 9 1. ["HIN-Acetylglucosamine alditol acetate from [3H]glucosamine-labeled glycopeptides 6--methyl 81 5. 3--methyl.3 4 3,6-Di-O-methyl 15 1. " The percentage of radiolabeled monosaccharide recovered in each derivative is shown. 'The ratio of monosaccharide derivatives for each of the three radiolabeled glycopeptide fractions was established by setting to 1. either the 2,4-di-O-methylmannose, the 2,3,4,6-tetra-O-methylgalactose, or the 3,6-di-O-methyl alditol acetate of N-acetylglucosamine, of methylation analysis, the sensitivity of [3H]mannose-labeled B-I glycopeptides to a1,3/1,4-fucosidase, and sensitivity of the defucosylated B-Ib glycopeptides to endo-p-galactosidase demonstrate that schistosomes synthesize antigenic N- linked oligosaccharides containing polyfucosylated po1y-nacetyllactosamine sequences with the repeating structure (3Galpl-4(Fucal-3)GlcNAcpl), which is illustrated in Fig. 6. Many Schistosome Glycoproteins Have Affinity for TPA, and These Are Immunoreactive with Anti" Antibody-The Triton X-1-solubilized schistosome glycoproteins were subjected to TPA affinity chromatography as described under "Experimental Procedures." The TPA-unbound and -bound fractions were pooled and the amount of protein was determined and the protein fractions were subjected to SDS-PAGE (Fig. 7). The results show that there were many glycoproteins (5-1 kda) bound by TPA that may have oligosaccharides containing fucose linked to N-acetylglucosamine. Next, we sought to find the reactivity of the TPA-bound glycoproteins with anti-le" monoclonal antibody. Fuc la1.3 [Gal p1,4glcnac] p1.3gal81.4 -- n { fglcnacpl$ \ GlcNAcp1,2Mana1,6 \Man~1,4GlcNAcp1.4GlcNAcAsn GlcNAcpl,PYana1.3' la1,6 1 f Fuc + GkNAc (31.4 FIG. 6. Proposed partial structure for the antigenic glycopeptides synthesized by adult S. mansoni.

222 Oligosaccharides Antigenic S. mansoni FIG. 7. SDS-PAGE of Triton X-I -solubilized schistosome glycoproteins. The solut)ilized schistosome glycoproteins were SUI)jected to TI A affinity chromatography a s described under Experimental Procedures. The total, ITA-unbound and TPA-hound glycoproteins (-26 p g ) were analyzed hy SDS-PACE. IAnr 1, Hio-liad high molecular weight standards a s indicated; lnnr 2. total ( 7 ) ; lnnr - I, TPA unhound ( I I H ) ; and lnnr 4, TI A hound ( H ). The proteins were visualized I ) v silver staining. had no effect indicating thatn-fucosvl residues are important antigenic determinants (data not shown). Terminal GalNAc residues in some of these glycopeptides, which we have shown to occur in schistosome glycopeptides (Nyame et af., 1989), may also contribute to their antigenicity since pretreatment of the [ Hlmannose-labeled glycopeptideswith ij-n-acetvlhexosaminidase reduced theirbindingtotheinfaffinity column. All these data are consistent with the interpretation that fucose and to alesser extent N-acetylgalactosamine may be part of the ant.igenic epitopes on the schistosomeoligosaccharides. DlSClJSSlON Previous studies (Nyame et af., 1989) have shown that the complex-typebiantennary oligosaccharides in schistosomederived glycoproteins contain significant amount of oligosaccharides with terminal N-acetylgalactosamine that bound to immobilized Wisteria floribunda agglutinin. Sucholigosaccharides are found in very few animal cell glycoproteins (Pierce TUBE 1 UB B and Parsons, 1981; Green et al., 1985: Donald and Feenev, 1986; Nakata et of., 1991; Chan et al., 1991) which are either sulfated or sialylated. The unusual structural feature of this set of oligosaccharides suggested that these could be antigenic in infected animals, and our studies on the affinitv of these oligosaccharides for the antibodiesfrom the serumof infected hamsters showed that less than 9Y of the [~ Hjglucosaminelabeledoligosaccharides bound to the INF affinity column whereasconsiderableamount of W. floribunda agglutininunbound oligosaccharides bound to it (data not shown) indianti Siolyl LeX Anti LeX cating that terminal N-acetylgalactosamine containing olifig. 8. Western blotting of the glycoproteins with anti-sia- gosaccharides are not significantly present in the antigenic lyl Le antibody and anti-le antibody. S I X - P A G E of the glyglycopeptides. coproteins (-26 pg), the protein transfer to the nitrocelluose memin our present study, we demonstrate that the antibodies h a n e, and the immunohlotting were performed a s described under recognize mainly the asparagine-linked tri-or tetraantennary Experimental Procedures. h n r 1, Rio-Rad high molecular weight complex-type oligosaccharides with polyfucosylated polylacstandards as indicated; lnnr 2, total (7 ): lanr 3, T P A unhound ( llh ); tosamine chains containing the Le antigenic blood ~ o u p. and lnnr 4, TPA hound ( H ). The removal of fucose considerably reduced the binding to antibodies showing that fucose may be part of the carbohyequal amounts of total, TPA-unbound and TPA-bound glycoproteins were subjected to SDS-PAGE and the proteins drate epitope recognized by the antibodies from the infected serum. were electrophoreticallytransferredtonitrocellulosemempoly-n-acetyllactosamine-containingoligosaccharides are brane and Ponceau-S stainwas used to observe the complete commonly found in animal cells, and they contain antigenic transfer. The stain was removed by washing the membrane structures such asblood group and Ii antigens and are carriers with deionized water and the membrane was immunoblotted with the anti-le antibody and anti-sialyl Le antibody was of Le antigens, also called stage-specific embryonic antigensequence Galrjlused as the negative control; the results are shown in Fig. 8. 1, which hastheterminaltrisaccharide T h e majority of the glycoproteins bound by TPA was immu- 4(Fucnl-3)GlcNAc-R (Gooi et al., 1981; Fukuda. 1985). The maturation of human myeloid cells is associated with IR and noreactive with anti-le antibody.thetotalglycoproteins Le.related oligosaccharides (Fukuda, 1985) and recent eviwere alsoimmunoreactivewiththeanti-le antibodybut requires proteinsin excess to observe high immunoreactivity. dence indicates thatfucosylated poly-n-acetyllactosamine sequences are importantfor human leukocyte interactions with None of the proteins were immunoreactive with the antit.he vascular endothelium and platelets (Zhou ~t af., 1991). sialyl Le antibody. Thus, i t is possible that these t-ypes of antigenic oligosacchathe results from the various experiments carried out on the structure of the schistosome-derived antigenic glycopro- rides described in the current study may interfere with or teins have demonstrated that these are complex-type tri- and alter in some way the inflammatory response of the host at tetraantennary N-linked oligosaccharides that are highly fu- the siteof parasite attachment. There is evidence that poly-n-acetyllactosamine-containcosylatedandcontainlactosaminechains of a t least four repeats of Gal[j-l,4GlcNAc. The TPA-bound glycoproteins ing oligosaccharides contribute to host-pathogen interactions, contain oligosaccharides with Le antigenic blood group con- and they may be synthesized by parasites. The interaction of is mefirming our structural analyses of the antigenic oligosaccha- Mycoplasmapneumoniae withhumanerythrocytes diated by sialyl oligosaccharides containing polv-n-acetvllacrides derived from schistosome glycoproteins. et of., 1984). Experiments to determine some of the structural features tosaminechains of I i antigentype(loome of the carbohydrate determinants required for binding to the Trypanosoma brucei has been shown to synthesize branched I N F affinity column showed that pretreatment of the glyco- poly-n-acetyllactosamine-containing oligosaccharides in Type 2 variantsurface glycoproteins(zamze et al.. 1991). peptides withbovinekidneyn-l-fucosidase causeda6% Recently, KO et af. (199) observed that a monoclonal antidecrease in their binding to the INF affinity column, whereas pretreatment with either8-galactosidase or neuraminidase body tostage-specificembryonicanitigen-1antigen recog-

nizes a determinant present in S. mansoni and that antibodies to this determinant bind to the schistosome larvae and mediate antibody-dependent cytotoxicity. Our data are consistent with the expression of the stage-specific embryonic antigen-1 antigen by schistosomes and furthermore provide the first chemical evidence for the synthesis of polyfucosylated poly-n-acetyllactosamine by an invertebrate. Recently, Levery et al. (1992) have characterized a series of fucose-containing glycosphingolipid immunogens from eggs of S. mansoni. These contain terminal fucose-linked a1,3 and a1,4 to GlcNAc and also an unusual internally substituted fucose with GalNAc-Glc-ceramide backbone. Makaaru et al. (1992) have identified the GalNAc-Glc-ceramide structure in schistosome glycolipids, and we have presently shown the presence of polyfucosylated oligosaccharides in schistosome glycoproteins. 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