The Human Blood Fluke Schistosoma mansoni Synthesizes ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biolopy, Inc.

Vol. 267, No. 28, lrrsue of October 5 , pp. 20196-20203, 1992 Printed in U.S.A.

The Human Blood Fluke Schistosoma mansoni Synthesizes Glycoproteins Containingthe Lewis X Antigen* (Received for publication, May 6, 1992)

Jayanthi Srivatsan, David F. Smith, and RichardD. Cummingst From the University of Georgia, Department of Biochemistry, Athens, Georgia 30602

Infectionofvertebrateswiththeparasiticblood unusual oligosaccharides (Makaaru et al., 1992; Levery et al., fluke Schistosoma mansoni induces a variety of host 1992). immuneresponses,which are directedagainstboth Most studies in recent yearshave 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 oligosaccharides synthesized by schistosomula and adult schisgosaccharides derivedfrom 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 suggested that these may be antigenic in infected animals. in glycoproteinsfromtheadultparasite.Structural analysis ofthemajor antigenic oligosaccharides re- Our present studies are focused on identifying the nature of the antigenic carbohydratedeterminants inglycoproteins synvealed that they have high amounts of fucose-linked thesized by schistosomes. In this report, we present our finda1,3 to N-acetylglucosamine residues within the linear repeating 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-acetyllactosaminesequence 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 EXPERIMENTALPROCEDURES of vaccines to prevent this disease in humans. Materials-Concanavalin A (ConA)’-Sepharosewas procured from

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 antigenicglycoproteins 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, 1980; Linder and Huldt, 1982; Weiss andStrand, 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; KOet al., 1990). Recent studies have shown that there areantigenic 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-8810164 (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, 940 S. L. Young Blvd., Oklahoma City, OK 73104. Tel.: 405-271-2546; Fax: 405-2713910.

Pharmacia LKB Biotechnology Inc. CNBr-activated Sepharose 4B, Protein A-Sepharose CLGB, Sephadex G-25-80, anti-mouse IgM alkaline phosphatase conjugate, galactose, lactose, fucose, a-methylglucoside, a-methylmannoside, Tetragonolobuspurpureas,Triton X-100, N-acetylgalactosamine, N-acetylglucosamine, MOPS, Tris-base,Amberlite MB-3, anti-mouse alkaline phosphatase-conjugatedIgM, neuraminidase, and P-D-galactosidase from jack beanwere purchased from Sigma. a-1,3/1,4-fucosidase from Streptomyces sp. was obtained from Takara Biochemicals Inc. (Berkeley, CA). Affi-Gel 10 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 Pronasewas obtained from Calbiochem (San Diego, CA). Anti-LeuM1 (CD15) monoclonal antibody was purchased from Becton Dickenson Immunocytometry Systems (San Jose, CA). /3-NAcetylhexosaminidase was purified from jack bean meal (Li and Li, 1972). The radiolabeled sugars [6-3H]glucosamine (40 Ci/mmol), [23H]mannose (21 Ci/mmol) and [6-3H]galactose (25 Ci/mmol) were purchased from ICN Biochemical Co (Irvine, CA). Dulbecco’s minimum essentialmedium 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 58920 gas chromatography system fitted with 30-m SP2330 or 30-m DB-1 columns for neutral and aminosugars, respectively. Metabolic Radiolabeling of Adult Schistosome Wormpairs with Radioactive 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.

20196

Oligosaccharides Antigenic S. mansoni described previously (Nyame etal., 1987). At least 12 wormpairswere 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-streptomycinsolution (10,000 units of penicillinG/ml and 10 mgof streptomycin/ml in 0.9% NaCI) in a final volume of 0.5 ml for 24 h. Incubations were conducted at 37 "C in a humidified incubator containing 5%CO,. At the endof the incubation, the worms were motile and viable, and the culture was devoid of bacterial and fungal contamination. Theworms were washed three times with phosphate-buffered saline and stored at -80 "C till further use. Preparation of Radiolabeled Glycopeptides from Adult Schistosome Wormpairs-The radiolabeled adult schistosomewormpairs were sonicated in lysis buffer (0.1 M Tris-base, pH 8.0, containing 1 mM CaCl,). Lipids were extracted with 20 volumes of chloroform/methanol/water (10:10:3) followed by chloroform/methanol (21).The protein extracts were dried under a stream of nitrogen and digested with 10 mg/ml Pronase in lysis buffer at 60 "C in toluene atmosphere for 24 b. After incubation, the samples were boiled for 5min and desalted o n a Sephadex G-25 (1 X 50-cm) column in water containing 7% npropyl 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 8week infected hamsters (INF) and normal hamsters (UNINF) were heat inactivated at 56 "C for 30 min and centrifuged a t 3000 X g for 30 min to remove any precipitate. Two 1.5-ml (0.7 X 4-cm) Protein A-Sepharose columns equilibrated with Tris-buffered saline, TBS-1 (10 mM Tris-base, pH 8.0, containing 1mM EDTA, 0.15 M NaCl, and 0.02% NaN3), were saturated with 1.0 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 1.0 ml were collected. The bound glycopeptides were eluted with 0.1 M sodium acetate, pH 2.8, containing 0.15 M NaC1. The fractions were neutralized by collecting in test tubes containing0.1 ml of 1.0 M Tris-base, pH 8.0. Aliquots of fractions (-50 ml) were mixed with Scintiverse BD (Fisher) for determining radioactivity in the scintillation counter. Coupling of ImmuneSerum from Infected Hamsters to CNBrSepharose 4B-The heat-inactivatedimmuneserumfrom 8-week infected hamsters was dialyzed against 30 volumes of coupling buffer (0.1 M NaHC03, 0.15 M NaCI, pH 8.3)overnight. The serumwas 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 withTBS1.A column of 25 ml was prepared in TBS-1, pH8.0. The radiolabeled glycopeptides were applied to thecolumn and fractions of 3.0 ml were collected and thebound 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 0.1 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 RadiolabeledAntigenic Glycopeptides-Immobilized column of T. purpureas lectin (TPA) in Affi-Gel 10 was prepared according to themanufacturer's instructions. Briefly, Affi-Gel (1-1.5 ml) is washed with 10 volumes of deionized water. Lectin solution (1.0 ml, 15-20 mg lectin/ml in 0.1 M MOPS buffer, p H 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 1.0 ml of 0.2M ethanolamine for a t least 2 hat 4 "C. The column was equilibrated with Tris-buffered saline-2, TBS-2 (10 mM Trisbase, pH 8.0, containing 1 mM CaC12, 1 mM MgCI2,0.15 M NaCl, and 0.02% NaN3). The radiolabeled antigenic glycopeptides were fractionated on 2.0ml columns (0.7 X 5-cm) of ConA-Sepharose equilibrated with TBS2 at room temperature (Cummings and Kornfeld, 1982; Merkle and Cummings, 1987). Glycopeptides bound to the columns were first eluted with 10 mM a-methylglucoside followed by 100 mM a-methylmannoside. Fractions of 2.0.ml were collected. The pooled glycopeptide fractions were dried, desalted, and were further fractionated on a 1.0-ml column (0.3 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 containing0.4% fucose. Fractions of 0.5 ml were collected. RCA-I agarose affinity chromatography was performed on a 20-ml column (0.8 X 40-cm) a t room temperature as described previously (Merkle and Cummings, 1987), and 1.0-ml fractions were collected. The bound material was elutedwith0.1 M

20197 lactose. Glycopeptides were desalted and separated from monosaccharides by chromatography on a column of Sephadex G-25 (1 X 50cm) 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 100 "C for 4 h,and thehydrolysates were dried by evaporation under reduced pressure, resuspended in 20 p1 of water, and analyzed by descending paper chromatography on Whatman No.1 paper insolvent I, ethylacetate/pyridine/water (8:2:1) for 24 and 48 h, respectively. [3H]Glucosamine-labeled glycopeptides were hydrolyzed in 4 N HCI at 100 "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 1cm segments of the strips in a liquid scintillation counter. Glycosidase Treatment-The radiolabeled total and antigenic glycopeptides were digested with exoglycosidases in 40 pl of 50 mM citrate buffer, pH 4.6, a t 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 0.25 unit/ml. The samples after digestion were boiled for 5 min and are subjected either to INF-Protein ASepharose 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 antigenicglycopeptides were digested before and after a-fucosidase treatment with 40 milliunits of endo-p-galactosidase in 50 g1 of 0.1 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 a t 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 chromatographyin 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-labeledantigenic 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 (250:3:1.5) 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-0-methylgalactosewere prepared from [3H]galactoselabeled glycopeptides from Chinese hamster ovary cells as described previously (Cummings and Kornfeld, 1984). The sample lanes were marked traversely into 0.5-cm sections and scraped into scintillation vials and mixed with Scintiverse BD (Fisher) for the determination of radioactivity in the scintillation counter. The 0-methylmannitols and 0-methylfucitols obtained after hydrolysis and reduction (Lindberg, 1972) of the [3H]mannose-labeled methylated species were analyzed by HPLCas described earlier (Szilagyi et al., 1985) using a C-18 Zorbax reverse-phase column with slight modifications. The sample (20 pl) was injected and the eluate collected every 12 s directly into a 5.0-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 0-methylalditolacetatesof amino sugars were prepared after hydrolysis, reduction, and reacetylation of the [3H]glucosamine-labeled methylated derivatives, and these were analyzed by a HewlettPackard 5710-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 170 "C for 2 min, then raised to 250 "C at a rate of 2 "C/min, and was held at 250 "c for 20 min. Injector temperature was a t 250 "C, and the detector temperature was also maintained at 250 "C with the flame ionizationdetectoron. The combustion of radioactive derivatives produces tritiated water which was collected by inverting a precooled vial (-80 "C) for periods of 10-15 s/fraction between 10 and 25 min

Antigenic S. mansoni

20198

after each injection. The condensate was mixed with 2.0 ml of Scintiverse BD and 0.2 ml of water to determine the radioactivity in the scintillation counter. SDS-PAGE and Western Blotting of Schistosome GlycoproteinsThe schistosome wormpairs were solubilized (three times volume) with TBS-2 containing 0.1% Triton X-100 and the protease inhibitors, aprotinin, pepstatin, anddichlorocoumarin (1 pg/ml), leupeptin (10 pg/ml), trypsin inhibitor(0.1%), andEDTA (1mM) by sonication for 3 X 10 s, and the worm extract was centrifuged at 15,000 X g for 30 min followed by 100,000 X g for 1 h. The supernatant was applied t o a column of TPA (0.3 X 20-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 0.4% 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 (50% MeOH, 20% acetic acid, 0.1% Coomassie Blue) or silver staining. Proteins were electrophoretically transferred to nitrocellulose according tothe 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

MetabolicRadiolabeling 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 instudying the structure of oligosaccharides in glycoproteins inminutequantities and avoids contamination of host glycoproteins during analyses (Cummings et al., 1989). The Adult SchistosomeGlycopeptides 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 seraof 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 0.4% 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, butnot totheUNINF affinity column, demonstrating that the antigenic glycopeptides contain complex-type N-linked oligosaccharides (Tarentino and Plummer, 1987). Similar studieswith 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 structuresof 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 chromatographyto separatethem 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-labeledantigenic glycopeptides (designated the B glycopeptides) were applied to ConA-Sepharose affinity columns, and their fractionation profiles are shown in Figs. 2 A , 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 10 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 Nlinked oligosaccharides which require 100 mM a-methylmannoside for elution. A major portion of [3H]glucosamine-and ['HH]mannose-labeled B glycopeptides and less than 50% of ['H]galactose-labeled glycopeptides did not bind to theConA column and were designated as B-I. The glycopeptides that were eluted with 10 mM a-methylglucoside and with 100 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 oligosaccharides containing fucose linked a1,3 to N-acetylglucosamine in the outer chain of N-linked oligosaccharides (Goldstein andHayes, 1978). The glycopeptides bound by TPA (BIb and B-IIb) were eluted with 0.4% fucose. Most of the B-I glycopeptides bound to theaffinity column (Figs. 2B, 3B, and 4B) and some of the [3H]mannose- and [3H]glucosaminelabeled B-IT glycopeptides also bound to thecolumn (Figs. 2C and 3C). The B-I1 glycopeptides metabolically radiolabeled with [3H]galactosewere not tested for their binding to TPASepharose. We found that theradioactivity 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 RCAI-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 Nacetylglucosamine residues (Baenziger and Fiete, 1979; Merkle and Cummings, 1987). A significant portion (60%) of the B-Ib glycopeptides bound to the column (Fig. 4C) suggesting that complex-type N-linked chains inB-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 GlycopeptidesThe [3H]glucosamine-labeled antigenic glycopeptides were hydrolyzed in strong acid, and the released monosaccharides were reacetylated and analyzed by descending paper chro-

20199

S. mansoni Antigenic Oligosaccharides

B

0

FIG. 1. Antibody affinity column

I

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 ( U N I N F )and immune ( I N F ) sera from hamstersas described under“Experimental Procedures.” Panel A , [3H]glucosamine-labeled glycopeptides; panel B , [“Hlmannose-labeled glycopeptides; panel C, [“Hlgalactose-labeled glycopeptides.

FRACTION

I

a

B0

0

r

‘ti I

0

10

I

20

30

40

FRACTION

01

10

30

20\

40

FIG. 2. Serial lectin affinity chromatography of [SH]glucosamine-labeled antigenic glycopeptides. [3H]

50

Glucosamine-labeled antigenic glycopeptides were applied to columns of immobilized lectins as described under “ExperimentalProcedures.” The indicatedfractions from the ConA-Sepharose column were pooled, dried, desalted, and applied to a column of TPA.

C

TPA 300.

B0

FRACTION

a”

100.

1 .

0,

0

10

20

30

FRACTION

40

50

0

, 10

.

, 20

.

, 30

.

, 40

.

, 50

FRACTION

matography (Table I). Thirty-two % of the radioactivity recovered from the B fraction was present in GalNAc and the remainder was present asGlcNAc. The B-Ifraction 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). Thebound fraction contained 77% fucose and theremainder 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 thecase of [3H]galactoselabeled antigenic glycopeptides (Table I). More than 50% 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 BIb 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 structuralanalyses were focused on the B-Ib antigenic glycopeptides.

Oligosaccharides Antigenic S. mansoni

20200

0 1 20 10

t

t

400,

600

L 0

:m

30

40

50

gwo

W

400

200 200

a

0

0 0

10

20

20

40

60

80

FRACTION

30

FIG.4. Serial lectin affinity chromatography of [3H]galacFRACTION FRACTION FIG.3. Serial lectin affinity chromatography of [3H]man- tose-labeled antibody-bound glycopeptides. [3H]Galactose-lanose-labeled antigenic glycopeptides. [3H]Mannose-labeled an- beled antigenic glycopeptideswere applied to columns of immobilized tigenic 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.

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 ( R C A ) .

TABLE I Compositional analyses of radiolabeled antigenic glycopeptides

L3H1

Glycopeptides

Mannose Man Fuc

% radioactivity released

GalNAc

GlcNAc

as“ Gal

Glc

B 16 23 77 32 68 85 B-I 6 94 6 94 60 40 51 3 97 B-I1 12 88 49 100 -‘ B-Ib ND ND ND ND’ ” 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.

Antigenic Glycopeptides (B-Ib) Contain Lactosamine Chains-The high molecular weight (eluted in the void volume in Sephadex G-50 size exclusion column), the high content of fucose inthese glycopeptides and the presence of terminal galactose linked @-1,4to N-acetylglucosamine as shown by affinity for immobilized RCA-I suggested that they might contain polyfucosylated poly-N-acetyllactosamine sequences composed of the repeating disaccharide (3Gal@14GlcNAcP-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-linkedgalactosyl 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-fucos-

1000

n ”

0

10 20 MIGRATION, CM

30

FIG.5. Endo-@-galactosidasetreatment 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 5000 cpm) were digested with 40 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.

idase. The native and the defucosylated glycopeptides were digested with the enzyme as described under “Experimental Procedures.’’ The released fragments were analyzed by paper chromatography, and the results are shown in Fig.5. The native glycopeptides were resistant to endo-@-galactosidase, whereas the defucosylated B-Ib glycopeptides were highly susceptible to thisendoglycosidase;80%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-3Galwhich were recovered in the digestion of the schistosome antigenic oligosaccharides inthe ratioof 4 1 , respectively (Fig. 5).The slowly


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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 arisebecause 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 andhence has tobe usedin excess to observe complete nally fucosylated poly-N-acetyllactosamine sequences (Fu- release of fucose. The fucose is present in a1,3linkage in the outer chainsof the glycopeptides and not the inner core since kuda et al., 1984). These results demonstrate that the antigenic glycopeptides contain poly-N-acetyllactosamine chains TPA does not bind glycopeptides obtained from wild type with several GalPl-4GlcNAc repeating units that arefucosy- CHO cells that contain a1,G-linked fucose in the inner core but appears to havehigh affinity for fucosylated polylactosalated. Linkage Analyses Demonstrated That B-Ib Glycopeptides mine chains. Methylation of the [3H]galactose-labeled B-Ib glycopepContain Terminal Fucose, and It Is Linked a1,3 to N-Acetylglucosamine with Terminal Galactose-The linkage patterns tides resulted in therecovery of mostly 2,4,6-tri-O-methylgalactose, indicating that most of the galactoseresidues are of mannose, galactose, and N-acetylglucosamine residues in 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 residuesin po1y-N0-methylmannitols of [3H]mannose-labeled B-Ib glycopep- acetyllactosamine sequences. This conforms with our obsertides were prepared and analyzed by HPLC asdescribed under vation above showing the affinity of the B-Ib glycopeptides "Experimental Procedures." The ratiosof various methylated forRCA-I and their susceptibility to endo-P-galactosidase whichwas set to 1.0 and, therefore, the presence of galactose substituted a t C-3 residues to 2,4-di-O-methylmannitol, residue, are shown in Table 11. A mixture of radioactive 3,4- position in /3 linkage to N-acetylglucosamine. The methyladi-0, 2,4-di-0,3,4,6-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 nativeglycopeptides (data not shown) small HPLC. The results of methylating the [3H]mannose-labeled indicatingthat galactose isnot fucosylated.Onlya amount of galactose was found in the terminal position by B-Ib glycopeptides indicate that theglycopeptides are a mixture of tri-andtetraantennaryN-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 wasrecovered as 2,3,4-tri-O-methylfucitol, the galactose (data not shown). Methylation of the [3H]glucosamine-labeled B-Ib glycopepindicating thatfucosyl residues are in terminal unsubstituted tides, the compositional analysisof which indicated that the positions. The presence of fucose in a1,3 linkage was shown by the radioactivity was primarily in N-acetylglucosamine, showed digestion of [3H]mannose-labeledB-I glycopeptides with that 81% of the radioactivity was recovered as 6-0-methylal,3/1,4-fucosidase from Streptomyces sp. (Sano etal., 1992), N-acetylglucosamine (Table 11). This derivative arises from and the amount of fucose released was analyzed by paper N-acetylglucosamineresidues that are substituted at both positionsC-3and C-4, as shownin Fig. 6. The3,6-di-0chromatography as described earlier.[3H]Mannose-labeled ConA-I fraction from wild type Chinese hamsterovary (CHO) methyl-N-acetylglucosamine may likely represent thecore N cells was used as negative control and that from NeoLewis acetyglucosamine present in N-linked sugar chains substito mannose in,6 linkage. The results cells (CHO cell line transfected withal,3-fucosyltransferase) tuted at C-4 position core (Zhou et al., 1991) as a positive control. Fucose was released of methylation analysis, the sensitivity of [3H]mannose-lafrom B-I and Neolewis glycopeptides but not from those of beled 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 TABLE I1 linked oligosaccharides containing polyfucosylated po1y-NMethylation analyses of radiolabeled B-Ib glycopeptides acetyllactosamine sequenceswith therepeatingstructure Methylated derivatives % radioactivity"Ratiob (3Galpl-4(Fucal-3)GlcNAcpl), which is illustrated inFig. 6. derived from B-lb glycopeptides Many Schistosome Glycoproteins Have Affinity for TPA, ["HlMannitols from ['HH]mannose-labeled and These Are Immunoreactive with Anti" Antibody-The glycopeptides Triton X-100-solubilized schistosome glycoproteinswere sub1.0 30 3,4-Di-O-methyl jected to TPA affinity chromatography as described under 18 0.6 3,6-Di-O-methyl 30 1.0 2,4-Di-O-methyl "Experimental Procedures." The TPA-unbound and -bound 22 0.7 3,4,6-Tri-O-methyl fractions were pooled and the amount of protein was determined and the protein fractions were subjected to SDS-PAGE ["HIGalactose from ['H]galactose-labeled (Fig. 7). The resultsshow that therewere many glycoproteins glycopeptides (50-100 kDa) bound by TPA that may have oligosaccharides 91 10.0 2,4,6-Tri-O-methyl 9 1.0 containing fucose linked to N-acetylglucosamine. Next, we 2,3,4,6-Tetra-O-methyl sought to find the reactivity of the TPA-boundglycoproteins ["HIN-Acetylglucosamine alditol acetate with anti-Le" monoclonal antibody. from [3H]glucosamine-labeled glycopeptides 6-0-methyl 3-0-methyl 3,6-Di-O-methyl

0.3

81 4 15

5.0 1.0

fGlcNAcpl$ Fuc la1.3 [Gal p1,4GlcNAc] p1.3Gal81.4

--

\ GlcNAcp1,2Mana1,6

\Man~1,4GlcNAcp1.4GlcNAcAsn " The percentageof radiolabeled monosaccharide recovered in each la1,6 GlcNAcpl,PYana1.3' derivative is shown. f Fuc 1 'The ratio of monosaccharide derivatives for each of the three + GkNAc (31.4 n { radiolabeled glycopeptide fractions was established by setting to 1.0 either the 2,4-di-O-methylmannose, the2,3,4,6-tetra-O-methylgalac- FIG. 6. Proposed partial structure for the antigenic glycotose, or the3,6-di-O-methyl alditol acetateof N-acetylglucosamine, peptides synthesized by adult S. mansoni.

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FIG. 7. SDS-PAGE of Triton X-I 00-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 causeda60% Recently, KO et af. (1990) 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-

Antigenic S. mansoni nizes a determinant present in S. mansoni and thatantibodies 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 terminalfucose-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. There are schistosome protein antigens that provide protection against experimental schistosomiasis and are being considered as vaccines for this disease (Colley and Colley, 1989; Xu et al., 1991). Earlier studies have also shown that antibodies reactive with surface glycoproteins on S. mansoni recognize carbohydrate epitopes, and some of these antibodies confer protection against parasitic infection (Dunne, 1990). Our present studieson the structureof the schistosome antigenic oligosaccharides suggest that theseoligosaccharides may be candidates for vaccines in future studies and also studies involving the mechanism of host-parasite interactions. Acknowledgments-We thank Dr. M. Pierce, Dr. K. Moreman, Dr. N. Stults, and P. Wilkins for critical reading of the manuscript and Sally Timberlake for maintenance of the hamster colony and preparation of schistosomes. REFERENCES Baenziger, J. U., and Fiete, D. (1979) J. Bid. Chem. 254,9795-9799 Bennett, J. L., and Seed, J. L. (1977) J. Parasitol. 6 3 , 250-258 Capron, A,, ,Dessaint, J. P., Capron, M., Ouma, J. H., and Butterworth, A. E. (1987) Sczence 2 3 8 , 1065-1072 Chan, A. L., Morris, H. R., Panico, M., Etienne, A. T., Rogers, M. E., Gaffney, P., Creighton-Kempsford, L., and Dell, A. (1991) Glycobiology 1 , 173-185 Cincanu, I., and Kerek, F. (1984) Carbohydr. Res. 1 3 1 , 209-213 Clark, G. F., Gorbea, C. M., Smith, D. F., Cummings, R. D., and Mattox, S. (1991) Carbohydr. Res. 2 1 3 , 155-168 Colley, D. G., and Colley, M. D. (1990) Parasitol. Today 5 , 350-354 Cummings, R. D., and Kornfeld, S. (1984) J. B-l. Chem. 2 5 9 , 6253-6260 Cummlngs, R. D., and Kornfeld, S. (1982) J. Bzol. Chem. 2 5 7 , 11235-11240 Cummings, R. D., Merkle, R. K., and Stults, N. L. (1989) Methods Cell Biol. 3 2 , 141-183 Dissous, C., Gryzch, J. M., and Capron, A. (1986) Nature 3 2 3 , 443-445 Donald, A. S. R., and Feeney, J. (1986) Biochem. J. 236,821-828 Dunne, D. (1990) Parasttol. Today 6 , 45-48

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