The Gne M712T mouse as a model for human glomerulopathy

Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation.     

Immunohistochemical Localization of Concanavalin A and Wheat Germ Lectin Receptors in the Normal Human Spermatozoa*

ABSTRACT: A semiquantitative immunohistochemical technique for the detection of N-acetyl α-D-neuraminic acid, N-acetyl β-D-glucosamine and its β-(1 ? 4)-linked internal chains, α-D-glucopyranosyl and α-D-mannopyranosyl and its α-(1 ? 2)-linked internal chains and sterically related, nonreducing, end-chain residues of oligosaccharide chains of glycoproteins or glycolipids on the surface of membranes was developed using Con A and wheat germ lectins. When this method was applied to the localization of carbohydrate receptors on the membrane of the normal human spermatozoa, it was found that the Con A and wheat germ lectin receptors were mainly located in the equatorial and post nuclear cap with few receptors located in the acrosome and neck. None of them were found in the intermediate segment plus tail. Con A receptors were α-D-mannopyranosyl end-chain residues and wheat germ lectin receptors were N-acetyl β-D-glucosamine (1 ? 4)-linked internal chains. These groups occur together in the oligomannosidic type of N-glycosidic-linked oligosaccharide chains of glycoproteins and so the use of both lectins on desialycated membranes or on those which contain nonclustered N-acetyl neuraminic acid residues may be of help to localize this type of glycoprotein oligosaccharide chains. Con A receptors were not removed after proteases digestion, suggesting the possibility that they are part of intrinsic spermatozoal antigens.     

Host cell-induced differences in O-glycosylation of herpes simplex virus gC-1. I. Structures of nonsialylated HPA- and PNA-binding carbohydrates

Lectins with narrow oligosaccharide specificities were established as probes to study the host cell influence on the biosynthesis of O-linked oligosaccharides of the herpes simplex virus type 1 (HSV-1)-specified glycoprotein C (gC-1). We found that only gC-1 and no other glycoprotein bound to the peanut lectin (PNA), with main specificity for Gal(beta 1-3)GalNAc. Previously, we have shown that only gC-1 binds to the Helix pomatia lectin (HPA), with main specificity for terminal GalNAc. The O-linked oligosaccharides binding to PNA and HPA were released by alkaline borohydride treatment and characterized. A structural determination of these oligosaccharides showed that the HPA-binding carbohydrates were monosaccharides (GalNAc), and that the PNA-binding oligosaccharides were disaccharides with the structure Gal-GalNAc. The PNA- and HPA-binding oligosaccharides were arranged as Pronase-resistant clusters on gC-1, consisting of about seven individual, adjacent oligosaccharides. In addition to these disaccharides, Pronase-resistant PNA-binding glycopeptides of gC-1 also contained neutral trisaccharides. Larger O-linked oligosaccharides, binding to the wheat germ lectin, were found in gC-1, but not in proximity to the PNA-binding ones. It was concluded that the lectins mentioned should be useful probes in screening HSV-infected cells of different lineages for differences in processing of O-linked oligosaccharides.     

Processing of virus-specific glycoproteins of varicella zoster virus

Monoclonal antibodies to varicella zoster virus (VZV) glycoproteins were used to study the processing of three glycoproteins with molecular weights of 83K-94K (gp 2), 64K (gp 3), and 55K (gp 5). Immunoprecipitation experiments performed with VZV-infected cells, pulse labeled with [3H]glucosamine in the presence of tunicamycin, suggest that O-linked oligosaccharide is present on the glycoprotein of gp 2. Use of the enzyme endo-beta-N-acetylglucosaminidase H revealed that the fully processed form of gp 3 had high-mannose type and that of gp 5 had only complex type of N-linked oligosaccharides. Experiments with monensin suggest that the precursor form (116K) of gp 3 is cleaved during the processing from Golgi apparatus to cell surface membrane. The extension of O-linked oligosaccharide chain and the complex type of N-linked oligosaccharide chains also occurs during this processing.     

Dietary lectins can stimulate pancreatic growth in the rat

Lectins are proteins or glycoproteins of nonimmune origin, which bind specifically to carbohydrate structures. They are widespread in the human diet, and many are resistant to digestion. High doses of lectins have been shown to stimulate intestinal and pancreatic growth. The aim of the present study was to investigate the long-term actions of low doses of lectins on the rat intestine and pancreas. A long-term carcinogenesis study was performed using low levels (40 micro g/rat/day) of peanut (PNA) or mushroom lectin (ABA) which bind to O-linked (mucin-type) oligosaccharides in the gut. While this was primarily designed as a colon carcinogenesis study, the pancreas was also investigated. No significant changes in colon carcinogenesis were seen, however, the colons were slightly heavier in the lectin treated groups. The weight of the pancreas was significantly greater (by 18 and 23%) in both lectin treated groups (P < 0.03/0.001). The weights of the acini and septal tissue were also increased by 39-46% in PNA and ABA fed animals, respectively (P < 0.002); there was no significant change in the endocrine pancreas. In conclusion, long-term feeding of low doses of lectin can influence pancreatic growth, and this trophic action may have potential adverse implications for the development of pancreatic cancer in humans.     

The two envelope membrane glycoproteins of Tomato spotted wilt virus show differences in lectin-binding properties and sensitivities to glycosidases

Tomato spotted wilt virus (TSWV, Genus: Tospovirus, Family: Bunyaviridae) is a major constraint to the production of several different crops of agronomic and horticultural importance worldwide. The amino acid sequence of the two envelope membrane glycoproteins, designated as G(N) (N-terminal) and G(C) (C-terminal), of TSWV contain several tripeptide sequences, Asn-Xaa-Ser/Thr, suggesting that the proteins are N-glycosylated. In this study, the lectin-binding properties of the viral glycoproteins and their sensitivities to glycosidases were examined to obtain information on the nature of potential oligosaccharide moieties present on G(N) and G(C). The viral proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by affinoblotting using a battery of biotinylated lectins with specificity to different oligosaccharide structures. G(C) showed strong binding with five mannose-binding lectins, four N-acetyllactosamine-binding lectins and one fucose-binding lectin. G(N) was resolved into two molecular masses and only the slow migrating form showed binding, albeit to a lesser extent than G(C), with three of the five mannose-binding lectins. The N-acetyllactosamine- and fucose-specific lectins did not bind to either molecular mass form of G(N). None of the galactose-, N-acetylgalactosamine-, or sialic acid-binding lectins tested showed binding specificity to G(C) or G(N). Treatment of the denatured virions with endoglycosidase H and peptide:N-glycosidase F (PNGase F) resulted in a significant decrease in the binding of G(C) to high mannose- and N-acetyllactosamine-specific lectins. However, no such differences in lectin binding were apparent with G(N). These results indicate the presence of N-linked oligosaccharides of high mannose- and complex-type on G(C) and possibly high mannose-type on G(N). Differences in the extent of binding of the two envelope glycoproteins to different lectins suggest that G(C) is likely to be more heavily N-glycosylated than G(N). No evidence was observed for the presence of O-linked oligosaccharides on G(N) or G(C).     

Lectin histochemistry on the dorsal epidermis of the Breton dog

Expression of sugar residues and the nature of oligosaccharide linkage during keratinocyte maturation in the epidermis of the Breton dog were studied with the use of lectin histochemistry. Thirteen lectins were used. Labelling was not observed with GSA I-B4, GSA II, UEA-I, and LTA. The cytoplasm of keratinocytes reacted with PNA, HPA, Con A, and WGA from the basal layer to the granular layer. PNA and Con A showed highest reactivity in the granular cell layer. The cell surface showed increased reactivity with PNA, HPA, and WGA with maturation of keratinocytes. KOH-neuraminidase treatment (KOH-Neu) increased PNA and RCA120 staining during keratinocyte differentiation thus indicating an increase in oligosaccharides terminating with sialic acid-Galbeta(1,3)GalNAc and sialic acid-Galbeta(1,4)GlcNAc, respectively. Labelling of the glycocalyx of basal and spinous keratinocytes with SNA and MAA revealed terminal Neu5acalpha(2,6)Gal/GalNAc and Neu5acalpha(2,3)Galbeta(1,4)GlcNAc. KOH-Neu-DBA showed oligosaccharides terminating with sialic acid-GalNAcalpha(1,3)GalNAc in the spinous and granular layers. A selective glycocalyx labelling of granular keratinocytes was observed with DBA and SBA. Reactions with MAA, PNA, DBA, RCA120, SBA, HPA, and WGA disappeared after the beta-elimination reaction. Our findings indicate that Breton dog epidermis contains more O-linked than N-linked oligosaccharides and confirm that different subpopulations of keratinocytes can be distinguished by lectin histochemistry.     

A structural analysis of the carbohydrate side chains on class I and class II histocompatibility antigens of the swine facilitated by heteroantisera specific for the denatured polypeptides

Rabbit heteroantisera specific for the denatured glycoprotein subunits of swine class I and class II major histocompatibility complex (MHC) antigens have been prepared and utilized to monitor changes in the mobilities of these polypeptides on SDS-polyacrylamide gel electrophoresis subsequent to various deglycosylation procedures. This information, in combination with lectin reactivity patterns for the glycoproteins bound to nitrocellulose, has made it possible to define specific structural features of the MHC antigen-associated carbohydrate side chains. Both the class I heavy (alpha) chain and the class II light (beta) chain bear a single, N-linked, complex-type oligosaccharide which reacts with lentil lectin (LcH), but not concanavalin A (Con A); a reactivity pattern suggesting the possibility of a special triantennary structure. In contrast, the class II heavy (alpha) chains appear to possess two carbohydrate units, one an N-linked, LcH-reactive, complex-type side chain, and the other, an N-linked, Con A-reactive, high-mannose-type of oligosaccharide. The data suggest considerable homology between the swine and human MHC antigens with respect to the structure of their carbohydrate side chains. The analysis also serves to illustrate how antibodies specific for the denatured polypeptide backbone of individual glycoproteins, along with lectin reactivity patterns, can be used to extract structural information about the attached carbohydrate moieties using minimal amounts of partially purified glycoproteins.     

Molecular identification of lectin binding sites differentiating related low and high metastatic murine lymphomas

We have previously shown that differences in cell surface carbohydrates can be detected in related murine tumor lines of varying metastatic capacity using plant lectins such as soybean agglutinin (SBA) orVicia villosa (VV) but not concanavalin A (ConA). Here we show that weakly metastatic Eb cells bind SBA via four glycoproteins and one GL2-like glycolipid. The major high-affinity SBA binding component of weakly metastatic ESb-MP cells was a glycoprotein of 210–220 kd. Highly metastatic ESb cells also expressed this protein but the oligosaccharide side chains were altered in such a way that SBA-binding was completely lost while ConA and peanut agglutinin (PNA) binding remained similar. Quantitative binding studies using iodinated lectins indicated that SBA binding of ESb cells could only be detected at lectin concentrations > 75g/ml. The role of altered carbohydrates in metastasis is discussed.     

Expression of cell surface lectins on activated human lymphoid cells

A cell surface lectin found on activated human lymphoid cells has been identified and characterized using membrane glycoprotein micelles as probes. These micelles, which are large, water-soluble aggregates, are composed of glycoproteins isolated from detergent-solubilized membranes of human B lymphoblastoid cell lines by Lens culinaris hemagglutinin affinity chromatography. The micelles have an average apparent molecular weight of 4 x 10(6) estimated by gel filtration and range in diameter from 25-100 nm. Micelles bind to B and T lymphoblastoid cell line cells and peripheral blood lymphocytes activated with concanavalin A or in a mixed lymphocyte response. Unactivated peripheral blood lymphocytes and red blood cells bind very low levels of the micelles. The binding is saturable, reversible, and temperature-dependent, with poor binding below 15 degrees C. Glycoproteins such as fetuin and porcine thyroglobulin, which contain complex oligosaccharide side chains, inhibit the binding, whereas glycoproteins containing only high mannose or simple serine-linked carbohydrate side chains do not. In addition, binding can be inhibited by complex asparagine-linked glycopeptides purified from pronase-digested fetuin, but not by the simple serine-linked glycopeptides. Membrane glycoprotein micelles are bound to the surface of the cells but are not internalized or degraded. The potential role of this cell surface lectin in lymphocyte function is discussed.     

Growth-inhibiting extracellular matrix proteins also inhibit electrical activity by reducing calcium and increasing potassium conductances

Inhibitionof neurite sprouting and electrical activity by extracellular matrix (ECM) glycoproteins was studied during neurite regeneration by using anterior pagoda (AP) neurons of the leech. Adult isolated neurons were plated in culture inside ganglion capsules, which among many ECM proteins, contain a group of inhibitory peanut lectin- (PNA) binding glycoproteins. These proteins inhibit neurite production and contribute to the formation of a bipolar outgrowth pattern by AP neurons. Addition of PNA lectin to the culture medium to block the inhibitory effects of ECM glycoproteins induced an increase of neurite sprouting, the loss of the bipolar pattern, and also an increase in the amplitude and duration of action potentials evoked by intracellular current injection. PNA lectin had independent effects on neurite sprouting and electrical activity, since there was no correlation between the total neurite length and the amplitude of the action potentials. Moreover, action potentials were increased by the presence of PNA lectin even in neurons that did not grow. The changes induced by PNA lectin on the active conductances underlying the action potentials were estimated by quantitative model simulations. We predict that the increases in the amplitude and duration of the action potential induced by PNA lectin were due to an increase in a calcium conductance and a reduction in the delayed rectifier potassium conductance. Our results suggest that inhibitory ECM glycoproteins may use independent signaling pathways to inhibit neurite sprouting and electrical activity. These proteins affect the action potential by changing the proportion of inward and outward active conductances.     

Changes in glycoconjugate expression during early chick embryo development: a lectin-binding study.

A selection of lectins was used to investigate developmentally regulated changes in the distribution of cell surface oligosaccharides during the gastrulation and neurulation stages of early chick embryo development. Lectins from three specificity classes were used: glucose/mannose specificity (concanavalin A [Con A], Lens culinaris agglutinin [LCA], Pisum sativum agglutinin [PSA]); N-acetylglucosamine specificity (Lycopersicon esculentum agglutinin [LEA], wheat germ agglutinin [WGA], succinylated WGA [sWGA]); N-acetylgalactosamine/galactose specificity (Dolichos biflorus agglutinin [DBA], soybean agglutinin [SBA], Sophora japonica agglutinin [SJA], Bandeiraea (Griffonia) simplicifolia lectin I [BSL I], peanut agglutinin [PNA], Artocarpus integrifolia lectin [Jacalin], Ricinus communis agglutinin-1 [RCA-1], Erythrina cristagalli lectin [ECL]). At gastrulation stages, patterns of lectin binding could be distinguished in the epiblast, mesoderm, and endoderm cell layers. The primitive streak failed to bind any of the lectins, but LEA and WGA bound to the epiblast in regions lateral to the streak, indicating the loss of some glucosamine residues medially in preparation for the ingression movements of gastrulation. Several lectins showed marked binding to the mesoderm cells after their passage through the primitive streak; these were LCA, PSA, WGA, sWGA, BSL, and most particularly PNA. Therefore, the epithelial-mesenchymal transformation from epiblast to mesoderm at the primitive streak is accompanied by cell surface oligosaccharide changes in the epiblast and mesoderm that involve all classes of lectins including the PNA-binding sequence Gal beta 1-3GalNAc. Ultrastructurally, PNA was shown to bind extracellularly to matrix fibrils. Jacalin, having the same sugar specificity as PNA, but binding to serine/threonine linked chains rather than asparagine linked chains showed no binding to the mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunology and Immunopathology of the Intestines: Crypt Cell Antigens (Cca): New Carbohydrate Markers for Human Colon Cancer Cells

A panel of monoclonal antibodies produced against surface membrane components of the human colon tumor cell line Caco-2 was found to define oncofetal crypt cell antigens (CCA) expressed by fetal intestinal cells, adult small intestinal crypt cells, and human and rat colonic adenocarcinomas. The epitopes recognized by these antibodies have been identified as O-linked oligosaccharide chains associated with specific glycoproteins in cultured intestinal cells and in colon tumors in vivo. Analysis of a large group of normal and diseased human intestinal specimens has demonstrated a marked heterogeneity in CCA expression which correlated with the degree of organization of the tumor cells in the tissue, suggesting that the CCA represent useful histological and clinical markers for colon cancer.     

Crypt cell antigens (CCA): new carbohydrate markers for human colon cancer cells

A panel of monoclonal antibodies produced against surface membrane components of the human colon tumor cell line Caco-2 was found to define oncofetal crypt cell antigens (CCA) expressed by fetal intestinal cells, adult small intestinal crypt cells, and human and rat colonic adenocarcinomas. The epitopes recognized by these antibodies have been identified as O-linked oligosaccharide chains associated with specific glycoproteins in cultured intestinal cells and in colon tumors in vivo. Analysis of a large group of normal and diseased human intestinal specimens has demonstrated a marked heterogeneity in CCA expression which correlated with the degree of organization of the tumor cells in the tissue, suggesting that the CCA represent useful histological and clinical markers for colon cancer.     

Peanut lectin-binding sites in polyps of the colon and rectum. Adenomas, hyperplastic polyps, and adenomas with in situ carcinoma

Peanut lectin (PNA) has a specificity for the disaccharide beta-D-Gal-(1 leads to 3)-D-GalNac which is the purported antigenic determinant for the T blood group antigen (TAg). This TAg is considered the immediate precursor of the MN blood group substance. In normal colonic epithelium, PNA binds to the supranuclear (stalk) portion of epithelial cells. This corresponds to the detection of beta-DGal-(1 leads to 3)-D-GalNac in nascent oligosaccharide chains in the Golgi cisternae prior to addition of terminal sialic acid. Colonic carcinomas bind PNA in the "region" of the glycocalyx or in the apical portion of the cell, which represents incomplete glycoprotein synthesis. Eighty-two percent of tubular adenomas, 80% of villous adenomas, and 91% of adenomas with in situ cancer expressed PNA in a supranuclear distribution, reminiscent of normal colonic epithelium. This stalk distribution was seen in goblet cells. Twenty-five percent of tubular adenomas, 43% of villous adenomas and 60% of adenomas with in situ cancer (adenoma portion) expressed PNA in an apical cytoplasmic and/or glycocalyx pattern among nonmucinous columnar cells. In 80% of the cases, the in situ cancer itself expressed PNA in an apical cytoplasmic and/or glycocalyx pattern. Fetal and most colon cancer cells fail to produce mucin goblets and make incomplete glycoproteins. The cytologic localization of TAg by PNA corresponds to the cells' ability to produce mucin goblets. Most adenomas consist of goblet cells, localize TAg to the stalk, and probably make complete MN glycoprotein as does normal colonic epithelium. However, in adenomas, nonmucinous columnar cells localize TAg to the apical cytoplasm and/or glycocalyx region and represent incomplete blood group glycoprotein synthesis.     

Poly[N-acetyl-lactosamine]-type sugar chains in CD45 antigens of abnormal T cells of lpr mice are different from those of normal T cells and B cells

The lymph node (LN) T cells from autoimmune MRL/MpJ-lpr/lpr (lpr) mice and control MRL/MpJ-+/+ (+/+) mice were compared as to their cell surface lectin binding sites and the glycoproteins responsible for the lectin binding. T cells from enlarged lymph nodes of lpr mice were found to express more binding sites for lectins which are reactive to poly[N-acetyl-lactosamine]-type sugar chains than normal +/+ mouse lymph node T cells. Furthermore, we found that high mol. wt (180,000-220,000) glycoproteins on lpr T cells were strongly stained with these poly [N-acetyl-lactosamine]-binding lectins on Western-blotting. These glycoproteins were found to belong to the CD45 family on immunoprecipitation and absorption with monoclonal anti-CD45 antibody. Thus, aberrant expression of high mol. wt CD45 (CD45R) antigens on lpr T cells may contribute greatly to the strong reaction of these cells with poly[N-acetyl-lactosamine]-binding lectins. We also found that poly[N-acetyl-lactosamine]-type sugar chains are more abundant on B cells than on lpr T cells, and that the molecular weights and the carbohydrate moieties of CD45R antigens on lpr T cells are different from those of CD45R antigens on +/+ spleen B cells.     

Use of porcine fibrinogen as a model glycoprotein to study the binding specificity of the three variants of K88 lectin.

Known glycoproteins were used to determine the differences occurring in the binding specificities of the three variants of the K88 lectin in an approach essentially based on lectin blotting. During the screening, it was demonstrated that each variant of the K88 lectin biotinylated via its amino groups (NbioK88) exhibited a characteristic binding to the three chains of porcine fibrinogen. NbioK88ab weakly bound to A alpha chains, NbioK88ac bound to B beta and gamma chains, and NbioK88ad bound only to the gamma chain. To validate this model, the oligosaccharide moieties of porcine fibrinogen were analyzed with glycosidases and by lectin blotting and sugar composition. Both the B beta chain and gamma chain carry biantennary N-glycans of the N-acetyllactosamine type that are not recognized by K88 lectins. A alpha chains are substituted by sialylated T antigen. O-glycans were also detected on B beta and gamma chains of porcine fibrinogen and contribute to the recognition of these chains by K88ac and K88ad fimbriae.     

Characterization of N- and O-linked oligosaccharides of glycoprotein 350 from Epstein-Barr virus

Glycoprotein 350 (gp350), the major Epstein-Barr Virus (EBV) envelope glycoprotein, has extensive N- and O-linked oligosaccharide chains. To characterize these oligosaccharide chains, [3H]glucosamine-labeled gp350 was isolated from an EBV transformed marmoset lymphoblastoid cell line (B95-8) induced to replicate EBV. Radiolabeled pronase-glycopeptides were fractionated by serial affinity chromatography and O-linked oligosaccharides released by mild alkaline borohydride treatment. Virtually all (99%) N-linked oligosaccharides were of complex type, with a predominance of tri-tetraantennary versus diantennary chains. A significant portion (28%, in term of radioactivity) of the tri-tetraantennary chains bound to leucoagglutinin-agarose, indicating an additional branch in beta(1-6)-linkage to the trimannosyl core. N-linked oligosaccharides with such a branching pattern have not been previously described in any herpesvirus glycoprotein, but have been associated with neoplastic transformation. Half of [3H]glucosamine incorporated into gp350 was recovered in O-linked oligosaccharides. The smallest chains have a core beta Gal-GalNAc disaccharide structure. Most O-linked chains have two to three N-acetylglucosamine and one N-acetylgalactosamine residues, besides the N-acetylgalactosamine residue located at the terminal reducing end, suggesting a di- or tri- N-acetyllactosamine structure. Consistent with such a structure, the size of these chains, after sialic acid removal, was that of an heptasaccharide or larger.     

Characterization of concanavalin A-binding glycoproteins from procyclic culture forms ofTrypanosoma congolense,T. simiae andT. brucei brucei

Concanavalin A-binding glycoproteins were obtained from procyclic culture forms (PCFs) ofTrypanosoma congolense, T. simiae, andT. b. brucei strains. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that glycoproteins of 38.5, 30.5, and 27 kDa were conserved between the different species and strains of the procyclic parasites. There were few similarities in the profiles of the high-molecular-weight glycoconjugates between the parasites. Monoclonal antibody analysis revealed that the 38.5- and 27-kDa glycoproteins were intracellular molecules and that they contained cross-reactive antigenic determinants. Surface biotinylation of PCFT. congolense K45/1 identified surface-accessible glycoproteins of 81.5, 59, and 38–42 kDa. By use of lectin blots and enzymatic deglycosylation studies, we demonstrated that the 81.5-, 59-, 38.5-, and 27-kDa glycoproteins contained N-linked oligosaccharide chains with both high-mannose-type and complex-type oligosaccharides, and the 81.5- and 59-kDa surface glycoproteins contained sialic acid residues. The glycoproteins identified in this study provide a starting point for further structure and function studies.     

Characterization of the high mannose asparagine-linked oligosaccharides synthesized by Schistosoma mansoni adult male worms

This report describes the structures of the high-mannose-type N-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult male worms. Adult male schistosomes were incubated in vitro in media containing either [2-3H]mannose, [6-3H]glucosamine or [6-3H]galactose to allow metabolic radiolabeling of the oligosaccharide moieties of newly synthesized glycoproteins. Glycopeptides were prepared from the radiolabeled glycoproteins by digestion with Pronase and fractionation by chromatography on concanavalin A-Sepharose. Eleven percent of [3H]mannose incorporated into the schistosome glycopeptides was recovered in high mannose-type Asn-linked oligosaccharides which bound to the immobilized lectin. Upon treatment of [3H]mannose-labeled glycopeptide with endo-beta-N-acetylglucosaminidase H, the high mannose-type chains were released and their structures were determined by high performance liquid chromatography, methylation analysis, acetolysis and exoglycosidase digestion. The major species of high mannose-type chains synthesized by S. mansoni adult males have the composition Man7GlcNAc2, Man8GlcNac2 and Man9GlcNA2. Structural analyses indicate that these oligosaccharides are similar to high mannose-type chains synthesized by mammalian cells.