Study of membrane glycoproteins of human erythrocytes using lectins

A method to study the glycoprotein composition of cell membranes, in particular of human red blood cells, has been developed. It includes the separation of membrane components by the SDS-polyacrylamide gradient slab gel electrophoresis, electroblotting of the phoretograms onto the nitrocellulose sheets and detection of glycoprotein fractions with FITC and peroxidase labeled lectins. PNA detected asialoglycoproteins with O-linked oligosaccharide chains, corresponding to all the PAS-positive bands of the phoretogram. SBA interacted more selectively and revealed only certain PAS-positive bands. Glycoproteins with N-linked carbohydrate chains were PAS-negative and can be identified only by the interaction with WGA, LCL, RCA. Group-specific agglutinins have shown that the ABO antigenic determinants are located in N-linked carbohydrate chains of membrane glycoproteins.     

Electrophoretic analysis of four high molecular weight sialoglycoproteins produced by metastatic human colon carcinoma cells

We have found that polyacrylamide gel electrophoresis in 3% gels in the presence of sodium dodecyl sulfate is suitable for the separation of cellular glycoproteins having molecular weights ranging from 200,000 to 1,000,000. The gels secured on a rigid support (Gelbond) allow blotting techniques with lectins and antibodies for the detection of glycoproteins. Using these methods we have separated lysates of HT-29 human colon carcinoma cells and detected at least four distinct high molecular weight sialoglycoproteins having molecular weights of 900,000, 740,000, 560,000, and 450,000. The expression of the 900,000 component, as revealed by wheat germ agglutinin binding, was much higher in a subline of HT-29 cells established from liver metastases in a nude mouse than it was in the parental cells. The relative intensity of wheat germ agglutinin binding to these four sialoglycoprotein components differs depending upon their growth phase in vitro. These glycoproteins were also detectable by the binding of peanut agglutinin, provided the glycoproteins were previously treated in the gels with mild acid to remove the sialic acid from their carbohydrate chains, suggesting that mucin-type carbohydrate chains are present on these glycoproteins. The same set of glycoproteins can be detected by metabolic labeling of the cells with [3H]glucosamine in tissue culture. Very similar glycoprotein profiles are revealed by metabolic labeling of fresh colon carcinoma tissues with [3H]glucosamine in vitro.     

Interactions of concanavalin A with glycoproteins: formation of homogeneous glycoprotein-lectin cross-linked complexes in mixed precipitation systems.

We have previously demonstrated that the interactions between branched chain oligosaccharides and glycopeptides isolated from glycoproteins and glycolipids with specific lectins lead to the formation of homopolymeric carbohydrate-protein cross-linked complexes, even in the presence of mixtures of the carbohydrates or lectins [cf. Bhattacharyya, L., Fant, J., Lonn, H., & Brewer, C. F. (1990) Biochemistry 29, 7523-7530]. Recently, we have shown that highly ordered cross-linked lattices are formed between the tetrameric glycoprotein soybean agglutinin (SBA), which possesses a Man9 oligomannose chain per monomer, and the Glc/Man-specific plant lectin concanavalin A (Con A) [Khan, M. I., Mandal, D. K., & Brewer, C. F. (1991) Carbohydr. Res. 213, 69-77]. Using radiolabeling and quantitative precipitation techniques, we show in the present study that Con A binds and forms unique cross-linked complexes with four different glycoproteins having different numbers and types of carbohydrate chains as well as different quaternary structures. The glycoproteins include quail ovalbumin, Lotus tetragonolobus isolectin A (LTL-A), Erythrina cristagalli lectin (ECL), and Erythrina corallodendron lectin (EcorL). The results show that a preparation of quail ovalbumin containing either one Man7 or Man8 oligomannose chain per molecule forms a 1:2 cross-linked complex with tetrameric Con A, thereby demonstrating bivalency of the single carbohydrate chain(s) on the glycoprotein. Tetrameric LTL-A and dimeric ECL, which possess two xylose-containing carbohydrate chains per monomer, both form 1:2 and 1:1 cross-linked complexes (per monomer) of glycoprotein to lectin, depending on their relative ratios in solution. However, dimeric EcorL, which has the same carbohydrate structure and number of chains as ECL, forms only a 1:2 cross-linked complex with tetrameric Con A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Capillary affinity electrophoresis for the screening of post-translational modification of proteins with carbohydrates

Glycosylation is one of the most important post-translational events for proteins, affecting their functions in health and disease, and plays significant roles in various information traffics for intracellular and intercellular biological events (Hancock, W. S. J. Proteome Res. 2002, 1, 297). We have attempted to obtain the information on the numbers and amounts of carbohydrate chains. Interaction between carbohydrate chains and proteins that recognize them is a target to understand the biological roles of glycosylation. To date, there have been a few strategies for simultaneous analysis of the interactions between complex mixtures of carbohydrates and proteins. Here, we report an approach to categorize carbohydrate chains using a few glycoprotein samples as models for the studies on the analysis of post-translational modification of proteins with carbohydrates. A combination of some specific lectins was used as carbohydrate-binding proteins. The method is based on high-resolution separation of fluorescent-labeled carbohydrates by capillary electrophoresis with laser-induced fluorescent detection in the presence of carbohydrate-binding proteins at different concentrations. The present technique affords (1) simultaneous determination of carbohydrate chains, (2) binding specificity of the constituent carbohydrate chains to specific proteins, and (3) kinetic data such as the association constant of each carbohydrate. We found that the lectins employed in the present study could discriminate subtle difference in linkages and resolved the carbohydrate mixtures. The results will be useful, for example, to understand the biological events expressed with carbohydrate changes on the cell surface.     

Binding and cross-linking properties of galectins

The galectins are a family of animal lectins that possess similar carbohydrate binding specificities and conserved consensus sequences. The biological properties of mammalian galectins include the regulation of inflammation, cell adhesion, cell proliferation and cell death. Evidence suggests that the biological activities of the galectins are related to their multivalent binding properties since most galectins possess two carbohydrate recognition domains and are therefore bivalent. For example, galectin-1, which is dimeric, binds and cross-links specific glycoprotein counter-receptors on the surface of human T-cells leading to apoptosis [J. Immunol. 163 (1999) 3801]. Different galectin-1 counter-receptors associated with specific phosphatase or kinase activities formed separate clusters on the surface of the cells as a result of the lectin binding to the carbohydrate chains of the respective glycoproteins. Importantly, monovalent galectin-1 is inactive in this system. This indicates that the separation and organization of signaling molecules that result from galectin-1 binding is involved in the apoptotic signal. The separation of specific glycoprotein receptors induced by galectin-1 binding was modeled on the basis of molecular and structural studies of the binding of lectins to multivalent carbohydrates resulting in the formation of specific two- and three-dimensional cross-linked lattices [Biochemistry 36 (1997) 15073]. In this article, the binding and cross-linking properties of galectin-1 and other lectins are reviewed as a model for the biological signal transduction properties of the galectin family of animal lectins.     

Structural characterization of glycoprotein carbohydrate chains by using diagoxigenin-labeled lectins on blots

The carbohydrate structures of blotted glycoproteins can be analyzed by probing them with lectins. Here we describe a method where lectins conjugated with digoxigenin are used in combination with an anti-digoxigenin antibody AP conjugate as a very sensitive detection system for this type of analysis. The specificity of the lectins used, and the sensitivity of the detection system, provide valuable conclusions on the glycan structures. Only small amounts of glycoproteins are required for the analysis. The binding specificity of a set of lectins is demonstrated with various glycoproteins of defined carbohydrate structure. The application of these labeled lectins in combination with specific glycosidases for the characterization of the carbohydrate chains of recombinant tissue plasminogen activator and erythropoietin is presented.     

Microassay analyses of protein glycosylation

To accurately characterize the carbohydrate moieties of oligosaccharide chains in glycosylated proteins, it is necessary to distinguish exactly which types of oligosaccharides are present at which site. We describe lectin overlay assays, which take advantage of the ability of lectins to distinguish between different types of glycoproteins via recognition of terminal sugars, thus allowing the chain type and peripheral antigenic components to be determined. Three microassays involving lectins are reported in this paper: non-proteasetreated intact glycoproteins; glycopeptides released by prior digestion of the glycoprotein and then separated by HPLC; and release of sugars from glycoproteins by hydrazinolysis and then coupling them to a multivalent support.     

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.     

Characterization and comparison of the major glycoprotein from three strains of Rous sarcoma virus.

The major glycoprotein g2 was purified from three strains of Rous sarcoma virus, representing subgroups A, B, and C. Carbohydrate analysis showed that glucosamine, mannose, galactose, fucose and sialic acid constitute 40% of the weight of the subgroup A glycoprotein and 15% of the subgroup B and C glycoproteins. The molar ratios of sugars were very similar and amino acid compositions were similar but not identical for the three glycoproteins. Glycosidase digestions of subgroup A and C glycoproteins suggested the presence of two types of oligosaccharide chains, the complex serum type, with terminal sequences sialic acidα-Galβ-GlcNAcβ- and the high mannose type with terminal α-linked mannosyl residues. After removal of 70% of the carbohydrate by glycosidases, subgroup A glycoprotein contained only glucosamine and mannose, in the molar ratio 2.0:1.3. The sequence of sugar release was consistent with oligosaccharide structures such as those which have been described for other glycoproteins. The plant lectins concanavalin A and wheat germ agglutinin were shown to interact strongly with the g2 glycoprotein from viruses of all three subgroups.     

Glycosylation characteristics of pigmentation-associated antigen (GP75): an intracellular glycoprotein of human melanocytes and malignant melanomas

Pigmentation-associated antigen (PAA) or gp75 is a glycoprotein localized to the melanosomes of human melanomas and melanocytes to which a mouse monoclonal antibody (AbTA99) has been produced (T. M. Thomson et al. (1985) J. Invest. Dermatol. 85, 169). Treatment of 3H-labeled immunoprecipitated melanoma PAA with alkaline-borohydride, hydrazinolysis, or N-glycanase released three families of carbohydrate chains (I, II, and III). Peak I consists of a major component (Ia) of sialylated triantennary N-linked chains which are partially substituted with fucose on terminal positions as well as on the chitobiose core and a minor component (Ib) which is a sialylated biantennary N-linked species. Peak II was not well characterized but may be a monoantennary complex chain species. Peak III consists of typical N-linked high mannose units with six to seven mannose residues. Melanocyte PAA carbohydrate chains have the same general features as melanoma PAA except that the biantennary complex chain predominates; this difference resembles that observed between the cell surface glycopeptides of transformed animal cells and their nontransformed counterparts. The glycosylation characteristics of this melanosomal glycoprotein are compared with those of glycoproteins from endoplasmic reticulum, Golgi, and lysosomes, and with tyrosinase. It is suggested that the glycosylation pattern is a reflection of the biosynthetic origin and cellular destination of a particular organelle and its constituents.     

LAMP-1 in CHO cells is a primary carrier of poly-N-acetyllactosamine chains and is bound preferentially by a mammalian S-type lectin

Recent studies indicate that some mammalian S-type lectins bind preferentially to oligosaccharides containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1]n or poly-N-acetyllactosamine (PL) sequence. We report here our investigation on the distribution of these sequences in glycoproteins in Chinese hamster ovary (CHO) cells and the interaction of glycoproteins containing PL chains with an immobilized S-type lectin (L14) from calf heart tissue. Our results demonstrate that PL chains are carried by a few high molecular weight glycoproteins which are bound by tomato-lectin Sepharose and one of these was precipitated by antibody to LAMP-1 (a lysosomal-associated membrane glycoprotein). More importantly, these high molecular weight glycoproteins, including LAMP-1, were bound with high affinity by L14. These results indicate that mammalian S-type lectins are highly specific in their interactions with glycoproteins and that LAMPs carry important recognition sequences for these lectins.     

Developmental regulation of the carbohydrate composition of glycoproteins associated with central nervous system myelin

Abstract: Glycoproteins from central nervous system myelin were evaluated for developmental alterations in their carbohydrate composition by autoradiographic analysis of radioiodinated lectin binding after separation by high-resolution sodium dodecyl sulfate-pore gradient slab gel electrophoresis (SDS-PGE). Sixteen lectin-binding components were assessed in highly purified myelin preparations from 15-day, 18-day, and adult rat brains, using the lectins Triticum vulgaris (wheat germ agglutinin) and Ulex europeus (gorse agglutinin I). Developmental changes in lectin binding for individual glycoproteins were evaluated semiquantitatively by comparing densitometric scans of the auto radiographs. Both increases and decreases in lectin binding for individual components were observed as a consequence of development, as well as the appearance and disappearance of lectin binding to three low-molecular-weight components. No changes in electrophoretic mobility and hence glycoprotein molecular weight were observed in any components when using these lectins. These developmental changes in lectin binding suggest that increases in glycoprotein (receptor) density occur, as well as an elaboration of oligosaccharide branching for individual glycoproteins. In addition, the appearance of a new glycoprotein in the adult myelin membrane could imply a new functional role not present in the immature membrane. These observations suggest that dynamic alterations of myelin-associated glycoproteins occur during development. Such developmental regulation of membrane glycoproteins increases the significance of their potential role in myelination and myelin maintenance.     

Schistosoma mansoni egg glycoproteins and C-type lectins of host immune cells: molecular partners that shape immune responses

Schistosome eggs and egg-derived molecules are potent immunomodulatory agents. There is increasing evidence that the interplay between egg glycoproteins and host C-type lectins plays an important role in shaping immune responses during schistosomiasis. As most experiments in this field so far have been performed using complex protein/glycoprotein mixtures or synthetic model glycoconjugates, it is still largely unclear which individual moieties of schistosome eggs are immunologically active. In this review we will discuss molecular aspects of Schistosoma mansoni egg glycoproteins, their interactions with C-type lectins, and the relevance to schistosome egg immunobiology.     

Glycoprotein hyposialylation gives rise to a nephrotic-like syndrome that is prevented by sialic acid administration in GNE V572L point-mutant mice

Mutations in the key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosamine kinase, result in distal myopathy with rimmed vacuoles (DMRV)/hereditary inclusion body myopathy (HIBM) in humans. Sialic acid is an acidic monosaccharide that modifies non-reducing terminal carbohydrate chains on glycoproteins and glycolipids, and it plays an important role in cellular adhesions and interactions. In this study, we generated mice with a V572L point mutation in the GNE kinase domain. Unexpectedly, these mutant mice had no apparent myopathies or motor dysfunctions. However, they had a short lifespan and exhibited renal impairment with massive albuminuria. Histological analysis showed enlarged glomeruli with mesangial matrix deposition, leading to glomerulosclerosis and abnormal podocyte foot process morphologies in the kidneys. Glycan analysis using several lectins revealed glomerular epithelial cell hyposialylation, particularly the hyposialylation of podocalyxin, which is one of important molecules for the glomerular filtration barrier. Administering Neu5Ac to the mutant mice from embryonic stages significantly suppressed the albuminuria and renal pathology, and partially recovered the glomerular glycoprotein sialylation. These findings suggest that the nephrotic-like syndrome observed in these mutant mice resulted from impaired glomerular filtration due to the hyposialylation of podocyte glycoproteins, including podocalyxin. Furthermore, it was possible to prevent the nephrotic-like disease in these mice by beginning Neu5Ac treatment during gestation.     

The influence of lectins on the aggregation of neutrophils and erythrocytes in healthy humans

A study was made of the influence of some plant lectins on the aggregation of neutrophils and erythrocytes in healthy humans, and the state of carbohydrate determinants of glycoprotein receptors of these cells was characterized. These carbohydrate determinants, containing D-mannose, N-acetyl-D-glucosamin, and bD-galactose, provide neutrophils aggregation in healthy persons. The surface receptors of erythrocytes have remains of bD-galactose, several N-acetyl-D-glucosamin, N-acetyl-neyranimic acid, N-acetyl-D-galactosamin, L-fucose. Thus, in neutrophils and erythrocytes of healthy persons there is a definite composition of carbohydrate determinants of glycoproteins. Changes in these carbohydrate determinants are able to increase cells aggregation and, consequently, to disturb reological property of the blood, and to impair processes of microcirculation and thrombose stimulation.     

Structure of a major yolk glycoprotein and its processing pathway by limited proteolysis are conserved in echinoids

To study the fate of the yolk glycoproteins found in eggs and embryos of the sea urchin, Strongylocentrotus purpuratus, a polyclonal antibody to a 90-kDa polymannose glycoprotein found in the embryo was prepared. Immunoblot analysis of total proteins over the course of development showed that this antibody recognized a family of glycoproteins. Concomitant with the disappearance of the major 160-kDa yolk glycoprotein of the egg during embryogenesis, glycoproteins with a lower molecular mass appeared. These glycoproteins (115, 108, 90, 83, and 68 kDa) were purified from S. purpuratus and analyzed by limited proteolysis and peptide mapping. This analysis revealed that these glycoproteins were cleavage products derived from the major yolk glycoprotein. The antibody to the 90-kDa glycoprotein in S. purpuratus embryos was used to identify a homologous set of yolk glycoproteins with similar molecular masses in the embryos of three other species in the class Echinoidea: Arbacia punctulata, Lytechinus pictus, and Dendraster excentricus. However, eggs from other echinoderm classes and from Xenopus laevis, Drosophila melanogaster, and the chicken did not contain any cross-reactive molecules. Cross-reactivity within the class Echinoidea was not due to a common carbohydrate epitope, because the antibody recognized the glycoproteins even after the N-linked carbohydrate side chains were enzymatically removed. The major yolk glycoprotein (160-170 kDa) from each of the three sea urchin species was purified and analyzed. Comparison of the physical and chemical properties of these glycoproteins revealed striking similarities in pI and in amino acid and monosaccharide composition. The results of peptide mapping also supported the conclusion that the 160- to 170-kDa glycoproteins from the four echinoids are structurally homologous glycoproteins containing N-linked polymannose chains. Immunolocalization by electron microscopy in S. purpuratus showed that the yolk glycoproteins remained within the yolk platelet throughout development, and that externalization of the 160-kDa glycoprotein or its cleavage products was not detectable.     

Comparative serum glycoproteomics using lectin selected sialic acid glycoproteins with mass spectrometric analysis: application to pancreatic cancer serum

A strategy is developed in this study for identifying sialylated glycoprotein markers in human cancer serum. This method consists of three steps: lectin affinity selection, a liquid separation and characterization of the glycoprotein markers using mass spectrometry. In this work, we use three different lectins (Wheat Germ Agglutinin, (WGA) Elderberry lectin,(SNA), Maackia amurensis lectin, (MAL)) to extract sialylated glycoproteins from normal and cancer serum. Twelve highly abundant proteins are depleted from the serum using an IgY-12 antibody column. The use of the different lectin columns allows one to monitor the distribution of alpha(2,3) and alpha(2,6) linkage type sialylation in cancer serum vs that in normal samples. Extracted glycoproteins are fractionated using NPS-RP-HPLC followed by SDS-PAGE. Target glycoproteins are characterized further using mass spectrometry to elucidate the carbohydrate structure and glycosylation site. We applied this approach to the analysis of sialylated glycoproteins in pancreatic cancer serum. Approximately 130 sialylated glycoproteins are identified using microLC-MS/MS. Sialylated plasma protease C1 inhibitor is identified to be down-regulated in cancer serum. Changes in glycosylation sites in cancer serum are also observed by glycopeptide mapping using microLC-ESI-TOF-MS where the N83 glycosylation of alpha1-antitrypsin is down regulated. In addition, the glycan structures of the altered proteins are assigned using MALDI-QIT-MS. This strategy offers the ability to quantitatively analyze changes in glycoprotein abundance and detect the extent of glycosylation alteration as well as the carbohydrate structure that correlate with cancer.     

Kinetic Analysis of Glycoprotein–Lectin Interactions by Label-Free Internal Reflection Ellipsometry

Introduction

Glycoproteomics is undergoing rapid development, largely as a result of advances in technologies for isolating glycoproteins and analyzing glycan structures. However, given the number and diversity of glycans, there is need for new technologies that can more rapidly provide differential carbohydrate–protein structural information on a large scale. We describe a new microarray platform based on a label-free imaging ellipsometry technique, which permits simultaneous detection of multiple glycoprotein–lectin interactions without the need for reporter labels, while still providing high throughput kinetic information at much lower cost. Our results demonstrate the utility of LFIRE? (Label-Free Internal Reflection Ellipsometry) for the rapid kinetic screening of carbohydrate–lectin recognition. The technology was also used to evaluate the benefits of the lectin immobilization format using multi-lectin affinity chromatography (M-LAC) to capture glycoproteins (with enhanced binding strength or avidity) from biological samples. Using a printed panel of lectins, singly or in combination, we examined the binding characteristics of standard glycoproteins.

Results and Discussion

Using kinetic measurements, it was observed that the binding strength of lectins to carbohydrates is enhanced using a multi-lectin strategy, suggesting that improved selectivity and specificity can lead to increased functional avidity. The data presented confirm that this label-free technology can be used to effectively screen single or combinations of lectins. Furthermore, the combination of LFIRE? and M-LAC may permit more rapid and sensitive identification of novel biomarkers based on carbohydrate changes in glycoproteins, and lead to a better understanding of the connections of glycan function in cellular mechanisms of health and disease.     

Mass spectrometric profiling of O-linked glycans released directly from glycoproteins in gels using in-gel reductive beta-elimination

Glycosylation is a widespread PTM of proteins; the carbohydrate moieties provide various functional, immunological and structural aspects of both eukaryotic and prokaryotic glycoproteins. Traditional strategies used to analyse glycoprotein O-glycans involve glycoprotein isolation, followed by glycan release using solution-phase base-catalysed beta-elimination. However, in a proteomics context, mixtures of proteins and glycoproteins are routinely separated using SDS-PAGE. We have therefore developed a method to enable the profiling of O-linked glycans directly from glycoproteins on gels. This is achieved using in-gel reductive beta-elimination followed by mass spectrometric analysis of the released glycans. Here we describe our demonstration of the feasibility of this approach, our development and optimisation of the procedure using bovine submaxillary gland glycoproteins as a standard, and then show its usefulness by applying the developed procedure to the analysis of the O-glycans from a glycoprotein band from a Coomassie-stained SDS-PAGE separation of a mixture of Mycobacterium avium capsular proteins and glycoproteins. The procedure has been shown to be applicable to both CBB- and silver-stained gels. The method offers a quick and easy way to identify the O-glycans from gel-separated glycoproteins within gel-based proteomics workflows.     

Myelin-Associated Glycoprotein Shares an Antigenic Determinant with a Glycoprotein of Human Melanoma Cells

A sulfated 100K-dalton glycoprotein has been shown to be released into the culture medium of melanoma cells. Monoclonal antibodies 10C5 and 11B5, which were raised to human melanoma cells, as well as HNK-1 bind to this glycoprotein. It is shown here that mouse anti-myelin-associated glycoprotein (MAG) carbohydrate antibodies raised to human MAG and a human IgM paraprotein associated with neuropathy also bind to the same 100K molecule. However, anti-MAG antibodies recognizing peptide epitopes do not appear to react with this glycoprotein of melanoma cells, a result suggesting that its similarity to MAG is restricted to shared carbohydrate moieties. The anti-melanoma antibodies (10C5 and 11B5) resemble HNK-1 in binding to MAG and to some 19-28K-dalton glycoproteins and sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, the anti-melanoma antibodies cross-react with neural cell adhesion molecule (N-CAM), an observation emphasizing the shared antigenicity between MAG and other adhesion molecules. The results demonstrate that the anti-melanoma antibodies fall into a class of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, anti-human MAG antibodies, and L2 antibodies) that are characterized by reactivity against related carbohydrate determinants shared by human MAG, N-CAM, and several protein and lipid glycoconjugates of the PNS.