Identification of a human endothelial cell antigen with monoclonal antibody 44G4 produced against a pre-B leukemic cell line

Staining of a variety of human tissue sections (lymph node, tonsil, spleen, thymus, kidney, lung, and liver) by the indirect immunoperoxidase method indicated that mAb 44G4, produced against a human pre-B leukemic cell line, was strongly reactive with vascular endothelium. All other cell types observed in these tissues were unreactive. Immunofluorescence staining of endothelial cells isolated from umbilical cord vein and grown in culture confirmed that mAb 44G4 recognized a surface membrane component of vascular endothelium. Granulocytes, monocytes, B and T lymphocytes, and T lymphocytes cultured in the presence of PHA for 72 h did not express the 44G4 Ag. mAb 44G4 reacted weakly with leukemic cells from 28 of 41 patients with non-T cell acute lymphocytic leukemia and 4 of 7 patients with acute myelocytic leukemia, whereas 8 of 10 cases of T cell acute lymphocytic leukemia were negative. Moderate reactivity with leukemic cell lines of pre-B and myelomonocytic origin was also observed. The level of 44G4 Ag on umbilical endothelial cells was three to five times that of leukemic cell lines and 25 times the average levels observed on leukemic cells isolated from patients. Immunoprecipitation of lysates prepared from surface-iodinated endothelial cells and the immunizing pre-B leukemic cell line revealed that the 44G4 Ag from both cell types was composed of two subunits of apparent m.w. 95,000 linked by disulfide bond(s). Comparison of the cellular localization and subunit structure of 44G4 to that of known Ag suggests that it represents a previously undescribed marker of endothelial cells.     

Differential localization within human kidney of five membrane proteins expressed on acute lymphoblastic leukemia cells

The reactivity of a panel of monoclonal antibodies (MAb) produced against non-T, non-B acute lymphoblastic leukemia cells was investigated by immunoperoxidase staining of sections of normal human kidney. The antigens of kidney reactive with the MAb were isolated by immunoaffinity chromatography and were purified further by immunoprecipitation. Two MAb, 44D7 and 44H9, reacted with determinants found exclusively on the basolateral membranes of proximal convoluted tubules. The 44D7 antigen isolated from kidney was biochemically similar to that isolated from leukemic cells. It was resolved as a multimeric complex with an apparent m.w. of 120,000 when analyzed by SDS-PAGE under nonreducing conditions. The 44H9 antigen has not yet been purified from kidney. MAb 50B4 reacted with components of the interstitium and with the mesangium of glomeruli. It immunoprecipitated a polypeptide chain of apparent m.w. 85,000, similar to that of the 50B4 antigen isolated from leukemic cells. MAb 44G4 also reacted with the mesangium of glomeruli and with the interstitium of the kidney. However, the endothelium of glomerular capillaries and of interstitial blood vessels has also reacted with MAb 44G4. The kidney antigen recognized by MAb 44G4 was characterized as a major polypeptide band, 95,000 m.w. (reduced) and 125,000 m.w. (nonreduced), a subunit structure analogous to the 44G4 antigen isolated from leukemic cells. MAb 44E3 reacted with all cellular elements of glomeruli, tubules, blood vessels, and interstitium. Two polypeptide chains of apparent m.w. 94,000 and 90,000 were immunoprecipitated from kidney by MAb 44E3, while a single polypeptide chain of 94,000 m.w. was precipitated from leukemic cells. Our results describe five new antigens with distinctive cellular distributions within kidney.     

Identification and in vitro reconstitution of lysosomal neuraminidase from human placenta

Lysosomal neuraminidase from human placenta has been obtained in its active form by association of an inactive neuraminidase polypeptide with beta-galactosidase and the protective protein. Using a specific antiserum, we have now identified a 66-kDa protein as the inactive neuraminidase polypeptide. It is specifically recognized on immunoblots only in its nonreduced state, and it coprecipitates with neuraminidase activity. The 66-kDa polypeptide is substantially glycosylated (38-kDa protein core with 7-14 N-linked oligosaccharide chains), a feature characteristic of lysosomal integral membrane proteins. Specific removal of the 66-kDa neuraminidase polypeptide from glycoprotein preparations prevents the generation of neuraminidase activity. Removal of beta-galactosidase or destruction of the protective protein also hinders the formation of active neuraminidase. Reconstitution of neuraminidase activity is observed after mixing glycoprotein preparations, depleted in different components of the beta-galactosidase-neuraminidase-protective protein complex, indicating that all three components of the complex are required for neuraminidase activity. Association of the neuraminidase polypeptide and the protective protein generates unstable neuraminidase activity, whereas association with beta-galactosidase is required for stability.     

Anti-CD9 monoclonal antibodies induce homotypic adhesion of pre-B cell lines by a novel mechanism

Anti-CD9 mAb are known agonists of platelet aggregation, but have not been implicated in cell-cell adhesion. We show here in an experimental system that the anti-CD9 mAb 50H.19, ALB6, and BA-2 can induce rapid, and irreversible, homotypic aggregation of the CD9-positive pre-B lymphoblastoid cell lines NALM-6 and HOON, but not of the CD9-negative B cell line Raji. The specificity of the response is indicated by the failure to effect aggregation with mAb directed to CD24, or to HLA class I Ag. The initiation of strong homotypic aggregates of lymphoid cells is a property ascribed to lymphocyte function-associated Ag-1 (LFA-1), a member of the beta 2 subfamily of leukocyte integrins. We show that CD9-induced aggregation is an active process which proceeds at 37 degrees C, but not at 4 degrees C, requires the expenditure of metabolic energy, and a functioning cytoskeleton, and is not inhibited by Arg-Gly-Asp-Ser peptide. These are properties described for LFA-1-mediated aggregation. However, because beta 2-integrins are not expressed on NALM-6 or HOON cells, they are not the mediators of CD9-induced aggregation. In contrast to LFA-1-mediated adhesion which is Mg2+ dependent, CD9-induced adhesion has an absolute requirement for Ca2+, but not Mg2+, indicating that a Ca2(+)-dependent event is sufficient for adhesion. However, Mg2+ enhances adhesion even at optimal concentrations of Ca2+, implicating an additional Mg2(+)-dependent event which requires Ca2+ to be effective. These findings suggest that CD9 Ag regulates a novel mechanism for promoting tight cell-cell adhesion which requires both Ca2+ and Mg2+ for optimal expression.     

Role of a vitelline layer-associated 350 kDa glycoprotein in controlling species-specific gamete interaction in the sea urchin

The process of sperm-egg binding is one of the barriers to cross-fertilization between related sea urchin species. A 350 kDa glycoprotein in the egg vitelline layer of Strongylocentrotus purpuratus has been shown to be a sperm-binding protein (SBP). Sulfated O-linked oligosaccharide chains on the 350 kDa glycoprotein, as well as domains of the polypeptide chain, serve as ligands for this binding process. The hypothesis that species-specific sperm-egg binding is attributed to the interaction between the sperm and the 350 kDa glycoprotein was tested using S. purpuratus and S. franciscanus. It was found that both species had a 350 kDa glycoprotein on the egg surface that cross-reacted immunologically using antibodies prepared against a recombinant form of the SBP. Because earlier studies had implicated the carbohydrate chains of the 350 kDa glycoprotein of S purpuratus in sperm binding, differences in carbohydrate chains on the 350 kDa glycoproteins of these species were examined. It was found that among the lectins tested only wheat germ agglutinin and Sambucus nigra agglutinin showed a significant difference in reactivity to the 350 kDa glycoproteins between species. Finally, using a bead-binding assay, it was shown that the isolated 350 kDa glycoproteins exhibited species-specific sperm-binding activity.     

Direct interaction between the catalytic subunit of the calmodulin-sensitive adenylate cyclase from bovine brain with 125I-labeled wheat germ agglutinin and 125I-labeled calmodulin

A calmodulin-sensitive adenylate cyclase has been purified to apparent homogeneity from bovine cerebral cortex using calmodulin-Sepharose followed by forskolin-Sepharose and wheat germ agglutinin-Sepharose. The final product appeared as one major polypeptide of approximately 135,000 daltons on sodium dodecyl sulfate-polyacrylamide gels. This polypeptide was a major component of the protein purified through calmodulin-Sepharose. The catalytic subunit was stimulated 3-4-fold by calmodulin (CaM) with a turnover number greater than 1000 min-1 and was directly inhibited by adenosine. The catalytic subunit of the enzyme interacted directly with 125I-CaM on a sodium dodecyl sulfate-polyacrylamide gel overlay system, and this interaction was Ca2+ concentration dependent. In addition, the catalytic subunit was shown to directly bind 125I-labeled wheat germ agglutinin using a sodium dodecyl sulfate-polyacrylamide gel overlay technique, and N-acetylglucosamine inhibited binding of the lectin to the catalytic subunit. Calmodulin did not inhibit binding of wheat germ agglutinin to the catalytic subunit, and the binding of calmodulin was unaffected by wheat germ agglutinin. These data illustrate that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is a glycoprotein which interacts directly with calmodulin and that adenosine can inhibit the enzyme without intervening receptors or G coupling proteins. It is concluded that the catalytic subunit of adenylate cyclase is a transmembrane protein with a domain accessible from the outer surface of the cell.     

Biochemical analysis of a novel H-2 class I-like glycoprotein expressed in five AKR-(Gross virus) derived spontaneous T cell leukemias

The H-2 class I Ag profiles of five spontaneous AKR (H-2K) Gross virus leukemic cell lines were analyzed. A novel H-2 class I, "alloantigen"-like glycoprotein was immunoprecipitated and isolated from all the tumor cell lines using an H-2Dd-specific mAb 35-5-8. The novel Ag was also recognized in vitro by anti-H-2Dd-specific CTL. In addition, DNA from all the thymomas, but not the DNA from normal adult AKR thymic cells showed a transcribed gene detectable with an H-2Dd-specific oligonucleotide probe. The molecular profile of the novel antigen was further studied by two-dimensional gel electrophoresis and analyzed by a computer based image analyzer system and reverse-phase HPLC tryptic peptide mapping. Its molecular pattern was different from the syngeneic H-2Kk, H-2Dk, and the allogeneic H-2Dd gene products. The two-dimensional gel pattern of the novel H-2 class I molecule had a different overall structure reflected in isoelectric point, number, and distribution of polypeptide spots. The tryptic peptide map analysis showed six peaks exclusively identified with the novel Ag. The calculated degree of homology with the corresponding H-2Dd, H-Dk, and H-Kk peptides was 41, 56, and 51%, respectively. In addition, an unusual cell surface distribution of the novel Ag was observed in most of the leukemic lines. The removal of sialic acid residues by neuraminidase treatment facilitated the detection of the allodeterminants by anti-H-2Dd-specific mAb and CTL. Furthermore, we showed that in one AKR tumor line, 424, there is a close association of the novel Ag with the syngeneic class I molecules. Prior preclearance of the syngeneic class I molecules revealed the presence of the H-2Dd-like allospecificity. The genetic and molecular relationship between the expression of this novel class I-like glycoprotein and the recently sequenced Q5 gene is under current investigation.     

Mapping of the low pH-sensitive conformational epitope of rabies virus glycoprotein recognized by a monoclonal antibody #1-30-44

Monoclonal antibody (mAb) #1-30-44 recognized an acid-sensitive conformational epitope of rabies virus glycoprotein (G). The antigenicity of G protein exposed on the cell surface was lost when the infected cells were exposed to pH 5.8. By comparing the deduced amino acid sequence of G protein between the HEP-Flury strain and the epitope-negative CVS strain as well as the mAb-resistant escape mutants, two distant sites that contained Lys-202 and Asn-336 were shown to be involved in the epitope formation. Lys-202 is located in the so-called neurotoxin-like sequence, while Asn-336 is included in antigenic site III and is very near the amino acid at position 333, which is known to affect greatly the neuropathogenicity of rabies virus when changed. Consistent with this finding, antigenicity of a neurovirulent revertant of the HEP-Flury strain, in which Gln-333 of G protein was replaced by Arg, was also affected as shown by its greatly decreased reactivity with mAb #1-30-44 compared to that of the original avirulent HEP virus. Based on these results, we hypothesize that the neurotoxin-like domain and some amino acids in antigenic site III come into contact with each other to form a conformational epitope for mAb #1-30-44, and such a configuration would be lost when exposed to acidic conditions to perform a certain low pH-dependent function of G protein.     

Selective staining of human platelet glycoproteins using nitrocellulose transfer of electrophoresed proteins and peroxidase-conjugated lectins

Platelet proteins (0.5-5 micrograms) were electrophoresed in a one-dimensional or an unreduced-reduced, two-dimensional sodium dodecyl sulfate gel system. The separated proteins were then transferred electrophoretically to nitrocellulose and reacted with peroxidase-conjugated lectins. Visualization of specific glycoproteins which bound the lectins was made by the chromogenic reaction catalyzed by peroxidase utilizing 3,3'-diaminobenzidine as the substrate. Wheat germ agglutinin specifically reacted with and allowed the visualization of glycoprotein Ib. Peanut agglutinin also specifically stained glycoprotein Ib after treatment of the nitrocellulose transferred proteins with neuraminidase. Ricinus communis agglutinin I stained thrombospondin, a 260 kDa protein, and factor VIII. Concanavalin A stained mainly glycoproteins IIb, III, IV, and V. Glycoproteins Ia, Ic, IIa, and other minor glycoproteins could be separated by unreduced-reduced, two-dimensional gel electrophoresis and were stained weakly with wheat germ agglutinin conjugates. These techniques were found to be reproducible as well as easily applied to the analysis and identification of platelet glycoproteins, particularly when dealing with a limited amount of platelets.     

Schistosoma mansoni: radiometric assay of lectin binding specificities of the major egg glycoprotein and its carbohydrate-rich fragment

The binding by lectins of the Schistosoma mansoni major egg glycoprotein and of a carbohydrate-rich fragment which is serologically cross-reactive with it was studied. The major egg glycoprotein was purified from a crude soluble egg antigen by a succession of affinity chromatography procedures on concanavalin A-sepharose and by ion-exchange chromatography. The carbohydrate-rich fragment was isolated by ultrafiltration of the crude glycoprotein fraction initially obtained from the crude soluble egg antigens. The major egg glycoprotein and the carbohydrate-rich fragment contain 77 and 92.5% carbohydrate, respectively. When radioiodinated and run on SDS-polyacrylamide gel electrophoresis, each of them exhibited a single peak with respective Rf values of 0.33 and 1.0, and their respective molecular weights were 70K and 10-13K. The binding of the radioiodinated major egg glycoprotein and the carbohydrate-rich fragment by peanut agglutinin, Ricinus communis agglutinin-60, wheat germ agglutinin, and lotus agglutinin was studied by double diffusion in agar, and by a radiometric solid-phase assay in which the lectins were used to coat microtiter plates. The latter assay was employed to determine the specificity of the binding by inhibition with the specific sugars. Both the major egg glycoprotein and the carbohydrate-rich fragment bound specifically to concanavalin A columns as indicated by their isolation procedure. They also bound specifically to peanut agglutinin, R. communis agglutinin 60, and lotus agglutinin, while binding by wheat germ agglutinin appeared not to be specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibody identifies a 200-kDa subunit of the dihydropyridine-sensitive calcium channel

A monoclonal antibody, mAb 1A, that immunoprecipitates the [3H]PN200-110-binding complex from rabbit skeletal muscle has been used to study the subunit structure of the dihydropyridine-sensitive, voltage-activated calcium channel. Digitonin-solubilized [3H]PN200-110-binding component, purified by wheat germ agglutinin chromatography, sediments as a 21 S complex. The sedimentation coefficient of the complex is increased to about 24 S after incubation with mAb 1A IgG. Four polypeptides with apparent molecular weights under nonreducing conditions of 220,000, 200,000, 61,000, and 33,000 co-sediment with the 21 S complex. mAb 1A recognizes the Mr 200,000 polypeptide, as shown by Western blotting analysis. [3H] PN200-110 complex purified by wheat germ agglutinin chromatography followed by immunoaffinity chromatography on an mAb 1A column is comprised primarily of the same four polypeptides. When analyzed by sodium dodecyl sulfate gel electrophoresis under reducing conditions, the Mr 220,000 protein migrates as a polypeptide of Mr 143,000; the mobility of the Mr 200,000 protein recognized by mAb 1A is unaffected by reduction. Thus, the Mr 200,000 polypeptide appears to be a previously undescribed component of the dihydropyridine-binding complex and, in association with the other polypeptides, may comprise the voltage-sensitive calcium channel.     

Comparison of brush border membrane glycoproteins and glycoenzymes in the proximal and distal rat small intestine

Brush border membranes isolated from the proximal and distal portions of the rat small intestine were examined to see whether qualitative differences exist in their glycoprotein constituents. After SDS-polyacrylamide gel electrophoresis distinct differences were observed, indicating that the protein and glycoprotein profiles of the distal intestine are less complex. A competitive radioassay of lectin receptors revealed that there are significantly more wheat germ agglutinin and succinylated wheat germ agglutinin receptors present on brush border membranes from proximal intestine as compared to distal intestine. However, binding of Ricinus communis agglutinin I to brush border membranes of distal intestine was 2-times higher than that of proximal intestine. These segmental differences were also reflected in the binding patterns of individual brush border membrane hydrolases to wheat germ agglutinin and R. communis agglutinin I. Carbohydrate analysis demonstrated that the overall sugar content of brush border membranes is higher in distal intestine, with more galactose and sialic acid residues. No difference was found in the content of N-acetylglucosamine between the two segments. When brush border membranes from both segments were used as acceptors for galactosyltransferase, those from proximal intestine were better acceptors. Neuraminidase treatment significantly enhanced galactose oxidase/sodium borotritide labeling of brush border membranes from distal intestine and altered the electrophoretic mobility of dipeptidyl aminopeptidase IV and aminopeptidase N. No significant changes in labeling or enzyme electrophoretic mobility were noted in brush border membranes from proximal intestine after neuraminidase treatment. These studies indicate that the glycoproteins from brush border membranes of proximal and distal intestine are qualitatively different and that the glycoproteins from distal intestine may have more completed oligosaccharide side chains.     

Several epitopes of p85 glycoprotein (CDw44) are dependent on intact disulphide bonds. Isolation of cDNA clones requires a polyclonal antibody raised against the reduced protein

Monoclonal antibodies 50B4 and 50E6 recognize two distinct epitopes of human p85 glycoprotein (CDw44). Both epitopes are destroyed by reduction of the purified gycoprotein as demonstrated by inhibition of cellular radioimmunoassay and Western blot analysis. Endoglycosidase F treated p85 glycoprotein, with an apparent molecular weight of 73,000 is still reactive with both monoclonal antibodies. Thus both epitopes are conformational determinats of the polypeptide chain. A rabbit antibody produced against purified native p85 glycoprotein also reacted only with the non-reduced form of p85. Repeated immunizations with SDS-dissociated and reduced p85 yielded a polyclonal antibody reactive by Western blot analysis with reduced and non-reduced forms of p85 glycoprotein. When a HOON leukemia cell line cDNA expression library was screened with this polyclonal antibody, two cDNA clones were isolated which reacted specifically with the antiserum and not with the control non-immune serum. Preliminary characterization of these clones indicates that they are p85-related.     

Carbohydrate specificity of IgM autoantibodies to CD45 in Systemic Lupus Erythematosus

Patients with SLE develop IgM autoantibodies to different isoforms of CD45, the major surface membrane protein tyrosine phosphatase on lymphocytes and other nucleated hemopoietic cells. Because such autoantibodies could have a potential role in the development of immune dysfunction in this disorder, we performed a series of experiments to characterize their antigenic specificity further. Blots of recombinantE. coli fusion proteins encoded by exons 3–7 of the p220 and p180 isoforms were uniformly non-reactive with SLE IgM, suggesting that anti-CD45 autoantibodies in SLE are directed against conformational and/or carbohydrate epitopes, rather than linear polypeptide epitopes. This issue was examined further using chemically and enzymatically modified CD45 purified from T cells by lectin affinity chromatography as substrates. Treatment of CD45 with 25 mM sodium-m-periodate, sufficient to abrogate binding to various lectins, abolished the reactivity with SLE anti-CD45 autoantibodies. On the other hand, digestion of CD45 with neuraminidase enhanced the binding of anti-CD45 autoantibodies from some of the SLE sera. This result probably reflects decreased steric hindrance or charge repulsion because the binding of mouse monoclonal antibodies directed against linear polypeptide epitopes of CD45 was similarly enhanced. Digestion of CD45 with N-glycosidase F had no effect on autoantibody staining. Taken together, these data suggest that IgM anti-CD45 autoantibodies in SLE recognize non-sialylated carbohydrate determinants in the highly O-glycosylated polymorphic domains of CD45.Abbreviations SLE systemic lupus erythematosus - SBA soybean agglutinin - RCAI Ricinus communis agglutinin - SNL Sambucus nigra lectin - MBP maltose binding protein - mAb monoclonal antibody - WGA wheat germ agglutinin     

Association of the CD59 and CD55 cell surface glycoproteins with other membrane molecules.

mAb against human glycosyl-phosphatidylinositol-linked leucocyte surface Ag CD59 and CD55 immunoprecipitated from detergent lysates of HPB ALL cell line in addition to the respective Ag a common 80-kDa glycoprotein component and (glyco)lipids. The 80-kDa glycoprotein is different from otherwise similar CD44 Ag. The CD59 immunoprecipitate contained also a small amount of the CD55 glycoprotein and the CD55 immunoprecipitate minute amount of the CD59 Ag. These results are interpreted in terms of existence of noncovalent complexes resistant to dissociation by mild detergents and consisting of the 80-kDa glycoprotein, CD59 and CD55 glycoproteins, relatively tightly bound (glyco)lipids and possibly other so far unidentified components. These complexes contain probably also other glycosyl-phosphatidylinositol-linked Ag, as an anti-CD48 mAb immunoprecipitated also an apparently very similar complex. The complexes immunoprecipitated by mAb against the CD55, CD59, and CD48 Ag also contain a protein kinase activity. This type of complexes could not be demonstrated in several other cell types such as RBC, PBMC, and HeLa cells. However, a qualitatively very similar set of components was immunoprecipitated from the murine thymoma EL-4 cell line by an anti-Thy-1 mAb.     

Exosome-driven antigen transfer for MHC class II presentation facilitated by the receptor binding activity of influenza hemagglutinin

The mechanisms underlying MHC class I-restricted cross-presentation, the transfer of Ag from an infected cell to a professional APC, have been studied in great detail. Much less is known about the equivalent process for MHC class II-restricted presentation. After infection or transfection of class II-negative donor cells, we observed minimal transfer of a proteasome-dependent "class I-like" epitope within the influenza neuraminidase glycoprotein but potent transfer of a classical, H-2M-dependent epitope within the hemagglutinin (HA) glycoprotein. Additional experiments determined transfer to be exosome-mediated and substantially enhanced by the receptor binding activity of incorporated HA. Furthermore, a carrier effect was observed in that incorporated HA improved exosome-mediated transfer of a second membrane protein. This route of Ag presentation should be relevant to other enveloped viruses, may skew CD4(+) responses toward exosome-incorporated glycoproteins, and points toward novel vaccine strategies.     

Murine monoclonal antibodies to the myelin-associated glycoprotein (MAG) recognize Leu-7-reactive molecules on human mononuclear cells

It is known that the antibody to human myelin-associated glycoprotein (MAG) reacts with a subset of human mononuclear cells (MNC) mediating a natural killer (NK) activity. The properties of the target molecule of the anti-MAG antibody, however, have not yet been elucidated. Three (GC-J4, MC-P2, and MC-P4) of five murine monoclonal antibodies (mAb) to MAG bound to human MNC. Moreover, MC-P2 and MC-P4 inhibited the binding of 125I-labeled anti-Leu-7 to MNC in a dose-dependent fashion. Conversely, anti-Leu-7 inhibited the binding of MC-P2 and MC-P4 to MNC, but did not inhibit the binding of GC-J4. Therefore, it is possible that MC-P2 and MC-P4 bind directly to or close to the Leu-7 epitope, and that GC-J4 binds to the epitope which is distinct from the Leu-7 epitope. The electrophoretic patterns of immunoprecipitates with GC-J4, MC-P2 and anti-Leu-7 from detergent lysates of surface-labeled human MNC were very similar. The target molecules of anti-Leu-7 and anti-MAG mAb have apparent m.w. of 205, 170, 150, 135, 110, 85, 65, and 55 kDa. All of the molecules precipitated by these mAb are monomeric or noncovalently associated proteins, because the electrophoretic mobilities of the proteins remained unchanged whether the samples were reduced or not. MC-P4 may have a higher affinity for the 65 kDa molecule than the other mAb, and precipitates the 58 kDa molecule as well. Therefore, the fine antigenic specificity of MC-P4 is slightly different from those of anti-Leu-7 or MC-P2. The implication of these results is that mAb, whose specificity is directed to the carbohydrate part of human MAG, reacts with the Leu-7 reactive molecules on human MNC, and that at least two epitopes detected by anti-MAG mAb coexist on the surface molecules with various apparent m.w.     

Expression of platelet glycoprotein Ib alpha in HEL cells

We have previously shown that platelet glycoprotein Ib is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio, A., Rosa, J. P., Testa, U., Kieffer, N., Nurden, A. T., Del Canizo, M. C., Breton-Gorius, J., and Vainchenker, W. (1984) EMBO J. 3, 453-459). In this report, we have selected a stable HEL subclone with increased expression of glycoprotein (GP) Ib as assessed by 6 different monoclonal antibodies in order to investigate the biochemical characteristics of this glycoprotein. A single polypeptide chain of apparent Mr = 60,000 was precipitated under reducing and nonreducing conditions by a specific polyclonal anti-platelet glycocalicin antibody and two anti-GPIb alpha monoclonal antibodies (AN51 and AP1), both from surface-labeled and metabolically labeled HEL cells. We were unable to demonstrate the presence of a polypeptide corresponding to the beta subunit of GPIb or GPIX which is closely associated with GPIb. Competitive immunoprecipitation performed in the presence of an excess amount of cold platelet glycocalicin completely displaced the Mr = 60,000 polypeptide. Synthesis of N-linked oligosaccharide chains on this Mr = 60,000 polypeptide was inhibited by the antibiotic tunicamycin, and a shift of the apparent Mr from 60,000 to 48,000 was observed. O-Linked oligosaccharide chains identical to platelet GPIb hexasaccharides were deficient or incomplete since no peanut agglutinin binding to the Mr = 60,000 polypeptide was observed after neuraminidase treatment of HEL cells. Thus, our results provide evidence that the Mr = 60,000 polypeptide expressed on the surface membrane of HEL cells is closely related to platelet GPIb and corresponds to an incompletely or abnormally O-glycosylated GPIb alpha subunit.     

Neutralization-sensitive epitopes are exposed on the surface of infectious Cryptosporidium parvum sporozoites

Cryptosporidiosis is a diarrheal disease of humans, calves, and other mammals caused by the coccidian parasite Cryptosporidium parvum. Immune bovine serum and two surface-reactive antisporozoite mAb with neutralizing activity were used to identify sporozoite surface Ag by radioimmunoprecipitation/SDS-PAGE and immunoblotting. When isolated sporozoites were incubated with mAb 18.44, 12 to 25 times the ID50 for mice was completely neutralized. This mAb binds diffusely to the sporozoite surface and recognizes a sporozoite surface Ag that eluted in the void volume of a Bio Gel A column with an exclusion limit of 500,000 daltons. The Ag recognized by mAb 18.44 was not radiolabeled with 125I or [35S] methionine, migrated with the dye front in SDS-PAGE, and was insensitive to proteinase K digestion, suggesting a non-protein composition. mAb 17.41 significantly neutralized 25 times the ID50 of sporozoites for mice. This mAb binds multifocally to the sporozoite surface and recognizes [35S] methionine-labeled sporozoite surface Ag of 28,000 m.w., 55,000 m.w., and 98,000 m.w. Immune bovine serum immunoprecipitated [35S] methionine- or 125I-labeled sporozoite Ag ranging from less than 14,300 m.w. to greater than 200,000 m.w., including surface Ag of 28,000 m.w. and 55,000 m.w. The results indicate that two different molecules capable of inducing neutralizing antibody are exposed on the surface of C. parvum sporozoites.     

Specific changes in the oligosaccharide moieties of VSV grown in different lectin-resistnat CHO cells.

The carbohydrate moieties of the G glycoprotein of vesicular stomatitis virus (VSV) grown in three distinct lectin-resistant (LecR) Chinese hamster ovary (CHO) cell lines have been compared by fine structural analysis of radiolabeled glycopeptides. The mutant WgaRIII, selected for resistance to wheat germ agglutinin (WGA), produces VSV containing G glycoprotein specifically lacking in sialic acid. The mutant PhaRI, selected for resistance to phytohemagglutinin (PHA) and previously shown to lack a particular glycoprotein N-acetyl-glucosaminyl-transferase activity, produces VSV containing G glycoprotein specifically lacking terminal N-acetylglucosamine-galactose-sialic acid sequences and possessing an increased number of mannose residues in the "core" region of its carbohydrate moieties. The mutant PhaRIConARII, a "double" mutant selected from PhaRI cells for resistance to concanavalin A (ConA), produces VSV containing G glycoprotein with a further alteration in the mannose residues of the "core" oligosaccharide region. We discuss the relevance of these findings to the mechanisms of glycoprotein biosynthesis in mammalian cells and to the biochemical bases of lectin resistance in CHO cells.