Identification of a novel ganglioside on erythrocytes with blood group Cad specificity

The blood group Cad antigen is a carbohydrate structure well characterized on the sialoglycoproteins of the red cell membrane from some rare individuals (Blanchard, D., Cartron, J. P., Fournet, B., Montreuil, J., Van Halbeck, H., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 7691-7695). However, protease treatment of whole cells did not destroy their antigenic activity which indicated that glycolipid might also be involved in the antigenic reaction. A crude ganglioside fraction was prepared from Cad cells and found to inhibit the hemagglutination reaction, whereas neutral glycolipids were inactive. Further analysis of the ganglioside extract from Cad erythrocytes by thin layer chromatography revealed an unusual profile characterized by a lower content of sialosylparagloboside and the presence of a novel ganglioside of slower mobility. Immunochemical studies demonstrate that this ganglioside binds Helix pomatia lectin and inhibits human anti-Sda antibody. In addition, a ganglioside with identical chromatographic mobility can be obtained by the enzymatic transfer of GalNAc from UDP-GalNAc to sialosylparagloboside using a microsomal preparation from human kidney. These results together with cell surface labeling experiments suggest that the major ganglioside of Cad erythrocytes might be derived from sialosylparagloboside by substitution with an additional N-acetylgalactosamine residue.     

An Arg-Gly-Asp-directed receptor on the surface of human melanoma cells exists in an divalent cation-dependent functional complex with the disialoganglioside GD2

The disialogangliosides GD2 and GD3 play a major role in the ability of human melanoma cells to attach to Arg-Gly-Asp-containing substrates such as fibronectin and vitronectin, since pretreatment of these cells with monoclonal antibodies to the oligosaccharide of GD2 and GD3 can inhibit their attachment and spreading on such adhesive proteins. This report demonstrates that human melanoma cells (M21) synthesize and express a glycoprotein receptor that shares antigenic epitopes with the vitronectin receptor on human fibroblasts and is capable of specifically recognizing the Gly-Arg-Gly-Asp-Ser-Pro sequence. In the presence of calcium, GD2, the major ganglioside of M21 cells, colocalized with this receptor on the surface of human melanoma cells and their focal adhesion plaques as demonstrated by double-label transmission immunoelectron microscopy and indirect immunofluorescence. Biochemical evidence is presented indicating that the vitronectin receptor on M21 human melanoma cells contains associated calcium and GD2. This ganglioside copurified with the glycoprotein receptor for vitronectin on affinity columns containing either an Arg-Gly-Asp-containing peptide, concanavalin A, or lentil lectin. This major Arg-Gly-Asp-directed receptor on M21 cells could be metabolically labeled with 45Ca2+. Chelation of this ion with EDTA caused the dissociation of GD2 from the receptor and rendered the remaining glycoprotein incapable of binding to an Arg-Gly-Asp-containing peptide. Reconstitution experiments demonstrated a requirement for calcium, and not magnesium, for receptor binding to Arg-Gly-Asp and indicated that addition of ganglioside can enhance this interaction.     

Rapid fractionation of bovine transferrin using immobilized gangliosides.

Bovine transferrin (BTF) was fractionated from bovine whey using ganglioside affinity chromatography. After loading the immobilized matrix with a 2% whey solution, the matrix was washed with sodium acetate buffer at pH 4 containing 1 M NaCl before elution of BTF with sodium phosphate buffers at pH 7. Concanavalin-A affinity and ion exchange chromatography were used for further purification. The ganglioside column showed a 74.2% BTF recovery from whey and BTF was enriched to 61% purity with ion exchange chromatography. Bovine transferrin was identified by SDS-PAGE and western analysis. The Concanavalin-A affinity and ion exchange chromatography steps enriched BTF in the samples and removed other whey proteins from ganglioside purified fractions. These results indicate that immobilized ganglioside can be used to fractionate BTF from bovine whey. Our novel ganglioside affinity chromatography is rapid and efficient for the fractionation of BTF from whey.     

Paramphistomum cervi: antigenic profile of adults as recognized by infected cattle sera

An antigenic profile of adult Paramphistomum cervi was revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cattle naturally infected with P. cervi, Fasciola gigantica and strongylids. SDS-PAGE of whole worm extracts exhibited 26 distinct protein bands. Immunoblotting analysis of these proteins showed five major antigenic bands which were recognized by serum of individual cattle naturally infected with P. cervi. These antigenic proteins had molecular weights ranging from 23 to 116kDa. One antigenic protein with a molecular weight of 52kDa exhibited a consistent reaction with sera from all infected cattle. It's diagnostic sensitivity, specificity and accuracy using this test were 100%, 98% and 98.9%, respectively. The positive and negative predictive values were 97.6% and 100%, respectively. This finding suggests that the 52kDa protein may be a diagnostic antigen for paramphistomosis.     

A photoreactive derivative of radiolabeled GM1 ganglioside: preparation and use to establish the involvement of specific proteins in GM1 uptake by human fibroblasts in culture

A new procedure was used to synthesize a derivative of ganglioside GM1 containing a photoreactive nitrophenyl azide group at the end of the fatty acyl moiety, using deAc-deAcyl-GM1 obtained by deacetylation of the sialic acid and deacylation of the ceramide portion of GM1. This deAc-deAcyl-GM1 was first acylated at the long chain base amino group with 12-aminododecanoic acid, which has the amino group protected by a fluorenyl residue, and tritium labeled at the sialic acid amino group with [3H]acetic anhydride of very high specific radioactivity. The fluorenyl group removed by ammonia treatment was substituted by a nitrophenyl azide group. Cultured human fibroblasts were exposed to mixtures of radioactive photolabeled GM1 and cold natural GM1 (1:10 by mol) for different times and then illuminated and the radioactive protein patterns studied by SDS-PAGE. After 2h of exposure, the photolabeled GM1 was stably associated to the cells and underwent almost no metabolic processing, behaving exactly as the underivatized natural GM1. Under these conditions very few proteins became radioactive: one, of about 30 kDa, interacted with the ganglioside molecules inserted into the outer membrane layer; three, in the region of 46 kDa, interacted with the portion of associated ganglioside able to be released by trypsin treatment. Thus, it is evident that the ganglioside binding to fibroblasts and insertion into the outer layer of the plasma membrane involve few individual proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and partial characterization of AgF-1:a rat lymphocyte membrane antigen.

A genetically defined, serologically identified antigen of the rat lymphocyte membrane (AgF-1) has been isolated. Viable spleen and lymph node cells, prepared by Ficoll-Hypaque density centrifugation from Fischer rats, were radioiodinated with soluble lactoperoxidase. Extracts obtained with Nonidet P-40 were shown to contain numerous radiolabeled proteins including cell-surface globulin. AgF-1 was isolated from these extracts by precipitation with a highly specific alloantibody in conjunction with xenospecific anti-globulin antibody and polyethylene glycol (PEG). The use of PEG greatly increased the efficiency of the double antibody technique. The putative antigenic peak was eluted from sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and specific antigenic activity was recovered. Removal of the SDS from these eluates was achieved by equilibration with urea and passage over an anion exchange resin. Renaturation, as evidenced by specific inhibition of complement-mediated cytotoxicity, occurred upon the removal of urea by dialysis. The m.w. of the purified antigen was estimated to be 35 to 40,000 daltons by SDS-PAGE and was unaffected by reduction with 2-mercaptoethanol. Amino acid composition was roughly similar to those reported for the major histocompatibility antigens of the rat and other species.     

Obtaining immunologically active bovine viral diarrhea virus (BVDV) proteins for enzyme linked immunosorbent assay (ELISA)

We report here the preparation of BVDV antigen (Ag) to obtain adequate quantities of pure active viral proteins from Madin Darby Bovine kidney (MDBK) cell culture infected with BVDV cytophatic Oregon C24V strain. SDS-PAGE, Immunodot, ECL-Western blot and ELISA showed the best specificity and activity of BVDV Ag obtained from infected cells only by mild experimental conditions. BVDV Ag preparations showed the peculiar BVDV sensitivity to proteases, nonionic surfactants and tend for protein degradation and irreversible loss of conformational antigenic determinants with subsequent inability to detect antibodies against viral Ag in hyperimmune sera. The widest panel of immunologically active and specific polypeptides, that elicit Ab production in hyperimmune sera, was obtained by ultrasonication and subsequent purification on 20%-50% sucrose cushion. We observed that BVDV tend to remain in the infected cells, to associate with components of serum and cellular origin--this is of crucial importance towards the specificity of ELISA. BVDV antigenic properties are determined by the labile conformational antigenic epitopes.     

Alteration of phosphotyrosine-containing proteins during differentiation of chicken erythroleukemia cells (HD3)

After treatment of HD3 cells with erythroid-inducing agents (hemin and butyric acid) at 42°C, the profile of phosphotyrosine-containing proteins was altered. Upon induction the overall level of phosphotyrosine-containing proteins increased. To examine the role of protein phosphorylation in HD3 cells differentiation, the cells were treated with specific inhibitors. In the presence of okadaic acid, cell proliferation was arrested and accompanied by a marked increase in haemoglobin synthesis, a differentiation marker of erythroid cells. Okadaic acid caused decrease of the phosphotyrosine-containing proteins, presumably to maintain a balance between phosphorylation/dephosphorylation processes in the cells. Addition of 3-isobutyl-l-methyl-xanthine, an activator of phosphatases, caused a decrease or disappearance of almost all phosphotyrosine-containing proteins and, at the same time, prevented the erythroid differentiation of HD3 cells. Sodium orthovanadate, a specific inhibitor of phosphotyrosine phosphatase, increased the level of phosphotyrosine proteins and induced differentiation of HD3 cells. These results indicate that phosphorylation of cellular proteins is coupled with a reaction(s) which is responsible for triggering the differentiation of HD3 cells. The phosphorylation/dephosphorylation processes are associated with an early event(s) during the differentiation of HD3 cells and may not be connected to tyrosine residues. Copyright © 1998 John Wiley & Sons, Ltd.     

Neospora caninum antigens defined by antigen-dependent bovine CD4+ T cells

Neosporosis is an important cause of pregnancy loss in cattle worldwide. The objective of the present study was to identify Neospora caninum antigens as vaccine candidates using antigen-specific, short-term CD4+ T cells established from N. caninum-immunized and -challenged cows. Whole N. caninum tachyzoite lysate was separated into 6 fractions by DEAE anion-exchange chromatography using high-pressure liquid chromatography (HPLC). The CD4+ T-cell proliferation assay results indicated that antigenic activity was associated with proteins from HPLC fractions 4-6, with fraction 5 exhibiting the highest antigenic activity. Also, SDS-PAGE analysis revealed a 16-kDa protein in fractions 4-6 that was recognized by anti-N. caninum antibodies. This 16-kDa protein was absent in other fractions, and it may be a target of a T-cell response in cattle. Further identification of immunogenic proteins of N. caninum may facilitate development of subunit vaccines against neosporosis.     

Molecular mimicry of phage displayed peptides mimicking GD3 ganglioside.

We have analyzed sequence homologies between nonimmunogenic phage displayed peptides mimicking GD3 ganglioside and human/mouse self-proteins. The GD3 ganglioside phagotopes showed homology to proteins involved in carbohydrate metabolism/transport. Besides this expected homology, molecular mimicry of critical regulatory proteins was found. These data contribute to our understanding of the structural relatedness of antigenic determinants defined by specific anti-GD3 monoclonal antibodies and, in addition, suggest that molecular mimicry might explain the nonimmunogenicity of these peptides otherwise characterized by specificity to the mAb counterpart. We conclude that construction of peptides harboring motifs absent or scarcely represented in endogenous self-proteins might be a useful approach in melanoma immunotherapy.     

Cautionary note on the use of the B subunit of cholera toxin as a ganglioside GM1 probe: detection of cholera toxin A subunit in B subunit preparations by a sensitive adenylate cyclase assay

The use of the B subunit of cholera toxin, a protein that binds specifically to ganglioside GM1, has provided a new paradigm for studying physiological functions of ganglioside GM1. The B subunit inhibited the growth of rat glioma C6 cells that had been pretreated with ganglioside GM1. In some preparations of the B subunit, the inhibition was independent of adenylate cyclase activation and was due to the binding of the B subunit to ganglioside GM1 inserted onto the cell surface. However, in other preparations of the B subunit, there was an additional inhibitory effect due to small contaminations with the A subunit, which caused increases in intracellular cyclic adenosine monophosphate (cAMP) levels and concomitant growth inhibition. This vanishingly small contamination with the A subunit could not be detected by conventional protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis but could be measured utilizing a sensitive adenylate cyclase activation assay. Thus caution must be used to ensure that any biological effects of the B subunit are not due to contaminating A subunit and are due solely to the binding of the B subunit to ganglioside GM1 exposed on the cell surface. This is especially important in cyclic nucleotide-sensitive systems.     

A monoclonal antibody recognizes an O-acylated sialic acid in a human melanoma-associated ganglioside

Monoclonal antibody D1.1 originally prepared against the B49 cell line derived from a rat brain tumor was shown to react with a ganglioside present in fetal rat brain. We have found that this antigen is also present in human malignant melanoma tumors as well as many melanoma cell lines. The ganglioside from human melanoma cell lines migrates between GM1 and GM2 on one-dimensional thin layer chromatography. Analysis by two-dimensional thin layer chromatography with intermediate ammonia treatment suggests that the ganglioside contains one or more base-labile O-acyl esters. Mild base hydrolysis under conditions known to remove O-acyl esters results in complete loss of antigenic reactivity. Thus, the alkali-labile moiety is a critical component of the epitope recognized by the antibody. Analysis of the sialic acids of total gangliosides from [6-3H]glucosamine-labeled melanoma cells showed that approximately 10% of these molecules are O-acylated. Similar analysis of the purified ganglioside showed that greater than 30% of the sialic acids comigrated with authentic 9-O-acetyl-N-acetylneuraminic acid. The antibody did not cross-react with normal human skin melanocytes nor with any of a large number of normal human adult and fetal tissues. The antibody also did not react with numerous other malignant cell lines studied. These findings suggest that the antigenic epitope defined by antibody D1.1 contains an O-acylated sialic acid and may arise from aberrant O-acetylation occurring in human malignant melanoma cells.     

Single step purification of potent antigenic protein from sparganum by gelatin-affinity chromatography

Out of many component proteins in crude saline extract of Spirometra mansoni plerocercoid (sparganum), 36 kDa and 29 kDa proteins were found to be the most antigenic and were already purified by immunoaffinity chromatography using monoclonal antibody as a ligand. In this study, a single step purification of these potent antigenic proteins of sparganum extract was investigated. When the crude saline extract was charged to gelatin-Sepharose 4B affinity column, 36 kDa and 29 kDa protein fractions were bound. SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE/immunoblot confirmed that the bound protein to gelatin was serologically pure. When evaluated by ELISA with patients sera, the purified protein of 36 and 29 kDa also showed improved antigenicity.     

空肠弯曲菌肠毒素理化特性的研究

经ＳＤＳＰＡＧＥ 分析发现,空肠弯曲菌细胞紧张性肠毒素（Ｃｙｔｏｔｏｎｉｃ ｅｎｔｅｒｏｔｏｘｉｎ ,ＣＥ） 的硫酸胺盐析粗提物除有一条６８ｋＤ 的带外,还有一些未分开的小分子物质,而经神经节苷脂ＧＭ１ 亲和层析后仅有６８ＫＤ 的一条带,即表明６８ｋＤ 的蛋白质为ＣＥ 的主要成分。ＣＥ 不耐热、ｐＨ 依赖和对胰酶有抗性。５６ ℃和６０ ℃加热３０ｍｉｎ 、１００ ℃加热１５ｍｉｎ 即可完全失活。其活性在ｐＨ６－０ 时最高,在ｐＨ３－０ 和９－０ 时均可使其完全丧失活性。在４ ℃保存超过３ｄ 后,其活性迅速降低。抗ＬＴ 血清能完全抑制ＣＥ 的活性。     

Expression of Hanganutziu-Deicher antigen in activated human T lymphocytes

Hanganutziu-Deicher (HD) antigen is a heterophile antigen that is widely distributed in many animals other than humans and chickens and is highly immunogenic in humans and chickens. In the present study, we demonstrated expression of HD-antigenic glycoproteins in activated T lymphocytes by SDS-PAGE and immunoblotting. Treatment with IL-2 plus PMA induced 29kD glycoprotein antigen detected under reducing condition. It contained sialic acid epitope of HD antigen because of the expression being neuraminidase-sensitive. Treatment with PMA plus A 23187 or PHA treatment and then PHA plus IL-2 treatment also induced two proteins of Mr 50kD and 70kD. These expressions were not detected in all individuals examined. These results indicate that HD antigen is an activated T cell antigen and expressed as an isoantigen as it is expressed in cancerous tissues from some patients.     

Proton NMR and fast-atom bombardment mass spectrometry analysis of the melanoma-associated ganglioside 9-O-acetyl-GD3

A glycolipid antigen, detected by a monoclonal antibody (ME 311) obtained by immunizing mice with a human metastatic melanoma cell line (WM 46), was isolated and structurally characterized. Using immunostaining on thin-layer chromatograms for monitoring, 1.0 mg of a pure alkali-labile disialoganglioside was obtained from 23 g of packed melanoma cells (WM 164). Fractionation of the lipid extract was done on DEAE-Sepharose columns into total disialogangliosides which were repeatedly separated by high-pressure liquid chromatography. On mild alkaline treatment, the ganglioside was converted to a slower migrating species identical with a ganglioside GD3 isolated from the same source (Neu5Ac alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-cer-amide) and specifically detected by monoclonal antibody R24. Comparison of the two gangliosides by fast-atom bombardment mass spectrometry (revealing an acetyl group on the terminal sialic acid on the alkali-labile species) and by 1H NMR (indicating the position of the acetyl group) suggested the following structure: Neu5,9Ac2 alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-ceramide. This is identical with a ganglioside proposed earlier to exist in melanoma cells (Cheresh, D. A., Varki, A. P., Varki, N. M., Stallcup, W. B., Levine, J., and Reisfeld, R. A. (1984) J. Biol. Chem. 259, 7453-7459). Immunostaining with ME 311 antibody of cell extracts on thin-layer chromatography chromatograms revealed only this ganglioside in the melanoma cells, while normal human brain was negative. However, in one of the total ganglioside extracts tested for presence of binding with antibody ME 311, three gangliosides were found to bind. No evidence was obtained for the presence of the antigenic epitope in mucins or glycoproteins of the melanoma cells.     

Characterization of ganglioside associated with the thyrotrophin receptor

The receptor protein for thyrotrophin (thyroid-stimulating hormone;TSH) is associated with a glycosphingolipid moiety. The proteinbelongs to the family of receptors that couple to guanine nucleotidebinding proteins; the glycosphingolipid contains sialic acidand belongs to the family of gangliosides. This report definesthe structure of the receptor ganglioside in the Fisher ratthyroid cell line (FRTL-5). Receptor protein was purified byTSH affinity chromatography from FRTL-5 cells, biosyntheticallylabelled with [³H]galactose and [³H]glucosamine, and resolvedby SDS-PAGE. A single radiolabelled band of M_r     

Specific ganglioside-cell protein interactions: a study performed with GM1 ganglioside derivative containing photoactivable azide and rat cerebellar granule cells in culture.

The incubation of cultured rat cerebellar granule cells with a photoreactive derivative of radiolabeled GM1 ganglioside, [3H]GM1(N3), followed by illumination, led to the specific association of ganglioside to cell proteins. After 30 min of incubation only a few out of the cell proteins became radiolabeled. Two of these, at apparent molecular weights of 95 and 112 kDa, are interacting with the portion of associated ganglioside that is released by trypsin treatment; others, in the region between 31 and 44 kDa, are probably bound to molecules of ganglioside inserted into the outer membrane layer, thus showing that the ganglioside association to the cell surface is a selective phenomenon, involving specific proteins. Increasing the incubation time up to 24 h resulted in a larger number of radiolabeled proteins, probably as a consequence of the internalization and metabolic processing of administered [3H]GM1(N3). In fact, photoreactive and radioactive metabolic derivatives of [3H]GM1(N3) can also interact with a number of proteins. After 24 h incubation, some radioactivity was also associated to cytosolic proteins. Again in this case the interaction with proteins seems to be a specific process involving only a few out of the total cytosolic proteins.     

Gnathostoma binucleatum: excretion-secretion antigen analysis obtained from advanced third-stage larvae in in vitro culture

The paper describes an introductory characterization of antigenic stimulation of excretion-secretion products (ESP) of Gnathostoma binucleatum advanced third-stage larvae cultured in vitro and proteinases present in this products. Excretory and secretory proteins were obtained after 10 larvae were maintained in 5% CO(2) RPMI medium. The supernatant was collected each week for two months. The proteins were dialyzed, concentrated, and separated in 10% SDS-PAGE gels under reducing conditions and transferred to nitrocellulose paper for immunoblot analyses. G. binucleatum immunized mice serum was used to determine protein antigenicity. Four proteins of 40, 80, 120, and 208 kDa persisted for two months and three proteins, 80, 120, and 208 kDa were recognized for antibodies of mice. In SDS-PAGE gelatin substrate gels ESP resolved as two proteins with molecular weight of 80 and 208 kDa that were sensitive to a metalloproteinase inhibitor, and thus it may be inferred that they might be used for diagnosis of gnathostomiasis.     

Cell-surface associated proteins of corneal fibroblasts: dissection with monoclonal antibodies

It is now generally accepted that the cell surface is involved in the interaction of the cells with the extracellular matrix. To identify and characterize cell-surface-associated components of corneal fibroblasts, several monoclonal antibodies were developed. Hybridomas were developed by fusing mouse myeloma cells SP2/OAg14 with spleen cells from mice immunized with membrane fractions of corneal fibroblasts grown in culture. Twenty-five hybridomas secreting monoclonal antibodies to cell-surface components were selected by an enzyme-linked immunosorbent assay using corneal fibroblasts grown in microtiter plates as the substrate. Immunohistochemical staining demonstrated that the antigenic determinants recognized by these antibodies were not present on corneal epithelial cells, but were present on skin fibroblasts. The antigenic determinants recognized by two of these antibodies, designated 10D2 and 716, were matrix components of the corneal stroma. Immunochemical characterization of the antigens was carried out by indirect precipitation of the radioactively labeled cellular proteins with the monoclonal antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the precipitates. Four antibodies were able to precipitate antigens from cell extract in detectable amounts. Antibodies designated 5E2, 9G2, and 10D2 recognized antigens consisting of polypeptides of approximate molecular weights 105K and 110K, while antibody 716 recognized an antigen of 100K molecular weight. However, based on the tissue distribution and cell-surface distribution, these antibodies reacted with different antigenic determinants. The antigen recognized by 716 was also secreted by cells in culture but consisted of 220K and 200K polypeptide chains. It was tentatively identified as cellular fibronectin, based on the reaction of this antigen with polyclonal antibodies to plasma fibronectin.