MB1, a quail leukocyte/vascular endothelium antigen: characterization of the lymphocyte-surface form and identification of its secreted counterpart as alpha 2-macroglobulin

A monoclonal antibody (alpha-MB1) binds to a cell surface marker expressed throughout ontogeny and adult life by vascular endothelial and hemopoietic cells of the quail, with the exception of erythrocytes, although it was raised against the heavy chain of quail immunoglobulin M. In addition to an 80 kDa polypeptide accounting for immunoglobulin mu chain, alpha-MB1 stains intensely a 180-kDa band on Western blots of reduced plasma proteins. We have previously characterized MB1 antigens of quail endothelial cells as glycoproteins of apparent molecular masses ranging from 80 to 200 kDa and provided evidence for the participation of vascular endothelium in the secretion of alpha-MB1-positive plasma components. We demonstrate here that this circulating material is the proteinase inhibitor alpha 2-macroglobulin. Furthermore, the MB1 antigens immunoprecipitated from lymphocytes are shown to be essentially similar to their endothelial counterparts, suggesting that the same molecular complex is expressed by all the elements of the hemangioblastic cell lineage. Finally, the cross reactivity of the alpha-MB1 antibody with immunoglobulin mu chain is confirmed and shown to occur via a carbohydrate epitope.     

The cell surface of a restrictive fenestrated endothelium

The choriocapillaris is one example of a capillary bed lined by a fenestrated endothelium that is restrictive to exogenous tracers and endogenous plasma proteins. In this study we have examined the distribution of cell-surface monosaccharides utilizing biotinylated lectin-avidin ferritin cytochemistry. Receptors for wheat germ agglutinin were localized to the plasmalemma and diaphragms of some fenestrae, vesicles, and channels at the luminal endothelial front in amounts greater than seen for the other lectins employed. The absence of labeling following inhibition with N-acetylglucosamine and after tissue digestion with N-acetylhexosaminidase, but not after neuraminidase indicated that this lectin marked N-acetylglucosamine residues and not sialic acid. Wheat germ agglutinin receptors were not affected by pronase E or trypsin digestion, but were partially removed by proteinase K. The latter also removed many fenestral diaphragms. Wheat germ agglutinin receptors were cleaved with endoglycosidase D. The combined results indicate that the wheat germ agglutinin receptor is of the low-mannose type and part of a protein with hydrophobic properties. Receptors for concanavalin A (mannose) and Ricinus communis agglutinin (galactose) were also localized to the plasmalemma and endothelial diaphragms. The examination of sections at different tilt angles revealed that these lectins bound to the endothelium in a non-random distribution, encircling diaphragms of fenestrae and channels. Soybean agglutinin (N-acetylgalactosamine) marked endothelial structures sparsely. Following digestion with pronase E or trypsin, receptor sugars for the latter three lectins were completely removed, indicating their presence on protease susceptible glycoproteins. These findings demonstrate that the endothelium of the choriocapillaris bears carbohydrate moieties that are different than those described for permeable fenestrated endothelia.     

The cell surface of a restrictive fenestrated endothelium

Summary The location and chemical composition of anionic sites on the endothelium of the choriocapillaris was investigated with cationic ferritin and enzyme digestion techniques. Cationic ferritin administered intravenously initially labeled essentially all fenestral diaphragms. Within 30 min after injection, no diaphrams remained labeled, but they could be relabeled by a second cationic ferritin injection. Following perfusion of cationic ferritin, the entire luminal front of the endothelium was labeled: the plasmalemma and fenestral, vesicle, and channel diaphragms. Perfusion of neuraminidase or chondroitinase did not affect subsequent cationic ferritin binding. In contrast, heparitinase removed anionic sites on all structures except fenestral diaphragms. Cationic ferritin did not mark the endothelium following heparinase digestion. All sites were cleaved with pronase E. These results indicate that heparin is the anionic moiety on fenestral diaphragms while the glycocalcyces of the plasmalemma and vesicle and channel diaphragms are rich in a heparan sulfate proteoglycan. Furthermore, since the heparan sulfate localized to these structures was digested by both heparinase and heparitinase, it is in a form similar to heparin. These findings demonstrate that the endothelium of the choriocapillaris bears cell-surface anionic components that are different than those described for fenestrated endothelia lining other vascular beds.Supported by NIH EY 03776     

Identification of a macrophage-specific cell surface antigen.

A mouse-specific macrophage antigen (MSMA) was identified in NP-40 extracts of 125I-radiolabeled mouse preitoneal macrophages by using a rabbit anti-mouse macrophage serum (AMS) and SDS-polyacrylamide gel electrophoresis. The antigen was shown to have a m.w. of 83,000 daltons and was present on both normal and "activated" peritoneal macrophages. MSMA was also present on syngeneic adherent spleen cells, allogeneic peritoneal macrophages, a mouse macrophage cell line (P388D1), and exhibited some cross-reactivity with peritoneal macrophages from closely related species (rats and hamsters). MSMA was not present on nonadherent peritoneal exudate cells, spleen cells, erythrocytes, thymocytes, or bone marrow cells. Extensive absorptions of AMS with thymocytes and erytrocytes from mice were necessary to remove other antibodies that reacted with other mouse membrane antigens. An antiserum directed against a specific membrane antigen has great potential in elucidating structure-function relationships with regard to a number of macrophage activities.     

A cell surface/plasma membrane antigen of Candida albicans.

Antibody from BALB/cByJ mice immunized against a membranous fraction of Candida albicans agglutinated whole cells as well as the membranous fraction. Hybridoma techniques were used to isolate an IgM monoclonal antibody (mAb) designated 10G which agglutinated whole cells and reacted with the subcellular fraction. Yeast cells of 15 additional C. albicans strains and isolates of C. stellatoidea, C. tropicalis, C. intermedia and C. lusitaniae were also agglutinated by mAb 10G. The antigen was not detected on other fungi, including Candida krusei, C. utilis, Cryptococcus neoformans, Cr. albidus, Torulopsis glabrata, Rhodotorula spp. and Saccharomyces cerevisiae. To determine the cellular location of the epitope to which mAb 10G is specific, freeze-substitution was compared with traditional chemical fixation methods in preparation of samples for immunocolloidal gold electron microscopy (IEM). With both fixation procedures, the antigen recognized by mAb 10G was found randomly and densely concentrated on the plasma membrane on exponential-phase yeast-form cells and had a patchy distribution on the cell wall surface. Association of the antigen with the plasma membrane was confirmed by IEM of isolated membranes. On developing hyphal cells, antigen appeared first on the plasma membrane and later on the cell wall surface. Treatment of yeast cells with beta-mercaptoethanol and Zymolyase before fixation removed the antigen from the surface but left the cytoplasmic antigen undisturbed. Treatment of yeast cells or solubilized antigen with heat or proteolytic enzyme (trypsin, Pronase B, proteinase K) did not remove or destroy the antigen, suggesting a non-protein nature of the epitope.     

Sequence analysis of the cloned streptococcal cell surface antigen I/II

The gene spa P (formerly designated as spa P1) encoding the Mr 185,000 surface antigen (I/II) of Streptococcus mutans, serotype c (NG5), has been sequenced. The gene (4683 bp) encodes a protein of 1561 amino acid residues including putative signal peptide (residues 1-38) and transmembrane (residues 1537-1556) sequences. The N-terminal region (60-550) has alanine-rich repeats and is predicted to be alpha-helical. However, the C-terminal region (800-1540) is proline-rich and favours an extended structure. Except for a short central variable region the sequences appear to be highly conserved for S. mutans serotype c. N-Terminal sequencing of separated antigen I and antigen II polypeptides suggests that the former represents the N-terminal and the latter the C-terminal portions of the intact antigen.     

Light microscopical detection of leukocyte cell surface antigens with a one-nanometer gold probe

Summary The potential of ultrasmall gold particles for the light microscopical detection of leukocyte cell surface differentiation antigens was investigated. Suspensions and cytocentrifuge preparations of peripheral blood leukocytes were first incubated with monoclonal antibodies and then with goat antimouse antibodies coupled to colloidal gold particles of 1-nanometer diameter. Cytocentrifuge preparations were made from the cell suspensions. Silver enhancement was performed on all preparations. Then they were counterstained with May-Grünwald Giemsa and examined in light microscopy. The immunostaining appeared as fine dark granules on the surface membrane of the cells. Labeling conditions were determined which gave a dense specific immunostaining and a low background. High dilutions of the ultrasmall gold probe could be used to detect all antigen expressing cells in the samples. The labeling efficiency of the IGSS method with the 1 nanometer probe was comparable to that described earlier for 5 nanometer gold particles. Lymphocyte subsets enumerated with this method in normal peripheral blood were similar to those found with immunofluorescence microscopy. We concluded that one nanometer probes do not offer a major advantage in comparison with 5 nanometer probes for the study of cell surface antigens.     

Light microscopical detection of leukocyte cell surface antigens with a one-nanometer gold probe.

The potential of ultrasmall gold particles for the light microscopical detection of leukocyte cell surface differentiation antigens was investigated. Suspensions and cytocentrifuge preparations of peripheral blood leukocytes were first incubated with monoclonal antibodies and then with goat antimouse antibodies coupled to colloidal gold particles of 1-nanometer diameter. Cytocentrifuge preparations were made from the cell suspensions. Silver enhancement was performed on all preparations. Then they were counterstained with May-Grünwald Giemsa and examined in light microscopy. The immunostaining appeared as fine dark granules on the surface membrane of the cells. Labeling conditions were determined which gave a dense specific immunostaining and a low background. High dilutions of the ultrasmall gold probe could be used to detect all antigen expressing cells in the samples. The labeling efficiency of the IGSS method with the 1 nanometer probe was comparable to that described earlier for 5 nanometer gold particles. Lymphocyte subsets enumerated with this method in normal peripheral blood were similar to those found with immunofluorescence microscopy. We concluded that one nanometer probes do not offer a major advantage in comparison with 5 nanometer probes for the study of cell surface antigens.     

Identification of a novel T cell surface disulfide-bonded dimer distinct from the alpha/beta antigen receptor

Hybridomas were prepared from the spleen of a BALB/c mouse immunized with EL-4 T lymphoma cells. One, designated A1, was found to secrete a monoclonal antibody that reacted with two T lymphoma cells of C57BL origin, EL-4 and C6VLB, but not with normal C57BL/6 splenocytes or thymocytes, C57BL/6 T cell clones, or other T or B lymphomas by complement-mediated cytotoxicity or indirect immunofluorescent staining. Monoclonal antibody (MAb) A1 precipitated a protein that migrated at 85 kD under nonreducing and 43 kD under reducing conditions. The fact that the antigen defined by MAb A1 was a disulfide-linked dimer, together with the essentially clone-specific distribution of the reactive epitope, raised the possibility that the antibody defined an epitope of the antigen receptor. However, several additional observations revealed that the antibody defined a distinct and novel T cell surface structure. MAb 124-40, previously shown to react with the antigen receptor of C6VLB cells, reacted with variants of C6VLB that failed to express the A1 epitope. Sequential immunoprecipitation indicated that MAb A1 and MAb 124-40 reacted with distinct molecular species on C6VLB cells. Endoglycosidase digestion showed that the structure reactive with MAb A1 was not derived from that reactive with MAb 124-40 by addition of N-linked oligosaccharide residues. Two-dimensional gel electrophoretic analysis of precipitates obtained from radioiodinated C6VLB cells with MAb 124-40 resolved the alpha and beta subunits of the antigen receptor. Similar analysis of precipitates obtained with MAb A1 revealed only a single basic chain under reducing conditions, although anomalous mobility suggestive of a second, more acidic chain was observed under nonreducing conditions. Two-dimensional maps of tyrosine-containing chymotryptic peptides of the proteins isolated with MAb A1 and MAb 124-40 were completely different, suggesting that the molecules shared no peptides and were distinct in primary structure. Finally, cross-linking studies performed with a cleavable reagent indicated that the A1 molecule, unlike the antigen receptor defined with MAb 124-40, was not associated with additional, T3-like structures on the surface of C6VLB cells. Although the MAb A1 was unreactive with normal cells in cytotoxicity or staining assays, a molecule of the appropriate size was immunoprecipitated in small amounts from lysates of radioiodinated normal spleen and thymus cells. These data indicate that MAb A1 defines a novel disulfide-linked T cell surface molecule distinct from the antigen receptor.     

Somatic variants of the level of expression of a cell surface antigen

Somatic variants with constitutive changes in the expression of the cortical thymocyte differentiation antigen HTA 1 were derived from the T-cell leukemia line Molt 4 using monoclonal antibody NA1/34 and the fluorescent activated cell sorter. Cells with the highest and lowest fluorescence were sorted and expanded. After several cycles, high expressor variants, with 10- to 12-fold increased surface HTA 1 and low expressors, with a level of one third relative to the wild-type, were obtained. The stability of the high expressors was improved by cloning. In spite of the large differences in the level of HTA 1 between the various mutants and the wild-type, the expression of HLA-A,B,C or its increase following interferon-alpha stimulation, remained unchanged. Although in HTA 1 there was only a trace of beta2-microglobulin (beta2m) detected by lactoperoxidase labelling of the high expressor variants, significant levels of both beta2m and HTA 1 light chain beta(t) were observed in gels stained with Coomassie blue. The physiological association of beta(t) with HTA 1 was confirmed by the substantial increase of beta(t) which parallels the increase of HTA 1.     

Role of cell surface carbohydrate moieties in monocytic cell adhesion to endothelium in vitro

Monocyte adhesion to endothelium represents the first step in the emigration of this leukocyte from blood to tissue during such pathologic and physiologic processes as atherosclerotic plaque development, wound healing, and inflammation. We have examined the role of carbohydrate moieties in the binding of mononuclear cells to endothelium in vitro. Wheat germ agglutinin (WGA) completely inhibited binding of the human monocytic cell line U937 to pig or human endothelial cells (EC). The inhibition was abolished by the presence of N-acetyl glucosamine, a preferred ligand for WGA. This sugar itself, however, had no effect on monocytic cell binding to EC, suggesting that WGA is inhibiting the cell-cell interaction by binding to a distinct sugar moiety. We tested a series of simple and phosphorylated sugars for the ability to inhibit U937 cell binding to EC. Two phosphorylated disaccharides, lactose-1-phosphate and maltose-1-phosphate, but not 14 other sugars, caused complete suppression of monocyte adhesion to EC. Among the inactive sugars were mannose-6-phosphate and fructose-1-phosphate, which have been shown by others to markedly suppress lymphocyte adhesion to EC. A nonionic detergent, n-octyl-beta-D-glucopyranoside (octyl glucoside), which contains a sugar group as a hydrophilic moiety, also inhibited U937 cell or human monocyte binding to human or porcine EC. The inhibition was observed at a nontoxic concentration of octyl glucoside and appeared to be due to an effect on the monocytic cell rather than the EC. When suboptimal doses of WGA and octyl glucoside were added in combination to the U937 cell-EC adhesion assay, the level of inhibition was greatly reduced when compared with either of the inhibitors alone, suggesting an interaction between these two blocking agents. Lactose-1-phosphate, but not octyl glucoside or WGA, blocked neutrophil adhesion to EC. In summary, our results indicate that specific cell surface carbohydrate groups are required for the adhesion of monocytes to the endothelium.     

Characterization of a 140-kD avian cell surface antigen as a fibronectin-binding molecule

A 140,000-D protein cell surface antigen (140k) complex has been implicated in fibronectin-mediated cell-substratum attachment. We have used three different experimental systems to evaluate the hypothesis that this 140k complex can function as a fibronectin receptor. A monoclonal antibody that binds to the 140k complex specifically inhibits the direct binding of 3H-labeled 75,000-D fibronectin cell-binding fragment (f75k) to chicken embryo fibroblasts in suspension. The 140k complex is retarded in its passage through an affinity column consisting of immobilized f75k, and this interaction is specifically inhibited by a synthetic peptide that contains the fibronectin cell-recognition signal sequence. Finally, exogenous purified 140k complex inhibits the attachment and spreading of chicken embryo fibroblasts on fibronectin-coated substrates. Thus, our results indicate that the 140k complex can bind directly to fibronectin and is likely to be a fibronectin receptor for chicken cells.     

Molecular cloning and characterization of a novel murine T cell surface antigen, YE1/48

YE1/48 is a murine cell surface disulphide-linked dimeric Ag consisting of two 45,000-50,000 Mr subunits. It is expressed on some T lymphoma lines at high levels but its expression on normal lymphocytes is very low. The functional significance of this Ag is currently unknown. We have now cloned a cDNA encoding the YE1/48. Sequence analysis revealed that it encodes a Type II membrane protein of 262 amino acids (30,500 MW), with 44 amino acids in the N-terminal cytoplasmic domain, 22 amino acids in the transmembrane domain and 196 amino acids in the C-terminal extracellular domain. There are three potential N-linked glycosylation sites in the extracellular domain all of which are probably used in the mature protein. No significant homology can be identified with other known protein sequences in the data base or with human CD28(T44), a human T cell activation antigen consisting of two 44,000 Mr subunits. The protein sequence includes in its extracellular domain the arginine-glycine-aspartic acid tripeptide, a potential cell-adhesive binding site, and a sequence similar to the consensus domain of any metal-binding proteins. However, whether these sequences are functional is unknown. Genomic Southern analysis of C57BL/6, BALB/c and C3H mice has demonstrated a restriction fragment length polymorphism. The analysis has also strongly suggested the existence of some other genes with sequences highly homologous to the YE1/48 gene. The YE1/48 gene appears to be expressed at very low levels in a wide range of lymphoid cells with no restriction to their differentiation stages. Interestingly, YE1/48 expression appears to be induced in pre-B cells after transformation by Abelson virus, suggesting an association of YE1/48 expression with the transformation of T and pre-B Cells.     

Study of a 53 kDa cell surface antigen of oligodendrocytes in culture

It was shown previously that pure oligodendrocytes release proteins when maintained in a chemically defined medium. Among these proteins, a 53 kDa glycoprotein was characterized as a component accessible from the external surface of these glial cells. Specific antibodies directed against this glycoprotein were obtained using two different procedures. They were tested on immunoblots of different cells; the protein was detected in C6 glioma cells and fibroblasts, but not in astrocytes. No immunoreactive band was observed on immunoblots of developing rat brain suggesting that this protein may be a minor constituent of the oligodendrocyte in vivo. These antibodies were also used on oligodendrocyte cultures to confirm our earlier finding that this glycoprotein is on the surface of the oligodendroglial plasma membrane. This protein appears to be a useful surface marker for oligodendrocytes in culture.