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R Bragado

Publications and source records attributed to R Bragado.

28 records · Page 2Linked to original sources
PubMed1985

Structural analysis of an HLA-B27 functional variant: identification of residues that contribute to the specificity of recognition by cytolytic T lymphocytes.

The structure of a variant HLA-B27 antigen, B27.2, that is distinguished from the HLA-B27.1 and HLA-B27.3 subgroups by specific cytolytic T lymphocytes has been established by comparative peptide mapping and sequence analysis. There are only three amino acid substitutions between B27.1 and B27.2: aspartate-77, threonine-80, and leucine-81 in HLA-B27.1 are changed to asparagine-77, isoleucine-80, and alanine-81 in HLA-B27.2. These changes account for their single charge difference detectable by isoelectric focusing. The three clustered substitutions of HLA-B27.2 are identical to the corresponding residues in HLA-A24, so that both molecules become identical in their amino acid sequence between residues 72 and 96. This suggests that gene conversion may have occurred during the diversification of the HLA-B27 antigens. HLA-B27.2 has no changes in the alpha 2 domain and is similar in its pattern of substitutions to the murine bm11 mutant. It is suggested that residues 77-81 are of major significance in determining the specificity of cellular recognition of class I HLA antigens. This study, together with the previous analyses of HLA-B27.1 and HLA-B27.3, completes the structural characterization of the three major HLA-B27 functional subtypes and establishes the molecular basis of their functional and serological differences.

Amino Acid Sequence↗
PubMed1985

Delineation of functional sites in HLA-B27 antigens. Molecular analysis of HLA-B27 variant Wewak I defined by cytolytic T lymphocytes.

An HLA-B27 positive, Epstein Barr virus-transformed cell line Wewak I was not lysed by Epstein Barr virus-specific cytolytic T lymphocytes restricted by HLA-B27. This line is weakly reactive with a B27-specific monoclonal antibody, M2, which recognizes a majority, but not all, of the HLA-B27-positive cells. To establish the molecular basis for this lack of recognition, the structure of the variant HLA-B27 antigen was compared with the known structure of HLA-B27 from the LG-2 cell line, which is representative of a major subtype of this antigen. Both molecules were almost indistinguishable by isoelectric focusing. However, comparative peptide mapping and sequencing of the difference peptides revealed two amino acid changes: At position 77, Asp in donor LG-2 had changed to Ser in the variant, and at position 152, Val in LG-2 had changed to Glu in the variant. The nature of these substitutions was consistent with the extreme similarity of the isoelectric focusing pattern. An evaluation of these findings in the context of studies on other HLA variants and H-2Kb mutants suggests that both positions 77 and 152 contribute to the determinants recognized by B27-specific cytolytic T lymphocytes. The change at position 152 adds to previous evidence suggesting that the segment 149-156 is critical for cellular recognition. In addition, it is proposed on the basis of structural comparisons that residue 77 may also participate in the epitope recognized by the B27M2 antibody.

Amino Acid Sequence↗
PubMed1984

Variability and conformation of HLA class I antigens: a predictive approach to the spatial arrangement of polymorphic regions.

Comparison of available sequences of HLA-A and HLA-B antigens shows that variable positions are predominantly localized in four segments spanning residues 63-85, 105-116, 138-156, and 177-194. The fourth segment is unique in that it contains no differences between antigens of the same locus. Secondary folding of HLA heavy chain was estimated by three independent predictive methods and areas of defined structure were correlated with the distribution of local hydrophobicity to outline putative internal and external portions. The three analyses each independently predict a high probability for beta structure in the alpha 1, alpha 2, and alpha 3 domains. A single alpha-helix is predicted within residues 146-160, a segment of likely importance in cytotoxic T cell recognition and graft rejection. Substitutions within this segment are spatially related by the helical turn. Variable residues usually lie in areas of high local hydrophilicity, and therefore they are probably on the surface of the molecule. The model predicts that they are frequently located in beta strands, beta-turns, or the above-mentioned alpha-helix, so that most substitutions would be accommodated within rigid frameworks that may impose structural constraints to variability. The secondary structure of alpha 1, alpha 2, and alpha 3 domains presents some analogies that suggest that they might share common features in their tertiary folding. The predicted structure of alpha 3 is strongly reminiscent of that of immunoglobulin constant domains. Possible arrangements of elements of secondary structure are discussed, as an attempt to situating the polymorphic regions of HLA class I antigens in a spatial context.

Amino Acid Sequence↗
PubMed1983

Primary structure of papain-solubilized human histocompatibility antigen HLA-B40 (-Bw60). An outline of alloantigenic determinants.

The primary structure of papain-solubilized human histocompatibility antigen HLA-B40 (-Bw60) has been determined. Its comparison with that of the cross-reactive HLA-B7 antigen allows for the first time a direct sequence comparison between two HLA-B locus products and an outline of the location of their alloantigenic sites. Overall sequence homology between HLA-B40 and HLA-B7 is 93%. Of 19 detected differences, 18 are located in the two amino-terminal domains (residues 1-182). Half of them are clustered in two short segments spanning residues 63-74 and 147-156, respectively, which suggests that they may encompass major sites of their alloantigenic determinants. The first of these segments is highly polymorphic in HLA and H-2 molecules. It is proposed that it may belong to a hypervariable region of class I histocompatibility antigens. The remaining substitutions are scattered through the N-terminal portions of the two external domains. Thus, in addition to the above-mentioned segments, other positions may contribute significantly to the antigenic polymorphism of these molecules.

Amino Acid Sequence↗
PubMed1982

The effect of the chemical modification in human Fc gamma fragment on protein A and Fc receptor binding.

The role of acidic side-chains on Fc gamma fragment in granulocyte receptor binding and in S. aureus protein A binding has been investigated by means of chemical modification. Alteration of a restricted number of carboxyl groups after 5 min of reaction is sufficient to abrogate the capacity of Fc to inhibit EA rosette formation by human neutrophils. More limited modification, which affects mainly the most exposed acidic chains, does not change receptor binding activity. In contrast, the interaction with protein A is largely unaffected, even under reaction conditions which are able to induce significant changes in the circular dichroism spectrum of Fc fragment. The results suggest that some acidic groups on Fc may be involved in the interaction with neutrophil receptor and that the binding to protein A and Fc receptor involves different sites.

Amino Acids↗
PubMedDate not supplied

Disruption of cadherin-related junctions triggers autocrine expression of vascular endothelial growth factor in bovine aortic endothelial cells : effects on cell proliferation and death resistance.

The mechanisms involved in the blockade of proliferation in confluent endothelial cells are insufficiently understood. In this regard, the continuity of intercellular junctions appears to be critical to the regulation of endothelial monolayer cell growth. The present study examined the hypothesis that the disruption of the intercellular adherens junctions will trigger both endothelial cell proliferation and autocrine production of growth factors. With this purpose, we assessed the changes in growth, death resistance, and expression of vascular endothelial growth factor (VEGF) under conditions of disruption of the intercellular junctions between endothelial cells. Disruption of cell junctions was produced by means of a specific anti-vascular endothelial cadherin monoclonal antibody, EGTA, or cytochalasin D. Our results disclosed that these maneuvers induce an increase in VEGF mRNA production, with transcription of the 121-, 165-, and 189-amino acid isoforms of VEGF. Further evidence of the relationship between endothelial cells monolayer continuity and VEGF protein expression was obtained by the demonstration of an increase in VEGF protein, as determined by Western blot, induced by the aforementioned maneuvers, as well as by immunocytochemical detection of increased VEGF staining in the areas surrounding a mechanical endothelial injury and in endothelial cells at subconfluence. In functional terms, the autocrine expression of VEGF was associated with growth-promoting and cytoprotective effects, as assessed by [(3)H]thymidine uptake, (51)Cr release, and flow cytometry. In conclusion, our results reveal that disruption of homophilic interendothelial junctions induces VEGF expression. Under these conditions, autocrine VEGF appears to have a relevant role in death inhibition and proliferation of endothelial cells.

Animals↗