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P Parham

Publications and source records attributed to P Parham.

At least 253 records · Page 14Linked to original sources
PubMed1990

Epitope map of the HLA-B7 CREG using affinity-purified human alloantibody probes.

Monoclonal antibodies (mAb) recognizing the B7 CREG have been used to construct an epitopic map of HLA-B7. Similar studies with human HLA alloantisera have been lacking due to the polyclonal nature of the alloantibodies (aAb). Detergent-solubilized HLA Class I antigens were purified and coupled to activated CH-Sepharose 4B. Sequential affinity isolation of aAb populations using a series of HLA antigen columns enabled us to produce a battery of aAb eluates against both the private B7, B13, B27, and B omega 60 determinants and the public B7-42, B7-60, B7-60-61, B7-27-13-60, B7-42-22-27, B7-8-42-60-41, and B omega 6 epitopes. The topographic relationship of the B7 family of determinants recognized by the Ab probes was derived using crosscompetition Ab blocking assays with quantitation by indirect immunofluorescence and FACS analysis. We have found that aAb and mAb of similar specificity crossblock; Ab of different specificity give complex patterns including both overlapping blocking between the alpha domains and Ab-induced conformational change of the molecule. From these investigations, we conclude that HLA Class I alloantigens bear both multiple, topographically distinct public epitopes and separate private determinants that can be distinguished using human aAb probes. At least four discrete epitopes are expressed by each molecule of the HLA-B7 CREG and can be ascribed to unique aa substitutions on the hydrophilic beta loops of the distal heavy chain domains and also on several exposed areas of the alpha helices. These findings are extremely similar to those of the HLA-A2 CREG and suggest that possibly all Class I molecules possess a comparable, complex degree of serologic polymorphism.

Antibodies, Monoclonal↗
PubMed1990

Concerted evolution of class I genes in the major histocompatibility complex of murine rodents.

Full-length cDNA sequences of two class I major histocompatibility complex molecules from the DA strain of Rattus norvegicus are reported. One codes for the classical class I restriction element RT1.Aa, which maps to the locus in the rat major histocompatibility complex homologous to H-2K in the mouse. The other probably codes for a soluble nonclassical class I molecule present in DA rat serum; a short deletion in the fifth exon implies that the translated product will terminate in the membrane-spanning region. These sequences have been compared with mouse classical class I sequences as well as with three published rat class I cDNA partial sequences. The results show, first, that "locus-specific" substitutions from the H-2K, H-2D, and H-2L data set are scrambled in the RT1.Aa molecule; a majority of these substitutions have H-2D/L-specific features. Second, the data show that the four rat sequences are strikingly similar to one another regardless of locus or haplotype of origin; they share a number of apparently species-specific features that distinguish them all from mouse classical class I sequences, which likewise share distinctive features of their own. The results suggest that segmental sequence exchange plays a major role in determining the evolution of sequence in class I major histocompatibility complex molecules.

Amino Acid Sequence↗
PubMed1990

Rapid cloning of HLA-A,B cDNA by using the polymerase chain reaction: frequency and nature of errors produced in amplification.

A method for cloning full-length HLA-A,B cDNA (1.1 kilobases) by using the polymerase chain reaction (PCR) is described. Six HLA-A,B alleles (HLA-A2, -A25, -B7, -B37, -B51, and -B57) were cloned, and their structures were determined. Multiple PCR clones for each allele were sequenced to obtain both an accurate consensus sequence and an "authentic" clone having that sequence. Sequences from 50 clones encoding five different alleles permit assessment of the frequency and nature of PCR-produced errors. These include recombinations, deletions, and insertions in addition to point substitutions. Authentic clones were obtained at a frequency of between 30% and 70%, and analysis of three or four clones generally should be sufficient for characterization of an allele.

Alleles↗
PubMed1990

Structure of class-I MHC molecules: HLA-B27 and disease.

In this introductory article, the structure and function of HLA class-I molecules is discussed. The differences between HLA-B27 and the other class-I molecules are described. It is suggested that HLA-B27 contributes to autoimmune phenomena, but little is known about the actual autoimmune mechanisms that cause the B27-associated disease.

Amino Acid Sequence↗
PubMed1990

Neuron-specific expression of high-molecular-weight clathrin light chain.

High-molecular-weight forms of clathrin light chains LCa and LCb contain inserted sequences and are expressed in brain tissue but have not been observed in peripheral tissues. Monoclonal antibodies specific for the high-molecular-weight form of LCb and all forms of LCa were used to analyze their expression in different species and different neuronal cell types. High-molecular-weight light chains were found in bovine, rat, mouse, chicken, and human brain, indicating a conserved pattern of expression. Neuron-specific expression of the high-molecular-weight light chains was suggested by analysis of human brain gray matter and white matter. The former contained a higher proportion of light chains with insertion sequences. Immunohistochemical analysis localized the high-molecular-weight form of LCb to synapses and neuronal perikarya, but not to glial cells. Immunofluorescent labeling of cultured chicken dorsal root ganglia confirmed expression in neurons but not Schwann cells. These results indicate that the high-molecular-weight forms of clathrin light chains are restricted in expression and found in neuronal cells.

Animals↗
PubMed1989

Rab 12 kD.

Explore the source record for details and available documents.

Amino Acid Sequence↗
PubMed1989

Diversity and diversification of HLA-A,B,C alleles.

The nucleotide sequences encoding 14 HLA-A,B,C and 5 ChLA-A,B,C molecules have been determined. Combining these sequences with published data has enabled the polymorphism in 40 HLA-A,B,C and 9 ChLA-A,B,C alleles to be analyzed. Diversity is generated through assortment of point mutations by recombinational mechanisms including gene and allelic conversions. The distribution and frequency of silent and replacement substitutions indicate that there has been positive selection for allelic diversity in the 5' part of the gene (exons 1 to 3) and for allelic homogenization and locus specificity in the 3' part of the gene (exons 4 to 8). These differences may correlate with the lengths of converted sequences in the two parts of the gene and frequency of the CpG dinucleotide. Locus-specific divergence of HLA-A,B, and C demonstrates that recombinational events involving alleles of a locus have been more important than conversion between loci. This contrasts with the predominance of gene conversion events in the evolution of mutants of the H-2Kb gene. However, a striking example of gene conversion involving HLA-B and C alleles of an oriental haplotype has been found. Comparison of human and chimpanzee alleles reveals extensive sharing of polymorphisms, confirming that diversification is a slow process, and that much of contemporary polymorphism originated in ancestral primate species before the emergence of Homo sapiens. There is less polymorphism at the HLA-A locus compared to HLA-B, with greater similarity also being seen between HLA-A and ChLA-A alleles than between HLA-B and ChLA-B alleles. Although greater diversity is seen in the 5' "variable" exons of HLA-B compared to HLA-A, there is increased heterogeneity in the 3' "conserved" exons of HLA-A compared to HLA-B.

Alleles↗
PubMed1989

Polymorphism in the alpha 3 domain of HLA-A molecules affects binding to CD8.

Cytotoxic T lymphocytes (CTL) expressing the CD8 glycoprotein recognize peptide antigens presented by class I major histocompatibility complex (MHC) molecules. This correlation and the absence of CD8 polymorphism led to the hypothesis that CD8 binds to a conserved site of class I MHC molecules. Using a cell-cell binding assay we previously demonstrated specific interaction between human class I MHC (HLA-A,B,C) molecules and CD8. Subsequent analysis of the products of 17 HLA-A,B alleles revealed a natural polymorphism for CD8 binding in the human population. Two molecules, HLA-Aw68.1 and HLA-Aw68.2, which do not bind CD8, have a valine residue at position 245 whereas all other HLA-A,B,C molecules have alanine. Site-directed mutagenesis shows that this single substitution in the alpha 3 domain is responsible for the CD8 binding phenotype and also affects recognition by alloreactive and influenza-specific CTL. Our results indicate that CD8 binds to the alpha 3 domain of class I MHC molecules.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

A panel of unique HLA-A2 mutant molecules define epitopes recognized by HLA-A2-specific antibodies and cytotoxic T lymphocytes.

HLA-A2.1 and HLA-A2.3, which differ from one another at residues 149, 152, and 156, can be distinguished by the mAb CR11-351 and many allogeneic and xenogeneic CTL. Site-directed mutagenesis was used to incorporate several different amino acid substitutions at each of these positions in HLA-A2.1 to evaluate their relative importance to serologic and CTL-defined epitopes. Recognition by mAb CR11-351 was completely lost when Thr but not Pro was substituted for Ala149. A model to explain this result based on the 3-dimensional structure of HLA-A2.1 is presented. In screening eight other mAb, only the substitutions of Pro for Val152 or Gly for Leu156 led to the loss of mAb binding. Because other non-conservative substitutions at these same positions had no effect, these results suggest that the loss of serologic epitopes is in many cases due to a more indirect effect on molecular conformation. Specificity analysis using 28 HLA-A2.1-specific alloreactive and xenoreactive CTL clones showed 19 distinct patterns of recognition. The epitopes recognized by alloreactive CTL clones demonstrated a pronounced effect by all substitutions at residue 152, including the very conservation substitution of Ala for Val. Overall, the most disruptive substitution at amino acid residue 152 was Pro, followed by Glu, Gln, and then Ala. In contrast, substitutions at 156 had little or no effect on allogeneic CTL recognition, and most clones tolerated either Gly, Ser, or Trp at this position. Similar results were seen using a panel of murine HLA-A2.1-specific CTL clones, except that substitutions at position 156 had a greater effect. The most disruptive substitution was Trp, followed by Ser and then Gly. In addition, when assessed on the entire panel of CTL, the effects of Glu and Gln substitutions at position 152 demonstrated that the introduction of a charge difference is no more disruptive than a comparable change in side chain structure that does not alter charge. Taken together, these results indicate that the effect of amino acid replacements at positions 152 and 156 on CTL-defined epitopes depends strongly on the nature of the substitution. Thus, considerable caution must be exercised in evaluating the significance of particular positions on the basis of single mutations. Nonetheless, the more extensive analysis conducted here indicates that there are differences among residues in the class I Ag "binding pocket," with residue 152 playing a relatively more important role in formation of allogeneic CTL-defined epitopes than residue 156.

Amino Acid Sequence↗