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P Parham

Publications and source records attributed to P Parham.

At least 271 records · Page 15Linked to original sources
PubMed1989

Direct binding of influenza peptides to class I HLA molecules.

Activation of T lymphocytes requires the intracellular fragmentation of foreign antigens and their presentation by class I or class II major histocompatibility complex (MHC) glycoproteins. The direct binding of peptides to class II molecules has been demonstrated using equilibrium dialysis, gel filtration and fluorescence energy transfer at planar membranes, and its specificity compared to that of T-cell activation. In contrast, direct binding of peptides to class I molecules has been difficult to detect; although peptide sensitization experiments and the crystallographic structure of HLA-A2 (ref. 9) persuasively argue for its occurrence and importance. Here we describe a gel filtration assay from which we derive direct evidence for selective binding of an influenza matrix peptide to HLA-A2 and for binding of an influenza nucleoprotein peptide to HLA-B37. These two peptides have previously been shown to act respectively as targets for certain HLA-A2 or HLA-B37 restricted influenza-specific cytotoxic T lymphocytes (CTL). In addition we demonstrate binding to some, but not all, HLA allospecificities that cannot present these peptides to CTL. We estimate that less than 0.3% of the HLA molecules present in any given purified preparation were able to bind the added peptides.

Amino Acid Sequence↗
PubMed1989

Cytotoxic T cell responses in HLA-A2.1 transgenic mice. Recognition of HLA alloantigens and utilization of HLA-A2.1 as a restriction element.

Previous studies have indicated that the frequency of murine CTL precursors (CTLp) for human class I molecules is one to two orders of magnitude lower than that for murine class I alloantigens, and that this is due to species-specific structural differences between these molecules. Transgenic mice expressing the human class I MHC Ag HLA-A2.1 were used to examine changes in the frequency of class I HLA-specific precursors after T cell differentiation in an HLA-A2.1 positive environment. The HLA-A2.1 gene product was expressed at levels comparable to those of the endogenous H-2Db molecule in thymus, bone marrow, and spleen. By limiting dilution analysis, it was observed that the frequencies of CTLp in transgenic mice responding to the human alloantigens HLA-B7 or HLA-A2.2 were comparable to or lower than those in normal C57BL/6 mice, regardless of whether the Ag was presented on human or murine cells. Thus, expression of a human class I molecule in these animals did not result in an expansion of the number of CTLp specific for other human class I Ag. In addition, the frequency of HLA-A2.1-restricted, influenza specific CTLp was substantially lower than the frequency of H-2b restricted CTLp, indicating a poor utilization of HLA-A2.1 as a restricting element. Finally, the frequencies of CTLp for HLA-A2.1 expressed on syngeneic murine tumor cells were decreased significantly. Thus, expression of HLA-A2.1 in these animals appeared to induced tolerance to this Ag. Interestingly, however, these mice were not tolerant to the HLA-A2.1 molecule expressed on human cells. This indicates that the HLA-A2.1 associated epitopes expressed on murine and human cells differ and suggests that, under these circumstances, HLA-A2.1 acts as a restricting element for human nominal Ag. These results are discussed in the context of current models of T cell repertoire development.

Animals↗
PubMed1989

The occurrence of disulphide bonds in purified clathrin light chains.

Three forms of clathrin light chain contain two cysteine residues. These are the predominant brain-specific forms of LCa and LCb and the non-brain form of LCb. After purification in the absence of thiols they contain intramolecular disulphide bonds. The reduced and the oxidized forms show differences in electrophoretic mobility, explaining the variable and heterogeneous patterns observed on electrophoresis. Accessibility of the thiol groups in the free light chains is greater than when they are associated with the heavy chain. In contrast the cysteine residues of the clathrin heavy chain are completely inaccessible in the absence of denaturants and are not found in disulphide bonds. The antigenic properties of the oxidized and the reduced forms of the clathrin light chains are similar, as is their capacity to bind to the clathrin heavy chain. After isolation in the presence of 10 mM-iodoacetamide, the light-chain cysteine residues are fully alkylated. The results are consistent with the reduced form being the native state and the light-chain disulphide bonds an artifact of isolation.

Adrenal Glands↗
PubMed1989

HLA-B51 and HLA-Bw52 differ by only two amino acids which are in the helical region of the alpha 1 domain.

Genes encoding the serologically cross-reactive HLA-B51 and HLA-Bw52 molecules were isolated and the exons sequenced. HLA-B51 genes obtained from Caucasian and Oriental individuals were identical. HLA-Bw52 differs from HLA-B51 by four nucleotide substitutions in exon 2 encoding the alpha 1 domain. These comprise one isolated silent substitution in codon 23 and a cluster of three coding substitutions in codons 63 and 67. Amino acid substitutions of N----E at position 63 and F----S at position 67 are the only differences between HLA-B51 and HLA-Bw52 and these residues are postulated to form HLA-B51 specific epitopes. HLA-B51 could have been formed from HLA-Bw52 by the combination of a genetic exchange with HLA-B8 and a point mutation. Similarity of HLA-B51 and HLA-Bw52 with HLA-Bw58 suggest they also share a common ancestor.

Amino Acid Sequence↗
PubMed1989

Alien antigens return to the fold.

Theories rationalizing major histocompatibility complex (MHC) class I polymorphism and the high frequency of alloreactive T lymphocytes initiated the search for expression of genetically inappropriate MHC class I molecules by tumour cells. Many examples of such 'Alien Antigens' have been reported in the past fifteen years. Some were previously shown to result from a variety of serological artefacts. Recent papers describe structural and genetic analysis of two extensively studied and outstanding systems. In both cases the Alien Antigens are also artefacts, resulting from genetic heterogeneity in either mice or cell lines. No Alien Antigens have survived rigorous evaluation and they are unlikely to constitute a significant biological phenomenon. It is also unlikely that the successive experimental plagues that created the Alien Antigens are unique to this corner of tumour immunology. Sophisticated innovation in experimental systems makes much immunological investigation increasingly dependent on the genetic integrity of mice and cell lines.

Animals↗
PubMed1988

Function and polymorphism of human leukocyte antigen-A,B,C molecules.

Human class I major histocompatibility complex, human leukocyte antigen (HLA)-A,B,C molecules are peptide-binding proteins that present degraded fragments of antigens to cytotoxic T lymphocytes. HLA-A,B,C loci are highly polymorphic and their products are strong alloantigens. Comparison of the primary structure of 39 HLA-A,B,C molecules shows that variation is found at many positions in the extracellular domains (alpha 1, alpha 2, and alpha 3). Positions with high variability are concentrated in and around the peptide-binding groove formed by the alpha 1- and alpha 2-domains and defined by crystallographic analysis of HLA-A2. It is likely that the polymorphic differences serve to alter both the peptide-binding specificity and the interaction with T cell receptors. This in turn may result in differences in immune responsiveness, susceptibility, and resistance to disease, and in alloantigenicity.

Amino Acid Sequence↗
PubMed1988

Cytotoxic T lymphocyte-defined epitope differences between HLA-A2.1 and HLA-A2.2 map to two distinct regions of the molecule.

Hemi-exon shuffling and site-directed mutagenesis have been used to determine which amino acid differences between HLA-A2.1 and HLA-A2.2 alter the CTL-defined epitopes on these two molecules. Two genes were constructed that encode novel molecules in which the effect of amino acid differences at residues 9, 43, and 95, or at residue 156 could be separately evaluated. Using both human and murine CTL that were specific for either HLA-A2.1 or HLA-A2.2, four types of epitopes were identified: 1) epitopes that were insensitive to substitutions at either residues 9, 43, and 95, or residue 156 but were lost when all four positions were changed; 2) epitopes that were dependent on the residues 9, 43, 95, but not residue 156; 3) epitopes that were dependent on residue 156, but not amino acid residues 9, 43, and 95; and 4) epitopes that were dependent on residues 9, 43, and 95, as well as amino acid residue 156. Overall, there was a roughly equal distribution of clones recognizing each of these types of epitopes. Additional molecules were constructed by hemi-exon shuffling between the HLA-A2.2 and HLA-A2.3 genes, and by site-directed mutagenesis, to analyze the epitopes recognized by two HLA-A2.2/A2.1 cross-reactive murine CTL that do not recognize HLA-A2.3. Although the epitopes recognized by these CTL were unaffected by changes occurring at residues 9, 43, and 95, or at residues 149, 152, and 156 alone, simultaneous changes in both of these regions acted in concert to destroy the epitopes. Both of the CTL recognized epitopes that were lost when substitutions were made at residues 9, 43, 95, 149, and 152. The epitope recognized by one of the CTL was also destroyed by the substitution of residues 9, 43, 95, 152, and 156. Overall, these results indicate that residues 9, 43, and 95, as well as residues in the alpha-helical region of the molecule, are all capable of contributing to the definition of the epitopes recognized by HLA-A2.1- and HLA-A2.2-specific CTL. They further indicate that some epitopes can be mapped to a particular region of the molecule, whereas other epitopes are formed through a complex interaction of residues in distant regions of the molecule.

Amino Acids↗
PubMed1988

Structure of human clathrin light chains. Conservation of light chain polymorphism in three mammalian species.

Complementary DNAs (cDNA) encoding the brain and non-brain forms of the human clathrin light chains LCa and LCb have been isolated, sequenced, and compared with their homologues in cow and rat. The significant differences that distinguish LCa from LCb and the brain from non-brain forms show remarkable preservation in all three species. These features include the position and sequence of the brain-specific inserts, a totally conserved region of 22 residues near the amino terminus, the LCb-specific phosphorylation site, the heavy chain binding site, and a distinctive pattern of cysteine residues near the carboxyl terminus. Unorthodox sequences for translation initiation and polyadenylation are found for LCb contrasting with LCa which exhibits orthodox regulatory sequences. Small insertions in human LCa revealed a duplicated sequence of 13 residues that flank the 22-residue conserved region. Only the carboxyl-terminal copy of this sequence is present in LCb. All sequences are consistent with the heavy chain binding site comprising an alpha-helical central region of the light chains. The hydrophobic face of this helix, which is presumed to interact with the heavy chain, is highly conserved between LCa and LCb, whereas the hydrophilic face shows considerable divergence. To help define the carboxyl-terminal limit of the heavy chain binding region, the epitope recognized by the CVC.6 monoclonal antibody was localized to residues 192-208 of LCa with glutamic acid 198 being of most importance. The faithful preservation of clathrin light chain polymorphism in three mammalian species provides evidence supporting a functional diversification of the brain and non-brain forms of LCa and LCb.

Amino Acid Sequence↗
PubMed1988

Cell-cell adhesion mediated by CD8 and MHC class I molecules.

CD4 and CD8 are cell-surface glycoproteins expressed on mutually exclusive subsets of peripheral T cells. T cells that express CD4 have T-cell antigen receptors that are specific for antigens presented by major histocompatibility complex class II molecules, whereas T cells that express CD8 have receptors specific for antigens presented by MHC class I molecules (reviewed in ref. 1). Based on this correlation and on the observation that anti-CD4 and anti-CD8 antibodies inhibit T-cell function, it has been suggested that CD4 and CD8 increase the avidity of T cells for their targets by binding to MHC class II or MHC class I molecules respectively. Also, CD4 and CD8 may become physically associated with the T-cell antigen receptor, forming a higher-affinity complex for antigen and MHC molecules, and could be involved in signal transduction. Cell-cell adhesion dependent CD4 and MHC II molecules has recently been demonstrated. To determine whether CD8 can interact with MHC class I molecules in the absence of the T-cell antigen receptor, we have developed a cell-cell binding assay that measures adhesion of human B-cell lines expressing MHC class I molecules to transfected cells expressing high levels of human CD8. In this system, CD8 and class I molecules mediate cell-cell adhesion, showing that CD8 directly binds to MHC class I molecules.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Identification by site-directed mutagenesis of amino acid residues contributing to serologic and CTL-defined epitope differences between HLA-A2.1 and HLA-A2.3.

Site-directed mutagenesis of HLA-A2.1 has been used to identify the amino acid substitutions in HLA-A2.3 that are responsible for the lack of recognition of the latter molecule by the HLA-A2/A28 specific antibody, CR11-351, and by HLA-A2.1 specific CTL. Three genes were constructed that encoded HLA-A2 derivatives containing one of the amino acids known to occur in HLA-A2.3: Thr for Ala149, Glu for Val152, and Trp for Leu156. Three additional genes were constructed that encoded the different possible combinations of two amino acid substitutions at these residues. Finally, a gene encoding all three substitutions and equivalent to HLA-A2.3 was constructed. These genes were transfected into the class I negative, human cell line Hmy2.C1R. Analysis of this panel of cells revealed that recognition by the antibody CR11-351 was completely lost when Thr was substituted for Ala149, whereas substitutions at amino acids 152 and 156, either singly or in combination, had no effect on the binding of this antibody. The epitopes recognized by the allogeneic and xenogeneic HLA-A2.1 specific CTL clones used in this study were all affected by either one or two amino acid substitutions. Of those epitopes sensitive to single amino acid changes, none were affected by the substitution of Thr for Ala149, whereas all of them were affected by at least one of the substitutions of Glu for Val 152 or Trp for Leu156. Overall, amino acid residue 152 exerted a stronger effect on the epitopes recognized by HLA-A2.1 specific CTL than did residue 156. Of those epitopes affected only by multiple amino acid substitutions, double substitutions at residues 149 and 152 or at 152 and 156 resulted in a loss of recognition, whereas a mutant with substitutions at residues 149 and 156 was recognized normally. This reemphasizes the importance of residue 152 and indicates that residue 149 can affect epitope formation in conjunction with another amino acid substitution. These results are discussed in the context of current models for the recognition of alloantigens and in light of the recently published three-dimensional structure of the HLA-A2.1 molecule.

Amino Acid Sequence↗
PubMed1988

HLA-A and B polymorphisms predate the divergence of humans and chimpanzees.

Major histocompatibility complex (MHC) glycoproteins bind processed fragments of proteins and present them to the receptors of T lymphocytes. The extraordinary polymorphism of class I MHC molecules in man (HLA-A, B and C) and mouse (H-2 K, D and L) poses many questions concerning their diversification and evolution. Comparison of allelic sequences within a species suggests diversity is generated by the assortment of point mutations into varied combinations by mechanisms of recombination and gene conversion. We have now compared class I MHC alleles in two closely related species: humans (Homo sapiens) and chimpanzees (Pan troglodytes). Chimpanzee homologues of HLA-A, HLA-B and a non-classical gene have been identified. No features distinguishing human and chimpanzee alleles could be found. Individual HLA-A or B alleles are more closely related to individual chimpanzee alleles than to other HLA-A or B alleles. These results show that a considerable proportion of contemporary HLA-A and B polymorphism existed before divergence of the chimpanzee and human lines. The stability of the polymorphism indicates that hyper-mutational mechanisms are not necessary to account for HLA-A, B and C diversity.

Alleles↗
PubMed1988

Molecular cloning of bovine class I MHC cDNA.

Two cDNA cloned from a Hereford cow B cell line (BL-3) have allowed the determination of the complete coding region for two class I molecules encoded by the bovine MHC (BoLA). The predicted protein sequences have all the features expected of expressed class I molecules that present peptide Ag to cytotoxic T cells. Comparison with class I molecules from other species strongly suggests these cDNA are derived from different genes and provides evidence for the existence of a second expressed class I BoLA locus. The BoLA proteins show greater similarity to HLA than to H-2 molecules, correlating with the cross-reactions of W6/32 and other murine anti-HLA-A,B,C mAb with BoLA molecules. The basis for the W6/32 epitope and the preferential association of H-2 class I H chains with bovine beta 2-m is examined.

Amino Acid Sequence↗
PubMed1988

Identification of the phosphorylation sites of clathrin light chain LCb.

Clathrin light chains, LCa and LCb, are products of two closely related genes whose mRNAs undergo differential splicing to result in at least four different light chain isoforms. The physiological significance of clathrin light chain diversity remains unclear. To date, the only evidence for a functional distinction of LCa and LCb is the preferential phosphorylation of LCb, which takes place at serine residues and is mediated by coated vesicle-associated casein kinase II. As a first step toward determining the function of light chain diversity, we have mapped the in vitro phosphorylation sites on LCb. We use [32P]ATP to phosphorylate LCb within coated vesicles, followed by sequencing of 32P-labeled chymotryptic peptides thereof, to identify serine residues at positions 11 and 13 as the phosphorylation sites. We find that phosphorylation of LCb within coated vesicles can be inhibited by four monoclonal antibodies specific for different epitopes of the clathrin light chains.

Adenosine Triphosphate↗
PubMed1988

Comparison of the structure of HLA-Bw47 to HLA-B13 and its relationship to 21-hydroxylase deficiency.

Adrenal 21-hydroxylase deficiency is strongly associated with HLA-Bw47. This rare HLA allele and the HLA-B13 allele are both found in positive genetic linkage disequilibrium with HLA-A3, -Cw6, -DR7 and also display serological cross-reactivity. To investigate the relationship between these two alleles at the structural level, the nucleotide sequences of the HLA-B13 and HLA-Bw47 genes have been determined. They differ by 28 nucleotides, resulting in 14 amino acid substitutions: 5 in the alpha 1 domain, 8 in the alpha 2 domain, and 1 in the transmembrane region. Comparison of HLA-Bw47 nucleotide sequence with other HLA-B sequences shows a segment of 228 bp identical with B44 in the alpha 1 domain and a segment of 218 bp identical with B27 in the alpha 2 domain, but only a 91 bp segment of identity with B13 in the alpha 1 domain. The complex pattern of substitutions and their degree of divergence indicate that HLA-B13 and HLA-Bw47 alleles are not related by a simple mutational event.

Adrenal Hyperplasia, Congenital↗