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Biomedical subjects

P Parham

Publications and source records attributed to P Parham.

At least 73 records · Page 4Linked to original sources
PubMed1998

The influence of exogenous peptide on beta2-microglobulin exchange in the HLA complex: analysis in real-time.

We used an optical biosensor to determine the relative binding affinity of peptides to purified HLA class I molecules. In this assay we monitor beta2-microglobulin (beta2m) exchange within the HLA-A2 molecule, whereby native beta2m in the complex is replaced by beta2m immobilized at the surface of the biosensor. Quantitative kinetic measurements permit us to obtain association rate (kass), dissociation rate (kdiss) and affinity constants (KA) for the beta2m exchange reaction, alone, (control) and in the presence of exogenous peptide. We tested a panel of six peptides which had been designed and synthesized with an HLA-A2 binding motif, and had also been tested by the T2-cell binding assay, along with control peptides. The biosensor results demonstrate that exogenous peptide influences the dynamics of beta2m exchange in a sequence-specific manner. Five of six peptides increased the association rate, decreased the dissociation rate, and significantly increased the affinity (KA=1. 55-1.88x10(9) M-1) of HLA-A2 for immobilized beta2m compared with the control (KA =1.14+/-0.04x10(9)M-1), demonstrating stabilization of the complex. One peptide was unable to stabilize the complex, as also shown in the T2 binding assay. However, analysis of peptide sequences demonstrated that the HLA-A2 secondary motif as well as primary motif residues are required for HLA-A2 stabilization. Further experiments demonstrated that beta2m exchange alone cannot stabilize the HLA class I complex at the cell surface until a peptide of sufficient binding affinity is bound. Hence kinetics equal to or below the control values in our biosensor assay probably represent an unstable complex in vivo. Unlike other methods described for the analysis of peptide stabilization, this approach is significantly faster, provides full kinetic analysis, and is simpler, since it requires no labeling of peptides. Furthermore, this may have important implications in the assessment of peptide vaccines.

Biosensing Techniques↗
PubMed1998

beta2-Microglobulin in neotropical primates (Platyrrhini).

Nucleotide sequences for the three exons of the beta2-microglobulin (beta2m) gene (B2m) were determined for 135 animals representing 37 species and all 16 genera of neotropical primates (Platyrrhini). Twenty-eight different nucleotide sequences, encoding for 26 different proteins, were obtained. In comparison with those of other primate species, the beta2-microglobulins of the Platyrrhini form a distinct clade. Individual genera of neotropical primates have distinctive B2m sequences, but within a genera species can have either the same or different B2m sequences. B2m polymorphism was found within three of the species sampled: Callicebus personatus, Saguinus midas, and Aotus azarae. Of these only the polymorphism in A. azarae has an effect upon the mature, functional beta2m protein: residue 4 being either alanine or threonine. The A. azarae B2m allele encoding alanine at position 4 is shared with another species of Aotus (A. infulatus). In pairwise comparison the mature beta2m proteins of neotropical primates differ by 1-9 amino acid substitutions which can occur at 18 positions within the sequence. The substitutions are distributed throughout the primary structure but are more commonly found in loops rather than beta strands of the tertiary structure. Of 17 residues of beta2m which hydrogen-bond with the class I heavy chain in human MHC class I molecules, 13 are conserved in the neotropical primates. The overall pattern of sequence variation in the B2m genes of the Platyrrhini is consistent with an evolution by successive selectively neutral events.

Animals↗
PubMed1998

Comparative gene assignment in Ateles paniscus chamek (Platyrrhini, Primates) and man: association of three separate human syntenic groups and evolutionary considerations.

Regional assignment of eight markers to chromosome 2 of Ateles paniscus chamek (APC) confirmed a syntenic association similar to human (HSA) 12q + 14q + 15q. Three HSA 12q markers (RAP1B, PAH and ALDH2) were allocated to a shortest region of overlap (SRO) in APC 2p and found to be syntenic to other HSA 12q markers (PEPB and TCF1). Five HSA 14q markers (CTLA, PAX9, NSP, FOS and CHGA) were allocated to APC 2q and found to be syntenic to other HSA 14q markers (NP, TGM1, and CALM1) and to four HSA 15q markers (THBS1, B2M, HEXA and MPI) but dissociated from markers close to HSA 14qter (CKB) and HSA 15qter (FES-IDH2). Karyotypic comparisons showed an evident homoeology between APC 2p and HSA 12q while APC 2q was similar to an HSA 14qter::HSA 15qter fusion product. Comparative gene mapping data show that the HSA 14q + HSA 15q syntenic association is an ancestral mammalian gene cluster that has been maintained in several primate taxa. Conversely, in Ateles, it has been further associated with HSA 12q while, in Hominoids and Cebus, it has been independently dissociated into two separate syntenic groups, similar to HSA 14q and HSA 15q.

Animals↗
PubMed1998

CK-1, a putative chemokine of rainbow trout (Oncorhynchus mykiss).

Chemokines are small inducible proteins that direct the migration of leukocytes. While chemokines are well characterised in mammals, they have yet to be identified in fish. We have isolated a cDNA clone from rainbow trout (Oncorhynchus mykiss) which encodes a protein (CK-1) having structural features typical of chemokines. Amino-acid residues that define the beta-chemokines of mammals are conserved in CK-1, including the paired cysteine motif, CC. Further similarities are shared with the C6 subfamily of beta-chemokines. In contrast, the organisation of the CK-1 gene is closer to that of mammalian alpha-chemokine genes than beta-chemokine genes. The CK-1 gene is present in all four salmonid species examined and the nucleotide sequences of the exons are highly conserved. CK-1 has characteristics in common with mammalian alpha and beta-chemokine genes, suggesting that this salmonid chemokine gene preserves traits once present in the ancestral chemokine gene from which modern mammalian chemokine genes evolved.

Animals↗
PubMed1997

Conserved and variable residues within the Bw4 motif of HLA-B make separable contributions to recognition by the NKB1 killer cell-inhibitory receptor.

Allotypes from four divergent HLA-B families (B8, B15, B16, and B27) were compared for their inhibition of cytolysis by NK cells expressing the NKB1 receptor. Allotypes differing solely at the Bw4/Bw6 region were examined as were a more divergent subset of B15 allotypes. The capacity to interact with NKB1 correlated precisely with possession of a Bw4 sequence motif at residues 77-83, whereas no correlation was made with the peptide-binding specificities of two Bw4 and four Bw6 allotypes of the B15 family. HLA-B allotypes having four different Bw4 motifs were examined and all interact with NKB1. In contrast, HLA-A allotypes, which have a Bw4 motif identical with one of those present in HLA-B, do not. Mutation at leucine 82 and arginine 83, the residues common to Bw4 motifs, shows they contribute to NKB1 interaction but are not essential. Three types of polymorphism are implicated in formation of the ligand recognized by NKB1: ones shared by Bw4 motifs; ones distinguishing Bw4 motifs; and ones outside the Bw4/Bw6 region that distinguish HLA-B from HLA-A.

Binding Sites↗
PubMed1997

Natural inactivation of a common HLA allele (A*2402) has occurred on at least three separate occasions.

HLA-A*2402 is common and widely distributed in human populations. Several individuals were identified who type genotypically for A*2402, but are serologically null for the HLA-A24 Ag. Sequencing and transfection of genomic DNA fragments containing null and wild-type A*2402 alleles, and the related A*2301 allele, revealed three different null alleles (A*2409N, A*2411N, and A*2402(low)), each of which differs from A*2402 by a single nucleotide change within the 6.7-kb sequence. The A*2301 and A*2402 sequences differ by no substitutions additional to those previously determined for the 1.1-kb cDNA. In exon 4, A*2409N has an in-frame stop codon, while A*2411N has a nucleotide insertion that alters the reading frame, causing premature termination. A*2402(low) has a nucleotide substitution near the splice acceptor site for intron 2 that impairs the production of correctly spliced mRNA. For A*2409N and A*2411N, mRNA is undetectable by Northern analysis, whereas A*2402(low) produces a low level of mRNA and a concomitant amount of normal A*2402 protein at the cell surface. The protein expressed from the A*2402(low) allele is sufficient to stimulate an alloreactive T cell response. On a background of unexpected sequence homogeneity, the single nucleotide changes in the A*2409N, A*2411, and A*2402(low) alleles have dramatic effects upon gene expression and are of likely importance for HLA matching in clinical transplantation. Segregation of at least three independently inactivated A*2402 alleles in human populations raises the possibility that loss of A*2402 may be the result of natural selection.

Alleles↗
PubMed1997

Polymorphism in the alpha 1 helix of the HLA-B heavy chain can have an overriding influence on peptide-binding specificity.

Previously, we reported overlap in the repertoires of peptides endogenously bound by a group of HLA-B allotypes related to HLA-B7. Extending such analysis to four members of the B17 family and seven members of the B15 family shows that allotypes that share sequence identity in the alpha 1 helix of the class I heavy chain possess markedly similar peptide-binding specificities. Members of the B17 family share a preference for peptides with serine, threonine, or alanine at position 2 and aromatic residues at the carboxyl terminus. Strikingly, the presence of a segment of the B17 alpha 1 helix in B*1516 and B*1517 confers the B17-like peptide-binding motif. The strong influence of natural variation in the alpha 1 helix is exemplified by the differences in peptide-binding specificity of B15 allotypes related by conversion events that replaced segments of the alpha 1 helix. In contrast, evolutionary changes that are confined to the alpha 2 domain confer less dramatic change. They do not perturb the primary anchors of the peptide-binding motif but can modulate the specificity through development and diversification of secondary anchors. Our results, in combination with those obtained previously for other HLA-B allotypes, suggest a general trend whereby polymorphism in the alpha 1 helix is the overriding influence on peptide-binding specificity of HLA-B allotypes, while amino acid substitutions in the alpha 2 domain play a more modulatory role.

Alleles↗
PubMed1997

Cw*1701 defines a divergent african HLA-C allelic lineage.

The complete sequence of a new HLA-C allele, Cw*1701, was determined from a South African Zulu individual. Unique features that distinguish Cw*1701 from other HLA-C alleles include multiple point substitutions and an 18 nucleotide insertion in exon 5, which encodes the transmembrane domain. In a phylogenetic analysis of HLA-C sequences, Cw*1701 forms a third, distinct allelic lineage. A comparison of the transmembrane domain of Cw*1701 with other HLA-B and -C alleles reveals that duplications and deletions have been common in the evolution of these loci. A polymerase chain reaction based typing method was used to determine the distribution of this unusual allele in human populations. In contrast to the other two lineages of HLA-C alleles, the Cw*17 lineage is found at high frequencies only in populations of African descent. In addition, the HLA-B/Cw*17 haplotype diversity is higher in Africa.

Africa↗
PubMed1997

Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors.

The expression of KIR and CD94:NKG2 receptors was determined for more than 100 natural killer (NK) cell clones obtained from two blood donors who differ in their HLA class I and KIR genes. More than 98% of the clones were inhibited by individual autologous class I allotypes, and every clone was inhibited by the combination of autologous allotypes. The patterns of inhibition correlate with expression of inhibitory receptors of defined specificity. One donor possesses three class I ligands for KIR, and a majority of NK cells use KIR as their inhibitory receptor; the second donor possesses only a single ligand for KIR, and a majority of NK cells use the more broadly reactive CD94:NKG2a as their inhibitory receptor. Because of these differences, the first donor has subpopulations of NK cells that kill cells of the second donor, whereas the NK cells of the second donor are universally tolerant of cells from the first donor.

Antigens, CD↗
PubMed1997

Human diversity in killer cell inhibitory receptor genes.

The presence and expression of killer inhibitory receptor (KIR) and CD94:NKG2 genes from 68 donors were analyzed using molecular typing techniques. The genes encoding CD94:NKG2 receptors were present in each person, but KIR gene possession varied. Most individuals expressed inhibitory KIR for the three well-defined HLA-B and -C ligands, but noninhibitory KIR genes were more variable. Twenty different KIR phenotypes were defined. Two groups of KIR haplotypes were distinguished and occurred at relatively even frequency. Group A KIR haplotypes consist of six genes: the main inhibitory KIR, one noninhibitory KIR, and a structurally divergent KIR. Allelic polymorphism within five KIR genes was detected. Group B comprises more noninhibitory KIR genes and contains at least one additional gene not represented in group A. The KIR locus therefore appears to be polygenic and polymorphic within the human population.

Antigens, CD↗
PubMed1997

HLA class I genotyping by cDNA sequence of a Vietnamese family expressing a weak B46 antigen.

Serological heterogeneity in HLA-B46 antigens has been described. Previous studies have identified B*4601 as the allele encoding the "strong" B46 antigen found in Chinese populations. Serological characterization of a Vietnamese family revealed a "weak" B46 antigen. Complementary DNA for the HLA-A, B and C alleles of three family members were cloned and the coding regions sequenced. The allele encoding the weak B46 antigen has the same coding sequence as B*4601, demonstrating that the antigenic differences are not due to polymorphism in the amino acid sequence of the HLA-B heavy chain. The recently described HLA-B*1525 and HLA-Cw*0403 alleles were also found to segregate in this Vietnamese family.

Alleles↗
PubMed1997

A new HLA-B44 variant (B44BO [B*4408]) identified by serology.

Using HLA serology, we detected a new variant of HLA-B44- B44BO- in two families. This antigen reacts with B44 antisera and is negative with over one-third of B12 (B44, B45) sera but reacts with 50% antisera with a B62 component, especially if they contain anti-B57. The variant, B*4408, differs from the common B*4402 by 4 nucleotide substitutions in exon 2: 193, 206 and 209, which produce changes in the the alpha 1 domain at positions 41, 45 and 46 (TKE in B*4402 and AMA in B44BO); and nucleotide 213, a silent substitution. At each of these positions, B*4408 is identical to B*46 B*57 and may B*15 alleles. As anticipated from its predicted iso-electric point (5.71), one-dimensional isoelectric focusing studies showed that B44BO focuses at the same position as B*4402. The sequence and serological reactivity of this rare antigen allowed the identification of two likely epitopes shared by two different groups of HLA-B antigens.

Alleles↗
PubMed1997

Episodic evolution and turnover of HLA-B in the indigenous human populations of the Americas.

Nucleotide sequences were determined for the HLA-A, B and C alleles of three populations of Amerindians: the Havasupai tribe from North America, and the Guarani and Kaingang tribes from South America. All 15 Havasupai alleles are found in Eastern Hemisphere populations, whereas the Guarani and Kaingang each have six alleles that appear to be present only in the Western Hemisphere. Nine of the "new" alleles come from HLA-B, one comes from HLA-A and one from HLA-C: ten appear to be the result of recombination and one the result of point substitution. Of the 14 Guarani alleles and 16 Kaingang alleles, only four are held in common. Despite their differences, the three tribes possess comparable numbers of HLA class I alleles, revealing a trend for "allele turnover", in which new alleles tends to supplant older alleles rather than supplement them. Although many new HLA-B alleles have been produced in Latin America, their net effect has been to differentiate populations, not to increase allele diversity within a population. From sequence comparisons, the Amerindian subset of HLA class I allotypes appears to cover the overall ranges of peptide binding specificity, natural killer-cell interactions, and CD8 interactions, that are found in all HLA class I. The recombinations that produced the new alleles of the Kaingang and Guarani class I are predicted to have modulated these functional properties rather than radically change them. Exchange of Bw4 and Bw6 motifs by recombination are noticeably absent in the events forming new alleles in America, whereas they have been the most common of recombinations elsewhere.

Alleles↗
PubMed1997

Interactions of HLA-B*4801 with peptide and CD8.

Functional properties of the B*4801 allotype were investigated using HLA class I-deficient 221 cells transfected with B*4801 cDNA. From pool sequence analysis of endogenously bound peptides, B*4801 was shown to select for nonamer peptides having glutamine or lysine at position 2 and leucine at the carboxyl-terminus. In an in vitro cell-cell binding assay, B*4801 binds CD8 alpha homodimers weakly due to the presence of a threonine residue at position 245 in the alpha 3 domain. A mutant B*4801 molecule in which alanine replaces threonine 245, binds CD8 alpha homodimers at levels comparable to those of other HLA class I allotypes. Despite the low affinity of B*4801 for CD8 alpha, alloreactive T-cells that recognize B*4801 molecules expressed by the 221 transfectant are inhibited by anti-CD8 monoclonal antibodies. Analysis of 25 B*48-expressing individuals from various populations showed threonine 245 was encoded by every B*48 allele.

Alleles↗