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

108

When human class I cDNA clones containing coding sequences are used to probe genomic DNA, [15][16][17][18][19][20] fragments, each containing a complete class I gene or pseudogene, are seen. Identification of which genomic DNA segments encode the HLA-A and -B antigens has to date required transfection of mouse L cells with cloned class I genes or analysis of HLA loss mutants. In this report we show that under highstringency conditions, probes constructed from the 3'-untranslated region can be used to specifically identify the segments of DNA that encode the HLA-A and -B antigens in the human lymphoblastoid cell line 721. Examination of DNA from unrelated individuals indicates that these probes are locus specific and will permit identification of HLA-A and -B genes in the population.The definition of the HLA system arose from the discovery of leukocyte agglutinins in the sera of polytransfused individuals (1) and multiparous women (2, 3). Two antigenic groups were initially described; the group Four, or HLA-B, and LA, or HLA-A. As more sera were described, it was found that the antigens fell into two series, HLA-A and -B, each of which behaved as if controlled by a set of alleles at two closely linked loci (4). Today, >20 HLA-A and >40 HLA-B alleles have been described (5). As well as providing a major barrier to transplantation, the HLA antigens are responsible for restriction of T-cell-mediated cytotoxicity to virus-infected target cells (reviewed in ref. 6).Both HLA-A and -B antigens are integral membrane glycoproteins of about 44,000 daltons expressed on the cell surface of most tissues in noncovalent association with p2-microglobulin (reviewed in ref. 7). Protein sequence analysis of products of the HLA-A and -B loci shows overall homology of 86% (8). However, few of the differences appear to be locus specific. Comparison of the protein sequences of HLA-A2 and -B7 shows that most of the differences between these two proteins are found clustered in three areas of their NH2-terminal regions. Between amino acid residues 65 and 80, homology drops to 30% and from residues 105-116 and 177-194, 50% homology is seen. However, these areas are also poorly conserved between the HLA-A2 and HLA-A28 heavy chains (9). Moreover, residues that distinguish HLA-B7 from IHLA-A2 are found in another -A allele, HLA-A28.

mentioning

confidence: 99%

Isolation of HLA locus-specific DNA probes from the 3'-untranslated region.

Koller

Sidwell

DeMars

et al. 1984

108

“…However, it is difficult to assess the relevance of such an amino acid substitution in view of the observation that a free cysteine occupies, for example, a similarly polymorphic site in the HLA-Cw3 sequence (position 9). In contrast, the appearance of a serine in position 131 is likely to have a more profound effect on the conformation of HLA-B27, because this nonconservative replacement occurs at a site invariably occupied by a species-associated arginine residue in human and lysine in mice (22). The species specificity of this residue is confirmed in all murine class I gene sequences so far known, which include H-2Kq, H-2Kwa (see ref.…”

Section: Resultsmentioning

confidence: 92%

“…4). The occupied by a potentially reactive cysteine that might influence the intermolecular interactions of HLA-B27 due to its location in an area thought to mediate the binding of monoclonal antibodies as well as of alloantibodies (22). However, it is difficult to assess the relevance of such an amino acid substitution in view of the observation that a free cysteine occupies, for example, a similarly polymorphic site in the HLA-Cw3 sequence (position 9).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Complete sequence of HLA-B27 cDNA identified through the characterization of structural markers unique to the HLA-A, -B, and -C allelic series.

Szöts¹,

Riethmüller²,

Weiß³

et al. 1986

Antigen HLA-B27 is a high-risk genetic factor with respect to a group of rheumatoid disorders, especially ankylosing spondylitis. A cDNA library was constructed from an autozygous B-cell line expressing HLA-B27, HLA-Cwl, and the previously cloned HLA-A2 antigen. Clones detected with an HLA probe' were isolated and sorted into homology groups by differential hybridization and restriction maps. Nucleotide sequencing allowed the unambiguous assignment of cDNAs to HL4-A, -B, and -C loci. The HLA-B27 mRNA has the structural features and the codon variability typical of an HLA class I transcript but it specifies two uncommon amino acid replacements: a cysteine in position 67 and a serine in position 131. The latter substitution may have functional consequences, because it occurs in a conserved region and at a position invariably occupied by a species-specific arginine in humans and lysine in mice. The availability of the complete sequence of HLA-B27 and of the partial sequence of HLA-Cwl allows the recognition of locus-specific sequence markers, particularly, but not exclusively, in the transmembrane and cytoplasmic domains.

“…Previously, we reported the production and characterization of an antibody to one of the most hydrophilic peptide regions of HLA-B7 antigen (7). These studies demonstrated that this region of the antigen did not encode for an allorecognition site and the conformation of this region was highly dependent upon the presence of bound A2-microglobulin.We report here on the characteristics of an antiserum produced to a synthetic peptide corresponding to residues 61-83 of the HLA-B7 antigen heavy chain amino acid sequence, which represents a region of high structural variability when compared to other class I antigen amino acid sequences (1,8,9). …”

mentioning

confidence: 99%

Human major histocompatibility complex class I antigens: residues 61-83 of the HLA-B7 heavy chain specify an alloreactive site.

Walker¹,

Ketler²,

Houghten³

et al. 1985

A chemically synthesized peptide (Asp-ArgAsn-Thr-Gln-Ile-Tyr-Lys-Ala-Gln-Ala-Gln-Thr-Asp-ArgGlu-Ser-Leu-Arg-Asn-Leu-Arg-Gly), homologous to residues 61-83 of the HLA-B7 heavy chain, induced antibodies that specifically recognized the HLA heavy chain-P2-microglobulin complex and the free heavy chain of the HLA-B7 antigen. These antibodies specifically immunoprecipitated the HLA-B7 (-microglobulin complex solubilized from human lymphoblastoid cells by nonionic detergents and reacted with free HLA-B7 heavy chains in blots on nitrocellulose. These observations suggest that the antigenic conformation of this region of the HLA-B7 molecule is independent of the presence of fmicroglobulin and that amino acid residues 61-83 mimic an alloreactive site expressed by the HLA-B7 antigen.Human class I histocompatibility antigens are a set of highlypolymorphic integral membrane glycoproteins consisting of two noncovalently associated polypeptides of Mr 45,000 and Mr 12,000. The smaller component light chain has been identified as the common serum protein f3A-microglobulin, which lacks allotypic determinants and is encoded on chromosome 15, which is outside the major histocompatibility complex (1). The Mr 45,000 heavy chain has been shown to contain all of the structural polymorphism associated with class I antigens and is therefore responsible for the serologically defined polymorphism of HLA-A, -B, and -C antigens (2). Although data obtained from protein amino acid sequence studies (3) and DNA sequence analyses (4-6) have answered many of the questions pertaining to the overall structure and molecular organization of these antigens, one of the key issues that remains to be resolved is the localization of the alloreactive site(s) in the HLA-A, -B heavy chain amino acid sequence. We have attempted to answer this question by producing antisera to synthetic peptides selected from the amino acid sequence of HLA-B7 antigen (7) followed by testing the ability of these antisera to bind to the native heavy chain-f32-microglobulin complex as well as the isolated heavy chain of class I antigens. Previously, we reported the production and characterization of an antibody to one of the most hydrophilic peptide regions of HLA-B7 antigen (7). These studies demonstrated that this region of the antigen did not encode for an allorecognition site and the conformation of this region was highly dependent upon the presence of bound A2-microglobulin.We report here on the characteristics of an antiserum produced to a synthetic peptide corresponding to residues 61-83 of the HLA-B7 antigen heavy chain amino acid sequence, which represents a region of high structural variability when compared to other class I antigen amino acid sequences (1,8,9). MATERIALS AND METHODSCell Lines. The human B-lymphoblastoid cell lines used in this study were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, and gentamycin at 50 ,g/ml.Isolation of Glycoprotein. Glycoproteins were isolated from the cell line GM3107 or LG-2 by affini...