[Show abstract][Hide abstract] ABSTRACT: The human major histocompatibility complex (MHC), or human leukocyte antigen (HLA) region, encompasses over 4 Mb of DNA on the short arm of chromosome 6 and is traditionally divided into the class I, class II and class III regions. The MHC has now been entirely sequenced and ~220 genes have been defined of which ~62 are in the class III region. It is becoming clear that many of the latter encode proteins that are likely to be involved in the immune and inflammatory responses. The MHC is known to contribute to a large number of immune-related disorders including insulin dependent diabetes mellitus, rheumatoid arthritis, common variable immunodeficiency and IgA deficiency and there is growing evidence that genes within the class III region are important in determining susceptibility to many of these complex conditions. Genes in the class III region have also been implicated in a number of non-immune-related diseases such as congenital adrenal hyperplasia and sialidosis. Now that the full gene content of the class III region is known the stage is set for the identification and characterisation of candidate disease genes, which will allow greater understanding of the causes of many MHC-linked diseases and thus aid the development of improved treatments.
Frontiers in Bioscience 09/2001; 6:D914-26. · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The three major histocompatibility complex (MHC)-linked hsp70s have been screened for variation in their 28 kDa C-terminal regions by direct nucleotide sequencing of the corresponding DNA fragments. No amino acid variation was detected in the major heat-inducible hsp70 (encoded by hsp70-1 and hsp70-2), although previously unreported silent mutations were identified in all three of the MHC-linked hsp70 genes. A novel coding polymorphism, a G to A transition, was identified at nucleotide 2763 of hsp70-hom (hom-2763). This dimorphism results in a glutamic acid to lysine alteration at position 602 in the C-terminal domain of hsp70-hom. The frequencies of the A-2763 and G-2763 alleles were calculated to be 27% and 73%, respectively. The hom-2763 dimorphism was characterised in 81 HLA-homozygous cell lines using an ARMS-PCR assay and A-2763 was found to be in strong linkage disequilibrium with DRB1*04 (Pc=1.31 x 10(-7), following Bonferoni's correction). Analysis of 60 rheumatoid arthritis (RA) families, each with an affected sib-pair, revealed an association between hsp70-hom A-2763 and RA using both the transmission disequilibrium test (TDT) and the transmission to sib-pair (Tsp) test (P=0.0038 and P=0.013, respectively). This association may be due to linkage disequilibrium with HLA-DR alleles, but could represent an additional risk factor for RA in the MHC class III region.
[Show abstract][Hide abstract] ABSTRACT: We previously demonstrated that lysis of tumor cells that express Hsp70, the highly stress-inducible member of the HSP70 family, on their plasma membrane is mediated by natural killer (NK) cells. Here, we studied the effects of different proteins of the HSP70 family in combination with interleukin 2 (IL-2) on the proliferation and cytotoxic activity of human NK cells in vitro. Proliferation of NK cells was significantly enhanced by human recombinant Hsp70 (rHsp70) and to a lesser extent by rHsp70homC, the recombinant C-terminal peptide-binding domain derived from Hsp70hom, but not by the constitutive Hsc70 or DnaK, the Escherichia coli analogue of human Hsp70. Even rHsp70 protein alone moderately enhances proliferation and cytolytic activity of NK cells, thus indicating that the stimulatory effect is not strictly dependent on IL-2. NK cells stimulated with rHsp70 protein also exhibit an increased secretion of interferon gamma (IFN-gamma). The phenotypic characterization of NK cells with specificity for Hsp70-expressing tumor cells revealed a CD16dim/CD56bright and increased CD57 and CD94 expression. The cytolytic activity of NK cells also was significantly reduced when a CD94-specific antibody or rHsp70 was added directly before the cytotoxicity assay, whereas other antibodies directed against CD57 and major histocompatibility complex class I molecules or Hsp70 proteins, including Hsc70 and DnaK, did not affect the NK-mediated killing. However, long-term incubation of NK cells with rHsp70 protein enhances not only the proliferative but also the cytolytic response against Hsp70-expressing tumor cells. Our results indicate that the C-terminal domain of Hsp70 protein affects not only the proliferative but also the cytolytic activity of a phenotypically distinct NK cell population with specificity for Hsp70-expressing tumor cells. 1999 International Society for Experimental Hematology.
[Show abstract][Hide abstract] ABSTRACT: The Neu1 locus, in the S region of the murine histocompatibility-2 complex, regulates the sialic acid content of several liver lysosomal enzymes. Three alleles, Neu1a, Neu1b, and Neu1c, have been described on the basis of differential sialylation of the enzyme liver acid phosphatase. The Neu1a allele occurs in a small number of mouse strains, e.g., SM/J and is associated with sialidase deficiency. We recently described G9, a sialidase gene in the human major histocompatibility complex (Milner et al. (1997) J. Biol. Chem., 272, 4549-4558), and we now report the characterization of the equivalent gene in mouse. The protein product of the murine G9 gene is 409 amino acids in length and is 83% identical to its human orthologue. Expression of the murine G9 protein in insect cells has confirmed that it is a sialidase, with optimal activity at pH 5. To elucidate the basis of sialidase deficiency in mouse strains carrying the Neu1a allele, we have sequenced the G9 coding regions from mice carrying the three Neu1 alleles and hence defined the amino acid sequence characteristic of each allotype. Of particular interest is a Leu-209 to Ile mutation that is unique to the Neu1a allotype and is associated with reductions in sialidase activity of approximately 68% and approximately 88% compared to the Neu1b and Neu1c allotypes, respectively, when these three protein variants are expressed in insect cells. Additional factors, such as differential expression, may also influence the activities of the Neu1 allotypes in vivo. We have observed that the level of G9 mRNA is substantially reduced in mice carrying the Neu1a allele compared to the Neu1b (85-95% reduction) and Neu1c (approximately 70% reduction) alleles.
[Show abstract][Hide abstract] ABSTRACT: Mammalian sialidases are important in modulating the sialic acid content of cell-surface and intracellular glycoproteins. However, the full extent of this enzyme family and the physical and biochemical properties of its individual members are unclear. We have identified a novel gene, G9, in the human major histocompatibility complex (MHC), that encodes a 415-amino acid protein sharing 21-28% sequence identity with the bacterial sialidases and containing three copies of the Asp-block motif characteristic of these enzymes. The level of sequence identity between human G9 and a cytosolic sialidase identified in rat and hamster (28-29%) is much less than would be expected for analogous proteins in these species, suggesting that G9 is distinct from the cytosolic enzyme. Expression of G9 in insect cells has confirmed that it encodes a sialidase, which shows optimal activity at pH 4.6, but appears to have limited substrate specificity. The G9 protein carries an N-terminal signal sequence and immunofluorescence staining of COS7 cells expressing recombinant G9 shows localization of this sialidase exclusively to the endoplasmic reticulum. The location of the G9 gene, within the human MHC, corresponds to that of the murine Neu-1 locus, suggesting that these are analogous genes. One of the functions attributed to Neu-1 is the up-regulation of sialidase activity during T cell activation.
[Show abstract][Hide abstract] ABSTRACT: Experimental allergic orchitis (EAO) is an autoimmune disease of the testis that is controlled by multple genes. The use of recombinant mouse strains has defined the map position of the H-2-associated locus controlling disease susceptibility, Orch-1, within the H-2S/H-2D interval. Over the last few years the definition of the structural organization of the C4-H-2D segment and identification of the recombination sites of the various intra-H-2 recombinations has reduced the map position of Orch-1 to the Hsp70.1-G7 interval. Three Hsp70 genes, Hsp70.1, Hsp70.3, and Hsc70t, and the genes G7b and G7a are located in this segment of DNA. In order to investigate whether Hsc70t is a suitable candidate for Orch-1 we have compared the sequence of the gene from a susceptible and a resistant haplotype.
[Show abstract][Hide abstract] ABSTRACT: In the last year progress has been made towards elucidating the roles of the MHC gene products in autoimmunity. A major advance has been the recent determination of the crystallographic structure of the human MHC class II molecule, which will be invaluable in delineating the minimum structural requirements for peptides that induce autoimmune disease. In addition, the use of animal models and transgenic mouse technology is continuing to increase our understanding of the involvement of the MHC gene products in immunopathogenesis.
Current Opinion in Immunology 01/1994; 5(6):887-93. DOI:10.1016/0952-7915(93)90101-W · 7.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The class III region of the human major histocompatibility complex spans approx. 1.1 Mbp on the short arm of chromosome 6 and is known to contain at least 36 genes. The complete nucleotide sequence of a 3.4 kb mRNA from one of these genes, G9a (or BAT8), has been determined from cDNA and genomic DNA clones. The single-copy G9a gene encodes a protein product of 1001 amino acids with a predicted molecular mass of 111,518 Da. The C-terminal region (residues 730-999) of the G9a protein has been expressed in Escherichia coli as a fusion protein with the 26 kDa glutathione S-transferase of Schistosoma japonicum (Sj26). The fusion protein has been used to raise antisera which, in Western-blot analysis, cross-react specifically with an intracellular protein of approx. 98 kDa. The function of the G9a protein is unknown. However, comparison of the derived amino acid sequence of G9a with the protein databases has revealed interesting similarities with a number of other proteins. The C-terminal region of G9a is 35% identical with a 149 amino acid segment of the Drosophila trithorax protein. In addition the G9a protein has been shown to contain six contiguous copies of a 33-amino acid repeat. This repeat, originally identified in the Notch protein of Drosophila and known as the cdc10/SW16 or ANK repeat, is also found in a number of other human proteins and may be involved in intracellular protein-protein interactions.
[Show abstract][Hide abstract] ABSTRACT: The restriction enzyme Pstl showed a two-allele restriction fragment length polymorphism (RFLP) marker when DNA polymorphism of the gene G8, a novel major histocompatibility complex (MHC) class III gene, was screened. The gene G8 is located ca. 4 kb centromeric of the heat-shock protein 70 (HSP70) gene cluster. A more detailed mapping indicated that the polymorphic restriction site is actually located in the coding region of the adjacent HSP70-2 gene, where it has been earlier reported to cause a silent mutation. To estimate the frequency of this polymorphism in the normal population, 95 blood donors were analyzed: The gene frequency of the 8.5 kb (designated 'L') allele was 0.45 and that of the 8.65 kb ('U') allele 0.55. However, when 19 families with patients suffering from celiac disease were studied, the gene frequencies in the affected haplotypes (L = 0.76, U = 0.24) significantly deviated from those observed in the normal population and in the non-affected MHC haplotypes of these families (L = 0.48, U = 0.52). However, the association results from a strong association between the allele 'L' and the MHC haplotype HLA B8 DR3, a known suspectibility marker of celiac disease. Only one patient, in fact, was negative for the well-established class II haplotype markers DR3 or DR7. The data therefore confirm the crucial role of MHC class II in suspectibility to celiac disease, but due to a strong linkage disequilibrium within MHC the role of MHC class III genes in disease associations can not be ruled out.
[Show abstract][Hide abstract] ABSTRACT: The human major histocompatibility complex (MHC), on the short arm of chromosome 6, represents one of the most extensively characterised regions of the human genome. This ∼4 Mb segment of DNA contains genes encoding the polymorphic MHC class I and class II molecules which are involved in antigen presentation during an immune response. Recently the whole of the MHC has been cloned in cosmids and/or yeast artificial chromosomes (YACs) and large portions have been characterised for the presence of novel genes. Many unrelated genes, both housekeeping and tissue specific, have been identified and the gene density in some regions is now approaching one gene every few kilobases. Some of the novel genes encode proteins involved in the intracellular processing and transport of antigens that are presented by MHC class I molecules. Others, however, have no obvious role in the immune response. The MHC is located in the chromosome band 6p21.3 which is a Giemsa (G)-light band. The detection of such a large number of functional genes (at least 70) in this region is compatible with the idea that both housekeeping and tissue-specific genes are localised predominantly in G-light bands.
[Show abstract][Hide abstract] ABSTRACT: The C4A6 allotype of the human complement component C4 is known to be defective in C5 binding within the C5 convertase. To characterize the position and nature of the molecular defect in the C4A6 allotype we have isolated the C4A6 gene from a cosmid genomic DNA library. Direct sequencing of a 4.4-kb region of the gene covering exons 17 to 31 and encoding the C4d fragment and most of the rest of the alpha chain of C4 revealed that the C4A6 allele encodes the A isotypic residues Pro Cys-Leu Asp at positions 1101, 1102, 1105, and 1106 and the same residues as the C4A3 alpha gene at the polymorphic positions 1054 (Asp), 1157 (Asn), 1182 (Thr), 1188 (Val), 1191 (Leu) and 1267 (Ala). In addition the C4A6 allele was shown to encode a Pro at the previously characterized polymorphic position 707 in the C4a peptide where the C4A3 alpha allele encodes a Leu. The remaining 26 exons of the C4A6 gene were analyzed by detecting nucleotide mismatches in C4A6/C4A3 and C4A6/C4B1 DNA heteroduplexes using the chemical cleavage of mismatch technique. The regions around detected mismatches were sequenced. In total seven nucleotide differences were defined on comparison of the C4A6 and other C4 sequences, of which three were present in exons. Two of these resulted in amino acid changes. One of the amino acid differences is a known polymorphism in C4, a Tyr/Ser substitution at position 328 in the beta-chain. The second amino acid difference caused by a C to T transition in the first base of the codon for amino acid residue 458 was the only one shown to be specific to the C4A6 allotype. The C4A6 allotype contains a Trp residue at this position in the beta-chain instead of the Arg residue found in all other C4A and C4B allotypes so far characterized. We propose that this Arg to Trp substitution at beta-chain residue 458 is responsible for the inability of C4A6 to bind C5 in the C5 convertase.
The Journal of Immunology 06/1992; 148(9):2795-802. · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Three genes encoding members of the M(r) 70,000 heat shock protein family (HSP70) are known to lie in the class III region of the human major histocompatibility complex. In order to determine whether these genes or their protein products exhibit any polymorphism the three genes have been specifically amplified from genomic DNA and sequenced. The HSP70-1 and HSP70-2 genes encode the major heat-inducible HSP70. A comparison of the nucleotide sequences of these genes from B8, SC01, DR3, B18, F1C30, DR3, and B7, SC30, DR2 haplotypes has revealed only very limited sequence variation which is not associated with any amino acid polymorphism. The HSP70-Hom gene encodes a protein that is highly related to HSP70-1, but which is not heat-inducible. Nucleotide sequence analysis of this gene from different haplotypes has revealed a Met----Thr amino acid substitution at residue 493 in a number of the haplotypes tested. This variable amino acid lies in the proposed peptide-binding site of the HSP70-Hom protein.
[Show abstract][Hide abstract] ABSTRACT: A duplicated locus encoding the major heat shock-induced protein HSP70 is located in the major histocompatibility complex (MHC) class III region 92 kilobases (kb) telomeric to the C2 gene. Nucleotide sequence analysis of the two intronless genes, HSP70-1 and HSP70-2, has shown that they encode an identical protein product of 641 amino acids. A third intronless gene, HSP70-Hom, has also been identified 4 kb telomeric to the HSP70-1 gene. This encodes a more basic protein of 641 amino acids which has 90% sequence similarity with HSP70-1. In order to investigate the expression of the three (MHC)-linked HSP70 genes individually by northern blot analysis, we have isolated locus-specific probes from the 3' untranslated regions of the genes. The HSP70-1 and HSP70-2 genes have been shown to be expressed at high levels as a approximately 2.4 kb mRNA in cells heat-shocked at 42 degrees C. HSP70-1 is also expressed constitutively at very low levels. The HSP70-Hom gene, which has no heat shock consensus sequence in its 5' flanking sequence, is expressed as a approximately 3 kb mRNA at low levels both constitutively and following heat shock.