Linkage of the gene controlling the synthesis of the fourth component of complement to the major histocompatibility complex of the guinea pig

C4-deficient (C4D) guinea pigs are lacking in C4 synthesis, a condition that appears to be caused by a structural gene defect. This defect is inherited as a simple autosomal recessive trait. We have demonstrated linkage between C4D and the major histocompatibility complex of the guinea pig (GPLA). Inbred C4D and inbred strain 13 guinea pigs appear to have the same GPLA haplotype. The use of these two strains should provide an animal model for reconstitution studies of C4 synthesis and for studied exploring the possible role of C4 in cellular and humoral immune responses.Abbreviations used in this paper are C4D deficiency of the fourth component of complement - MHC major histocompatibility complex - GPLA major histocompatibility complex of the guinea pig - MLC mixed lymphocyte culture     

Mapping of class I DNA sequences within the human major histocompatibility complex

Mutants that had lost expression of alleles of one or more HLA loci were isolated with immunoselection after gamma-irradiation of a human lymphoblastoid cell line LCL 721. DNAs from the mutants were digested with restriction endonucleases and analyzed by Southern blotting using probes for class I HLA genes. Eight polymorphic cut sites for HindIII and PvuII were discovered in class I-associated sequences of LCL 721. Losses of specific fragments generated by restriction enzymes could be associated with losses of specific antigenic expressions and it was possible in this way to assign HLA-A1, HLA-A2, and HLA-B8 to specific DNA fragments. Patterns of gamma-ray-induced segregations of DNA fragments permitted rough linkage alignment of about 30% of the fragments generated by PvuII. The resultant map showed that there are class I HLA genes on the telomeric side of the HLA-A locus. Restriction enzyme site polymorphisms were also examined in a panel of DNAs isolated from peripheral blood lymphocytes (PBLs) of HLA-typed individuals. This panel of PBL DNA complemented the analysis using the HLA deletion mutants.     

Mapping of a candidate region for susceptibility to inclusion body myositis in the human major histocompatibility complex

 Inclusion body myositis (IBM) is a form of idiopathic inflammatory myopathy of unknown aetiology. A strong association with HLA class II (HLA-DR3) suggested a role for genes in the human major histocompatibility complex (MHC) in the predisposition to this disease. In this study, we have taken advantage of the ancestral haplotype (AH) concept and historical recombinations to map for a possible susceptibility gene(s) in the MHC. We performed detailed typing of three MHC-related HSP70 genes and defined allelic combinations in the context of MHC AH. We also modified existing methods to give a simple and accurate method for typing two TNF microsatellites. Using the HSP70 and TNF markers and HLA-DR, –B, and C4 typing of our patients with IBM, we defined a potential site for the MHC-associated susceptibility gene(s) in the region between HLA-DR and C4. Received: 16 July 1998 / Revised: 14 January 1999     

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s.

The major histocompatibility complex (MHC) class I antigens contain a light chain, beta 2-microglobulin, non-covalently associated to the transmembrane heavy alpha-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with beta 2-microglobulin, and we recently found that activated C1s complement was capable of cleaving beta 2-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22,000 and 24,000 g/mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the alpha 2-and alpha 3-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-Ad). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the alpha 1-and alpha 2-domains which represent the binding site for antigenic peptides.     

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s

The major histocompatibility complex (MHC) class I antigens contain a light chain β₂-microglobulin, non-covalently associated to the transmembrane heavy α-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with β₂-microglobulin, and we recently found that activated C1s complement was capable of cleaving β₂-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22 000 and 24 000 g/ mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the α₂-andα₃-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-A^d). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the α₁andα₂-domains which represent the binding site for atnigenic peptide.     

The purification and properties of the second component of human complement.

The second component of human complement (C2) was purified by a combination of euglobulin precipitation, ion-exchange chromatography, (NH4)2SO4 precipitation and affinity chromatography. The final product was homogeneous by the criterion of polyacrylamide-gel electrophoresis and represents a purification of about 4000-fold from serum with 15-20% yield. Component C2 comprises a single carbohydrate-containing polypeptide chain, with an apparent mol.wt. of 102000; alanine is the N-terminal amino acid. The molecule is rapidly cleaved by activated subcomponent C1s with the loss of haemolytic activity to yield two fragments with apparent mol.wts. of 74000 and 34000. These fragments are not linked by disulphide bonds and can be easily separated. A second protein isolated during the purification of component C2 was identified by its haemolytic and antigenic properties as complement Factor B, the protein serving an analogous function to component C2 in the alternative pathway. The protein, which is also a single carbohydrate-containing polypeptide chain, has an apparent mol.wt. of 95000 and threonine as N-terminal amino acid. The amino acid analyses of component C2 and Factor B are compared.     

Purification and structural analysis of the fourth component of human complement.

The fourth component of human complement (C4) has been purified in 20% yield from fresh plasma using as starting material the 5-12% poly(ethylene glycol) precipitate which had been depleted of plasminogen by an affinity adsorbent. Sequential ion-exchange chromatography on diethylaminoethylcellulose, QAE-Sephadex, and DEAE-Bio-Gel A resulted in C4 homogeneous by immunological criteria and by polyacrylamide gel electrophoresis, the last chromatographic step achieving separation of native from inactivated C4. Reduction with 20 mM dithiothreitol for 2 h at 37 degrees C in 0.25 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride, pH 8.6, effected cleavage of the interchain disulfide bonds. A three-chain structure for C4 was confirmed, and molecular weight estimates of 93 000 +/- 9300, 75 000 +/- 7500, and 30 000 +/- 3000 determined for the alpha, beta, and gamma chains, respectively. The effects of known inactivators of C4 upon the chains of C4 were investigated, confirming that the inactivations by C1s and trypsin were accompanied by the fragmentation of the alpha chain. Inactivation of C4 by hydrazine, on the other hand, produced no detectable change in chain size. Separation of the chains was accomplished by gel filtration in the presence of 1 M acetic acid. Amino acid compositions of native C4 and the constitutive chains have been performed, and N-terminal sequences of the latter established by automated Edman degradation.     

Molecular analysis of human complement component C5: localization of the structural gene to chromosome 9

A human C5 clone (pC5HG2) was isolated from a cDNA library constructed from Hep G2 mRNA. The DNA sequence showed that the pC5HG2 insert was comprised of 3309 base pairs of pro-C5 coding sequence and 404 base pairs of 3'-untranslated sequence. The derived amino acid sequence contained the entire coding sequence of the C5 alpha-chain, the beta-alpha-chain junction region, and 100 amino acids (approximately 50%) of the beta-chain. Protein sequences of four C5 tryptic peptides were aligned exactly to this sequence and demonstrated that C5 synthesized and secreted by Hep G2 cells is probably identical with plasma-derived C5. Coding sequence alignment of the human C5 sequences with those of murine C5 indicated that 80% of the nucleotides and 79% of the amino acids were placed identically in the two species. Amino acid sequence alignment of the homologous family members C3, C4, and alpha 2-macroglobulin with that of C5 demonstrated 27%, 25%, and 19% identity, respectively. As was found in murine C5, the corresponding thiol ester region of human C5 contained several conserved amino acids, but the critical cysteine and glutamine residues which give rise to the intramolecular thiol ester bond in C3, C4, and alpha 2-macroglobulin were absent in C5, having been replaced by serine and alanine, respectively. With the use of a panel of hamster-human somatic cell hybrids, the C5 gene was mapped to human chromosome 9. In situ chromosomal hybridization studies employing metaphase cells further localized the gene to bands 9q32-34, with the largest cluster of grains at 9q34.1.     

Sequence analysis of the human major histocompatibility gene SX alpha.

The DP subregion of the human major histocompatibility complex contains two closely linked gene pairs, DP alpha, DP beta and SX alpha, SX beta. The exon-intron organization and the complete DNA sequence of the SX alpha gene are reported here. There are several mutations within the SX alpha gene which strongly suggest that it is a pseudogene. These include two frameshift mutations, one in the alpha 1 domain and the other in the cytoplasmic domain. A 5' splice site mutation at the end of the alpha 1 exon also exists. DNA sequence homology between DP alpha and SX alpha suggests that these genes arose through a gene duplication event.     

Enzymatic activity of the second component of complement.

Isolated C2 and C2i preparations were able to hydrolyze a number of synthetic esters containing basic amino acids, among which N-alpha-acetylglycyl-L-lysine methyl ester (AcGlyLysOMe) was most susceptible. The cleaving activity was a property of the C2 molecule, since it correlated with the presence of C2 on analyses of C2 preparations by ultracentrifugation in sucrose gradients, filtration through Sephadex G-200 columns, and on electrophoresis in acrylamide gels. Furthermore, acrylamide gel electrophoretic studies showed a shift in hydrolytic activity from the position occupied by C2 to that characteristic of C2i after incubation of C2 with C1s. The action was enzymatically mediated as evidenced by a bell-shaped pH activity curve, a linear dependence on C2 concentration, and the presence of Michaelis-Menten kinetics. The Michaelis constant for cleavage of AcGlyLysOMe by C2 was 1.8 X 10(-2) mol. Cleavage of C2 by C1s increased C2 enzymatic activity, yet chemical oxidation of the molecule, although enhancing hemolytic acitivity, failed to increase C2 hydrolytic activity. The observed enzymatic activity of C2 was found to be relevant to the function of C2 in the C42 complex, since AcGlyLysOMe competitively inhibited the C42 mediated cleavage of C3 in free solution and the C42 dependent binding of C3 to cells.     

Identification of the Tapasin gene in the chicken major histocompatibility complex

 The Tapasin molecule plays a role in the assembly of major histocompatibility complex (Mhc) class I molecules in the endoplasmic reticulum, by mediating the interaction of class I-β₂-microglobulin dimers with TAP. We report here the identification of the Tapasin gene in the chicken Mhc (B complex). This gene is located at the centromeric end of the complex, between the class II B-LBI and B-LBII genes. Like its human counterpart it comprises 8 exons, but features a significantly reduced intron size as compared to the human gene. Chicken Tapasin codes for a transmembrane protein with a probable endoplasmic reticulum retention signal. Exons IV and V, and possibly exon III, code for separate domains that are related to the immunoglobulin (Ig) superfamily (this relationship was so far unrecognized for human Tapasin domain IV which has lost its two cysteines). Two different cDNAs corresponding to the Tapasin gene were isolated, possibly related to alternative splicing events; the Ig-like domain encoded by exon IV is missing in one of the cDNAs, suggesting either that this domain is not necessary for the protein to perform its function, or that the two alternatively spliced cDNAs are translated into two functionally different forms of the protein. Received: 8 July 1998 / Revised: 5 October 1998     

The complement components of the major histocompatibility locus

Polymorphism of complement components, recognized by differences in either their antigenic specificity or their electrophoretic mobility, together with studies of inherited deficiencies, has enabled many of their structural genes to be mapped. In humans, three genes (for C2, C4, and factor B) have been placed between HLA-D and HLA-B on chromosome 6 and in mice, C4 between H2-I and H2-D, chromosome 17. Structural studies show that these components have exceptional features. C2 and factor B which contain the proteolytic active site of the C3 and C5 convertases are of the classical and alternative pathway respectively and are similar in structure and function. Both are novel types of serine proteases. C4 (as C3) contains an intrachain thioester bond essential for hemolytic activity. Molecular genetic investigations are determining the relative positions of these genes, and their precise structure, and should clarify their relation to the inherited diseases which are associated with defects in this section of the human genome.     

An integrated haplotype map of the human major histocompatibility complex

Numerous studies have clearly indicated a role for the major histocompatibility complex (MHC) in susceptibility to autoimmune diseases. Such studies have focused on the genetic variation of a small number of classical human-leukocyte-antigen (HLA) genes in the region. Although these genes represent good candidates, given their immunological roles, linkage disequilibrium (LD) surrounding these genes has made it difficult to rule out neighboring genes, many with immune function, as influencing disease susceptibility. It is likely that a comprehensive analysis of the patterns of LD and variation, by using a high-density map of single-nucleotide polymorphisms (SNPs), would enable a greater understanding of the nature of the observed associations, as well as lead to the identification of causal variation. We present herein an initial analysis of this region, using 201 SNPs, nine classical HLA loci, two TAP genes, and 18 microsatellites. This analysis suggests that LD and variation in the MHC, aside from the classical HLA loci, are essentially no different from those in the rest of the genome. Furthermore, these data show that multi-SNP haplotypes will likely be a valuable means for refining association signals in this region.     

Reproductive failure and the major histocompatibility complex.

The association between HLA sharing and recurrent spontaneous abortion (RSA) was tested in 123 couples and the association between HLA sharing, and the outcome of treatment for unexplained infertility by in vitro fertilization (IVF) was tested in 76 couples, by using a new shared-allele test in order to identify more precisely the region of the major histocompatibility complex (MHC) influencing these reproductive defects. The shared-allele test circumvents the problem of rare alleles at HLA loci and at the same time provides a substantial gain in power over the simple chi 2 test. Two statistical methods, a corrected homogeneity test and a bootstrap approach, were developed to compare the allele frequencies at each of the HLA-A, HLA-B, HLA-DR, and HLA-DQ loci; they were not statistically different among the three patient groups and the control group. There was a significant excess of HLA-DR sharing in couples with RSA and a significant excess of HLA-DQ sharing in couples with unexplained infertility who failed treatment by IVF. These findings indicate that genes located in different parts of the class II region of the MHC affect different aspects of reproduction and strongly suggest that the sharing of HLA antigens per se is not the mechanism involved in the reproductive defects. The segment of the MHC that has genes affecting reproduction also has genes associated with different autoimmune diseases, and this juxtaposition may explain the association between reproductive defects and autoimmune diseases.     

Cobra venom factor: structural homology with the third component of human complement

The functional analogy between cobra venom factor (CVF), the complement-activating protein in cobra venom, and C3b, the activated form of the third complement component, prompted us to conduct a comparative analysis of structural properties of the two proteins derived from two phylogenetically distant species. We subjected CVF and human C3 and its physiologic cleavage products, C3b and C3c, to a variety of biochemical analyses. We report here structural similarities of these proteins, which include similarities in amino acid composition, far and near UV circular dichroism spectra, secondary structure, band patterns and pI values from isoelectric focusing, immunochemical cross-reactivity, ultrastructural morphology, and amino-terminal amino acid sequences. Analysis of these data reveals that, structurally, CVF resembles C3c more than C3b. We conclude that CVF is not the product of a convergent evolution, but is, in all likelihood, derived from a common C3 ancestor protein.