Comparison and partial characterization of guinea pig Ia alpha- and beta-chain oligosaccharides

The structures of the N-linked oligosaccharides of mature guinea pig Ia molecules were partially characterized by serial lectin affinity analysis. Those Ia antigens that are thought to be allelic products (Ia.3,5 and Ia.4,5) were found to bear identical oligosaccharides, whereas differences in glycopeptide distribution were found for Ia antigens known to be products of separate I subregions (Ia.2 and Ia.4,5). The two predominant oligosaccharides present on alpha-chains from all three Ia molecules were of the high mannnose type and the triantennary or tetraantennary complex type. Two structurally distinct beta-chains were isolated from Ia.3,5 and Ia.4,5 molecules; beta 1 bore primarily triantennary or tetraantennary complex oligosaccharides, and beta 2 had predominantly biantennary complex-type carbohydrate chains. The composition and distribution of the oligosaccharide moieties of guinea pig Ia molecules indicate that there are structural features shared among guinea pig, murine, and human Ia antigens.     

Tryptic peptide disparity between serologically indistinguishable guinea pig Ia.3,5 antigens

Serological studies have suggested that the DHCBA strain guinea pig expresses an I region which is identical to that of strain 13. However, chemical characterization of Ia.3,5 molecules isolated from these two strains has indicated that these serologically indistinguishable Ia molecules are actually chemically distinct. Ia.3,5 molecules biosynthetically labeled with either [3H]leucine, [3H]arginine, or [3H]lysine were purified by ricin affinity chromatography and isolated by indirect immunoprecipitation with specific alloantisera. Initial examination of the two Ia.3,5 molecules by SDS-PAGE, isoelectric focusing, and two-dimensional gel analyses revealed no strain-specific differences. Furthermore, comparative peptide mapping of the DHCBA and strain 13 radiolabeled Ia.3,5 alpha-chains demonstrated complete peptide homology. In contrast, tryptic peptide maps of amino acid radiolabeled beta-chains revealed two peptides unique to the strain 13 beta-chain and one peptide unique to the DHCBA beta-chain. Analysis of [3H] mannose-labeled beta-chain tryptic peptides verified that the peptide differences observed using 3H-amino acids were not due to variation in N-linked glycosylation. However, strain-specific variability was also noted in the profiles of [3H]mannose-labeled beta-chains. These data indicate that the strain 13 and DHCBA alpha-chains are probably structurally identical, while the beta-chains show strain specific alterations in their chemical structure.     

Subunit structure of guinea pig Ia.1 antigens

Since their discovery in 1976, the guinea pig Ia.1 and Ia1,6 antigens were thought to be borne on a single protein species of approximately 26,000 m.w. This report demonstrates that the Ia.1 and Ia.1,6-bearing molecules are typical Ia.1 heterodimeric structures, consisting of acidic alpha-chain and basic beta-chain subunits. The mature Ia.1 and Ia.1,6 alpha-chains have an apparent size of 29,000 m.w., less than the 33,000 m.w. observed for the mature Ia.3,5 alpha-chain. Furthermore, pulse-chase studies and studies with tunicamycin demonstrate that the precursors of the Ia.1,6 alpha- and Ia.3,5 alpha-chains are clearly distinct. On the basis of their association with Ir genes and on their structural features, we conclude that the Ia.1 and Ia.1,6-bearing molecules are similar to other Ia antigens in subunit organization and biochemical properties.     

A novel guinea pig macrophage-specific polymorphic molecule. I. Biochemistry, genetics, and tissue distribution

In the course of studying Ia molecules from strain 2 and strain 13 guinea pig macrophages, with the intent of comparing them to B cell Ia molecules, it was observed that guinea pig alloserum prepared by cross-immunization of guinea pig lymphocyte Ag non-identical inbred guinea pigs immunoprecipitated not only conventional class I and class II molecules, but also a 98,000-Da molecule, termed gp98. Two different forms of the molecule were detected, indicating it is polymorphic. The genes encoding gp98 were shown not to be linked to the guinea pig lymphocyte Ag complex. The molecule gp98 was found on macrophages within populations of peritoneal exudate cells, resident peritoneal cells, bone marrow cells, and spleen. All gp98-bearing macrophages were also Ia-positive. However, only a subpopulation of macrophages bore gp98. The gp98 was not found on Ly-1 or Ig-bearing cells, indicating that B and T cells do not bear Ia. Thus, gp98 appears to be a highly immunogenic polymorphic macrophage-specific molecule that allows the characterization of guinea pig macrophage subsets.     

Structural identity of the TL antigen homologues derived from strain 2 and strain 13 guinea pigs

Strain 2 and strain 13 guinea pig thymocytes have been shown to bear a molecule that by several criteria appears to be a homologue of the murine TL antigen. The existence of a TL polymorphism in the mouse system as evidenced by TL- strains and various TL phenotypes in TL+ strains prompted a study to determine if a similar polymorphism could be demonstrated in the guinea pig system. By using two-dimensional gel electrophoresis, the thymocytes of a third inbred strain, DHCBA, were shown to bear a TL antigen, and the TL antigens of strains 2 and DHCBA were shown to give identical patterns of spots. A biochemical comparison of the strain 2 and strain 13 TL antigen heavy chains by tryptic and chymotryptic peptide mapping demonstrated that these molecules have identical peptides. Thus, no polymorphism could be demonstrated within the guinea pig TL system for the three inbred strains studied. Comparative tryptic peptide mapping of the guinea pig TL and class I B.1+S antigens demonstrated 43% homology, significantly higher than that reported for murine H-2 and TL antigens. These results provide suggestive evidence that the gene duplication giving rise to the genes determining the class I and TL antigens may have occurred more recently in the guinea pig than in the mouse.     

Transplantation in miniature swine. V. Characterization of Ia antigens.

The complexity of the I region analog associated with the MHC of miniature swine has been probed by sequential antibody precipitation studies of Ia antigens. Treatment of solubilized lymphocyte preparations from MSLA homozygotes of the DD haplotype with excess AA anti-DD alloantiserum led to precipitation of only a portion of the total Ia antigens, as determined by secondary precipitation of the remaining material with CC anti-DD serum. The presumed I region of miniature swine must therefore code for more than one Ia antigen-bearing polypeptide chain. In addition, certain mouse alloantisera that had previously been shown to react with rat Ia antigens were tested for reactivity with swine Ia antigens. Anti-Iak mouse alloantisera precipitated Ia molecules from every swine extract tested, regardless of MHC type, precluding genetic mapping studies. However, sequential precipitation studies demonstrated that the cross-reactive mouse alloantisera reacted only with a subclass of swine Ia antigens, again suggesting genetic complexity of the pig Ia locus.     

Fatty acylation of murine Ia alpha, beta, and invariant chains

Labeling of murine spleen cells with [3H]palmitate followed by analysis of immunoprecipitated Ia molecules indicated that Ia alpha- and beta-chains and their associated invariant chain contain covalently bound fatty acid. This modification is present in I-A and I-E molecules and has been found in all haplotypes examined. The 3H label was not dissociated from the glycoproteins by detergents or under the denaturing conditions of SDS-polyacrylamide gel electrophoresis. The fatty acid linked to Ii is released by treatment with neutral hydroxylamine, which indicates thioester linkage. The acylation of alpha- and beta-chains appears to involve attachment of palmitoyl groups via an ester linkage sensitive to alkaline hydrolysis. The radioactive species released from the isolated chains by treating with KOH/methanol co-migrated with palmitic acid and palmitic acid methyl ester on thin-layer chromatography.     

Structurally interdependent and independent regions of allelic polymorphism in class II MHC molecules. Implications for Ia function and evolution.

Possible interactions between regions of allelic polymorphism in the alpha- and beta-chains of class II MHC molecules were examined by measuring the efficiency of surface expression and the reactivity with mAb of wild-type and recombinant A alpha A beta-chain pairs from the b, d, and k haplotypes. These studies revealed regions of polymorphism within the alpha- and beta-chains that interact with complementary regions in the other chain. Unexpectedly, almost all the variable segments of both the class II MHC alpha- and beta-chains either directly contributed to or were near sites of interchain interactions. The exception was the beta HV3 (hypervariable (HV] segment (residues 61-71), which appeared to neither participate in nor be affected by interchain interactions. This division of the MHC molecule into interacting vs independent regions of allelic structural variation suggests that mutagenesis experiments involving the beta HV3 segment can be analyzed in a straightforward manner, as such mutations appear unlikely to alter the conformation of other molecular segments. Furthermore, functions attributed to the beta HV3 segment either experimentally or by population analysis should have a high probability of transfer by beta HV3 exchange (either experimentally or evolutionarily), because epitopes assigned to this region of the molecule are not affected by sequences outside this segment. This is of special importance because of the apparent involvement of this region in defining a potential site of interaction with antigenic peptides and TCR. In contrast, almost all other variable segments of the MHC molecule appear to have the capacity to contribute to interactions involving at least one other variable segment. This suggests not only that the experimental analysis of the contributions of these regions to various functions requires a consideration of inter- and intrachain interaction, but also that the transfer of function by genetic exchange of these structurally dependent regions is unpredictable. Selection must therefore operate on these interacting HV segments in the context of the complete alpha beta heterodimer. These results support our earlier arguments for cis-co-evolution of alpha- and beta-chain polymorphism and the absence of selection for F1 (hybrid) class II molecules. Finally, asymmetries observed in the contributions of particular pairs of HV segments to the efficient expression of Ia alpha beta heterodimers provide a basis for understanding mechanistically how cis-co-evolution may have occurred.     

The major histocompatibility complex of the guinea pig. I. Serologic and genetic studies.

Serologic and genetic studies of the antigens which comprise the guinea pig MHC have demonstrated three distinct but linked genetic regions. Antisera to the B region were raised by cross-immunization of random-bred animals; this region controls antigens B.1, B.2, B.3, and B.4 which behave as alleles at a single locus and which resemble the products of the murine D or K region genes in their tissue distribution and molecular characteristics. Cross-immunization of inbred strain 2 and strain 13 animals, both of which bear the B.1 antigen, leads to sera which identify antigens which resemble the products of the I region of the murine MHC. Specific absorption experiments have demonstrated four distinct I region antigens. In addition to the B and I regions, inbred strain 2, strain 13, and some outbred animals bear an antigen (S.1) which is the product of a third genetic region and which also resembles the murine D or K region gene products in molecular size. The results of these studies should facilitate the use of the guinea pig as an experimental model for studies of genetic control of the immune response and the function of the histocompatibility-linked Ir genes.     

Guinea pig homologues of TL and QA-2 antigens.

The H-2, thymus-leukemia (TL), and Qa-2 antigens of mice are encoded by closely linked genes on murine chromosome 17, and have structural similiarity in that each antigen is borne on a approximately 44,000 dalton molecule associated with beta2 microglobulin (beta2mu). The extensive homology of major histocompatibility complex (MHC) products that exists for the mouse and guinea pig suggested that a similar homology might exist for products of genetic regions closely linked to the MHC. By taking advantage of the selective association of beta2mu with H-2, Qa-2, and TL antigens, and by using the technique of sequential immunoprecipitation, we demonstrated two previously undescribed guinea pig molecules reactive with anti-guinea pig beta2mu. The first molecule was composed of a 36,000 dalton glycoprotein associated with beta2mu and was found on guinea pig thymocytes, but not lymphocytes. The second molecule was composed of a 40,000 dalton glycoprotein associated with beta2mu, and was found on both guinea pig thymocytes and lymphocytes. By structure, chemical composition, association with beta2mu, and tissue distribution, the first molecule is an attractive candidate for the guinea pig homologue of TL antigen, whereas the second fits the criteria for the guinea pig homologue of Qa-2 antigen.     

Sharing of Ia antigens between species. IV. Interspecies cross-reactivity of monoclonal antibodies directed against polymorphic mouse Ia determinants

The specificity of interspecies Ia cross-reactions has been analyzed by testing a panel of monoclonal antibodies (mAb) to mouse I-E and I-A antigens for reactivity with pig Ia antigens. Our earlier studies showed that mouse anti-I-E alloantisera recognized common determinants on Ia antigens of other species, whereas anti-I-A alloantisera showed much more limited cross-reactivity. These results were confirmed using a panel of 17 anti-I-E mAb, 10 of which were cytotoxic to pig cells. 2D gel electrophoretic analyses of precipitates with these mAb of 35S-labeled, NP40 solubilized pig cells revealed a limited set of protein spots that appeared to be identical to the subset of pig Ia antigens precipitated by A.TH anti-A.TL alloantiserum. Because the cross-reactive mouse sera were produced in mouse strains that do not express an I-E molecule (H-2b and H-2s), it was anticipated that the cross-reacting antibodies would be reactive with the monomorphic determinant of the I-E molecule, Ia.7. However, comparison of the reactivity of these mAb with pig cells and mouse cells revealed that the cross-reactivity on pig cells correlated not with Ia.7 but rather with detection of epitope(s) of the I-E molecule associated with inter-strain polymorphism. Anti-I-A cross-reactions were also detected, but were weaker and more limited. These findings may have implications for the evolution of Ia antigens in mammalian species.     

Identity of molecules bearing murine Ia alloantigenic specificities Ia.7 and Ia.22: evidence for a single type of I-E molecule in homozygotes

In homozygous mice bearing I regions derived from haplotype k, only a single type of Ia molecule bearing the alloantigenic specificities Ia.7 and Ia.22 was found using techniques of sequential immune precipitation and tryptic peptide analysis. As suggested at the fourth Ir Gene Workshop (Sachs 1978), Ia.7 is considered here to be an antigenic determinant associated with I-E-subregion-encoded molecules, i.e., it is excluded from the I-C subregion. The I-C subregion is currently defined mainly by functional traits. It is now known that the I-E molecules are composed of an alpha chain encoded in the I-E subregion, and a beta chain encoded in the I-A subregion. Since the I-C subregion is not involved with the determination of these Ia molecules, and since in homozygotes there is apparently only a single type of molecule bearing both specificities Ia.7 and Ia.22, the term "I-E/C" molecule should probably be dropped in favor of the simpler designation I-E.     

Expression of class II genes in regulatory T-lymphocyte populations in mice and the possible mechanism of genetic restriction of region I of the H-2 complex

Some contradictions exist in molecular and classical mapping of genes controlling I-J and I-AT (Iat) determinants of the T suppressor and T helper murine lymphocytes. There is strong evidence in favour of the hypothesis that lat molecules function as components of the T receptor for self Ia proteins in a I-restricted manner. These data are discussed from the point of view that beta-genes of the I region of the H-2 complex may control Iat determinants of regulatory T lymphocytes' receptors. A hypothesis is proposed that the I-region-determined restriction is based on interactions between alpha-chains of Ia molecules on A cells and beta-chains of receptors on T lymphocytes.     

Lead influences translational or posttranslational regulation of Ia expression and increases invariant chain expression in mouse B cells.

The molecular mechanisms governing the increased cell surface expression of major histocompatibility complex (MHC) class II molecules (Ia) on lead-treated mouse B cells was investigated. Lead has been shown to directly cause a selective, two-fold increase in the B cell's surface density of both products of the I region of the mouse MHC, I-A and I-E. In the present study, Western blot analysis showed that Pb increases the total cellular pool of I-A beta-chain by twofold. The increase in cellular I-A was not found to be due to increased messenger RNA (mRNA) for either the alpha- or the beta-chain of I-A. Biosynthetic labeling studies showed that Pb increases the translation or the stability of the Ia-associated invariant chain (Ii or gamma) and possibly the beta-chain of Ia. Collectively these results suggest that Pb increases the B cell's surface Ia by influencing translational or posttranslational regulation of Ia and/or Ia-associated chains.     

Invariant chain influences post-translational processing of HLA-DR molecules

HLA class II MHC molecule alpha- and beta-chains are normally synthesized in the presence of a third molecule, the invariant chain (Ii). Although Ii is not required for surface expression of HLA class II molecules, the influence of Ii on post-translational processing and maturation HLA class II molecules has not been thoroughly studied. In the present study, BALB/c 3T3 cells were transfected with HLA-DR alpha- and beta-chains with or without co-transfection with human Ii. Although Ii had no effect on the surface expression of DR, Ii did have a profound effect on the post-translational processing of both the alpha- and beta-chains. In the absence of Ii, the major species of alpha- and beta-chains were of lower m.w. than when expressed in the presence of Ii. The differences in m.w. were shown to be caused by differences in glycosylation with the majority of alpha- and beta-chains remaining unprocessed and endo H sensitive in the absence of Ii. The small proportion of alpha-chains that were processed in the absence of Ii showed an altered m.w. and altered sensitivity to treatment with endo H relative to alpha-chains processed in the presence of Ii. Pulse/chase studies demonstrated that although the majority of the alpha- and beta-chains remained unprocessed in the absence of Ii, the small amount that was processed was done so at a rate similar to that observed for alpha- and beta-chains processed in the presence of Ii. These studies demonstrate that Ii influences the post-translational processing of human class II molecules by affecting the proportion of alpha- and beta-chains that are processed and by determining the degree of processing of oligosaccharides on mature alpha-chains.     

Clonal analysis of B- and T-cell responses to Ia antigens

Thirty-five Ia^k-specific monoclonal alloantibodies, derived from hybridomas constructed by fusion between mouse myeloma and spleen cells from A.TH alloimmune mice (I ^S anti-I ^k ), have been used to estimate the allotypic polyporphism of the I^k-gene products. Cross-blocking studies using 17 mAb specific for the I-A molecule indicated that six determinants, which were associated with the conventional specificities Ia.2 and Ia.19, were organized in at least three distinct polymorphic areas of the I-A^k molecules. Similarly, another group of six determinants, which did not correspond to previously described conventional Ia specificities, were found to be topologically heterogeneous. By contrast, the five epitopes associated with the Ia. 1 specificity were clustered into a single region of this molecule. In addition the potentiation of binding observed between mAb specific for topologically distinct epitope regions of the I-A^k molecule, suggested that the latter may undergo conformational changes after binding of a given mAb. A similar analysis of 17 mAb specific for the I-E^k molecule indicated that specificity Ia. 7 of the E chain (as defined in this series by eight mAb) was composed of three topologically distinct polymorphic areas, one of which is also spatially related to a complex cluster of eight new determinants of the I-E^k molecule. Finally, one mAb identified a so far undescribed shared determinant of the I-A^k and I-E^k molecules. The present results, which provide a new estimate of the allotypic polymorphism of the Ia^k antigens, are discussed with regard to their functional, biochemical, and evolutionary implications.Abbreviations used in this paper mAb monoclonal antibodies - FCS Fetal calf serum - Con A concanavalin A - H-2 mouse major histocompatibility complex - NMS normal mouse serum - SaCI Staphylococcus aureus Cowan I strain - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Structural definition of a family of Ld-like molecules distributed among four of seven haplotypes compared

Comparative tryptic peptide analyses were performed on 12 different D region molecules representing seven different haplotypes. The Dd, Dq, and Dw16 regions were shown to encode multiple, antigenically distinct molecules (Dd Ld, Dq Lq Rq, and Dw16 Lw16, respectively). In addition, each of these molecules was found to have a unique primary structure, implying that they are the products of separate genes. However the previously described Rd molecule, which was identified by sequential immuno-precipitation and 2-D gel analyses, was indistinguishable from Ld by tryptic peptide mapping, implying that these two molecules may be products of the same gene. The Db, Ddx, Dk, and Dp regions were found to determine a single molecule with the reagents tested. Intra- and/or inter-haplotype comparisons of the peptide maps of each of these D region molecules revealed widely disparate structural relationships. For example, the Db, Dq, Lq, Rq, Dw16, and Lw16 molecules all showed striking homology with the Ld molecule. Members of this family share between 43 to 55% peptide homology with Ld, indicating a high conservation of primary structure (greater than 90%). However, because Dq and Dw16 region-encoded molecules show no exceptional relationship to each other, the portion of the conserved sequence is not the same for each of these Ld-like molecules. By contrast, comparisons of the Dk, Dd, Ddx, and Dp molecules with Ld or with each other revealed tryptic peptide homologies ranging from 22 to 38%, suggesting a sequence homology of 70 to 85%. When compared with the Kb molecule, each of the D region molecules showed between 21 to 36% peptide map homology (70 to 85% sequence homology). These studies indicate, therefore, that there is a family of Ld-like molecules representing several distinct haplotypes. This definition of a highly homologous family of D region molecules suggests that many D-region molecules have evolved from an Ld-like primordial gene and that in different haplotypes different portions of this prototypic structure have been maintained.     

Biochemical characterization of murine Ia antigens with xenoantisera

Rabbit anti-Ia sera was produced by immunization with detergentsolubilized extracts from splenic, lymph-node and thymus cells. The antisera contained activity against H-2 as well as Ia molecules. By a sequential immunoprecipitation assay it was shown that the rabbit anti-mouseH-2 ^s serum precipitated a second Ia molecule in theH-2 ^s haplotype. Previous studies with alloantisera have shown only one Ia molecule associated with this haplotype. Sequential precipitations with alloantiserum against the wholeI region were used to show that this second Ia molecule is coded by genes within theI region. Since only I-A- and I-E-region coded molecules are immunoprecipitable in most haplotypes, we presume that the rabbit antiserum could be identifying the I-E-subregion coded molecule in theH-2 ^s haplotype. The rabbit antiserum reacts with an isotypic specificity on the molecule. The studies suggest that theI-E subregion does exist in theH-2 ^s haplotype even though alloantiserum cannot be produced to identify allotypic variants associated with this subregion.     

Partial amino acid sequences of marine Ia antigens of the I-ECd subregion

Partial N-terminal amino acid sequences of the Ia molecule encoded by theI-E orI-C (I-EC) subregion of theH- 2^d haplotype are presented. Several homology relationships are apparent when these sequences are compared to the gene products of the mouseI- EC^k andI-A subregions and to their human and guinea pig counterparts. The polypeptide differs from the EC ^k polypeptide in two of the seven positions at which they can be compared and shows moderate homology with its human p29 counterpart. The polypeptide is identical to the EC ^k polypeptide in the eight positions which can be compared and is highly homologous to the human p34 polypeptide. The genetic implications of these observations are discussed.Abbreviations used in this paper are SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - PTH-amino acid phenylthiohydantoin amino acid - I-EC I-E or I-C (subregion of the I region)