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.     

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.     

The molecular basis for the difference in immune hemolysis activity of the Chido and Rodgers isotypes of human complement component C4

Human C4 displays a structural polymorphism which is consistent with there being two closely linked genetic loci coding for this protein. These give rise to two C4 isotypes, designated C4A and C4B, which can be distinguished by charge and apparent m.w. differences in their respective alpha-chains and by the presence or absence of the Chido/Rodgers blood group antigens. Previous qualitative studies of C4 immune hemolysis activity in whole plasma had suggested that the C4B isotype was functionally more active. By using purified C4A and C4B isolated from individual donors known serologically to possess only one of the C4 isotypes, we examined the molecular basis for the differences in their respective hemolytic activities. It was found that the C4B:C4A hemolytic activity ratio was approximately 4:1. This fourfold difference could not be accounted for by a commensurate difference in the cleavage rate of the two isotypes by C1s by differences in the kinetics of assembly or intrinsic decay of the respective C3 convertase enzymes, or by differences in the rate of isotypic C4b cleavage by factor I in the presence of C4bp . However, the fourfold greater deposition efficiency of nascent C4b of the C4B isotype onto the surface of C1-bearing sheep erythrocytes quantitatively accounted for the observed difference in immune hemolysis function. It was further found that the thioester bond of nascent C4b of the C4A isotype preferentially transacylates onto amino group nucleophiles, whereas in the C4B isotype, acylation of hydroxyl groups is strongly preferred. Thus, the difference in immune hemolysis activity between the two C4 isotypes does not necessarily indicate an impairment of function in C4A; it may merely be a reflection of the relative abundance at the surface of a C1-bearing target of hydroxyl and amino groups capable of being acyl acceptors for nascent C4b. Finally, we also present evidence showing that the apparent m.w. difference between the alpha-chains of the C4A and C4B isotypes is not due to differences in protein glycosylation.     

The distribution of Ia antigens on the surfaces of lymphocytes.

The distribution of Ia antigens was studied on murine spleen lymphocytes by an ultrastructural technique employing deep freeze-etched replicas. Ia antigens were labeled on cells from appropriate congenic and recombinant strains of mice by incubating the cells with FITC-conjugated anti-Iak antibody, followed by ferritin-coupled Fab anti-FITC. Ia antigens were detected predominantly on immunoglobulin (Ig)-bearing B lymphocytes. Antigens coded for by the entire Ik region were present on the surfaces of 95% of the positive cells (from B10.BR mice) in densely packed microclusters. Ia specificities coded for by the I-A and I-C subregions (on 4R and B10.HTT mice) exhibited a more variable pattern, with 30 to 35% of the labeled cells having sparsely distributed Ia antigens in relatively discrete microclusters. Binding of anti-Iak antibody at 37 degrees C led to patch formation but not to capping. Modulation of surface Ig left Ia antigens diffusely distributed on the cell surface, indicating that these two membrane proteins are independent molecules.     

Variation in the glycosylation of the murine sex-limited protein: comparison with the fourth component of murine complement

The murine sex-limited protein (Slp) is a hemolytically nonfunctional homologue of the fourth component of complement (C4). Two congenic mouse strains, B10.BUA1 (H-2w16) and B10.KPB128 (H-2w19), which have been previously shown to share a variant form of C4 (Karp et al., J. Biol. Chem., 257: 7330-7335), were examined and found to also produce a variant form of Slp. Slp molecules isolated from the plasma or peritoneal macrophage cultures from these strains have an alpha-chain approximately 2,000 daltons smaller than the alpha-chain of Slp from H-2d or H-2w7 mice as judged by sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis. Expression of this Slp was constitutive, i.e., not regulated by androgen, and is cis-dominant in F1 hybrid mice. Autolysis of the different relative molecular mass (Mr) alpha-chains at the internal thiolester produced similar Mr amino-terminal fragments and different Mr carboxy-terminal fragments. Deglycosylation of the alpha-chains with trifluoromethanesulfonic acid eliminated most, if not all, the Mr difference. The Mr difference was also manifested by the intracellular precursors of Slp and could be eliminated by endoglycosidase H (endo H) treatment. The number of oligosaccharides on the Slp alpha-chain was deduced by limited endo H treatment of Slp synthesized in the presence of swainsonine, a plant alkaloid that prevents maturation of complex-type oligosaccharides. This method is a simple way to enumerate the complex-type, N-linked oligosaccharides on glycoproteins. The genetic variation in the glycosylation of Slp was compared with the known variation in glycosylation of C4, and a scheme depicting some of the structural differences among these molecules was developed.     

Age-dependent TCR revision mediated by interaction between alphabeta TCR and self-antigens

Interactions between TCR and self-peptide/MHC complex play an important role in homeostasis and Ag reactivity of mature peripheral T cells. In this report, we demonstrate that the interactions between mature peripheral T cells and endogenous Ags have a negative impact on the maintenance of foreign Ag-specific T cells in an age-dependent manner. This is mediated by RAG-dependent secondary rearrangement of the TCR alpha-chain (receptor revision). The TCR revision in mature T cells is readily observed in mouse expressing transgenic TCR alpha-chain inserted into the physiological locus (knockin mouse) but not in conventional transgenic mouse with an identical TCR alpha-chain. Thus, our results suggest that under physiological conditions in which all TCR alpha-chains are susceptible to deletion by secondary rearrangement, TCR revision in mature peripheral T cells is an ongoing process in adult animals and contributes to age-dependent changes in T cell function and repertoire.     

Ag-B-linked analogue of Ia antigens in the rat.

The reactions of Lewis rat lymphocyte membrane antigens with two alloantisera, BN anti-Lewis and BN anti-Fischer have been studied. Three lines of evidence indicated that these antisera reacted with cell surface antigens homologous to Ia antigens of the mouse. 1) After absorption with Lewis platelets, the antisera killed only 40 to 50% of Lewis spleen cells. The majority of such cells were shown to be Ig-positive B cells by the examination of reaction patterns on lymphocytes after separation on nylon wool into T cell- and B cell-enriched subpopulations. 2) SDS-PAGE analysis of solubilized Lewis spleen cell antigens precipitated with these antisera revealed that the platelet-absorbed antisera reacted with molecules comparable in size to mouse Ia antigens (mw approximately equals 35,000 and 28,000). The unabsorbed sera reacted with these molecules and with additional molecules corresponding in size to mouse K and D antigens (m.w. = 45,000). 3) Neither of these antisera killed significant numbers of spleen cells from the partially congenic strain F.BN (seventh backcross homozygotes), a Fischer rat to which the Ag-B.3 allele is being transferred by repetitive backcrossing, indicating that the genes coding for these Ia-like antigens in the rat are linked to the Ag-B locus.     

Identification of two Ia-like alloantigens on rabbit B lymphocytes

Alloimmunizations with rabbit lymphoid cells have resulted in the identification of two cell-surface alloantigens, Ia1 and Ia2. These antigens reside on nearly all B cells; few, if any thymus cells or T cells of mesenteric lymph nodes bear these antigens. Genetic studies showed that Ia1 and Ia2 molecules appear to be controlled by allelic genes at a locus closely linked to the MHC. Immunochemical analyses revealed that Ia1 and Ia2 are glycoproteins and that each is composed of two polypeptide chains of molecular weights of 28 000 and 30 000–32 000. Thus, the alloantigens identified by these two antisera appear to be Ia-like molecules.     

Xenopus MHC class II molecules. I. Identification and structural characterization

Class II antigens from the Xenopus laevis MHC (f haplotype) were identified by using a rabbit antihuman class II beta-chain serum (anti-p29boost). This xenoantiserum inhibits bidirectional Xenopus MLR (but not PHA-stimulation), recognizes the same molecules as certain MHC-linked Xenopus alloantisera, and immunoprecipitates class II molecules from Xenopus cells consistent with the tissue distribution of mammalian class II molecules. The Xenopus class II molecules are composed of two different chains, both of which are 30 to 35kD transmembrane glycoproteins. The alpha-chains have some N-terminal sequence homology with mammalian class II alpha-chains (both I-E/DR and I-A/DC); the beta-chains are directly recognized by anti-p29boost and have a markedly increased SDS gel mobility under nonreducing conditions. During biosynthesis, they are noncovalently associated with a number of other chains, including ones at 25kD, 33kD, and 40 to 45kD. The alpha-chains bear three N-linked glycans (two Endo H insensitive in mature material) and the beta-chains bear two (one Endo H insensitive). Unlike most mammalian class II molecules, the deglycosylated beta-chains are significantly larger and more acidic than the alpha-chains.     

The analysis with monoclonal antibodies of the heterogeneity of Ia glycoproteins on chronic lymphocytic leukemia cells

The accessible Ia molecules on the surface of chronic lymphocytic leukemia (CLL) cells were quantitated in the cellular radioimmunoassay with saturating concentrations of monoclonal antibodies. Monoclonal antibody 21w4, like DA/2 antibody, recognizes monomorphic determinants of human Ia antigens. The amount of 21w4 or DA/2 bound to CLL cells derived from eight patients (varying from 2.6 to 13.9 x 10(5) molecules/cell) appears to be the maximum observed with the antibodies studied. Two other antibodies, 18d5 and 21r5, although also directed at nonpolymorphic Ia determinants, bind differentially to CLL cells, with the ratios of 21r5/21w4 and 18d5/21w4 varying from 0.08 to 0.90. Sequential immmunoprecipitation studies have established that the four epitopes 18d5, 21r5, 21w4, and DA/2 were present on the same molecules. All Ia molecules express 21w4 and DA/2 epitopes, whereas only certain subsets of Ia molecules carry accessible 21r5 or 18d5 epitopes. Competitive binding studies showed that the epitopes recognized by the four monoclonal antibodies were different. Monoclonal antibodies 21r5 and 21w4 did not inhibit each other's binding. Furthermore, binding of 21w4 to CLL cells potentiated the binding of 125I-21r5 IgG to the same cells, suggesting that binding of 21w4 antibody induces a conformational change in the molecule that renders 21r5 epitopes more accessible.     

The Ia.2 epitope defines a subset of lipid raft-resident MHC class II molecules crucial to effective antigen presentation

Previous work established that binding of the 11-5.2 anti-I-A(k) mAb, which recognizes the Ia.2 epitope on I-A(k) class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-A(k) mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-A(k) molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-A(k) class II molecules is critically necessary for effective B cell-T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-A(k) class II molecules possessing a β-chain-tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2(-) tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell-T cell interactions.     

Cytofluorometric isolation of I937, an Ia antigen-bearing variant of the Ia-negative human monocytic cell line U937

Cells of the human monocyte cell line U937 are generally considered devoid of any Ia antigens on their surface. In analyzing U937 cells with a large panel of monoclonal anti-human Ia antibodies by flow cytometry, we detected a small number of cells that appeared to react with antibodies to HLA-DR and HLA-DS/DC molecules. These Ia-positive cells were isolated and were cloned, resulting in a human monocyte cell line that expresses high levels of Ia antigens. We analyzed these antigens by one- and two-dimensional polyacrylamide gel electrophoresis, after radiolabeling and immunoprecipitation. Three distinct Ia molecules, alpha 1 beta 1, alpha 1 beta 3 (HLA-DR-like), and alpha 2 beta 2 (HLA-DC/DS-like) are synthesized by I937 cells, alpha 1 beta 3 molecule being the predominant species. The Ia antigen-bearing human monocyte cell line is expected to be useful for studying events involved in antigen presentation.     

Xenopus MHC class II molecules. II. Polymorphism as determined by two-dimensional gel electrophoresis

The class II antigens from four inbred strains of Xenopus laevis (r, f, g, and j haplotypes) and six gynogenetic LG clones (two Xenopus laevis, two Xenopus gilli haplotypes) with functionally well-defined MHC types have been immunoprecipitated with the rabbit anti-human class II beta-chain serum anti-p29boost and analyzed by two-dimensional gel electrophoresis. The glycosylated material from 15-hr biosynthetically labeled cells runs as a broad fuzzy band around 33kD that, upon removal of N-linked glycans by Endo F, resolves into upper beta-chain bands and lower alpha-chain bands. Both the glycosylated and deglycosylated class II antigens give rise to multiple IEF spots in evenly spaced arrays (alpha-chain: two to eight spots in one to three arrays, beta-chain: two to 12 spots in one to five arrays). Both chains are polymorphic and both map to the functionally defined MHC. The large number of spots argues for multiple class II antigens; by radioactive N-terminal sequencing, two homologous alpha-chains and five beta-chains are present in the f haplotype. By comparison with MHC-linked alloantisera, anti-p29boost recognizes all major polymorphic class II molecules in Xenopus laevis. A selection of outbred animals were typed by using an IEF procedure requiring only a million PHA-stimulated blood cells.     

A human alloantiserum precipitates Ia-like molecules from chronic lymphocytic leukemia cells.

Alloantigens specific for human B lymphocytes can be identified with selected antisera. These antigens have similarities to murine Ia antigens in that they are found on human B lymphocytes and are controlled by genes linked to genes controlling HLA. Chronic lymphocytic leukemia cells bearing B cell antigens were labeled with 3H leucine and the membrane components reacting with the B cell antisera isolated by immunoprecipitation. These membrane components had m.w. of 33,000 and 24,000 daltons similar to the murine Ia antigens. The results complete the homology of murine Ia and human B cell alloantigens.     

Carnivora: the primary structure of the alpha-chains of ferret (Mustela putorius furo, Mustelidae) hemoglobins

Ferret erythrocytes contain two hemoglobins differing only by their alpha-chains. The primary structure of the common beta-chain has been previously described; the complete sequence of the two alpha-chains are reported in this paper. The globin chains were separated by ion-exchange chromatography; the alpha-chains (42 steps), their tryptic peptides as well as the prolyl-peptides were subjected to automatic liquid- and gas-phase Edman degradation. The two alpha-chains are very similar, differing at only one position (Asp15----Gly15). Comparison with human hemoglobin alpha-chain shows 16 and 17 exchanges, for alpha 1 and alpha II chains, respectively; two substitutions involve alpha 1/beta 1 contacts and one the heme contacts. A high degree of homology was noted when the alpha-chains were compared to the corresponding chains of other representatives of the Carnivora order.     

Transplantation in minature swine. IV. Chemical characterization of MSLA and Ia-like antigens

Immunochemical analyses of radioactively labeled lymphocyte antigens from miniature swine of three different homozygous major histocompatibility (MHC) types, AA, CC, and DD, have been performed. Anti-MHC sera were incubated with lentil lectin purified Nonidet P-40 swine lymphocyte extracts. Antigen-antibody complexes were then precipitated with protein A bearing staphylococci, eluted, and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Analysis of antigens from AA or DD cells revealed peaks of 42,000, 31,000, 25,000, and 11,000 dalton m.w. Platelet absorption of the anti-MHC sera yielded antibodies that only precipitated the intermediate m.w. molecules and lysed a subpopulation of swine peripheral blood lymphocytes, suggesting that these molecules were the miniature swine analogues of murine Ia antigens. Antibodies eluted from platelets lysed all lymphocyte populations and precipitated only the 42,000 and 11,000 dalton peaks, indicating that these molecules represent the analogue of murine H-2 histocompatibility antigens, containing a heavy chain and putative swine beta2-microglobulin.     

The spectrin super-family

The review is focused on recent data on the primary sequences of erythroid and non-erythroid spectrins. As in other fields, the techniques of molecular genetics have allowed great advances in our knowledge of the structure and the genetic story of these molecules. Comparison of alpha-chains sequences of the non-erythroid (fodrin) and erythroid spectrin demonstrated that the fodrin alpha-genes are strictly conserved across species, while the mammalian spectrin genes have diverged rapidly. Spectrin and fodrin alpha-chains are largely composed of homologous 106-amino-acid repeat units. Spectrin alpha-chain is lacking a 37 amino-acid sequence which bears the calmodulin-binding site of the fodrin alpha-chain. The highest degree of homology between the spectrin alpha-chain and the fodrin alpha-chain lies in a central atypical segment unrelated to the canonical repeat sequence. This region is closely related to the N-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. Like the spectrin alpha-chain, the major central part of the spectrin beta-chain is made up of repeat units of 106 amino-acids. The N-terminal domain of the beta-chain, and especially the actin binding site, is the region of greatest homology among members of the spectrin super-family, including Drosophila spectrin beta-chain, dystrophin and alpha-actinin. The C-terminal extremity of the erythroid beta-chain is also of great interest, since tissue-specific differential processing of 3'beta-spectrin gene pre-mRNA generates a beta spectrin-isoform with a unique C-terminus in human skeletal muscle.     

Guinea pig lymphocyte antigen molecules Ia.3,5 and Ia.8 share structural homology expected for alleles

Because of the lack of I region recombinant guinea pig strains, rigorous comparative chemical analyses of guinea pig Ia alloantigens have been utilized to gather structural information that would allow tentative assignment of I region alleles. In this study, the B/Lac Ia.8 molecule was chemically compared to the strain 13 Ia.3,5 molecule and the strain 2 Ia.2 and Ia.4,5 molecules. SDS-PAGE, IEF, and Cleveland peptide mapping demonstrated a significant degree of homology between the Ia.8 and Ia.3,5 alpha-chains. HPLC mapping of trypsin/chymotrypsin cleavage products revealed an overall coincidence of peptides of 57 and 68% for the comparisons of the Ia.8 and Ia.3,5 alpha- and beta-chains respectively. In contrast, comparisons of Ia.8 and Ia.2 alpha- and beta-chains exhibited a significantly lower degree of similarity--39 and 47% respectively. The degree of homology seen in the Ia.8 and Ia.3,5 comparisons is consistent with those values seen in comparisons between the Ia.3,5 and Ia.4,5 molecules (putative allelic products of the guinea pig I region) and with those values obtained for allelic products of the mouse H-2 system. The results of this investigation strongly suggest that the Ia.8, Ia.3,5, and Ia.4,5 molecules are the products of alleles at the Ia alpha- and beta-chain loci.     

Macrophage Ia antigens. I. macrophage populations differ in their expression of Ia antigens

By indirect immunofluorescence and microcytotoxicity it was demonstrated that different populations of murine macrophages bear different amounts of Ia antigens on their membranes. At least three subpopulations could be distinguished: those that lack Ia antigens and predominate in peritoneal exudate; cells bearing I-A antigens that are the majority of splenic macrophages and a minor population in the peritoneum; and cells bearing I-C antigens that are a minor population in both spleen and peritoneum. Internal radioisotope labeling studies confirmed that the I region molecules are synthesized by the macrophages. It is suggested that these different macrophage subpopulations may play distinct roles in the immune response.