A role for calnexin (IP90) in the assembly of class II MHC molecules.

Major histocompatibility complex (MHC) class II antigens consist of alpha and beta chains that associate intracellularly with the invariant (I) chain. The HLA-DR alpha beta I complex assembles in the endoplasmic reticulum (ER) into a nonameric structure via progressive addition of three alpha beta dimers to a core invariant chain trimer. We have examined intracellular association of alpha beta I complexes with the resident ER protein calnexin. Calnexin associates rapidly (within 3 min) with newly synthesized alpha, beta and I chains, and remains associated with the assembling alpha beta I complex until the final alpha beta dimer is added, forming the complete nonamer. Dissociation of calnexin parallels egress of alpha beta I from the ER. These results suggest that calnexin retains and stabilizes both free class II subunits and partially assembled class II-I chain complexes until assembly of the nonamer is complete.     

Analysis by molecular cloning of the human class II genes

The HLA class II genes control immune responsiveness to defined antigens; they encode cell surface heterodimers composed of alpha and beta glycopeptides. Recently, cDNA and genomic clones encoding these chains have been isolated, which allows molecular analysis of the class II genes. cDNA clones encoding the alpha chain of the HLA-DR antigen as well as that of another HLA class II antigen have been identified and characterized by nucleotide sequence analysis. These clones have been used as probes to isolate additional class II alpha cDNA clones in cDNA libraries and to identify polymorphisms in genomic DNA. Polymorphic restriction sites have been localized within the HLA-DR alpha gene and used as genetic markers in the analysis of families and of disease (insulin-dependent diabetes mellitus) and control populations. In addition, cDNA clones encoding the DR beta and DC beta chains were used as hybridization probes to identify DNA polymorphism. cDNA clones encoding the DR gamma (Ii) chain have also been identified; unlike the DR alpha and DR beta loci, the DR gamma gene is located on some chromosome other than chromosome 6. The genetic complexity of the human class II alpha and beta loci, as revealed by analysis with cDNA and genomic clones, is greater than that of the murine class II genes. The extent of that complexity will be defined by future work in this area.     

N-terminal amino acid sequence analyses of the alpha and beta polypeptides from four distinct subsets of sheep major histocompatibility complex class II molecules

Four monoclonal antibodies, SBU.II 28-1, 37-68, 38-27, and 42-20, each recognizing a distinct, non-overlapping subset of sheep class II molecules, were used to purify class II molecules from a single sheep. Four class II alpha subunits designated 28-1 alpha, 37-68 alpha, 42-20 alpha, and 38-27 alpha and five class II beta subunits designated 28-1 beta, 37-68 beta 1, 37-68 beta 2, 42-20 beta, and 38-27 beta were compared by N-terminal sequence analyses. Two distinct alpha subunits were identified; the 28-1 alpha, 37-68 alpha, and 42-20 alpha subunits all had identical N-terminal amino acids sequences, which exhibited about 75% homology with HLA-DR alpha and mouse E alpha polypeptides. In contrast, the 38-27 alpha sequence exhibited about 80% sequence homology with HLA-DQ alpha and mouse A alpha polypeptides. In general, sheep beta subunits displayed insufficient sequence homology to enable correlation with human beta-chain sequences; however, the 38-27 beta-chain sequence showed homology with the HLA-DQ beta sequence. The conserved sequence surrounding the site for N-linked glycosylation within human/mouse beta polypeptides (residues 19 to 21) was not present in sheep beta sequences and in contrast with the beta-chains of mouse and man, sheep beta polypeptides contained between 1 and 3 positionally variable cysteine residues (residues 13 to 15 inclusive). Individual sheep beta subunits exhibited extensive sequence heterogeneity and each consisted of a unique population of beta polypeptide species. At least 16 different beta polypeptide sequences were identified from a single sheep and the existence of no fewer than nine non-allelic beta genes was inferred from the sequence data. We have previously provided evidence suggesting that the sheep has multiple major histocompatibility complex class II alpha and beta genes related to those of all three HLA-D subregions. The present results suggest that a number of these genes encode HLA-DQ-like heterodimers and that a sheep DR-like alpha gene product is shared with the products of a large and heterogeneous sheep beta gene family.     

Chemical, physical-chemical, and immunological properties of papain-solubilized human transplatation antigens.

Papain-solubilized HLA-A, -B, and -C antigens have been isolated from cadaveric spleens. The isolated material was homogeneous and comprised subunits with the apparent molecular weights 33,000 and 12,000. Amino acid analyses of a mixture of HLA antigen heavy chains obtained from a great number of spleens with different HLA antigen phenotypes revealed a composition that is very similar to that of individual HLA-A and -B antigens. Likewise, the NH2-terminal 30 residues of the HLA-antigen heavy chain mixture were virtually identical with that recorded for individual specificities. The circular dichroism spectra for the isolated HLA antigens and for free beta2-microglobulin revealed similarities with spectra recorded for immunoglobulin chains and domains. The HLA-antigen heavy chain may contain an appreciable amount of beta structure. Antibodies raised against free beta2-microglobulin react better with beta2-microglobulin in free form than when bound to the HLA-A, -B, and -C antigen heavy chains. This is due to the fact that free beta2-microglobulin can bind a maximum of four Fab fragments simultaneously, whereas the HLA-antigen-associated beta2-microglobulin can bind only two Fab fragments without dissociating from the heavy HLA-antigen subunit.     

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.     

The molecular structure of different polymorphic forms of canine C3 and C4

The subunit composition of different electrophoretic forms of canine C3 and C4 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of reduced immune precipitates. Canine C4 comprises alpha, beta, and gamma chains of approximate molecular weight 90 000–95 000, 72 000, and 33 000, respectively. The molecular weight of the alpha chain of the C4 1 allelic product was approximately 95 000, but 90 000 for the C4 2 and C4 4 allotypes. No differences were observed in the molecular weights of the beta or gamma chains of any canine C4 phenotype tested. Canine C3 appears to be encoded by a single locus. The subunit composition comprises an alpha and beta chain with molecular weights of approximately 106 000 and 71000, respectively. Unlike C4, no differences in the molecular weights of the subunits were observed in different electrophoretic forms of canine C3.     

Class III human liver alcohol dehydrogenase: a novel structural type equidistantly related to the class I and class II enzymes

The primary structure of class III alcohol dehydrogenase (dimeric with chi subunits) from human liver has been determined by peptide analyses. The protein chain is a clearly distinct type of subunit distantly related to those of both human class I and class II alcohol dehydrogenases (with alpha, beta, gamma, and pi subunits, respectively). Disregarding a few gaps, residue differences in the chi protein chain with respect to beta 1 and pi occur at 139 and 140 positions, respectively. Compared to class I, the 373-residue chi structure has an extra residue, Cys after position 60, and two missing ones, the first two residues relative to class I, although the N-terminus is acetylated like that for those enzymes. The chi subunit contains two more tryptophan residues than the class I subunits, accounting for the increased absorbance at 280 nm. There are also four additional acidic and two fewer basic side chains than in the class I beta structure, compatible with the markedly different electrophoretic mobility of the class III enzyme. Residue differences between class III and the other classes occur with nearly equal frequency in the coenzyme-binding and catalytic domains. The similarity in the number of exchanges relative to that of the enzymes of the other two classes supports conclusions that the three classes of alcohol dehydrogenase reflect stages in the development of separate enzymes with distinct functional roles. In spite of the many exchanges, the residues critical to basic functional properties are either completely unchanged--all zinc ligands and space-restricted Gly residues--or partly unchanged--residues at the coenzyme-binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha chain of HLA-DR transplantation antigens is a member of the same protein superfamily as the immunoglobulins

Four cDNA clones, pDR-α-1, pDR-α-2, pDR-α-3 and pDR-α-4, corresponding to the alpha chain of HLADR antigens, have been sequenced. Restriction maps and sequences suggest that all clones are identical apart from a single-base substitution present in pDR-α-1. Amino acid sequence data, together with the nucleotide sequence data, allowed the complete amino acid sequence to be predicted. The alpha chain is composed of 229 amino acids, of which 191 are exposed on the outside of the plasma membrane. The membrane-embedded portion of the chain consists of 23 hydrophobic amino acids. The succeeding 15 amino acids form the cytoplasmically localized hydrophilic tail. The extracellular portion, with carbohydrate moieties linked to Asn⁷⁸ and Asn¹¹⁸, seems to be organized into two domains. The second domain, which contains the only disulfide bond of the alpha chain, displays amino acid sequence homology to immunoglobulin constant regions, to the second domain of the beta chain of a class II antigen, to the third domain of heavy chains of class I antigens and to β2-microglobulin. Thus the subunits of immunoglobulins, class I antigens and class II antigens are related evolutionarily.     

An oligosaccharide of the O-linked type distinguishes the free from the combined form of hCG alpha subunit

JAR malignant trophoblast cells produce a free alpha subunit in addition to an alpha combined with beta subunit as hCG. The free alpha is larger by gel chromatography and SDS-PAGE than combined alpha and is unable to associate with beta subunit to form hCG. A tryptic fragment, representing amino acid residues 36-42, derived from free alpha was larger than the corresponding fragment from combined alpha. After neuraminidase treatment, the fragment from free alpha bound peanut lectin agarose, which is specific for Gal beta 1-3GalNAc as found in O-linked oligosaccharides. The fragment also contained Gal and GalNAc (and a lesser amount of GlcNAc) as determined by glycosidase sensitivity and amino sugar analyses. Removal of this tryptic fragment ablated the size difference between free and combined alpha subunits.     

Structure, biosynthesis, and polymorphism of chicken MHC class II (B-L) antigens and associated molecules

Chicken MHC class II (B-L) antigens were immunoprecipitated by the monoclonal antibody TaP1 from inbred chicken splenic leukocytes and a lymphoblastoid B cell line (RP9), and were studied by two dimensional gel electrophoresis. B-L antigens are composed of one alpha and one beta chain that are noncovalently bound at the cell surface. In all haplotypes studied, a single acidic 34,000 dalton non-polymorphic chain was observed, whereas two polymorphic chains could be distinguished, differing in both pH and m.w. The alpha-beta heterodimer is associated during its maturation in the cytoplasm with several basic invariant molecules with m.w. ranging from 30,000 to 42,000 daltons. Treatment of cells with tunicamycin and treatment of immunoprecipitated molecules with several glycosidases revealed a complex process of maturation for all of these molecules. The alpha and beta chains undergo a N-glycosylation of complex type, whereas the invariant molecules bear N-linked high mannose glycans, and perhaps also O-linked glycans in the RP9 lymphoblastoid line. Overall, the B-L antigens appear very similar to the HLA-DR and I-E antigens.     

A new gene structure of the disintegrin family: a subunit of dimeric disintegrin has a short coding region

Disintegrin is a potent platelet aggregation inhibitor isolated from various snake venoms. The cDNA of the snake venom disintegrin family precursor is well-known to encode pre-peptide, metalloprotease, spacer, and disintegrin domains. Recently, new types of disintegrins, dimeric disintegrins, have been isolated, and their amino acid sequences were determined to be approximately 65 amino acid residues in each subunit. We isolated a novel heterodimeric disintegrin, acostatin, from the venom of Agkistrodon contortrix contortrix, which consisted of 63 and 64 amino acid residues in the alpha chain and beta chain, and both chains had the Arg-Gly-Asp (RGD) sequence for binding platelet GPIIb/IIIa. The cDNA lengths of the alpha chain and the beta chain of acostatin were 902 bp and 2031 bp, respectively. The acostatin alpha chain precursor, surprisingly, has the only disintegrin domain alone and lacked almost all of the pre-peptide and metalloprotease domains. The precursor of the acostatin beta chain belongs to a well-known motif of disintegrin precursors. Furthermore, both precursors of alpha and beta chains of another heterodimeric disintegrin, piscivostatin, also have the same domain structures as those of acostatin subunits. These results indicate that the cDNAs of heterodimeric disintegrin subunits have quite a different length of coding region and their precursors have a novel domain structure of disintegrin-family proteins.     

Human thyrotropin and its alpha and beta subunits.

A new procedure is described for the isolation of human thyrotropin using ion exchange chromatography and gel filtration only. Thyroid stimulating activity of the final preparation of our human thyrotropin amounted to 0.5 IU/mg by bioassay. The alpha and beta subunit of the hormone were also obtained by a new procedure. In this method the native hormone was incubated in an acidified 8 M urea solution and the chains were then separated by ion exchange chromatography and gel filtration. The amino-terminal residues of the alpha and beta chains were valine and phenylalanine respectively. The beta chain appears shorter at its carboxy-terminal end by one methionine residue than its bovine counterpart. Cross-contamination of the subunit preparations were measured by radioimmunoassay. The beta chain exhibited a contamination of about 3 percent of the alpha subunit by weight. The alpha subunit is contaminated by about one percent of the beta chain by weight.     

Galactose-binding lectin from the seeds of champedak (Artocarpus integer): sequences of its subunits and interactions with human serum O-glycosylated glycoproteins

Our group has previously reported the isolation, partial characterisation, and application of a Galbeta1-3GalNAc- and IgA1-reactive lectin from the seeds of champedak (Artocarpus integer). In the present study, we have subjected the purified lectin to reverse-phase high performance liquid chromatography and sequenced its subunits. Determination of the N-terminal sequence of the first 47 residues of the large subunit demonstrated at least 95% homology to the N-terminal sequence of the alpha chains of a few other galactose-binding Artocarpus lectins. The two smaller subunits of the lectin, each comprised of 21 amino acid residues, demonstrated minor sequence variability. Their sequences were generally comparable to the beta chains of the other galactose-binding Artocarpus lectins. When used to probe human serum glycopeptides that were separated by two-dimensional gel electrophoresis, the lectin demonstrated strong apparent interactions with glycopeptides of IgA1, hemopexin, alpha2-HS glycoprotein, alpha1-antichymotrypsin, and a few unknown glycoproteins. Immobilisation of the lectin to Sepharose generated an affinity column that may be used to isolate the O-glycosylated serum glycoproteins.     

Primary structures of carp gonadotropin subunits deduced from cDNA nucleotide sequences

The alpha and beta subunits of carp gonadotropin (cGTH) were isolated by high performance liquid chromatography. They were identified to be the subunits of cGTH by bioassay and by partial N-terminal amino acid sequence analysis. To elucidate the complete primary structures of the alpha and beta subunits of cGTH, cDNA cloning technique was employed. The alpha and beta subunits consist of 95 and 115 amino acid residues, respectively. Homology of the alpha subunit of cGTH to those of mammalian GTH is around 70%. In comparison, the extent of homology of the beta subunit between carp and salmon GTH (75%) is higher than that between fish and mammalian GTH (39-47%). Such comparative data suggest that the alpha subunit is highly conserved while the beta subunit is diversified during the molecular evolution of vertebrate GTH.     

Human luteinizing hormone. Isolation and characterization of the native hormone and its alpha and beta subunits.

A new procedure is described for the isolation of the alpha and beta chains of the hormone. In this method, thenative hormone is incubated in acidic urea and the chains are then separated by ion-exchange chromatography. The amino-terminal residue of the alpha subunit is valine. The carboxy-terminal end of the alpha subunit is of variable length. No amino-terminal residue was detected for the beta chain; glycine was found at its carboxy-terminal end by the selective titration method. The amino acid and carbohydrate compositions of the hormone and both subunits are presented. The beta chain contains sialic acid and is devoid of galactosamine in contrast to the beta subunits of other species. Contamination of our human lutenizing hormone preparation by other pituitary glycoprotein hormones such as thyroid-stimulating hormone and follicle-stimulating hormone amounted to 0.5 and 0.25 percent by weight respectively. Cross-contamination of the initial alpha and beta subunit preparations was measured by specific radioimmunoassays and amounted to 4.1 and 2 percent by weight respecitively. Further extensive purification of these subunit preparations was then performed by means of affinity chromatography using immunosorbants. The final preparations exhibited a residual cross-contamination amounting to 0.2 and 0.02 percent by weight for the alpha and beta subunits respectively.     

Differential regulation of HLA-DR mRNAs and cell surface antigens by interferon

Human interferons-alpha, -beta and -gamma enhance HLA-DR mRNAs in all the human lymphoblastoid and melanoma cell lines studied. The increase concerns both alpha and beta chain mRNAs. Moreover, we show that immune interferon-gamma preferentially enhances class II MHC mRNA. This effect of IFN-gamma on the synthesis of alpha and beta HLA-DR chains has been also analysed by immunoprecipitation. It is abolished by a monoclonal antibody directed against human IFN-gamma. The effect of interferon on the cell surface level of HLA-DR molecules does not always correspond to the enhancement of HLA-DR mRNA. Our experiments suggest that this discrepancy between the enhancement of HLA-DR mRNA and cell surface antigen might be due to a constitutively high level of the corresponding antigens on several of the human cells studied.     

Direct identification of class II histocompatibility DR proteins in preparations of human T-cell lymphotropic virus type III

Class II histocompatibility DR antigen alpha and beta chains were isolated from preparations of human T-cell lymphotropic virus type III grown in human H-9 cells. The proteins were purified by reversed-phase high-pressure liquid chromatography and identified by direct N-terminal amino acid sequence analysis of each chain. The purified DR alpha chain had an N-terminal amino acid sequence identical to the known sequence of human DR alpha chain through the first 37 residues. The N-terminal amino acid sequence of the purified DR beta chain was identical to that of human DR4 beta chain. The DR alpha and beta chains appeared to be identical to the p34-36K and p30-32K proteins, respectively, concentrated in immunostimulatory complexes prepared from unfractionated virus and were the major immunogens in these complexes. These proteins represent a ready source of antigens which can cause false-positive enzyme-linked immunosorbent assay reactions in individuals previously exposed to allogenic histocompatibility antigens. The removal of the DR chains from virus preparations by use of available monoclonal antibodies or other means should result in a lower rate of initial false-positive enzyme-linked immunosorbent assay reactions.     

Purification and properties of citrate lyase from Escherichia coli

Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction. The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis. The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons. An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones. The polypeptide subunits have been isolated in pure state and their biological functions characterize. The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid. The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A. The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits. The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases. A ligase which acetylates the deacetyl[citrate lyase] in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural relationships among class I isozymes of human liver alcohol dehydrogenase

The alpha subunit of human liver alcohol dehydrogenase has been submitted to structural analysis. Together with earlier work on the beta and gamma subunits, the results allow conclusions on the relationship of all known forms of the class I type of the enzyme. Two segments of the alpha subunit were determined; one was also reinvestigated in the beta and gamma subunits. The results establish 11 residue replacements among class I subunits in the segments analyzed and show that the alpha, beta, and gamma protein chains each are structurally distinct in the active site regions, where replacements affect positions influencing coenzyme binding (position 47; Gly in alpha, Arg in beta and gamma) and substrate specificity (position 48; Thr in alpha and beta, Ser in gamma). Residue 128, previously not detected in beta and gamma subunits, corresponds to a position of another isozyme difference (Arg in beta and gamma, Ser in alpha). The many amino acid replacements in alcohol dehydrogenases even at their active sites illustrate that in judgements of enzyme functions absolute importance of single residues should not be overemphasized. Available data suggest that alpha and gamma are the more dissimilar forms within the family of the three class I subunits that have resulted from two gene duplications. The class distinction of alcohol dehydrogenases previously suggested from enzymatic, electrophoretic, and immunological properties therefore also holds true in relation to their structures.