Showing papers by "Per A. Peterson published in 1983"

HLA-D region β-chain DNA endonuclease fragments differ between HLA-DR identical healthy and insulin-dependent diabetic individuals

Abstract: The human HLA-D histocompatibility region encodes class II antigens each of which consists of two polypeptide chains (α and β) inserted in the plasma membrane. These molecules are implicated in the regulation of the immune response1-3 but several human diseases are also found to be associated with certain HLA-DR antigens4. The occurrence of insulin-dependent (type I) diabetes (IDDM) is strongly associated with HLA-DR3 and/or 4 (ref. 5). The class II antigens, however, show a marked genetic polymorphism1 associated with the β-chains6,7 which seem, from hybridization studies, to be encoded by several genes8,9. We have therefore used the β-chain cDNA probe, pDR-β-1 (refs 8, 10) to test whether there are differences in hybridization pattern between DNA from healthy individuals and diabetic patients, after digestion with restriction endonucleases. Among the HLA-DR 4 and 3/4 individuals, the IDDM patients showed an increased frequency of a PstI 18 kilobase (kb) fragment. A BamHI 3.7 kb fragment, frequent among controls (30-40%), was rarely detected in the IDDM patients (0-2%). These differences may be related to susceptibility to develop the disease. (Less)

Exon-intron organization and complete nucleotide sequence of a human major histocompatibility antigen DC beta gene.

Abstract: We have determined the complete nucleotide sequence of a human class II histocompatibility antigen DC beta gene. The gene spans more than 7 kilobases and contains five exons corresponding to the different domains of the DC beta polypeptide. The exon-intron organization is thus analogous to that of class II antigen alpha-chain genes, class I antigen heavy chain genes, and the constant parts of immunoglobulin genes, emphasizing further the evolutionary relationship among these molecules. The mature polypeptide deduced from the DC beta gene shows 93% and 88% homology, respectively, to sequences derived from two DC beta cDNA clones of other haplotypes. The allelic polymorphism of DC beta chains resides predominantly in the first extracellular domain, whereas the rest of the polypeptide is virtually constant. The exons of the DC beta gene display high homology to the corresponding exons of a murine I-A beta gene. Also, the introns show significant homology. The DC beta chains lack eight amino acids in the cytoplasmic tail, as compared to DR and I-A beta chains. This is probably due to a nonfunctional splice junction of DC beta genes, causing a separate cytoplasmic exon to be nonexpressed.

cDNA clone for the human invariant gamma chain of class II histocompatibility antigens and its implications for the protein structure.

Abstract: The invariant gamma chain is transitorily associated with class II histocompatibility antigens during intracellular transport. We have isolated and sequenced a cDNA clone corresponding to the human gamma chain. mRNA hybridizing to the cDNA clone translated into a 33,000-dalton chain that associated specifically with class II antigen alpha and beta chains. The gamma chain consists of 216 amino acids. The two N-linked carbohydrates are attached to asparagines 114 and 120. A continuous stretch of hydrophobic and neutral amino acids occurs in positions 31-56 from the NH2 terminus. This region seems to constitute the transmembrane portion of the polypeptide chain. The positions of the carbohydrate moieties and the putative transmembrane segment indicate that the NH2 terminus of the gamma chain resides on the cytoplasmic side of the membrane. Cell-free translations in conjunction with radiochemical amino acid sequence analyses suggest that the gamma chain lacks an NH2-terminal signal sequence.

Ligand-dependent regulation of intracellular protein transport: effect of vitamin a on the secretion of the retinol-binding protein.

Abstract: As a model of ligand-dependent protein secretion the biosynthesis, intracellular transport, and release of the retinol-binding protein (RBP) were studied in primary cultures of rat hepatocytes pulse-labeled with [35S]methionine. After various periods of chase RBP was isolated by immunoprecipitation and identified by SDS PAGE. Both normal and vitamin A-deficient hepatocytes synthesized RBP. The normal cells secreted the pulse-labeled RBP within 2 h. RBP synthesized by deficient cells was not secreted, and intracellular degradation of the protein appeared to be slow. Deficient cells could be induced to secrete RBP on the addition of retinol to the culture medium. This occurred also after protein synthesis had been blocked by cycloheximide. Since retinol induces the secretion of RBP, accumulated in the endoplasmic reticulum (ER), it seems reasonable to conclude that the transport of RBP from the ER to the Golgi complex is regulated by retinol.

Effects of the glucosidase inhibitors nojirimycin and deoxynojirimycin on the biosynthesis of membrane and secretory glycoproteins.

Abstract: The glucosidase inhibitors nojirimycin (NM) and 1-deoxynojirimycin (dNM) interfere with N-linked glycosylation. The effects of NM and dNM on the biosynthesis of secretory glycoproteins (IgD and IgM) and membrane glycoproteins (HLA-A, B, C and -DR antigens) have been examined. Whereas treatment of IgD- and IgM-producing cells with NM results in the transfer of drastically shortened oligosaccharide side chains, treatment with dNM inhibits trimming, most probably through interaction with glucosidase I and/or II. A comparison of NM and dNM with tunicamycin and the mannosidase inhibitor swainsonine (SW) show that each of the inhibitors interferes with N-linked glycosylation in a distinct manner. For both Ig and HLA antigens, the effects of SW are discernible at the final stages of glycan maturation only, whereas the effects of dNM are observed quite early in the biosynthetic process. The secretion of IgD, but not IgM, was blocked in dNM-treated cells. The HLA-A, B, C heavy chains synthesized by the Daudi cell line were degraded in an accelerated fashion in dNM-treated cells, but no effects were seen on the HLA-DR antigens in these cells. Although both SW and dNM interfere with trimming, further modifications of the oligosaccharide side chains occur, and show that the two processes are not obligately coupled. Glucosidase inhibitors such as NM and dNM, as well as the mannosidase inhibitor SW, allow modification of glycan structure, and may be used to study the biological role of glycoprotein oligosaccharides and their modifications.

The complete nucleotide sequence of the I-E alpha d immune response gene.

Abstract: We have isolated and sequenced the complete murine I-E alpha immune response gene of the H-2db haplotype. The I-E alpha d gene consists of 5300 basepairs and is organized into five or possibly six exons that correspond to different domains of the alpha chain. The amino acid sequence deduced from the I-E alpha gene shows 75% homology to its human counterpart, the HLA-DR alpha chain. The absence of I-E antigen in H-2 mice is due to lack of E alpha chain synthesis. We show here that this defect is caused by a deletion in the 5' end of the I-E alpha b gene.

Human class II major histocompatibility antigen β -chains are derived from at least three loci

Abstract: Class II antigens of the major histocompatibility complex (MHC) consist of two glycosylated, membrane-integrated polypeptide chains1. These cell surface-expressed molecules are involved in several immunobiological events involving cell-cell interactions2,3, most of which seem to require that genetically identical class II antigens, or other molecules controlled by the same region of the MHC, are expressed on the interacting cells4. The extensive genetic polymorphism of the class II antigens5 has rendered analyses in the human system of the number of non-allelic species of class II antigens difficult, although several laboratories have reported the existence of at least two types of human class II antigens6-9. Here we present the results of experiments using restriction enzyme digestions and separation of DNA from individuals homozygous for the MHC followed by hybridization to human class II antigen α-10,11 and β-12-14 chain cDNA probes. While the α -chain probe gave only a single hybridization band, the various β -chain probes revealed a more complex pattern that is consistent with the existence of at least three separate β -chain genes or pseudogenes in the human MHC. (Less)

Detection of HLA-D/DR-related DNA polymorphism in HLA-D homozygous typing cells.

Abstract: Sequences of different sizes are generated when DNA from homozygous HLA-Dw/DR typing cells are digested with restriction endonuclease and analyzed by hybridization with a HLA-D region class II antigen beta-chain cDNA probe. The patterns of hybridization were highly polymorphic but one endonuclease, BamHI, defined sequences unique to all HLA-Dw/DR specificities 1-8 except HLA-Dw/DR 2 and 6; however, these two specificities were resolved with the enzyme EcoRI. Digestion with other endonucleases such as Pst I results in patterns of restriction fragments that differ between homozygous typing cells of the same HLA-Dw/DR specificity. HLA-D region beta-chain probes permit HLA-D region genotyping at the DNA level and may allow detection of genes controlling the association of HLA specificities with a wide variety of diseases.

Association of human gamma chain with class II transplantation antigens during intracellular transport.

Abstract: Cell surface expressed human and murine class II transplantation antigens are composed of two polypeptide chains called alpha and beta. During intracellular transport an invariant chain, provisionally called gamma, is associated with the class II antigen chains. Since gamma chains leave the endoplasmic reticulum only when associated with alpha and beta chains, we have studied the intracellular transport of the gamma chain and its possible cell surface expression. Modifications of the carbohydrate moieties of the gamma chain during intracellular transport were also examined. The gamma chain appears to contain two Asn-linked carbohydrate moieties and maybe also one or more Ser/Thr-linked carbohydrates. At all times during the pulse-chase experiments core glycosylated gamma chains resolved into two distinct spots on two-dimensional gel electrophoresis. The occurrence of core-glycosylated gamma chains was expected since more gamma chains than alpha and beta chains exist in the endoplasmic reticulum. Terminally glycosylated, alpha, beta, and gamma chains emerged simultaneously supporting the idea that the three types of chains are brought to the Golgi complex bound to each other. However, terminal glycosylation is temporally related to the dissociation of the gamma chain from the alpha and beta chains. Since isolated plasma membranes contained molecules indistinguishable from gamma chains, it is concluded that gamma chains are transported together with class II antigens from the endoplasmic reticulum to the Golgi complex. After dissociation, class II antigens and some, if not all, gamma chains seem to become independently integrated into the plasma membrane.

[22] Glycophorin A: In vitro biogenesis and processing

Abstract: Publisher Summary This chapter emphasizes on the synthesis of the glycophorin A polypeptide in the K562 cell line and its intriguing posttranslational modifications, including N- and O-glycosylation, phosphorylation, and sulfation. The chapter also discusses its cell-free synthesis using glycophorin A mRNA obtained from K562 cells. The methods involve radioactive cell surface labeling of erythrocytes, radioactive metabolic labeling of K562 Cells, isolation of mRNA from K562 Cells, translation of mRNA in vitro, preparation of Lectin-Sepharose columns, preparation of anti-glycophorin A antiserum, Lectin-Sepharose affinity chromatography and immune precipitations, treatment of glycophorin A with endoglycosidase H, and polyacrylamide slab gel electrophoresis. The illustrative data and interpretation shows that tunicamycin inhibits N-glycosylation of proteins, and endoglycosidase H has no effect on either the lentil lectin- or wheat germ lectin-adsorbed molecules. The tunicamycin experiments clearly shows that the absence of the N-glycosidic oligosaccharide does not affect the intracellular migration of glycophorin A.

Growth factor-binding proteases in the murine submaxillary gland: isolation of a cDNA clone.

Abstract: The submaxillary gland of the adult male mouse contains a number of serine proteases, several of which are involved in the proteolytic processing of precursors to growth factors and other biologically active polypeptides. Here we report the isolation and identification of a cDNA clone corresponding to one of the proteases, the type B of the epidermal growth factor-binding protein. A pronounced sequence homology was found between the predicted activation peptide of this protease and the NH2-terminal extension of the nerve growth factor alpha subunit, suggesting that the latter protein has an uncleaved activation peptide attached to its NH2 terminus.

The complete nucle0tide sequence of the i-e-alpha-d immune response gene.

Abstract: We have isolated and sequenced the complete murine I-E alpha immune response gene of the H-2db haplotype. The I-E alpha d gene consists of 5300 basepairs and is organized into five or possibly six exons that correspond to different domains of the alpha chain. The amino acid sequence deduced from the I-E alpha gene shows 75% homology to its human counterpart, the HLA-DR alpha chain. The absence of I-E antigen in H-2 mice is due to lack of E alpha chain synthesis. We show here that this defect is caused by a deletion in the 5' end of the I-E alpha b gene.

Identification and Localization of Two Boar Sperm-Membrane Proteins

Abstract: The plasmalemma of the mammalian spermatozoon contains a number of specific proteins, some of which have a clear regional distribution (see ref. 1–5). It is reasonable to assume that one or more sperm membrane proteins are involved in the initial recognition events of fertilization. We have isolated a number of boar sperm membrane glycoproteins and in this communication two of them will be described in some detail.

Procede de determination du type de tissus codes par des genes mhc

Abstract: Un nouveau procede permet de determiner le polymorphisme d'alleles humains MHC (complexe majeur d'histocompatibilite) pour determiner le type de tissus, de preference de tissus du type HLA-D (Leucocytes humains, systeme A, type D). Une substance revelatrice qui s'hybride avec une sequence de base dans le MHC est additionnee a des fragments d'ADN resultant d'analyse par restriction et provenant de differents individus. La distribution par grandeur des fragments d'ADN qui s'hybrident avec la substance revelatrice et qui proviennent d'un premier individu est comparee avec la distribution correspondante de fragments d'ADN d'un deuxieme individu ou avec la distribution correspondante refletant un type particulier de polymorphisme. Les comparaisons sont effectuees afin de determiner a) la compatibilite genetique dudit deuxieme individu a des fins de greffe ou de transfusion de sang, b) les risques relatifs pour le premier individu de contracter une maladie particuliere liee a un ou plusieurs alleles du MHC, c) la paternite ou la maternite biologique du premier individu ou d) si le premier et le deuxieme individus presentent le meme polymorphisme d'un ou plusieurs alleles particuliers du MHC, c'est-a-dire, le procede peut etre utilise en medecine legale.