Abstract
Oligosaccharyltransferase (OST) is an oligomeric protein complex which catalyses the transfer en bloc of Glc3-Man9-GlcNAc2 from Dol-PP to specific asparagine residues in the nascent polypeptide chain. In order to study the function of the pig enzyme subunits, we have cloned OST48, ribophorin I and ribophorin II and characterized these proteins after in vitro translation as well as after expression in COS-1 cells. The individual full-length cDNAs contained open reading frames (ORFs) encoding polypeptides with calculated molecular masses of ∼48.9[emsp4 ]kDa (OST48), ∼68.7[emsp4 ]kDa (ribophorin I) and ∼69.3[emsp4 ]kDa (ribophorin II), respectively. A Kyte and Doolittle hydrophobicity analysis revealed that OST48, ribophorin I and ribophorin II possess a type I membrane topology with the bulk of their polypeptide chains directed towards the ER-lumen. In contrast to OST48, ribophorin I and II contain, respectively, three or two potential N-glycosylation sites of the Asn-Xaa-Thr/Ser type; only one is found to function as the acceptor site in each protein.
Transfection of COS-1 cells with vector constructs encoding either OST48, ribophorin I, or a ribophorin I variant tagged with a myc-peptide sequence, resulted in the over-expression of polypeptides whose molecular masses were similar to those calculated from the respective cDNA ORFs. None of these three polypeptides, or ribophorin II, were found to display OST activity when over-expressed alone. By contrast, a modest but reproducible ∼25% increase of activity was observed when OST48 together with ribophorin I, or OST48 and myc-tagged ribophorin I, were co-expressed, indicating that these two subunits are probably responsible for the catalytic activity in the hetero-oligomeric OST complex. The only modest over-expression of transferase activity suggests that either the dimeric enzyme complex is catalytically unstable, or that the OST48 and ribophorin I polypeptides are unable to fold properly when other subunit components of the hetero-oligomeric OST complex are lacking. OST48 as well as ribophorin I are expressed in COS-1 cells as ER-resident proteins. Whereas OST48 carries a double-lysine motif in the −3/−5 position of its cytosolic C-terminal domain, ribophorin I does not contain recognizable ER-retention information. Replacing the lysine residue in the −3 position by leucine resulted in plasma membrane expression of the OST48-Leu polypeptide, indicating that this sequence motif may be able to influence OST48 localisation. No cell surface staining was observed when OST48-Leu was co-expressed with ribophorin I. This suggests that localisation of OST48 in the ER is mediated by interaction with ribophorin I rather than by the double-lysine motif.
Similar content being viewed by others
References
Kornfeld R, Kornfeld S, Assembly of asparagine-linked oligosaccharides, Annu Rev Biochem 54, 631–664 (1985).
Bause E, Studies on the acceptor specifity of asparagine-N-glycosyltransferase from rat liver, FEBS Lett 103, 296–299 (1979).
Bause E, Structural requirements of N-glycosylation of proteins: studies with proline peptides as conformational probes, Biochem J 209, 331–336 (1983).
Bause E, Breuer W, Peters S, Investigation of the active site of oligosaccharyltransferase from pig liver using synthetic tripeptides as tools, Biochem J 312, 979–985 (1995).
Imperiali B, Protein glycosylation: The clash of the Titans, Acc Chem Res 30, 452–459 (1997).
Kelleher DJ, Kreibich G, Gilmore R, Oligosaccharyltransferase activity is associated with a protein complex composed of ribophorin I and II and a 48 kd protein, Cell 69, 55–65 (1992).
Kumar V, Heinemann FS, Ozols J, Purification and characterization of avian oligosaccharyltransferase. Complete amino acid sequence of the 50 kDa subunit, J Biol Chem 269, 13451–13457 (1994).
Kumar V, Heinemann FS, Ozols J, Purification and characterization of hepatic oligosaccharyltransferase, Biochem Mol Biol Int 36, 817–826 (1995).
Kumar V, Korza G, Heinemann FS, Ozols J, Human oligosaccharyltransferase: isolation, characterization, and the complete amino acid sequence of 50-kDa subunit, Arch Biochem Biophys 320, 217–223 (1995).
Breuer W, Bause E, Oligosaccharyl transferase is a constitutive component of an oligomeric protein complex from pig liver endoplasmatic reticulum, Eur J Biochem 228, 689–696 (1995).
Kelleher DJ, Gilmore R, DAD1, the defender against apoptotic cell death, is a subunit of the mammalian oligosaccharyltransferase, Proc Natl Acad Sci (USA) 94, 4994–4999 (1997).
Bause E, Wesemann M, Bartoschek A, Breuer W, Epoxyethylglycyl peptides as inhibitors of oligosaccharyltransferase: double-labelling of the active site, Biochem J 322, 95–102 (1997).
Yan Q, Prestwich GD, Lennarz WJ, The Ost 1p subunit of yeast oligosaccharyltransferase recognizes the peptide glycosylation site sequence, Asn-X-Ser/Thr, J Biol Chem 274, 5021–5025 (1999).
Crimaudo C, Hortsch M, Gansepohl H, Meyer, DI, Human ribophorin I and II: the primary structure and membrane topology of two highly conserved rough endoplasmic reticulum-specific glycoproteins, EMBO J 6, 75–82 (1987).
Harnik-Ort V, Prakash K, Marcanto E, Colman DR, Rosenfeld MG, Adesnik M, Sabatini DD, Kreibich G, Isolation and characterization of cDNA clones for rat ribophorin I: complete coding sequence and in vitro synthesis and insertion of the encoded product into endoplasmic reticulum membranes, J Cell Biol 104, 855–863 (1987).
Pirozzi G, Zhou Z, D'Eustachio P, Sabatini DD, Kreibich G, Rat ribophorin II: Molecular cloning and chromosomal localization of a highly conserved transmembrane glycoprotein of the rough endoplasmic reticulum, Biochem Biophys Res Commun 176, 1482–1486 (1991).
Silberstein S, Kelleher DJ, Gilmore R, The 48-kDa subunit of the mammalian oligosaccharyltransferase complex is homologous to the essential yeast protein WBP1, J Biol Chem 267, 23658–23663 (1992).
Bieberich E, Bause E, Man9-mannosidase from human kidney is expressed in COS cells as a Golgi resident type II transmembrane N-Glycoprotein, Eur J Biochem 233, 644–649 (1995).
Schweden J, Bause E, Characterization of trimming Man9-mannosidase from pig liver. Purification of a catalytically active fragment and evidence for the transmembrane nature of the intact 65 kDa enzyme, Biochem J 264, 347–355 (1989).
Laemmli UK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227, 680–685 (1970).
Sakai K, Gelfand DH, Stoffels S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA, Primer-directed enzymatic amplification of DNA with a thermostabile DNA polymerase, Science 239, 487–491 (1988).
White BA, PCR-Protocols — Current Methods and Applications, Ottawa, Humana Press (1993).
Treml K, Meimaroglou D, Hentges A, Bause E, The α-and β-subunits are required for expression of catalytic activity in the hetero-dimeric glucosidase II complex from human liver, Glycobiology 10, 493–502 (2000).
Sanger F, Nicklen S, Coulson AR, DNA sequencing with chain termination inhibitors, Proc Natl Acad Sci 74, 5463–5467 (1977).
Kyte J, Doolittle RF, A simple method for displaying the hydropathic character of a protein, J Mol Biol 152, 105–132 (1982).
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, Current Protocols in Molecular Biology, New York, John Wiley & Sons (1987).
Sambrook J, Fritsch EF, Maniatis T, Molecular Cloning: A Laboratory Manual, New York, Cold Spring Habor Laboratory Press (1989).
Von Heijne G, Pattern of amino acids near signal-sequence cleavage sites, Eur J Biochem 133, 17–21 (1983).
Cossen P, Letourneur F, Coatomer interaction with di-lysine endoplasmic reticulum retention motifs, Science 263, 1629–1631 (1994).
Nilsson T, Warren G, Retention and retrieval in the endoplasmic reticulum and the Golgi apparatus, Current Opinion in Cell Biology 6, 517–521 (1994).
Fu J, Kreibich G, Retention of subunits of the oligosaccharyl-transferase complex in the endoplasmic reticulum, J Biol Chem 275, 3984–3990 (2000).
Albright CF, Orlean P, Robbins PW, A 13-amino acid peptide in three yeast glycosyltransferases may be involved in dolichol recognition, Proc Natl Acad Sci (USA) 86, 7366–7369 (1989).
Pathak R, Hendrickson TL, Imperiali B, Sulfhydryl Modification of the Yeast Wbplp Inhibits Oligosaccharyl Transferase Activity, Biochemistry 34, 4179–4185 (1995).
Hentges A, Bause E, Affinity purification and characterization of glucosidase II from pig liver, Biol Chem 378, 1031–1038 (1997).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hardt, B., Aparicio, R. & Bause, E. The oligosaccharyltransferase complex from pig liver: cDNA cloning, expression and functional characterisation. Glycoconj J 17, 767–779 (2000). https://doi.org/10.1023/A:1010980524785
Issue Date:
DOI: https://doi.org/10.1023/A:1010980524785