Characterization of N- and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris
| Autor: | Charles R. Hauer, Trent R. Gemmill, Catherine Lubowski, Robert B. Trimble, S. Anand Kumar, Lynn McNaughton, Robert F. Stack |
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| Rok vydání: | 2003 |
| Předmět: |
Glycosylation
Magnetic Resonance Spectroscopy Molecular Sequence Data Alpha (ethology) Biochemistry Pichia law.invention Pichia pastoris chemistry.chemical_compound Sugar Alcohols Polysaccharides law Humans Phosphorylation Lipase chemistry.chemical_classification biology Sterol Esterase biology.organism_classification Molecular biology Recombinant Proteins carbohydrates (lipids) Carbohydrate Sequence chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Recombinant DNA biology.protein O-linked glycosylation Glycoprotein |
| Zdroj: | Glycobiology. 14:265-274 |
| ISSN: | 1460-2423 |
| DOI: | 10.1093/glycob/cwh036 |
| Popis: | Recombinant human bile salt-stimulated lipase (hBSSL) was expressed in and secreted by Pichia pastoris, an organism exploited for the large-scale production of recombinant (glyco)proteins by bioprocessing technology. The 76.3-kDa glycoprotein was associated with 75-80 Man and a small amount of GlcNAc. hBSSL has one N-glycosylation site at Asn187, which was 38-40% occupied with a Man(10)GlcNAc(2) structure defined previously in Pichia as the oligosaccharide-lipid form of Man(9)GlcNAc(2) trimmed of the middle-arm terminal alpha 1,2-Man and elongated with Man alpha 1,2Man alpha 1,6-disaccharide attached to the lower-arm core alpha 1,3-Man (Trimble et al. [1991], J. Biol. Chem., 266, 22807-22817). The C-terminal 192 residues of hBSSL contain 16 Pro-rich 11-amino-acid repeats, which include 32 Ser/Thr residues as potential O-glycosylation sites. Using hBSSL as a platform to study Pichia's O-glycosylation capabilities, we found that nearly all of these sites were occupied by mannose-containing O-glycans, whose structures, after beta-elimination and purification, were assigned by (1)H NMR and, in some cases, by linkage-specific exoglycosidases and methylation analysis. The most abundant O-glycan was alpha 1,2-mannobiitol (55%), followed by alpha 1,2-mannotriitol (16%) and mannitol (10%) and a lesser amount was alpha 1,2-mannotetraitol. Unexpectedly, Man(5) and Man(6) O-glycans were present, which had the structure Man beta 1,2Man beta 1,2Man alpha 1,2(Man alpha 1,2)(1,2)mannitol. Also a small amount of a phosphorylated Man(6) O-glycan was characterized by MALDI-TOF MS postsource decay analysis as having the reducing-end mannitol disubstituted with a glycosidically linked phosphorylated Man and an unbranched Man(4) polymer elongated from a different mannitol carbon. This is the first report of the synthesis of beta-Man- and phosphate-containing O-linked constituents on glycoproteins synthesized by P. pastoris. |
| Databáze: | OpenAIRE |
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