title: Characterization of N- and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris.
abstract: 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.
authors.affiliation: Wadsworth Center C-547, New York State Department of Health, PO Box 509, Albany, NY 12201-0509, USA. trimble@wadsworth.org
authors.forename: Robert B
authors.initials: RB
authors.lastname: Trimble
authors.forename: Catherine
authors.initials: C
authors.lastname: Lubowski
authors.forename: Charles R
authors.initials: CR
authors.lastname: Hauer
authors.forename: Robert
authors.initials: R
authors.lastname: Stack
authors.forename: Lynn
authors.initials: L
authors.lastname: McNaughton
authors.forename: Trent R
authors.initials: TR
authors.lastname: Gemmill
authors.forename: S Anand
authors.initials: SA
authors.lastname: Kumar
mesh_terms: D002240:Carbohydrate Sequence; D006031:Glycosylation; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010843:Pichia / Q000378:metabolism*; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D002787:Sterol Esterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013402:Sugar Alcohols / Q000737:chemistry
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D013402:Sugar Alcohols; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase