Glycobiology Advance Access originally published online on December 23, 2003
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Glycobiology vol 14 no 3 pp. 265-274, 2004
Glycobiology vol. 14 no. 3 © Oxford University Press 2004; all rights reserved.
Characterization of N- and O-linked glycosylation of recombinant human bile salt–stimulated lipase secreted by Pichia pastoris
2 Wadsworth Center C-547, New York State Department of Health, PO Box 509, Albany, NY 12201-0509; and 3 Astra Zeneca Research Foundation India, Bellary Road, Hebbal, Bangalore 560 024, India
Received on September 25, 2003; revised on November 5, 2003; accepted on November 17, 2003
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 Man10GlcNAc2 structure defined previously in Pichia as the oligosaccharide-lipid form of Man9GlcNAc2 trimmed of the middle-arm terminal 
1,2-Man and elongated with Man1,2Man1,6-disaccharide attached to the lower-arm core 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 ß-elimination and purification, were assigned by 1H NMR and, in some cases, by linkage-specific exoglycosidases and methylation analysis. The most abundant O-glycan was 1,2-mannobiitol (55%), followed by 1,2-mannotriitol (16%) and mannitol (10%) and a lesser amount was 1,2-mannotetraitol. Unexpectedly, Man5 and Man6 O-glycans were present, which had the structure Manß1,2Manß1,2Man1,2(Man1,2)1,2mannitol. Also a small amount of a phosphorylated Man6 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 Man4 polymer elongated from a different mannitol carbon. This is the first report of the synthesis of ß-Man- and phosphate-containing O-linked constituents on glycoproteins synthesized by P. pastoris.
1 To whom correspondence should be addressed; e-mail: trimble{at}wadsworth.org
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