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Glycobiology Advance Access published online on December 23, 2003
Glycobiology, doi:10.1093/glycob/cwh036
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Submitted on September 25, 2003
Revised on November 5, 2003
Accepted on November 17, 2003

© 2003 Oxford University Press

ORIGINAL ARTICLES

Characterization of N- and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris

Robert B. Trimble 1*, Catherine Lubowski 1, Charles R. Hauer III1, Robert Stack 1, Lynn McNaughton 1, Trent R. Gemmill 1, and S. Anand Kumar 2

1 Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA
2 Astra Zeneca Research Foundation India, Bellary Road, Hebbal, Bangalore 560 024, India

* To whom correspondence should be addressed. E-mail: trimble{at}wadsworth.org.

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 Man10GlcNAc2 structure defined previously in Pichia as the oligosaccharide-lipid form of Man9GlcNAc2 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 1H 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%), mannitol (10%), and a lesser amount was {alpha}1,2-mannotetraitol. Unexpectedly, Man5 and Man6 O-glycans were present, which had the structure, Man{beta}1,2Man{beta}1,2Man{alpha}1,2Man{alpha}1,2(Man{alpha}1,2)1,2Mannitol. Also a small amount of a phosphorylated Man6 O-glycan was characterized by MALDI TOF MS post source decay analysis as having the reducing-end mannitol di-substituted 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 {beta}-Man- and phosphate-containing O-linked constituents on glycoproteins synthesized by Pichia pastoris.


bile salt-stimulated lipase, glycan NMR, N-glycosylation, O-glycosylation, P. pastoris
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