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 Man10GlcNAc2 structure defined previously in Pichia as the oligosaccharide-lipid form of Man9GlcNAc2 trimmed of the middle-arm terminal α1,2-Man and elongated with Manα1,2Manα1,6-disaccharide attached to the lower-arm core α1,3-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 β-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 α1,2-mannobiitol (55%), followed by α1,2-mannotriitol (16%) and mannitol (10%) and a lesser amount was α1,2-mannotetraitol. Unexpectedly, Man5 and Man6 O-glycans were present, which had the structure Manβ1,2Manβ1,2Manα1,2(Manα1,2 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 β-Man-and phosphate-containing O-linked constituents on glycoproteins synthesized by P. pastoris. © Oxford University Press 2004; all rights reserved.