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 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