Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha.
Presently almost no information is available on the oligosaccharide structure of the glycoproteins secreted from the methylotrophic yeast Hansenula polymorpha, a promising host for the production of recombinant proteins. In this study, we analyze the size distribution and structure of N-linked oligosaccharides attached to the recombinant glycoprotein glucose oxidase (GOD) and the cell wall mannoproteins obtained from H. polymorpha. Oligosaccharide profiling showed that the major oligosaccharide species derived from the H. polymorpha-secreted recombinant GOD (rGOD) had core-type structures (Man(8-12)GlcNAc(2)). Analyses using anti-alpha 1,3-mannose antibody and exoglycosidases specific for alpha 1,2- or alpha 1,6-mannose linkages revealed that the mannose outer chains of N-glycans on the rGOD have very short alpha 1,6 extensions and are mainly elongated in alpha 1,2-linkages without a terminal alpha 1,3-linked mannose addition. The N-glycans released from the H. polymorpha mannoproteins were shown to contain mostly mannose in their outer chains, which displayed almost identical size distribution and structure to those of H. polymorpha-derived rGOD. These results strongly indicate that the outer chain processing of N-glycans by H. polymorpha significantly differs from that by Saccharomyces cerevisiae, thus generating much shorter mannose outer chains devoid of terminal alpha 1,3-linked mannoses.
Kim
Moo Woong
MW
Metabolic Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology, Oun-dong 52, Yusong-gu, Daejeon 305-600, Korea.
Rhee
Sang Ki
SK
Kim
Jeong-Yoon
JY
Shimma
Yoh-ichi
Y
Chiba
Yasunori
Y
Jigami
Yoshifumi
Y
Kang
Hyun Ah
HA
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005656:Fungal Proteins / Q000378:metabolism*; D005949:Glucose Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; C025363:mannoproteins; D005949:Glucose Oxidase; D006026:Glycoside Hydrolases