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J. Biol. Chem., Vol. 281, Issue 10, 6261-6272, March 10, 2006

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Functional Characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 Genes as Members of the Yeast OCH1 Mannosyltransferase Family Involved in Protein Glycosylation^*

Moo Woong Kim, Eun Jung Kim, Jeong-Yoon Kim, Jeong-Seok Park, Doo-Byoung Oh, Yoh-ichi Shimma^¶, Yasunori Chiba^¶, Yoshifumi Jigami^¶, Sang Ki Rhee, and Hyun Ah Kang¹

From the Metabolic Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-600, Korea, the Department of Microbiology, Chungnam National University, Daejeon 305-764, Korea, and ^¶Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, 1-1-4 Higashi, Tsukuba, Ibaraki, 305-8566, Japan

The -1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 (ScOCH1) is responsible for the outer chain initiation of N-linked oligosaccharides. To identify the genes involved in the first step of outer chain biosynthesis in the methylotrophic yeast Hansenula polymorpha, we undertook the functional analysis of three H. polymorpha genes, HpHOC1, HpOCH1, and HpOCR1, that belong to the OCH1 family containing seven members with significant sequence identities to ScOCH1. The deletions of these H. polymorpha genes individually resulted in several phenotypes suggestive of cell wall defects. Whereas the deletion of HpHOC1 (Hphoc1) did not generate any detectable changes in N-glycosylation, the null mutant strains of HpOCH1 (Hpoch1) and HpOCR1 (Hpocr1) displayed a remarkable reduction in hypermannosylation. Although the apparent phenotypes of Hpocr1 were most similar to those of S. cerevisiae och1 mutants, the detailed structural analysis of N-glycans revealed that the major defect of Hpocr1 is not in the initiation step but rather in the subsequent step of outer chain elongation by -1,2-mannose addition. Most interestingly, Hpocr1 showed a severe defect in the O-linked glycosylation of extracellular chitinase, representing HpOCR1 as a novel member of the OCH1 family implicated in both N- and O-linked glycosylation. In contrast, addition of the first -1,6-mannose residue onto the core oligosaccharide Man₈GlcNAc₂ was completely blocked in Hpoch1 despite the comparable growth of its wild type under normal growth conditions. The complementation of the S. cerevisiae och1 null mutation by the expression of HpOCH1 and the lack of in vitro -1,6-mannosyltransferase activity in Hpoch1 provided supportive evidence that HpOCH1 is the functional orthologue of ScOCH1. The engineered Hpoch1 strain with the targeted expression of Aspergillus saitoi -1,2-mannosidase in the endoplasmic reticulum was shown to produce human-compatible high mannose-type Man₅GlcNAc₂ oligosaccharide as a major N-glycan.

Received for publication, August 3, 2005 , and in revised form, December 16, 2005.

^* This work was supported by grants from the Korean Ministry of Science and Technology (Microbial Genomics and Applications Research and Development Program, Korea-Japan International Cooperative Program) and the Korean Ministry of Commerce, Industry, and Energy (Next Generation New Technology Development Program). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF540063 [GenBank] , AF490971 [GenBank] , AY502025 [GenBank] , DQ249343 [GenBank] , DQ249344 [GenBank] , DQ249345 [GenBank] , and DQ249346 [GenBank] .

¹ To whom correspondence should be addressed: Metabolic Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology, Oun-dong 52, Yusong-gu, Daejeon, 305-600, Korea. Tel.: 82-42-860-4378; Fax: 82-42-860-4594; E-mail: hyunkang{at}kribb.re.kr.

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