Functional characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 genes as members of the yeast OCH1 mannosyltransferase family involved in protein glycosylation

The alpha-1,6-mannosyltransferaseencoded 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 Delta) did not generate any detectable changes in N-glycosylation, the null mutant strains of HpOCH1 (Hpoch1 Delta) and HpOCR1 (Hpocr1 Delta) displayed a remarkable reduction in hypermannosylation. Although the apparent phenotypes of Hpocr1 Delta were most similar to those of S. cerevisiae och1 mutants, the detailed structural analysis of N-glycans revealed that the major defect of Hpocr1 Delta is not in the initiation step but rather in the subsequent step of outer chain elongation by alpha-1,2-mannose addition. Most interestingly, Hpocr1 Delta 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 alpha-1,6-mannose residue onto the core oligosaccharide Man(8)GlcNAc(2) was completely blocked in Hpoch1 Delta 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 alpha-1,6-mannosyltransferase activity in Hpoch1 Delta provided supportive evidence that HpOCH1 is the functional orthologue of ScOCH1. The engineered Hpoch1 Delta strain with the targeted expression of Aspergillus saitoi alpha-1,2-mannosidase in the endoplasmic reticulum was shown to produce human-compatible high mannose-type Man(5)GlcNAc(2) oligosaccharide as a major N-glycan.