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

Abstract

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 Man 8 GlcNAc 2 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 mannosetype Man 5 GlcNAc 2 oligosaccharide as a major N-glycan.

α-1,6-Mannosylation of N-Linked Oligosaccharide Present on Cell Wall Proteins Is Required for Their Incorporation into the Cell Wall in the Filamentous Fungus Neurospora crassa

“…In S. cerevisiae, C. albicans, K. lactis, P. angusta, Y. lipolytica, S. pombe, and P. pastoris, loss of OCH1 gives rise to morphologies that correlate with alterations in the cell wall (2,3,9,17,33,35,37,38). In contrast with these yeasts, knockout mutations of the OCH1 genes in the filamentous fungus A. fumigatus do not result in a cell wall-defective phenotype (18).…”

“…OCH1 mutants have also been identified in Pichia angusta, Yarrowia lipolytica, Pichia pastoris, and Schizosaccharomyces pombe, and these mutants have cell wall-related phenotypes (2,9,17,38). However, a recent report of OCH1 knockout mutants of Aspergillus fumigatus indicates that these mutants do not have a cell wall-defective phenotype (18).…”

Engineering of the Yeast Yarrowia lipolytica for the Production of Glycoproteins Lacking the Outer-Chain Mannose Residues of N-Glycans

“…Other yeast species, including Pichia pastoris, Hansenula polymorpha, and Schizosaccharomyces pombe, also use the Och1 protein to extend the mannose outer chain of N-glycans (14, 24, 26, 28). Therefore, the elimination of the Och1 protein was performed to block the yeast-specific outer-chain mannosylation, followed by further engineering of yeast N-glycosylation pathways for the production of glycoproteins with human-compatible oligosaccharides (14,17,26).Yarrowia lipolytica, a heterothallic yeast, is presently considered to be a good potential host for heterologous gene expression due to its ability to secrete large amounts of extracellular proteins (4, 12, 21) and the simplicity with which it is cultivated to a high cell density (13). Despite the potential of Y. lipolytica as a valuable host, little information on the structural characteristics of N-linked oligosaccharides of Y. lipolytica glycoproteins is available.…”

Hansenula polymorpha Hac1p Is Critical to Protein N -Glycosylation Activity Modulation, as Revealed by Functional and Transcriptomic Analyses

“…In recent decades, H. polymorpha has also been used as a host for heterologous protein production (12). In particular, glycoengineered H. polymorpha strains have been developed to produce glycoproteins with human-compatible N-glycans on the basis of information on the host-specific structure and biosynthesis pathway of N-linked glycosylation in H. polymorpha (13)(14)(15). Likewise, a recent study on the identification and functional analysis of protein O-mannosyltransferases (PMTs) in H. polymorpha provided some unique features of PMT proteins in H. polymorpha (16).…”