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

The α-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 (Hphoc1Δ) did not generate any detectable changes in N-glycosylation, the null mutant strains of HpOCH1 (Hpoch1Δ) and HpOCR1 (Hpocr1Δ) displayed a remarkable reduction in hypermannosylation. Although the apparent phenotypes of Hpocr1Δ were most similar to those of S. cerevisiae och1 mutants, the detailed structural analysis of N-glycans revealed that the major defect of Hpocr1Δ is not in the initiation step but rather in the subsequent step of outer chain elongation by α-1,2-mannose addition. Most interestingly, Hpocr1Δ 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 α-1,6-mannose residue onto the core oligosaccharide Man8GlcNAc2 was completely blocked in Hpoch1Δ 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 α-1,6-mannosyltransferase activity in Hpoch1Δ provided supportive evidence that HpOCH1 is the functional orthologue of ScOCH1. The engineered Hpoch1Δ strain with the targeted expression of Aspergillus saitoi α-1,2-mannosidase in the endoplasmic reticulum was shown to produce human-compatible high mannose-type Man5GlcNAc2 oligosaccharide as a major N-glycan.

Glycosylation is one of the most ubiquitous forms of post-translational modification, and the early stages of N-linked glycosylation are highly conserved among eukaryotes. The formation of N-linked oligosaccharides assembled on glycoproteins begins in the endoplasmic reticulum (ER), 2 where an identical Glc 3 Man 9 GlcNAc 2 oligosaccharide is transferred to the Asn residues on nascent proteins by oligosaccharyltransferase complex (1). Subsequent trimming by glucosidases I and II and a specific ER-residing ␣-1,2-mannosidase led to the formation of a core oligosaccharide (Man 8 GlcNAc 2 ). Glycoproteins containing Man 8 GlcNAc 2 are then collected into transport vesicles and delivered to the Golgi apparatus. It is in the Golgi that the diversity of N-glycan structures is generated by a series of glycosidases and glycosyltransferases acting in a manner that varies enormously between species, and even between individual proteins within a species (2).
In the traditional yeast Saccharomyces cerevisiae, where the N-linked glycosylation has been most studied, the maturation of N-linked oligosaccharides in the Golgi often leads to hypermannosylated glycoproteins possessing a large structure called the outer chain. This outer chain biosynthesis is initiated by the addition of the first ␣-1,6-mannose onto the Man 8 GlcNAc 2 core structure in the early Golgi, a process mediated by the OCH1 gene product in S. cerevisiae (3). After the action of the S. cerevisiae OCH1 gene product (ScOch1p), the extension of an ␣-1,6linked polymannose backbone occurs by the sequential action of two enzyme complexes, mannan polymerase (M-Pol) I and II (2). The linear backbone of the outer chain is often extended with 50 or more ␣-1,6linked mannoses, highly branched by the addition of ␣-1,2-linked mannoses and terminally capped by ␣-1,3-linked mannoses (4). The mannosyltransferase activity and the substrate specificity of the S. cerevisiae Och1p are well characterized and shown to require the intact structure of Man 8 GlcNAc 2 for efficient mannose outer chain initiation (5,6). The functional homologues of the S. cerevisiae OCH1 gene in other yeast species, including Schizosaccharomyces pombe (7) and Pichia pastoris (8), have been reported and shown to be involved in the initiation of ␣-1,6-linked mannose outer chain biosynthesis.
The S. cerevisiae HOC1 gene (Homologous to OCH1), isolated as a high copy suppressor of a protein kinase C mutant, encodes a putative ␣-1,6-mannosyltransferase that strongly resembles ScOch1p. However, no obvious defects in N-linked or O-linked glycosylation were detected in the null mutation of HOC1, and the overexpression of HOC1 cannot suppress an och1 mutation (9). The S. cerevisiae HOC1 gene product (ScHoc1p) was found to reside in the M-Pol II complex, but whether ScHoc1p directly contributes to the ␣-1,6-mannosyltransferase activity is not yet proven (2). At present, the function of ScHoc1p is unclear, although the S. cerevisiae hoc1 mutants show phenotypes associated with glycosylation defects, including sensitivity to Calcofluor White and hygromycin B. ScHoc1p might be a protein-specific mannosyltransferase or have a function overlapping with another glycosyltransferase or function only under certain conditions.
The thermotolerant methylotrophic yeast, Hansenula polymorpha, has emerged as a promising host for the high level expression of heterologous genes, because of its well established expression toolboxes along with the feasibility of its high cell density culture in methanolcontaining media (10,11). Furthermore, the less extensive hyperglycosylation of glycoproteins from H. polymorpha than from S. cerevisiae has been suggested to be another factor that favors the production of mammalian cell-originated proteins in this yeast (12,13). Our recent study on the structural characterization of the oligosaccharides assembled on glycoproteins secreted by H. polymorpha showed that most N-linked glycans synthesized in H. polymorpha have core-type structures (Man 8 -12GlcNAc 2 ) and that they are mainly branched by ␣-1,2-linkages without hyper-immunogenic terminal ␣-1,3-linked mannose residues. More interestingly, the outer chains of H. polymorpha N-glycans were shown to have very short ␣-1,6-extensions, mainly composed of single ␣-1,6-linked mannose (14). To identify H. polymorpha genes coding for glycosyltransferases involved in the first step of ␣-1,6-linked mannose addition in N-linked outer chain biosynthesis, we isolated and characterized three H. polymorpha genes, HpHOC1, HpOCH1, and HpOCR1 (OCH1-related), that share significant homology with S. cerevisiae HOC1 and OCH1 genes. Here we report that HpOCH1 codes for the key enzyme responsible for initiating N-linked outer chain biosynthesis by adding the first ␣-1,6-mannose residue onto the core oligosaccharide, whereas the functions of HpHOC1 and HpOCR1 are closely associated with the subsequent elongation step by ␣-1,2-mannose addition. Furthermore, we evaluated the potential of HpOCH1 deletion as a first step toward the N-glycan engineering of H. polymorpha to produce humancompatible N-linked oligosaccharides.
Cloning of the H. polymorpha OCH1 Family Genes-Two pairs of primers, R1for/R1rev and R2for/R2rev, were designed using information on two random sequenced tags of H. polymorpha (GenBank TM accession numbers AL435940 and AL433499), which shares significant homology with S. cerevisiae HOC1 and OCH1 genes. Two PCR products of 1.0 kb were amplified from H. polymorpha DL1-L chromosomal DNA using the primers R1for/R1rev and R2for/R2rev and used as probes for Southern blot analysis. Strong hybridization signals at the 7.5-kb HindIII and 5.0-kb BglII fragments of H. polymorpha DL1-L genomic DNA were detected using these probes, respectively. HindIII fragments of 6.7-7.7-kb and BglII fragments of 4.5-6.0-kb of H. polymorpha DL1-L genomic DNA were gel-eluted and ligated into the Hin-dIII and BamHI sites of pBluescript (Stratagene, La Jolla, CA), respectively. To screen H. polymorpha HindIII and BglII genomic DNA libraries, colony PCR was carried out using the same primers used for synthesizing the two probes mentioned above. Plasmids from each positive clone, designated pH305 and pB52, were partially sequenced. Plasmid pH305 contained a 7.5-kb HindIII insert with a full-length open reading frame (ORF) highly similar to that of the S. cerevisiae HOC1 gene product, and thus the ORF was designated as H. polymorpha HOC1 (HpHOC1). However, plasmid pB52 containing a 5.0-kb BglII insert was found to contain only the 3Ј portion of a putative ORF, which showed relatively low sequence similarity with the S. cerevisiae HOC1 and OCH1 gene products. To clone the full length of the ORF, the 3Ј region of ORF obtained from pB52 was amplified using the primers R3for and R3rev from H. polymorpha DL1-L genomic DNA, and this was then used as a probe in Southern blot analysis. A strong hybridization signal was detected at an ϳ2.3-kb BamHI fragment, and the BamHI fragments of 1.8 -3.0 kb of H. polymorpha DL1-L genomic DNA were gel-eluted and subcloned into the BamHI site of pBluescript. After screening the partial H. polymorpha genomic DNA library by colony PCR, positive clones were isolated and sequenced. Plasmid pBA302 from a positive clone was found to contain a 2.3-kb BamHI insert carrying the 5Ј-upstream region and a portion of an ORF, which partially overlapped with the ORF from pB52. The full length of an intact ORF, showing relatively low sequence identity (22%) with S. cerevisiae OCH1, was generated by combining two ORFs from pBA302 and pB52 and was designated as H. polymorpha OCH1-related gene 1 (HpOCR1). The nucleotide sequences of HpHOC1 and HpOCR1 have been submitted to GenBank TM under accession numbers AF540063 and AF490971, respectively. H. polymorpha OCH1 (HpOCH1) was isolated by PCR from H. polymorpha DL1-L genomic DNA using two primers, 168Not-N and 168Not-C, that were designed based on information obtained from the whole genome sequence of H. polymorpha (17). The nucleotide sequence of the resulting PCR product was determined and submitted to GenBank TM under accession number AY502025. The other members of H. polymorpha OCH1 family, named HpOCR2, HpOCR3, HpOCR4, and HpOCR5, were also isolated by PCR from H. polymorpha DL1-L genomic DNA using the primer sets, ORF288F/ ORF288R, ORF580F/ORF580R, ORF100F/ORF100R, and ORF576F/ ORF576R, respectively, which were designed based on information obtained from the whole genome sequence of H. polymorpha. The nucleotide sequences of the resulting PCR product were submitted to GenBank TM under accession numbers DQ249343, DQ249344, DQ249345, and DQ249346, respectively. The sequences of the primers used for the gene cloning are available on request.
Deletion of the H. polymorpha HOC1, OCR1, and OCH1 Genes-Deletional disruptions of the HpHOC1, HpOCR1, and HpOCH1 genes were carried out using fusion PCR and in vivo recombination as described previously (18,19) with slight modification. Briefly, in the fusion PCR stage, two DNA fragments containing the promoter (H1N) and terminator (H1C) regions of HpHOC1, respectively, were amplified by using two pairs of primers, HNfor/HNrev and HCfor/HCrev. The PCR primers HNrev and HCfor were 20-bp oligonucleotides and corresponded to the upstream and downstream sequences of the directed repeat region of the HpURA3 pop-out cassette in plasmid pLacUR3. 6 The N-and C-terminal fragments (UR3N and UR3C) of the HpURA3 pop-out cassette containing the overlapped internal sequence of HpURA3 (100 bp) were amplified from pLacUR3 by PCR using two pairs of primers UNfor/UNrev and UCfor/UCrev. Subsequently, the UR3N and UR3C fragments were fused with the H1N and H1C fragments by PCR using two pairs of primers (HNfor/UNrev and UCfor/ HCrev) to generate the fusion products HN-UR3N and HC-UR3C, respectively. In the in vivo recombination stage, these two PCR fusion products were introduced into H. polymorpha DL-LdU (leu2 ura3⌬::lacZ) and converted into one linear gene disruption cassette via in vivo homologous recombination at the overlapping sequence. Ura ϩ colonies were selected and screened for the Hphoc1⌬::HpURA3 strain, which was generated by the double homologous crossover of the disruption cassette at the HpHOC1 gene locus. Gene disruption was confirmed by PCR and Southern hybridization analysis (data not shown). Disruptions of the genomic copies of HpOCR1 and HpOCH1 were performed using the same procedure. To construct double deletion Hphoc1⌬Hpocr1⌬ or Hphoc1⌬Hpoch1⌬ mutant strains, the HpURA3 pop-out cassette was removed from the Hphoc1⌬::HpURA3 strain to recover the Ura Ϫ auxotrophic marker by cultivation on YPD plates containing 5-fluoroorotic acid (0.5 mg/ml), generating the DL-LdUdH1 (leu2 ura3⌬::lacZ hoc1⌬::lacZ) strain. Subsequent gene disruptions of HpOCR1 and HpOCH1 were performed in the DL-LdUdH1 strain using the same procedure described above. The sequences of the primers used in this study are available on request.
HPLC Analysis of N-Linked Oligosaccharides-N-Linked oligosaccharides were released from 200 g of purified rGOD or HpYps1p by PNGase F treatment, and the oligosaccharides obtained were labeled with 2-aminopyridine (PA) using the commercially available reagent kit (Takara Shuzo Co., Shiga, Japan). After pyridylamination, the samples were purified using Sephadex G-15 spin columns (Amersham Biosciences) to remove residual PA. Size fractionation HPLC was performed using a Shodex Asahipak NH2P-50 column (Showa Denko K. K., Tokyo, Japan, 0.46 ϫ 25 cm) at a flow rate of 1.0 ml/min (6). After sample injection, the proportion of solvent B was increased linearly up to 45% over 42 min. PA-oligosaccharides were detected by fluorescence ( ex ϭ 320 nm and em ϭ 400 nm) using a Waters 2475 fluorescence detector. PA-oligosaccharides were digested sequentially with ␣-1,2mannosidase (from A. saitoi, Seikagaku Corp., Tokyo) and ␣-1,6-man-nosidase (from Xanthomonas manihotis, New England Biolab, Beverly, MA) according to the manufacturer's instructions.
Preparation of Membrane Proteins and ␣-1,6-Mannosyltransferase Activity Assay-Yeast membrane fractions were obtained as described previously (5). Yeast cells were cultivated in YPD medium and harvested at mid-logarithmic phase (A 600 ϭ 3-4). Cells were collected by centrifugation at 3,000 ϫ g for 5 min, washed with 1% KCl, and resuspended in 5 ml of PMS buffer (50 mM Tris-HCl (pH 7.5), 10 mM MnCl 2 , 1 mM phenylmethylsulfonyl fluoride, 5% glycerol, and 2 g/ml each protease inhibitor (antipain, chymostatin, leupeptin, and pepstatin A)). Glass beads (425-600 m) were added to half of the cell suspension volume and homogenized four times for 1 min at 4°C. Homogenates were centrifuged at 10,000 ϫ g for 20 min, and the supernatant obtained was further centrifuged at 100,000 ϫ g for 1 h. High speed pellets were collected and resuspended in PMS buffer, and protein concentrations were determined using Bio-Rad protein assay agent (Bio-Rad). ␣-1,6-Mannosyltransferase activity was assayed as described by Nakajima and Ballou (20). 100 g of high speed pellet proteins was incubated in 100 l of 50 mM Tris-HCl (pH 7.5) buffer containing 10 mM MnCl 2 , 1 mM GDP-mannose, 0.5 mM 1-deoxymannojirimycin, and 100 pmol of Man 8 GlcNAc 2 -PA acceptor at 30°C for 2 h. The reaction was terminated by boiling at 99°C for 5 min, and the reaction mixture was filtered through an Ultrafree-MC membrane (10,000 cutoff; Millipore, Bedford, MA), and the filtrate was submitted for HPLC.
O-Glycosylation Analysis of Chitinase-Native chitinases from supernatants of saturated cultures were isolated, and their degrees of O-glycosylation were analyzed by SDS-PAGE as described previously (21). Thirty ml of culture supernatants of H. polymorpha cells grown in YPD for 24 h were collected and transferred into centrifuge tubes. After adding 40 mg of chitin, the tubes were rotated end-over-end at 4°C for 4 h. The chitin was then pelleted by centrifugation and washed three times with cold phosphate-buffered saline buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 (pH 7.4)). Chitinase was eluted from the chitin in 100 l of SDS-PAGE sample buffer by boiling for 5 min and then separated in 6% SDS-PAGE, which was visualized by silver staining.

RESULTS
Isolation of HpHOC1, HpOCR1, and HpOCH1 in H. polymorpha-In an effort to identify the gene(s) coding for ␣-1,6-mannosyltransferase contributing to the initiation of outer chain elongation on Man 8 GlcNAc 2 in H. polymorpha, we had initially isolated two H. polymorpha genes, designated HpHOC1 and HpOCR1 (OCH1-related gene 1), using sequence information from random sequenced tags of H. polymorpha (22). Sequence analysis revealed that the HpHOC1 gene product (HpHoc1p) showed 40 and 24% overall identities with the S. cerevisiae HOC1 and OCH1 gene products, respectively. On the other hand, the HpOCR1 gene product (HpOcr1p) showed relatively low sequence identity with both the ScHOC1 and ScOCH1 gene products (21 and 22% identity, respectively). A hydropathy analysis performed using PSORT II (www.psort.org/; see Ref. 23) predicted that HpHoc1p and HpOcr1p The alignment was performed using the program ClustalW 1.8 (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and shaded using the program Boxshade 3.21 (www.ch.embnet.org/software/BOX_form.html). Identical residues and conservative amino acid substitutions in proteins are shaded with black or gray. Putative membranespanning regions, which act as signal-anchor domains, are indicated by boxes, and the DXD motif is shown above a sequence as hhhhDXD.
possess a single potential transmembrane-spanning region, which acts as a signal-anchor domain (24) near the N terminus of the proteins, suggesting that they are type II membrane proteins. In addition, they possess a DXD motif well conserved in many glycosyltransferase families (25). An alignment of amino acid sequences of HpHoc1p and HpOcr1p with Och1p homologues from other yeast species reveals that several conserved regions are shared by Och1p homologues, although N-terminal portion is largely unique to each protein (Fig. 1). Most interestingly, HpOcr1p has a long C-terminal region that is absent in other Och1p homologues.
While we were analyzing the functions of HpHOC1 and HpOCR1, the entire genome of H. polymorpha was completely sequenced (17). Thus we searched this H. polymorpha whole genome data base to identify other possible H. polymorpha ORFs showing significant similarity to the S. cerevisiae OCH1 and HOC1 genes. In addition to the ORFs identified as HpHOC1 and HpOCR1, at least five other ORFs were found to encode putative mannosyltransferases with significant sequence identities to ScOch1p, ranging from 15 to 37% (Table 1). These additional ORFs were also predicted to have a potential transmembrane domain at the N-terminal region and a conserved region encompassing a DXD motif. Of these, an ORF with the highest homology to ScOCH1 was designated HpOCH1, and its functions in cell growth and N-linked glycosylation were investigated.
Effect of HpHOC1, HpOCR1, and HpOCH1 Deletion on Cell Growth and N-Glycosylation-To investigate the effect of HpHOC1, HpOCR1, or HpOCH1 gene deletion on cell growth and N-linked glycosylation, the single deletion (Hphoc1⌬, Hpocr1⌬, or Hpoch1⌬) or the double deletion (Hphoc1⌬Hpocr1⌬ or Hphoc1⌬Hpoch1⌬) mutant strains were constructed and analyzed. Disruptions of either of these genes in H. polymorpha resulted in hypersensitivity to hygromycin B or sodium deoxycholate (Fig. 2, D and E), which are characteristic phenotypes of cell wall and N-linked glycosylation defects (26). In particular, the Hpocr1 null mutant strain showed slow-growing and temperature-sensitive phenotypes (Fig. 2, A and B), as was reported previously in Scoch1 mutant strain. In addition, the growth of Hpocr1⌬ was completely inhibited by Calcofluor White, which binds to chitin and disrupts cell wall assembly (27) (Fig. 2F). The growth defect and temperature sensitivity of the Hpocr1⌬ strain were partially recovered by supplementing an osmotic stabilizer, 1 M sorbitol (Fig. 2C). Although Hphoc1⌬ cells displayed no obvious growth defects besides hygromycin B sensitivity, the introduction of the HpHOC1 deletion into the background of the Hpocr1⌬ strain (Hphoc1⌬Hpocr1⌬) resulted in more significant growth retardation but restored hygromycin B sensitivity (Fig. 2, A and D). Most interestingly, the HpOCH1 deletion mutant strain (Hpoch1⌬) also exhibited a temperature-sensitive growth phenotype, which was complemented by the presence of 1 M sorbitol, but did not show a slowgrowing phenotype under normal growth conditions (Fig. 2, A-C). The Hpoch1⌬ strain was less sensitive to Calcofluor White but was more sensitive to hygromycin B than the Hpocr1⌬ strain (Fig. 2, D and F). Moreover, unlike the Hpocr1⌬ strain, additional inactivation of HpHOC1 in the Hpoch1⌬ strain background did not cause any further changes in the growth characteristics of the Hpoch1⌬ strain. These

TABLE 1 Sequence identities and similarities between the Och1p homologs in S. cerevisiae and H. polymorpha
The degree of relatedness between protein pairs is shown as the percentage identity and similarity calculated from pairwise sequence alignments with gaps to maximize homology. results indicate that HpHoc1p, HpOcr1p, and HpOch1p are required for the maintenance of cell wall integrity, and that the function of HpHoc1p appears either to partially overlap or to be closely related with that of HpOcr1p. We could not obtain the double deletion strain of HpOCR1 and HpOCH1 even in the presence of osmotic stabilizers, possibly because of a severe growth defect in the absence of both genes.
The effect of the deletions of the HpHOC1, HpOCR1, and HpOCH1 genes on protein N-glycosylation was preliminarily investigated by analyzing the electrophoretic mobility of a recombinant A. niger glucose oxidase (rGOD) protein secreted by H. polymorpha wild type, Hphoc1⌬, Hpocr1⌬, Hphoc1⌬Hpocr1⌬, Hpoch1⌬, and Hphoc1⌬Hpoch1⌬ mutant strains (Fig. 3). The Hphoc1⌬ mutant strain secreted heterogeneous forms of rGOD with high molecular weights similar to those of rGOD secreted by the wild type, whereas the Hpocr1⌬ mutant strain produced much less heterogeneous forms of rGOD, which had a higher electrophoretic mobility than that of the wild type (Fig. 3, lanes 1-3). Slightly more homogeneous forms of rGOD were detected in the Hphoc1⌬Hpocr1⌬ double mutant strain, although their mobility was still slower than that of authentic GOD from A. niger (Fig. 3, lanes 4 and  7). The electrophoretic mobility of rGOD secreted by the Hpoch1⌬ mutant was also increased compared with rGOD from the wild type strain, even though a certain extent of heterogeneity still existed (Fig. 3, lane 5). No difference in the mobility of rGODs was detected between the Hpoch1⌬ single and the Hphoc1⌬Hpoch1⌬ double mutant strains (Fig. 3, lane 5 versus lane 6). These results suggest that the functions of HpOcr1p and HpOch1p are strongly associated with the N-linked glycosylation of rGOD, whereas HpHoc1p appears to play a minor role in mannose outer chain elongation.
Structural Analysis of N-Linked Oligosaccharides Produced in the Hphoc1⌬, Hpocr1⌬, Hphoc1⌬Hpocr1⌬, and Hpoch1⌬ Strains-To gain more detailed information on the size and structure of N-glycans attached to rGOD expressed by the Hphoc1⌬, Hpocr1⌬, Hphoc1⌬Hpocr1⌬, or Hpoch1⌬ strains, the N-linked oligosaccharides of rGOD secreted by these mutant strains were analyzed by size-fractionation HPLC (Fig. 4A). Comparisons of the oligosaccharide profiles of the wild type and of the deletion mutant strains revealed a dramatic reduction in the sizes of major oligosaccharides assembled on the proteins secreted by the mutant strains, except the Hphoc1⌬ single deletion mutant strain. In the case of oligosaccharides derived from the wild type, four major peaks of Man 8 -11 GlcNAc 2 were detected with several minor peaks of Man 12-15 GlcNAc 2 (Fig. 4A, panel a). An almost identical oligosaccharide profile to that of the wild type, but with a slight decrease in the portion of oligosaccharides larger than Man 8 GlcNAc 2 , was observed in the Hphoc1⌬ single mutant (Fig. 4A, panel d). However, oligosaccharides from Hpocr1⌬, Hphoc1⌬Hpocr1⌬, or Hpoch1⌬ mutant cells were mainly composed of a single predominating species, corresponding to the core-glycosylated form Man 8 GlcNAc 2 , although larger structures of Man 9 -14 GlcNAc 2 were present as minor components (Fig. 4A, panels g, j, and m). In the Hphoc1⌬Hpocr1⌬ mutant strain, the relative proportions of the oligosaccharides larger than Man 8 GlcNAc 2 appeared to be reduced versus the Hpocr1⌬ strain. These results indicate that mannose outer chain elongation is significantly inhibited in the Hpocr1⌬, Hphoc1⌬Hpocr1⌬, and Hpoch1⌬ mutant strains, although small fractions of oligosaccharides (Man 9 -14 GlcNAc 2 ) larger than Man 8 GlcNAc 2 were observed.
To investigate in more detail the structure of the oligosaccharide species produced by the mutant strains, sequential digestion experiments with ␣-1,2and ␣-1,6-mannosidases were carried out. After digestion of the oligosaccharides with ␣-1,2-mannosidase from A. saitoi, which is highly specific for nonreducing terminal ␣-1,2-mannose linkages, most of the oligosaccharides species from the wild type strain were shifted to two major species, i.e. Man 5 GlcNAc 2 and Man 6 GlcNAc 2 (Fig. 4A, panel b). Subsequent digestion with ␣-1,6-mannosidase from X. manihotis, which is highly specific for terminal ␣-1,6-linked mannose residues that are linked to a nonbranched sugar, converted all the Man 6 GlcNAc 2 oligosaccharide species generated by ␣-1,2-mannosidase digestion into Man 5 GlcNAc 2 (Fig. 4A, panel c) . Our previous study indicated that Man 5 GlcNAc 2 (M5) is the final product of specific ␣-1,2mannosidase digestion of the core oligosaccharide Man 8 GlcNAc 2 or of the larger oligosaccharide species extended with only ␣-1,2-mannose linkages, whereas Man 6 GlcNAc 2 (M6) oligosaccharide is the final product of the large oligosaccharides elongated by a single ␣-1,6-linked mannose addition and branched with a variable number of ␣-1,2-linked mannose units (14). After digestion with ␣-1,2-mannosidase, the oligosaccharides from the Hphoc1⌬, Hpocr1⌬, and Hphoc1⌬Hpocr1⌬ strains were shifted to two major species, i.e. M5 and M6, like the wild type (Fig.  4A, panels e, h, and k). Moreover, subsequent digestion with ␣-1,6mannosidase converted all M6 oligosaccharide species into M5 (Fig. 4A, panels f, i, and l) . These results indicate that a significant portion of large oligosaccharides from Hphoc1⌬, Hpocr1⌬, and Hphoc1⌬Hpocr1⌬ strains contain an additional ␣-1,6-linked mannose attached to the core oligosaccharide, which is believed to be mediated by the ␣-1,6-initiating mannosyltransferase activity of S. cerevisiae Och1p homologue. Even though the relative proportions of M6 to M5 appeared to gradually decrease from the wild type to Hpocr1⌬ and to Hphoc1⌬Hpocr1⌬ cells, our results strongly suggest that the major roles of HpHoc1p and HpOcr1p are not in the addition of the first ␣-1,6-linked mannose. However, in contrast to the N-glycans from the wild type, Hphoc1⌬, Hpocr1⌬, and Hphoc1⌬Hpocr1⌬ strains, all oligosaccharide species from the Hpoch1⌬ strain were converted to M5 just after ␣-1,2-mannosidase treatment (Fig. 4A, panel n), indicating that the ␣-1,6-linked mannose addition to the core oligosaccharide was completely blocked in the Hpoch1⌬ strain. This result strongly suggests that HpOch1p plays a key role as ␣-1,6-mannosyltransferase during the first step of outer chain biosynthesis.
The effect of HpOCH1 gene deletion on the structure of oligosaccharides attached to secreted glycoproteins was further analyzed using the N-glycans released from the endogenous H. polymorpha glycoprotein, HpYPS1 protein, which is a glycosylphosphatidylinositol-anchored aspartic protease with four potential N-linked glycosylation sites (Fig.  4B). The secreted form of HpYps1p was obtained by deleting a C-ter-  1 and 8), Hphoc1⌬ (lanes 2 and 9), Hpocr1⌬ (lanes 3 and 10), Hphoc1⌬Hpocr1⌬ (lanes 4 and 11), Hpoch1⌬ (lanes 5 and 12), Hphoc1⌬Hpoch1⌬  (lanes 6 and 13), and authentic GOD from A. niger (lanes 7 and 14). Lanes 8 -14, culture supernatants treated with PNGase F. minal region containing a glycosylphosphatidylinositol-anchoring motif. 7 Oligosaccharide profiles of HpYps1p secreted by the wild type and Hpoch1⌬ mutant strains displayed greater differences in terms of the relative proportions of M5 to M6 oligosaccharides than in those of rGOD secreted by these two strains. In the wild type strain, oligosaccharides containing 9 -10 mannoses (Man 9 -10 GlcNAc 2 ) were present as predominant species attached to HpYps1p (Fig. 4B, panel a). After digestion with ␣-1,2-mannosidase, most of the oligosaccharide species were converted to M6, which was then completely converted to M5 by subsequent digestion with ␣-1,6-mannosidase (Fig. 4B, panels b and c). This indicates that most oligosaccharides assembled on HpYps1p contained a single ␣-1,6-linked mannose attached to the core oligosaccharide in the wild type strain. In contrast, in the Hpoch1⌬ mutant strain, core form oligosaccharide Man 8 GlcNAc 2 predominated; oligosaccharides larger than Man 8 GlcNAc 2 were detected as minor fractions (Fig.  4B, panel d). Moreover, digestion with ␣-1,2-mannosidase converted all Hpoch1⌬ strain-derived oligosaccharides to M5 (Fig. 4B, panel e). These results clearly indicate that a defective addition of ␣-1,6-mannose residue onto the core oligosaccharide Man 8 GlcNAc 2 is a general phenotype of the Hpoch1⌬ strain and not a protein-specific phenotype.
Involvement of HpOCR1 in O-Glycosylation-To examine whether HpHOC1, HpOCR1, and HpOCH1 gene products are involved in O-linked glycosylation, we analyzed the electrophoresis mobility of an endogenous O-modified glycoprotein, H. polymorpha chitinase, that is reported to lack N-linked oligosaccharides (21), secreted by the wild type, Hphoc1⌬, Hpocr1⌬, Hpoch1⌬, Hphoc1⌬Hpocr1⌬, and Hphoc1⌬Hpoch1⌬ mutant strains (Fig. 5). The Hpoch1⌬, Hphoc1⌬, and Hphoc1⌬Hpoch1⌬ strains secreted heterogeneous forms of chitinase with high molecular weights similar to those of chitinase secreted by the wild type (Fig. 5, lanes 1, 3, 4, and 6). Most interestingly, compared with the wild type strain, the Hpoch1⌬ strains showed a rather increased extent of O-glycosylation in chitinase (Fig. 5, lanes 3 and 6 versus lane 1), probably reflecting a kind of compensatory mechanism to maintain the cell integrity caused by the loss of HpOch1p function. In contrast, sig- nificantly homogeneous forms of chitinase were detected in the Hpocr1⌬ and Hphoc1⌬Hpocr1⌬ strains, indicating that the O-glycosylation of extracellular chitinase appeared to be severely impaired in the absence of HpOcr1p (Fig. 5, lanes 2 and 5). The results strongly implied that HpOcr1p is involved in O-linked chain elaboration in addition to N-linked outer chain biosynthesis.
HpOCH1 as a Functional Homologue of ScOCH1 Encoding an Initiating ␣-1,6-Mannosyltransferase-To investigate whether HpOCH1 is a functional homologue of ScOCH1, we transformed an S. cerevisiae och1 null (Scoch1⌬) mutant with the plasmid YEp352GAPII-HpOCH1 carrying HpOCH1 under the control of the S. cerevisiae GAPDH promoter. The Scoch1⌬ mutant strain transformed with the null vector YEp352GAPII showed temperature sensitivity, but the Scoch1⌬ strain transformed with YEp352GAPII-HpOCH1 grew well at 30°C (Fig. 6A, row 2 versus row 5), indicating that the temperature-sensitive growth defect of Scoch1⌬ was recovered by the expression of HpOCH1. In contrast, Scoch1⌬ cells transformed with YEp352GAPII-HpHOC1 or YEp352GAPII-HpOCR1 displayed the same temperature-sensitive phenotype as the Scoch1⌬ mutant (Fig. 6A, rows 3 and 4). Moreover, the electrophoretic mobility of invertase secreted from the Scoch1⌬ cells transformed with the HpOCH1 expression plasmid decreased to the same extent as that observed from the Scoch1⌬ cells transformed with the ScOCH1 expression plasmid (Fig. 6B, lanes 5 and 6). Together with the observed complementation of the temperature-sensitive growth defect, the restoration of the hyperglycosylation defect of Scoch1⌬ suggests that HpOCH1 encodes a functional homologue of ScOch1p that plays a key role in the initiation of outer chain elongation.
To measure the ␣-1,6-mannosyltransferase activity of HpOch1p, solubilized membrane fractions were prepared from the S. cerevisiae mutant strains with OCH1, MNN1, and MNN4 deletions (Scoch1⌬mnn1⌬mnn4⌬), but harboring YEp352GAPII, YEp352GAPII-HpOCH1, or YEp352GAPII-ScOCH1 plasmids, and these were used as enzyme sources for ␣-1,6-mannosyltransferase assays (Fig. 6C). The Scoch1⌬mnn1⌬mnn4⌬ strain was shown to be defective in adding mannose residues to N-linked core oligosaccharide (16). PA-labeled Man 8 GlcNAc 2 , which has the structure of the core oligosaccharide formed in the ER of S. cerevisiae (3), was used as an acceptor, and the reaction products were analyzed by HPLC. Although the acceptor oligosaccharide, Man 8 GlcNAc 2 -PA (M8), was not converted into any other form by the membrane fraction of Scoch1⌬mnn1⌬mnn4⌬ transformant harboring the null vector (Fig. 6C, panel a), a peak corresponding to Man 9 GlcNAc 2 -PA (M9) was detected as a reaction product in the membrane fraction of Scoch1⌬mnn1⌬mnn4⌬ transformants harboring YEp352GAPII-HpOCH1 or YEp352GAPII-ScOCH1 (Fig. 6C, panels b  and c). These results strongly indicate that HpOCH1, like ScOCH1, encodes the initiation-specific ␣-1,6-mannosyltransferase acting on the core oligosaccharide. Solubilized membrane fractions prepared from the H. polymorpha wild type, Hpocr1⌬, and Hpoch1⌬ mutant strains were also analyzed for ␣-1,6-mannosyltransferase activity (Fig. 6D). The M9 peak corresponding to Man 9 GlcNAc 2 -PA was generated by the reaction between the acceptor oligosaccharide and the membrane fractions from the wild type and Hpocr1⌬ mutant strains but was hardly detected in the reaction with the membrane fraction of the Hpoch1⌬ mutant (Fig. 6D, panels a and b versus panel c). After ␣-1,2-mannosidase digestion, the M9 product was converted to Man 6 GlcNAc 2 -PA (M6), whereas the M8 core oligosaccharide was converted to Man 5 GlcNAc 2 -PA (M5) (Fig. 6D, panels d-f). These results consistently support the notion that HpOch1p is a major key enzyme in the initiation of outer chain biosynthesis, i.e. in the addition of ␣-1,6-mannose on the lower arm of the core oligosaccharide, as reported for ScOch1p.
Expression of the ER-targeted ␣-1,2-Mannosidase in Hpoch1⌬-The Hpoch1⌬ mutant strain, with a defect in the yeast-specific outer chain initiation step, was further evaluated as a starting strain for the genetic engineering of the N-linked glycosylation pathway to produce humantype sugars in H. polymorpha. To trim off the core oligosaccharide by removing ␣-1,2-mannose residues as in mammalian cells, we heterologously expressed ER-targeted A. saitoi ␣-1,2-mannosidase in H. polymorpha. Briefly, the msdS gene cassette fused with the signal sequence  Hpocr1⌬ (panel b), and Hpoch1⌬ mutant (panel c) strains, and the reaction products of (panels a-c) treated with ␣-1,2-mannosidase (panels d-f, respectively) were analyzed by HPLC.
of aspergillopepsin and the ER retention signal, HDEL (16), was further modified at its C terminus to contain the HA sequence to monitor its expression (Fig. 7A). Western blot analysis using HA antibody showed that the recombinant ␣-1,2-mannosidase-HA-HDEL was primarily expressed as core-glycosylated forms and retained inside H. polymorpha cells. No secreted form of the recombinant ␣-1,2-mannosidase was detected in the culture supernatant, which strongly indicated the proper localization of the recombinant ␣-1,2-mannosidase in the ER (Fig. 7B). Cell extracts of recombinant H. polymorpha containing the ␣-1,2-mannosidase-HA-HDEL construct were able to convert Man 6 GlcNAc 2 -PA into Man 5 GlcNAc 2 -PA in the in vitro ␣-1,2-mannosidase activity assay for A. saitoi ␣-1,2-mannosidase (16), whereas no such activity was detected in extracts of recombinant H. polymorpha transformed with the null vector (Fig. 7C). The result of this in vitro assay indicated that ␣-1,2-mannosidase-HA-HDEL was expressed as an active form in H. polymorpha.
To monitor N-glycan modification by the targeted expression of A. saitoi ␣-1,2-mannosidase in the ER of the Hpoch1⌬ mutant strain, we analyzed the structures of N-glycans on rGOD. Compared with the oligosaccharides synthesized in the Hpoch1⌬ mutant without ␣-1,2mannosidase-HA-HDEL, the oligosaccharides produced in the recombinant Hpoch1⌬ mutant strain expressing the active ␣-1,2-mannosidase-HA-HDEL were much shorter in length (Fig. 7D, panels a and b).
The fraction corresponding to Man 5 GlcNAc 2 , the smallest structure of human-compatible type high mannose oligosaccharide, was detected as a major component in the recombinant Hpoch1⌬ mutant strain expressing the active ␣-1,2-mannosidase-HA-HDEL. However, larger structures containing up to 10 mannoses were also detected as minor components, and all of these were converted into Man 5 GlcNAc 2 after in vitro ␣-1,2-mannosidase treatment (Fig. 7D, panel c). The incomplete trimming to Man 5 GlcNAc 2 in the recombinant Hpoch1⌬ strain might have been due to the low expression level or activity of ␣-1,2-mannosi-dase-HA-HDEL. Alternatively, the activity of endogenous ␣-1,2-mannosyltransferases in the Golgi might have subsequently added ␣-1,2linked mannoses to Man 5 GlcNAc 2 , to generate larger oligosaccharides extended with ␣-1,2-mannose linkages.

DISCUSSION
The yeast-specific outer chain biosynthesis of N-glycans is initiated by the addition of ␣-1,6-linked mannose to the Man 8 GlcNAc 2 coreoligosaccharide in the Golgi apparatus, and this process is mediated by the activity of the OCH1 gene product in S. cerevisiae. The three genes OCH1, HOC1, and SUR1 comprise the OCH1 gene family in S. cerevisiae, implying that one interesting feature of yeast Golgi glycosyltransferases is their redundancy in terms of function and structure (4). Seven ORFs, showing significant sequence homologies to ScOCH1 and the topologic characteristics of glycosyltransferases, were identified from the H. polymorpha genome sequence (Table 1). All seven members of the H. polymorpha OCH1 gene family, including HpHOC1, HpOCR1, and HpOCH1 analyzed during this study, were predicted to encode type II membrane proteins with a short cytoplasmic N-terminal domain, a single membrane-spanning region, and a C-terminal catalytic domain, suggesting their function as glycosyltransferases localized in the Golgi lumen (data not shown). To clarify sequence relationships and to functionally group the different members of the H. polymorpha OCH1 family, phylogenetic analysis was carried out together with the OCH1 family genes and homologues from other yeasts and fungi (Fig. 8). The tree shows that the OCH1 family genes would be grouped into several subfamilies such as OCH, HOC, OCR, and SUR groups. As expected from relatively high sequence homologies with those of S. cerevisiae, HpOCH1 and HpHOC1 are classified into OCH and HOC groups, respectively. Interestingly, the other members of the H. polymorpha OCH1 gene family are grouped together remotely from the OCH and HOC groups, constituting their own subfamily (HpOCR group). The HpOCR subfamily appears to be evolved from the common origin of the OCH1 and HOC1 genes before their split occurred. The genes belonging to HpOCR group were named as HpOCR2, HpOCR3, HpOCR4, and HpOCR5, in which the numbering was made based on their homologies to H. polymorpha OCH1 ( Table 1). None of the HpOCR subfamily was able to complement the defects of S. cerevisiae OCH1 and HOC1 mutation (data not shown), which is consistent with their relatively remote relationship with the OCH1 and HOC1 subfamily.
As generally observed in glycosylation defective mutant strains of S. cerevisiae (26), the deletion of HpHOC1, HpOCR1, or HpOCH1 resulted in characteristic phenotypic defects in cell wall integrity, such as hypersensitivities to hygromycin B or sodium deoxycholate, thus indicating that the functions of HpHOC1, HpOCR1, and HpOCH1 are associated with cell wall biosynthesis (Fig. 2). In particular, the null mutant strain of HpOCR1 had a slow growing and temperature-sensitive phenotype as was observed in the deletion strain of ScOCH1, whereas the Hpoch1⌬ and Hphoc1⌬ mutant strains showed growth rates that were comparable with that of the wild type under normal growth conditions. Analysis of the recombinant glycoprotein GOD expressed in the deletion mutants of HpOCR1 and HpOCH1 revealed a significant reduction in the size of N-linked oligosaccharides (Fig. 4), indicating that HpOcr1 and HpOch1 proteins have major roles in H. polymorpha-specific outer chain biosynthesis. No apparent defect in N-glycosylation was detected on disrupting HpHOC1, as is the case for S. cerevisiae HOC1 (9). However, the Hphoc1⌬Hpocr1⌬ double deletion mutant showed a more marked N-glycosylation defect than the single Hpocr1⌬ mutant strain (Figs. 3 and 4), suggesting that HpHoc1p might be partly involved in N-glycosylation in a redundant fashion with HpOcr1p.
Although the Hpocr1⌬ mutant strain displays apparent phenotypes quite similar to those of the Scoch1⌬ strain (5), i.e. a retarded growth rate and a dramatic reduction in the size of N-glycans, detailed structural analysis revealed that the initiation step of ␣-1,6-mannose addition to the core oligosaccharide was not impaired in the Hpocr1⌬ mutant strain (Fig. 4A, panels h and i). Rather, the noticeable reduction in large oligosaccharide species branched with ␣-1,2-linked mannose units led us to speculate that the severe defect of outer chain biosynthesis shown by the Hpocr1⌬ mutant strain might be due to a defect of outer chain extension by ␣-1,2-linked mannose addition. In contrast, most N-oligosaccharide species derived from the recombinant GOD and the endogenous glycoprotein HpYps1p secreted by the Hpoch1⌬ mutant strain were shown to be devoid of additional ␣-1,6-mannose residues attached on the core oligosaccharide Man 8 GlcNAc 2 (Fig. 4, A , panel n, and B, panel e), suggesting that HpOch1p functions as a critical element during the first addition of an ␣-1,6-mannose to the core oligosaccharide Man 8 GlcNAc 2 . In addition, the physiological role of HpOch1p as a functional homologue of ScOch1p was further confirmed by its ability to complement the temperature-sensitive growth phenotype (Fig. 6A) and the hyperglycosylation defect (Fig. 6B) of Scoch1⌬ cells. Furthermore, the result of in vitro assay on ␣-1,6-mannosyltransferase activity showed that HpOch1p has an initiating ␣-1,6-mannosyltransferase activity (Fig. 6, C and D). Taken together, our data provide evidence that HpOch1p is a key ␣-1,6-mannosyltransferase, which is responsible for the first step of outer chain biosynthesis in H. polymorpha. At present, only single ␣-1,6-mannosyltransferase has been found to be responsible for adding initiating ␣-1,6-mannose to the core oligosaccharide in outer chain biosynthesis in S. cerevisiae, S. pombe, and P. pastoris (5,7,28). The overexpressions of HpOCR1 and HpHOC1 in Scoch1⌬ mutant (Fig.  6) and in Hpoch1⌬ mutant cells (data not shown) did not complement the temperature-sensitive growth and N-glycosylation defects of these mutant strains, indicating that HpOcr1p and HpHoc1p cannot substitute for the function of ScOch1p and HpOch1p as an initiating ␣-1,6mannosyltransferase. Interestingly, no direct N-glycosylation defect was reported in the null mutation of Yarrowia lipolytica OCH1 homologue (YlOCH1), which led to speculation that either YlOCH1 has a different role in the N-linked glycosylation pathway in Y. lipolytica (29) or that redundant genes may encode a functional homologue of ScOch1p in Y. lipolytica.
At present, the functions of HpOcr1p and HpHoc1p have yet to be defined, although they appear to be involved in the elongation step of outer chain biosynthesis. The apparent decrease of ␣-1,2-mannose extension in the Hpocr1⌬ mutant strain strongly suggests that the function of HpOcr1p is closely related to ␣-1,2-mannosyltransferase activity. Moreover, the drastic decrease in the extent of O-glycosylation in the Hpocr1⌬ mutant strain (Fig. 5) strongly indicates that HpOcr1p might encode ␣-1,2-mannosyltransferase, because a large class of glycan additions that are found on both O-and N-linked oligosaccharides are ␣-1,2-linked mannoses. However, little homology (below 8% sequence identity) exists between HpOCR1 and S. cerevisiae KTR (Kre-Two-Related) family, which encodes ␣-1,2-mannosyltransferase involved in both N-and O-linked protein glycosylation in S. cerevisiae (30), excluding the possibility that HpOcr1p could be classified as a member of the KRE2/MNT1 family. Thus, we propose HpOCR1 as a novel member of the OCH1 family that is implicated in both N-and O-linked glycosylation. As suggested for S. cerevisiae Mnn9p, which was shown to have ␣-1,2-mannosyltransferase activity in addition to ␣-1,6mannosyltransferase activity (31), it could be that HpOcr1p has ␣-1,2mannosyltransferase activity and acts on N-glycans in which the first ␣-1,6-mannose was added by HpOch1p. The question of whether HpOcr1p is an enzyme directly involved in N-and O-linked oligosaccharide elaboration has to be addressed by further biochemical analysis.  (SpOCH1, CAD24818), and H. polymorpha SUR1 (HpSUR1) were included for phylogenetic analysis, and the result was graphically presented using ClustalW (align.genome.jp/).
As in the case of ScHoc1p (9), the function of HpHoc1p is unclear. The deletion of HpHOC1 generates cell wall defect, which is characterized by the sensitivity to hygromycin B and Calcofluor White. However, HpHOC1 could not complement the hypersensitivity of the Schoc1⌬ strain to hygromycin B (data not shown). The deletion effect of HpHOC1 on N-glycosylation became more manifest in the absence of HpOcr1p, indicating that the function of HpHoc1p might be partially overlapped with HpOcr1p. Our recent study on transcriptome analysis using a H. polymorpha partial genome microarray (32) showed a significant induction of HpHOC1 after administering the superoxide-generating drug, menadione, which suggests that the function of HpHoc1p might be associated with stress response. The elucidation of the specific roles of HpHOC1, HpOCR1, and other members of the HpOCR1 subfamily in the N-glycosylation pathway and other cellular processes in H. polymorpha remains an intriguing issue, which requires further genetic and biochemical studies.
Based on structural information of the N-linked oligosaccharides of H. polymorpha and the data presented in this study, we propose a putative N-linked outer chain biosynthetic pathway in H. polymorpha, as shown in Fig. 9. As reported in other yeasts, the core oligosaccharide of Man 8 GlcNAc 2 originating from a dolichol-linked Glc 3 Man 9 GlcNAc 2 is elongated at the ␣-1,3-branch by the addition of an ␣-1,6-linked mannose unit by an initiating ␣-1,6-mannosyltransferase, a process that is mainly catalyzed by HpOch1p. In S. cerevisiae, after the addition of the first ␣-1,6-mannose, the outer chain backbone is intensively elongated by the action of Mnn9p-containing M-Pol I and M-Pol II complexes, generating a mannan-type structure (2). S. cerevisiae strains mutated at MNN9 show severe underglycosylation and osmotic fragility (33). However, most surprisingly, the deletion of the MNN9 homologue in H. polymorpha (34) did not lead to a serious alteration in growth phenotype and caused no apparent hypermannosylation reduction, 8 indicating that the activity of HpMnn9p is not critical for outer chain elongation in H. polymorpha. This is in a good agreement with our previous report (14) and present data (Fig. 4) on the structures of N-glycans attached to the recombinant GOD, the cell wall mannoproteins, and the HpYps1 protein obtained from H. polymorpha, which reveal that the outer chains of H. polymorpha N-linked oligosaccharides have very short ␣-1,6-extensions and are mainly elongated by ␣-1,2-mannose addition. Therefore, in this methylotrophic yeast, the activities of ␣-1,2mannosyltransferases appear to out-compete those of ␣-1,6-mannosyltransferases for outer chain elongation, to generate mainly core-type glycans that lack the extended ␣-1,6-mannose backbone structure (Fig.  9, ii versus iii). Moreover, ␣-1,2-linked mannoses are exposed as terminal residues without further decoration with ␣-1,3-linked mannoses in H. polymorpha N-glycans (Fig. 9, iv), which is different from S. cerevisiae N-glycans in which the addition of ␣-1,3 mannoses acts as a stop signal for further extension.
It is noteworthy that the Hpoch1⌬ strain, despite its defect in outer chain initiation on the core glycan Man 8 GlcNAc, did not show severe growth retardation under normal conditions, unlike the Hpocr1⌬ and Scoch1⌬ mutant strains, although it displayed a temperature-sensitive growth phenotype. Differing from the outer chains of S. cerevisiae N-glycan with extensive ␣-1,6-extensions, those of H. polymorpha N-glycans were shown to have very short ␣-1,6-extensions, mainly composed of single ␣-1,6-linked mannose. This might explain the mild effect of HpOCH1 deletion on cell growth of H. polymorpha. On the contrary, the deletion of HpOCR1, which generated dramatic defects in both N-and O-linked glycosylation, would cause severe growth retardation. The wild type comparable growth of the Hpoch1⌬ strain under normal growth conditions warrants that this strain can be developed as a starting strain for the production of recombinant glycoproteins mimicking humanized N-glycans in H. polymorpha. In addition, the lack of the immunogenic terminal ␣-1,3-mannose linkage and the extremely low level of phosphomannose residues in H. polymorpha N-glycans (14) represent additional advantages of this yeast over S. cerevisiae in terms of glycan engineering with a view toward glycoprotein production. The engineered Hpoch1⌬ strain with the targeted expression of A. saitoi ␣-1,2-mannosidase in the ER was able to synthesize Man 5 GlcNAc 2 as a major N-glycan (Fig. 7D). These results demonstrate that H. polymorpha has the potential to be developed as a host for the production of therapeutic glycoproteins containing humanized oligosaccharides, although further optimization of mannose removal and the addition of other sugars are required to generate complex N-glycans of therapeutic value.