Originally published In Press as doi:10.1074/jbc.M508507200 on January 10, 2006
J. Biol. Chem., Vol. 281, Issue 10, 6261-6272, March 10, 2006
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
Received for publication, August 3, 2005
, and in revised form, December 16, 2005.
 |
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 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.
|
INTRODUCTION
|
|---|
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 Glc3Man9GlcNAc2 oligosaccharide is transferred to the Asn residues on nascent proteins by oligosaccharyl-transferase 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 (Man8GlcNAc2). Glycoproteins containing Man8GlcNAc2 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 Man8GlcNAc2 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 -1,6-linked 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 -1,6-linked 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 Man8GlcNAc2 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 methanol-containing 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 (Man8–12GlcNAc2) 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 human-compatible N-linked oligosaccharides.
|
EXPERIMENTAL PROCEDURES
|
|---|
Yeast Strains, Plasmids, and Media—H. polymorpha DL1-L (leu2) and DL-LdU (leu2 ura3::lacZ) strains were derivatives of DL-1 (ATCC26012) (15). S. cerevisiae och1 (MATa leu2 ura3 trp1 ade2 his3 och1::TRP1) and S. cerevisiae TOY137 (MATa leu2 ura3 trp1 ade2 his3 och1::hisG mnn1::hisG mnn4::hisG) were derived from S. cerevisiae W303-1A.3 The vector pDLMOX-GOD(H) (14) was used for the secretory expressions of Aspergillus niger glucose oxidase tagged with 6 residues of histidine (GOD-His) in H. polymorpha. pMOX-YPS1ct-His vector was constructed for the secretion of C-terminally truncated H. polymorpha Yps1p tagged with histidine residues.4 The plasmid YEp352GAPII, containing the S. cerevisiae GAPDH promoter and terminator,5 was used as a backbone vector for H. polymorpha gene expression in S. cerevisiae. To construct the HA-HDEL-tagged Aspergillus saitoi -1,2-mannosidase, the HA-HDEL tag sequence was introduced by PCR using the sense oligonucleotide primer, Msd-N (containing an EcoRI restriction site), and the antisense oligonucleotide primer, Msd-C (containing the coding sequences of the HA tag and the HDEL signal, followed by a stop codon and an NcoI restriction site), using pGAMH1 as a template (16). The resulting PCR product was digested with EcoRI and NcoI and used to substitute the 1.8-kb EcoRI/NcoI GOD-His fragment in pDLMOX-GOD(H) to generate the plasmid pDLMOX-MsdS(HA-HDEL). The auxotrophic marker HpLEU2 in pDLMOX-MsdS(HA-HDEL) was exchanged with HpURA3 to generate pDUMOX-MsdS(HA-HDEL). Drug sensitivity was assayed by spotting serially diluted yeast culture onto YPD (1% yeast extract, 2% peptone, and 2% glucose) solid media containing 40 µg/ml hygromycin B, 7 mg/ml Calcofluor White, or 0.4% sodium deoxycholate. Secretory expression and immunoblot analysis of A. niger GOD-His in H. polymorpha were performed as described previously (14). For deglycosylation, glycoproteins were digested with peptide:N-glycosidase F (PNGase F) according to the supplier's instructions (New England Biolab, Beverly, MA).
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 (GenBankTM accession numbers AL435940
[GenBank]
and AL433499
[GenBank]
), 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 HindIII 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 3' 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 3' 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 
2.3-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 5'-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 GenBankTM under accession numbers AF540063
[GenBank]
and AF490971
[GenBank]
, 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 GenBankTM under accession number AY502025
[GenBank]
. 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 GenBankTM under accession numbers DQ249343
[GenBank]
, DQ249344
[GenBank]
, DQ249345
[GenBank]
, and DQ249346
[GenBank]
, 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+ 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 Hphoc1Hpocr1 or Hphoc1Hpoch1 mutant strains, the HpURA3 pop-out cassette was removed from the Hphoc1::HpURA3 strain to recover the Ura– 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 µg 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 x 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 = 320 nm and em = 400 nm) using a Waters 2475 fluorescence detector. PA-oligosaccharides were digested sequentially with -1,2-mannosidase (from A. saitoi, Seikagaku Corp., Tokyo) and -1,6-mannosidase (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 (A600 = 3–4). Cells were collected by centrifugation at 3,000 x 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 MnCl2, 1 mM phenylmethylsulfonyl fluoride, 5% glycerol, and 2 µg/ml each protease inhibitor (antipain, chymostatin, leupeptin, and pepstatin A)). Glass beads (425–600 µm) 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 x g for 20 min, and the supernatant obtained was further centrifuged at 100,000 x 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 µg of high speed pellet proteins was incubated in 100 µl of 50 mM Tris-HCl (pH 7.5) buffer containing 10 mM MnCl2, 1 mM GDP-mannose, 0.5 mM 1-deoxymannojirimycin, and 100 pmol of Man8GlcNAc2-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 Na2HPO4, 2 mM KH2PO4 (pH 7.4)). Chitinase was eluted from the chitin in 100 µl of SDS-PAGE sample buffer by boiling for 5 min and then separated in 6% SDS-PAGE, which was visualized by silver staining.
-1,2-Mannosidase Assay—For in vitro A. saitoi -1,2-mannosidase activity assay, yeast cell extracts were prepared and analyzed as described by Chiba et al. (16) with slight modification. Soluble cell extracts were incubated with 30 pmol of Man6GlcNAc2-PA at 37 °C for 30 min and analyzed by reversed-phase HPLC using a TSK-GEL ODS-80Ts column (Tosoh, Tokyo, Japan, 0.46 x 25 cm3) equilibrated with a mixture of solvents A (100 mM acetic acid/triethylamine (pH 4.0)) and B (solvent A + 0.5% 1-butanol) (98:2, v/v). The ratio of solvent B was increased linearly up to 20% over 60 min at a flow rate of 1.0 ml/min.
|
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 Man8GlcNAc2 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 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.
View larger version (102K):
[in this window]
[in a new window]
|
FIGURE 1.
Overall sequence comparison of Och1p homologues in yeast. The amino acid sequences of gene products of S. cerevisiae HOC1 (ScHoc1p, NCBI protein accession number NP_012609
[GenBank]
), S. cerevisiae OCH1 (ScOch1p, NCBI protein accession number NP_011477
[GenBank]
), H. polymorpha HOC1, OCR1, and OCH1 (HpHoc1p, NCBI protein accession number AAQ1191; HpOcr1p, NCBI protein accession number AAQ06498
[GenBank]
; and HpOch1p, NCBI protein accession number AAS77488
[GenBank]
), Candida albicans OCH1 (CaOch1p, NCBI protein accession number XP_716632
[GenBank]
), P. pastoris OCH1 (PpOch1p, NCBI nucleotide accession number E12456
[GenBank]
), and S. pombe OCH1 (SpOch1p, NCBI protein accession number CAD24818
[GenBank]
) are shown. 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 membrane-spanning regions, which act as signal-anchor domains, are indicated by boxes, and the DXD motif is shown above a sequence as hhhhDXD.
|
|
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.
View this table:
[in this window]
[in a new window]
|
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.
|
|
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 (Hphoc1Hpocr1 or Hphoc1Hpoch1) 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 (Hphoc1Hpocr1) 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 slow-growing 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 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, Hphoc1Hpocr1, Hpoch1, and Hphoc1Hpoch1 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 Hphoc1Hpocr1 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 Hphoc1Hpoch1 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, Hphoc1Hpocr1, and Hpoch1 Strains—To gain more detailed information on the size and structure of N-glycans attached to rGOD expressed by the Hphoc1, Hpocr1, Hphoc1Hpocr1, 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 Man8–11GlcNAc2 were detected with several minor peaks of Man12–15GlcNAc2 (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 Man8GlcNAc2, was observed in the Hphoc1 single mutant (Fig. 4A, panel d). However, oligosaccharides from Hpocr1, Hphoc1Hpocr1, or Hpoch1 mutant cells were mainly composed of a single predominating species, corresponding to the core-glycosylated form Man8GlcNAc2, although larger structures of Man9–14GlcNAc2 were present as minor components (Fig. 4A, panels g, j, and m). In the Hphoc1Hpocr1 mutant strain, the relative proportions of the oligosaccharides larger than Man8GlcNAc2 appeared to be reduced versus the Hpocr1 strain. These results indicate that mannose outer chain elongation is significantly inhibited in the Hpocr1, Hphoc1Hpocr1, and Hpoch1 mutant strains, although small fractions of oligosaccharides (Man9–14GlcNAc2) larger than Man8GlcNAc2 were observed.
To investigate in more detail the structure of the oligosaccharide species produced by the mutant strains, sequential digestion experiments with -1,2- and -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. Man5GlcNAc2 and Man6GlcNAc2 (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 Man6GlcNAc2 oligosaccharide species generated by -1,2-mannosidase digestion into Man5GlcNAc2 (Fig. 4A, panel c). Our previous study indicated that Man5GlcNAc2 (M5) is the final product of specific -1,2-mannosidase digestion of the core oligosaccharide Man8GlcNAc2 or of the larger oligosaccharide species extended with only -1,2-mannose linkages, whereas Man6GlcNAc2 (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 Hphoc1Hpocr1 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 -1,6-mannosidase 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 Hphoc1Hpocr1 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 Hphoc1Hpocr1 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 Hphoc1Hpocr1 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-terminal 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 (Man9–10GlcNAc2) 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 Man8GlcNAc2 predominated; oligosaccharides larger than Man8GlcNAc2 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 Man8GlcNAc2 is a general phenotype of the Hpoch1 strain and not a protein-specific phenotype.
View larger version (35K):
[in this window]
[in a new window]
|
FIGURE 4.
HPLC analyses of N-linked oligosaccharides assembled on glycoproteins secreted by H. polymorpha mutant strains. A, analysis of N-glycans attached to rGOD secreted by H. polymorpha wild type (panels a–c), Hphoc1 (panels d–f), Hpocr1 (panels g–i), Hphoc1Hpocr1 (panels j–l), and Hpoch1 (panels m–o) strains. Chromatograms of the N-glycan profiles released from rGOD before any treatment (panels a, d, g, j, and m), after -1,2-mannosidase treatment (panels b, e, h, k, and n), and after subsequent-1,6-mannosidase treatment (panels c, f, i, l, and o) are shown. The HPLC column was equilibrated with 80% of solvent A (200 mM acetic acid/triethylamine (pH 7.3), acetonitrile = 1:9) and 20% of solvent B (200 mM acetic acid/triethylamine (pH 7.3), acetonitrile = 9:1). B, analysis of N-glycans assembled on Yps1p secreted by H. polymorpha wild type (panels a–c) and Hpoch1 (panels d–f) strains. Chromatogram of the N-glycans released from HpYps1p before any treatment (panels a and d), after -1,2-mannosidase treatment (panels b and e), and after subsequent -1,6-mannosidase treatment (panels c and f). The HPLC column was equilibrated with 75% of solvent A and 25% of solvent B. The elution times of peaks were compared with those of authentic Man5GlcNAc2-PA (M5), Man6GlcNAc2-PA (M6), and Man8GlcNAc2-PA (M8) of known structure (indicated by arrows). The unidentified peak (indicated by arrowhead) is detected in some samples during the HPLC analysis. We observed that the peak is resistant to digestion by jack bean (-1,2-, -1,3-, and-1,6-) mannosidase and becomes more dominant in the old samples compared with the fresh samples after PA labeling.
|
|
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, Hphoc1Hpocr1, and Hphoc1Hpoch1 mutant strains (Fig. 5). The Hpoch1, Hphoc1, and Hphoc1Hpoch1 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, significantly homogeneous forms of chitinase were detected in the Hpocr1 and Hphoc1Hpocr1 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 (Scoch1mnn1mnn4), but harboring YEp352GAPII, YEp352GAPII-HpOCH1, or YEp352GAPII-ScOCH1 plasmids, and these were used as enzyme sources for -1,6-mannosyltransferase assays (Fig. 6C). The Scoch1mnn1mnn4 strain was shown to be defective in adding mannose residues to N-linked core oligosaccharide (16). PA-labeled Man8GlcNAc2, 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, Man8GlcNAc2-PA (M8), was not converted into any other form by the membrane fraction of Scoch1mnn1mnn4 transformant harboring the null vector (Fig. 6C, panel a), a peak corresponding to Man9GlcNAc2-PA (M9) was detected as a reaction product in the membrane fraction of Scoch1mnn1mnn4 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 Man9GlcNAc2-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 Man6GlcNAc2-PA (M6), whereas the M8 core oligosaccharide was converted to Man5GlcNAc2-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.
View larger version (51K):
[in this window]
[in a new window]
|
FIGURE 6.
Functional complementation and in vitro activity analysis of H. polymorpha OCH1 gene product. A, spotting analysis of growth phenotype. Yeast cultures were spotted onto YPD plates, which were then incubated at 25 and 30 °C for 3 days. S. cerevisiae wild type strain (row 1) and Scoch1 mutant strains transformed with the control vector YEp352GAPII (row 2), YEp352GAPII-HpHOC1 (row 3), YEp352GAPII-HpOCR1 (row 4), YEp352GAPII-HpOCH1 (row 5), and YEp352GAPII-ScOCH1 (row 6). B, activity staining of invertase, carried out by the method of Gabriel and Wang (35). Analyzed samples are in the same order as in A. C, analysis of -1,6-mannosyltransferase activity in S. cerevisiae triple null strains Scoch1mnn1mnn4 carrying YEp352GAPII (panel a), YEp352GAPII-HpOCH1 (panel b), or YEp352GAPII-ScOCH1 (panel c). Solubilized membrane fractions were prepared as enzyme sources, and Man8GlcNAc2-PA was used as an acceptor. The reaction products were analyzed by HPLC. D, analysis of -1,6-mannosyltransferase activity in H. polymorpha wild type, Hpocr1, and Hpoch1 mutant strains. Reaction products by the membrane fractions of the wild type (panel a), 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.
|
|
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 human-type 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 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 Man6GlcNAc2-PA into Man5GlcNAc2-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.
View larger version (33K):
[in this window]
[in a new window]
|
FIGURE 7.
Expression of ER-targeted -1,2-mannosidase in Hpoch1 mutant strain. A, construct of the gene cassette for A. saitoi -1,2-mannosidase (MsdSp) tagged with the HA epitope and the ER retention signal HDEL. The signal sequence of aspergillopepsin I (apnS), which was used to direct secretion of MsdSp in yeast, and 8 potential N-linked glycosylation sites of MsdSp (36) are shown. The arrowhead indicates the fusion site between apnS and MsdSp. B, immunoblot analysis of A. saitoi -1,2-mannosidase expressed in H. polymorpha. Total cell lysates (corresponding to 0.5 A600, lanes 1 and 2) and the culture supernatant (16 µl, lanes 3 and 4) were fractionated on 10% polyacrylamide gel and analyzed using antibody raised against HA. Lanes 5 and 6 contain cell lysates treated with PNGase F. C, in vitro assay for A. saitoi -1,2-mannosidase activity in H. polymorpha. The standard Man6GlcNAc2-PA and Man5GlcNAc2-PA (panel a), the reaction products by the extracts prepared from H. polymorpha cells transformed with the null vector (panel b) and with the -1,2-mannosidase-HA-HDEL expression plasmid (panel c) were analyzed by reversed-phase HPLC. D, N-glycan profiles of the Hpoch1 mutant strain expressing A. saitoi -1,2-mannosidase. N-glycans obtained from the rGOD secreted by the Hpoch1 strain transformed with the null vector (panel a) and from Hpoch1 transformed with -1,2-mannosidase-HA-HDEL expression plasmid (panel b) were analyzed by HPLC. The N-glycans of b were further treated with -1,2-mannosidase (panel c).
|
|
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 -1,2-mannosidase-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 Man5GlcNAc2, 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 Man5GlcNAc2 after in vitro -1,2-mannosidase treatment (Fig. 7D, panel c). The incomplete trimming to Man5GlcNAc2 in the recombinant Hpoch1 strain might have been due to the low expression level or activity of -1,2-mannosidase-HA-HDEL. Alternatively, the activity of endogenous -1,2-mannosyltransferases in the Golgi might have subsequently added -1,2-linked mannoses to Man5GlcNAc2, 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 Man8GlcNAc2 core-oligosaccharide 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.
View larger version (27K):
[in this window]
[in a new window]
|
FIGURE 8.
Neighbor-joining phylogenetic tree of the OCH1 family and homologues from yeasts and fungi. The sequences of the OCH1 family and homologues were collected by Blast searches with S. cerevisiae OCH1 sequence. C. albicans HOC1, OCH1 (CaHOC1, NCBI protein accession number XP_716693
[GenBank]
; CaOCH1, XP_716632
[GenBank]
), Ashbya gossypii HOC1, OCH1 (AgHOC1, AAS53806
[GenBank]
; AgOCH1, AAS53836
[GenBank]
), Kluveromyces lactis HOC1, OCH1 (KlHOC1, XP_452168
[GenBank]
; KlOCH1, XP_456072
[GenBank]
), S. cerevisiae HOC1, OCH1, CSH1, and SUR1 (ScHOC1, NP_012609
[GenBank]
; ScOCH1, NP_011477
[GenBank]
; ScCSH1, NP_009719
[GenBank]
; ScSUR1, NP_015268
[GenBank]
), Candida glabrata HOC1, OCH1, and SUR1 (CgHOC1, XP_445987
[GenBank]
; CgOCH1, XP_444841
[GenBank]
; CgSUR1, XP_449590
[GenBank]
), Y. lipolytica OCH1 (YlOCH1, CAD91643
[GenBank]
), Aspergillus fumigatus OCH1 (AfOCH1, XP_753779
[GenBank]
), S. pombe OCH1 (SpOCH1, | |
TOP
ABSTRACT
INTRODUCTION
