Cloning and Characterization in Pichia Pastoris of PNO1 Gene Required for Phosphomannosylation of N-Linked Oligosaccharides

Gene 324 (2004) 129–137 www.elsevier.com/locate/gene

Cloning and characterization in Pichia pastoris of PNO1 gene required for phosphomannosylation of N-linked oligosaccharides

Masami Miura*, Masaaki Hirose, Taeko Miwa, Shinobu Kuwae, Hideyuki Ohi

Protein Research Laboratory, Research and Development Division, Mitsubishi Pharma Corporation, 2-25-1, Shodai-Ohtani, Hirakata, Osaka 573-1153, Japan Received 16 May 2003; received in revised form 25 August 2003; accepted 16 September 2003

Received by B. Dujon

Abstract

The yeast Pichia pastoris PNO1 ( Phosphomannosylation of N-linked Oligosaccharides) gene, which is involved in phosphomannosylation of N-linked oligosaccharides, was cloned using the Saccharomyces cerevisiae MNN4 gene [Glycobiology 6 (1996) 805] as a probe. The PNO1 open reading frame (ORF) encodes a type II membrane protein composed of 777 amino acid residues. Only in the short region extending from amino acid position 450 to 606 of Pno1p, sequence homology to S. cerevisiae Mnn4p was observed at a level of 45%. The tandem repeat sequence of Lys-Lys-Lys-Lys-Glu-Glu-Glu-Glu characteristic of the C-terminal region of S. cerevisiae Mnn4p is not present in Pno1p. To investigate the function of the PNO1 gene, we constructed a PNO1 gene disruptant by replacement with an expression cassette of human antithrombin (AT), a glycoprotein in plasma. The cell growth and recombinant human antithrombin (rAT) production levels of the disruptant were similar to those of recombinant human antithrombin-expressing wild-type strains. Moreover, the level of alcian blue dye cell staining, which shows the presence of acidic sugar chains on the cell surface, was also similar. However, the phosphomannosylation ratio of N-linked oligosaccharides on recombinant human antithrombin decreased dramatically from 20% in wild-type strains to less than 1% in the PNO1 disruptant. When the PNO1 gene was re-introduced into the disruptant, the phosphomannosylation ratio recovered to the original level. These results suggest that the newly cloned PNO1 gene promotes phosphomannosylation only to core-like oligosaccharides, and not to the hypermannosylated outer chain, and that it has a different function from the MNN4 gene, which promotes the phosphomannosylation of both core and outer sugar chains. D 2003 Elsevier B.V. All rights reserved.

Keywords: Antithrombin; Core oligosaccharides; Methylotrophic yeast; N-glycosylation

1. Introduction sis of the N-linked core oligosaccharide Man8GlcNAc2 in the endoplasmic reticulum is almost identical in manner to Yeasts have the ability to produce glycoproteins in a that in mammalian cells, but the yeast-specific hyperman- similar manner to mammals. However, the structure of nosylated outer chain consisting of 30–150 mannose resi- yeast-derived oligosaccharides is different from that of dues extends into the pre-Golgi and Golgi apparatus mammalian cells. In Saccharomyces cerevisiae, the synthe- (Kukuruzinska et al., 1987). Moreover, mannosylphosphate is often transferred to both the core and outer sugar chains (Ballou, 1990). In a related study of the phosphomannosy- Abbreviations: AT, antithrombin; bp, base pair; DU, dextrose unit; lation of N-linked oligosaccharides in S. cerevisiae,the ELISA, enzyme-linked immunosorbent assay; GlcNAc, N-acetylglucos- MNN4 and MNN6 genes were cloned and analyzed (Odani amine; HPLC, high-performance liquid chromatography; kb, kilobase; kDa, et al., 1996; Wang et al., 1997); disruption and overexpres- kilodalton; Man, mannose; ORF, open reading frame; PA, pyridylaminated; sion of MNN4 led to a decrease and increase, respectively, in PAGE, polyacrylamide-gel electrophoresis; PCR, polymerase chain reac- the mannosylphosphate content of cell-wall mannans tion; rAT, recombinant antithrombin; SDS, sodium dodecyl sulfate. * Corresponding author. Tel.: +81-72-856-9215; fax: +81-72-864- (Odani et al., 1996). Since it has been confirmed in vitro 2341. that phosphomannosylation of the core oligosaccharides and E-mail address: [email protected] (M. Miura). Man5GlcNAc2 is dependent on Mnn6p, it has been sug-

0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2003.09.023 130 M. Miura et al. / Gene 324 (2004) 129–137 gested that the MNN6 gene encodes the enzyme for phos- and phosphomannosylation to occur at the level of 22% phomannosylation and that the MNN4 gene product is the (Hirose et al., 2002). positive regulator of Mnn6p (Jigami and Odani, 1999). In the present study, we tried to clone the counterpart of Non-S. cerevisiae yeasts have been developed as heter- the MNN4 gene in P. pastoris to identify the cause of ologous gene expression systems (Romanos et al., 1992).In phosphomannosylation and to suppress it by disruption of particular, the methylotrophic yeast Pichia pastoris has the responsible gene. In attempting to do so, however, we been extensively developed and widely used as a high-level cloned a novel functional gene encoding 777 amino acids expression host for heterologous protein production (Cer- which is also involved in the phosphomannosylation of the eghino and Cregg, 2000). It is known that high-mannose N-linked oligosaccharides of glycoproteins in P. pastoris. type oligosaccharides are attached to proteins produced by The phosphomannosylation ratio of Man9–12GlcNAc2 on P. pastoris as well as other yeasts, but the oligosaccharides rAT dramatically decreased by disruption of the new gene, are generally shorter than in S. cerevisiae (Bretthauer and but the level of acidic oligosaccharides in the cell-wall outer Castellino, 1999). In most cases, P. pastoris produces glycans did not decrease. We considered that the cloned oligosaccharides with 8–18 mannose residues as major gene is not the counterpart of the MNN4 gene, but a novel components, e.g., Man8–14GlcNAc2 in S. cerevisiae inver- regulatory gene, and designated it PNO1 (Phosphomanno- tase (Grinna and Tschopp, 1989) and Man9–12GlcNAc2 in sylation of N-linked Oligosaccharides). the kringle 2 domain of tissue-type plasminogen activator (Miele et al., 1997b). Hypermannosylation is however observed in some cases, for instance in HIVgp120 (Scorer 2. Materials and methods et al., 1993) and the neuraminidase of the A/Victoria/3/75 influenza virus (Martinet et al., 1997). As minor compo- 2.1. Strains nents, phosphomannosylation is observed in a few P. pastoris-derived recombinant proteins. For example, 20% P. pastoris GTS115 (his4), provided by the Phillips of the kringle 2 domain of tissue-type plasminogen activator Petroleum and identical with strain GS115 (Cregg et al., is phosphomannosylated to Man10 –14GlcNAc2 (Miele et al., 1985), was used as the host strain for gene cloning, gene 1997a), and one-third of the N-linked oligosaccharides in disruption and rAT expression. P. pastoris RH101 (Mochi- invertase are negatively charged (Grinna and Tschopp, zuki et al., 2001) is a GTS115-derived rAT expression strain 1989). Phosphomannosylation was also detected in the integrated with the plasmid pAT101, which carries the Man9–14GlcNAc2 of aspartic protease, but not in the five mature AT cDNA (Yamauchi et al., 1992) placed under the other proteins examined in the same study (Montesino et control of the truncated and mutated AOX2 promoter (Ohi et al., 1998). These findings suggest that, while the probability al., 1994), the S. cerevisiae SUC2 secretion signal (pre- of hypermannosylation and phosphomannosylation is rela- peptide) and the AOX1 terminator. S. cerevisiae AH22 (a, tively low, the nature of the oligosaccharides in each P. leu2, his4, can1) (Hinnen et al., 1978) was used for cloning pastoris-derived glycoprotein should be analyzed. In this of the S. cerevisiae MNN4 gene. S. cerevisiae LB6-5D (a, respect, genetic studies relating to phosphomannosylation in mnn4-1) was purchased from the American Type Culture P. pastoris have advanced little. Collection. Escherichia coli XL-1 Blue MRF’ (D(mcrA)183, Human antithrombin (AT) is synthesized in the liver and D(mcrCB-hsdSMR-mrr)173, endA1, supE44, thi-1, recA1, has a plasma level of approximately 125 mg/l (Murano et gyrA96, relA1, lac[F’ proAB, laclqZDM15, Tn10(tetr)]) and al., 1980). It is a single-chain glycoprotein consisting of 432 SOLR (e14À(mcrA), D(mcrCB-hsdSMR-mrr)171, sbcC, amino acid residues with molecular weight of 58 kilodalton recB, recJ, umuCDTn5(kanr), uvrC, lac, gyrA96, relA1, (kDa) and has four N-linked sugar chain additional sites thi-1, endA1, ER [F’ proAB, laclqZDM15]SuÀ) were used (Bock et al., 1982). The content of oligosaccharide chains as bacterial host strains for gene cloning. E. coli DH5 of biantennary complex type is estimated at around 15% (supE44, hsdR17, recA1, endA1, gyrA96, thi-1, relA1) was (Franze´n et al., 1980; Mizuochi et al., 1980). AT is a plasma used for plasmid construction. protease inhibitor with wide-ranging ability to inhibit the activity of trypsin-type serine proteases, including thrombin 2.2. Cloning of the P. Pastoris PNO1 gene and coagulation factor Xa, and plays the most important role in the control of the blood-coagulation cascade. When First, the S. cerevisiae MNN4 gene was cloned by recombinant AT (rAT) was expressed using the yeasts S. polymerase chain reaction (PCR). The 1.5 kilobase (kb) cerevisiae or Schizosaccharomyces pombe, high mannose- EcoRI fragment of the S. cerevisiae MNN4 gene coding type sugar chains were added (Bro¨ker et al., 1987), al- region was used as a probe for P. pastoris genomic Southern though the study did not mention phosphomannosylation of hybridization. P. pastoris genomic DNA was prepared from oligosaccharides. In previous studies, we produced rAT in P. strain GTS115 by standard methods (Sherman et al., 1986). pastoris (Mochizuki et al., 2001), and analyzed the N- The genomic DNA was digested with various restriction linked oligosaccharides of the rAT, finding the major endonucleases, fractionated by agarose gel electrophoresis, component of the sugar chains to be Man9–12GlcNAc2, and transferred to a nylon membrane. Southern hybridization M. Miura et al. / Gene 324 (2004) 129–137 131 was performed with the DIG-ELISA kit (Roche Diagnostics) as a selectable marker. First, the pTM004 was digested and the band was detected after washing twice in 0.5 Â SSC with PstI and SmaI to eliminate the 5V non-coding region (75 mM NaCl, 7.5 mM trisodium citrate), 0.1% sodium and the 5V end of the PNO1 ORF,andthe2.7kb dodecyl sulfate (SDS) solution at 42 jC for 15 min. fragment was ligated to the PstI and SmaI site of the For cloning of the 7.5 kb SpeI fragment, which was vector pUC18 (Takara Bio). Next, the plasmid was positive in Southern hybridization, the SpeI-digested ge- digested with EcoRV and HpaI to remove the 0.4 kb nomic DNA was fractionated on an agarose gel and frag- region of the PNO1 ORF and the 4.9 kb fragment was ments around 7.5 kb were recovered. The isolated DNA obtained. On the other hand, pAT101 (Mochizuki et al., fragments were ligated to a SpeI digested EZAP II vector 2001) was digested with DraI and NaeI, and the fragment (Stratagene) and introduced into phage using the Gigapack containing the rAT-expressing construct and the HIS4 gene III Gold in vitro packaging kit (Stratagene). The phage was was isolated. The two fragments were then blunted and then absorbed to E. coli XL-1 Blue MRF’ and cultured on ligated. The resulting plasmid was named pTM009 (Fig. an NZY agarose plate (1% NZ amine, 0.5% NaCl, 0.5% 2). For disruption of the PNO1 gene, the PstI–SpeI yeast extract, 0.2% MgSO4, 1.5% agar) for plaque forma- fragment of pTM009 was introduced into the chromosom- tion. The plaques were transferred to a nylon membrane and al PNO1 gene locus of the strain GTS115. The transfor- plaque hybridization was performed as described elsewhere mation was performed using the lithium acetate method (Sambrook et al., 1989) using the 1.5 kb EcoRI fragment of (Ito et al., 1983) with slight modifications for P. pastoris. the S. cerevisiae MNN4 gene as a probe. Positive plaques The PNO1 gene disruptant was screened out of His+ were identified and the pBluescript SK( À )-based plasmids transformants using genomic Southern hybridization. The were rescued. A newly cloned gene was screened out of restriction map around the pno1 gene of the PNO1 gene these plasmids and the plasmid was designated pTM004 disruptant is shown in Fig. 2. (Fig. 1). The DNA sequence was determined by the dideoxy method (Sanger et al., 1977) and the nucleotide 2.4. The PNO1 gene reversion sequence was analyzed with the DNASIS computer software (Hitachi Software Engineering). To reverse the pno1 gene of the PNO1 gene disruptant, the plasmid pMM127 (Fig. 2), containing the full length of 2.3. The PNO1 gene disruption the PNO1 gene and the S. cerevisiae SUC2 gene (Taussig and Carlson, 1983), which functions as a selectable marker PNO1 gene disruption was performed by gene replace- for P. pastoris transformants (Sreekrishna et al., 1987), was ment of the PNO1 open reading frame (ORF) with an rAT constructed as follows. The pTM004 was digested with expression construct, with the P. pastoris HIS4 gene used SpeI, the 7.5 kb fragment containing the PNO1 gene was subcloned to the XbaI site of the pUC18, and the SUC2 gene was inserted into the SalI and SphI sites. The pMM127 was digested and linearized with PstI, and introduced into the remaining portion of the chromosomal pno1 gene in the PNO1 gene disruptant using the lithium acetate method (Ito et al., 1983). The PNO1 gene revertants were screened out of sucrose-utilizing transformants (Suc+) regenerating on Msu plates (0.67% yeast nitrogen base, 0.5% sucrose, 1.5% agar) using genomic Southern hybridization. The restriction map around the pno1 gene of the PNO1 revertant is shown in Fig. 2.

2.5. Cultivation of transformants and purification of rAT

The fed-batch fermentation culture and purification of rAT was performed by a method described previously (Mochizuki et al., 2001). Briefly, the yeast cells were cultured using a 3-l jar fermentor (BMD-3, Able), prolif- erated in a basal medium containing glycerol, and then fed a fed-batch medium containing methanol as the carbon source. Cell growth was monitored at 540 nm. The concentration of rAT in the culture-supernatant was mea- Fig. 1. Restriction map of the PNO1 cloned plasmid pTM004. The 7.5 kb region containing the PNO1 ORF is cloned to E. coli vector pBluescript sured by enzyme-linked immunosorbent assay (ELISA) SK( À ) including the ampicillin resistance gene. The direction of the using rabbit anti-human AT antibody (Dako), sheep anti- transcription of the PNO1 ORF is indicated with an arrow. human AT antibody horse radish peroxidase conjugate 132 M. Miura et al. / Gene 324 (2004) 129–137

Fig. 2. Genomic restriction map of wild-type strain, the PNO1 disruptant and the PNO1 revertant. The plasmid pTM009 for disruption of the PNO1 gene was constructed and digested with PstI and SpeI; an 8.2 kb fragment including the AT gene was isolated and introduced into the chromosomal PNO1 locus of P. pastoris GTS115 by homologous recombination and the PNO1 disruptant was screened up by Southern blot analysis. The plasmid pMM127 for reversion of the PNO1 gene was constructed and digested with PstI; the linearized plasmid was integrated into the chromosomal pno1 gene locus of the PNO1 disruptant by homologous recombination and the PNO1 revertant was screened up by Southern blot analysis. The restriction maps of the plasmids pTM009 and pMM127 are also shown. mAOX2 p., truncated and modified AOX2 promoter; SUC2 s., SUC2 secretion signal; AOX1 t., AOX1 terminator.

(Cedarlane) and 2,2V-azino-bis(3-ethylbenzthiazoline-6-sul- flask culture using YPD (1% yeast extract, 2% peptone and fonic acid) peroxidase substrate (Kirkegard and Perry 2% dextrose) were collected by centrifugation and stained in Laboratories). The commercially available human plas- 200 Al of 0.02 N HCl containing 0.1% alcian blue 8GX ma-derived AT NeuartR (Mitsubishi Pharma) used as a (Sigma) for 1 h at room temperature. The cells were again measurement standard. rAT secreted into the culture-su- collected by centrifugation, washed once with 0.02 N HCl, pernatant was purified by chromatography on a heparin- and suspended with 200 Al of 0.02 N HCl. The OD620 and affinity column and then hydroxyapatite and cation-ex- OD492 values of the suspension were measured and the change columns. Finally, contaminants with molecular difference was calculated as alcian blue binding value. weights higher than 60 kDa were removed by gel-perme- ation chromatography on a Sephacryl S-200 HR column 2.7. HPLC analysis of oligosaccharides (Amersham Pharmacia Biotech). The purity of rAT estimated by polyacrylamide-gel electrophoresis (SDS-PAGE) The method of oligosaccharide analysis of rAT was was over 99%. described previously (Hirose et al., 2002). First, N-linked oligosaccharides were released by PNGase F (Takara Bio) 2.6. Alcian blue staining of yeast cells and purified with a cellulose cartridge (Takara Bio). Next, the oligosaccharides were labeled with 2-aminopyridine The standard method for alcian blue staining of yeast using a pyridylamination kit (Takara Bio). The pyridylami- cells was described elsewhere (Friis and Ottolenghi, 1970). nated (PA)-oligosaccharides were then separated on an Yeast cells (OD540 = 3) cultured for 48 h at 30 jC in a shake- anion exchange column TSKgel DEAE-5PW (7.5 Â 75 M. Miura et al. / Gene 324 (2004) 129–137 133 mm) (Tosoh). Finally, neutral and acidic PA-oligosacchar- of the MNN4 gene as a probe, the genome restriction map ides were analyzed using the amide adsorption column of the P. pastoris homologue was determined and the TSKgel Amide-80 (4.6 Â 250 mm) (Tosoh). In the high- positive 7.5 kb SpeI fragment was cloned using the plaque performance liquid chromatography (HPLC) system, PA- hybridization method. The fragment was inserted into the oligosaccharides were detected by monitoring fluorescence SpeI site of the pBluescript, and the resulting plasmid was (excitation at 320 nm and emission at 400 nm). designated pTM004. The restriction map of the pTM004 is shown in Fig. 1, with the ORF determined as described below. 3. Results The nucleotide sequence and deduced amino acid sequence of the protein coding region are shown in Fig. 3. 3.1. Cloning and sequencing of the PNO1 gene There is no typical TATA box (TATAAA) in the 5V flanking region, but the TATA-like sequence TAATAA is found at In order to clone the gene involved in the phospho- position À 85 upstream of the ATG. The ORF consists of mannosylation of N-linked glycans in P. pastoris, we had 2331 base pair (bp) encoding a protein of 777 amino acids, initially planned to clone a P. pastoris homologue of the S. while S. cerevisiae Mnn4p consists of 1178 amino acids. cerevisiae MNN4 gene. Using the 1.5 kb EcoRI fragment The predicted amino acid sequence contains a transmem-

Fig. 3. Nucleotide sequence of the P. pastoris PNO1 gene and deduced amino acid sequence. The nucleotides are numbered relative to the translation initiation codon of the PNO1 gene. The deduced amino acid sequence is given beneath the nucleotide sequence of the PNO1 coding region. The TATA-like sequence (TAATAA, À 85) is in italic. A hydrophobic region consisting of 13 hydrophobic amino acids located near the N-terminal is indicated by bold and boxed. The region with high homology to the S. cerevisiae MNN4 gene is underlined. Sequence data have been deposited with the DDBJ/EMBL/GenBank database under accession number AB099514. 134 M. Miura et al. / Gene 324 (2004) 129–137 brane domain composed of 13 hydrophobic amino acids at the N-terminal side as well as the MNN4 gene. Only in the short region extending from amino acid position 450 to 606, sequence homology to Mnn4p is observed at a level of 45%, but no homology is recognized in the other regions (Fig. 4). The tandem repeat sequence of four lysine and four glutamic acid residues (KKKKEEEE) found in the C- terminal region of Mnn4p and thought to be important for its function (Odani et al., 1996) was not present in Pno1p. The cloned gene, which thus appeared to be novel and have a different function from the MNN4 gene, was designated PNO1.

3.2. The PNO1 gene disruption and sugar chain analysis of Fig. 5. Typical time-course of changes in cell density (OD540) and rAT secreted rAT concentration in fed-batch fermentation. The open circle and open triangle

indicates the OD540 of the wild-type RH101 and the PNO1 disruptant, To characterize the function of the PNO1 gene, we respectively. The closed circle and closed triangle indicates the concen- disrupted it by introducing a human AT expression construct tration of rAT in the culture-supernatant of the RH101 and the PNO1 into the PNO1 ORF of P. pastoris GTS115 chromosomal disruptant, respectively. DNA, and screened a PNO1 gene disruptant. In the disruptant, a 0.4 kb HpaI–EcoRV region of the PNO1 ORF is alcian blue staining on the P. pastoris PNO1 disruptant, and deleted and replaced with the human AT gene and the P. it stained blue to the same degree as the wild-type P. pastoris HIS4 gene (Fig. 2). Fermentation of the PNO1 pastoris RH101, while the S. cerevisiae mnn4 mutant disruptant and, as a control, the rAT-expressing wild-type LB6-5D remained white (Table 1). This means that the strain RH101, was performed using a jar-fermentor. As PNO1 gene product has no involvement in the phospho- shown in Fig. 5, the cell growth and rAT expression level mannosylation of the cell-surface mannoproteins. Therefore, of the two strains were similar. we thought that the function of Pno1p is different from that Alcian blue is a phthalocyanine dye whose intensity of Mnn4p. depends on the amount of phosphomannosylation to cell- Next, we analyzed the phosphomannosylation ratio of surface mannoproteins and which is therefore used to assay oligosaccharides on secreted rAT. We have already dem- the phosphate of N-linked oligosaccharides in the cell- onstrated that the major component of N-linked oligosac- surface mannoproteins. The wild-type strain of S. cerevisiae charides attached to the rAT produced by P. pastoris is stains with alcian blue, but the haploid strain LB6-5D Man9–12GlcNAc2, and that 22% of oligosaccharides are carrying the mnn4 mutation does not (Odani et al., 1996), phosphomannosylated (Hirose et al., 2002). Using the indicating that the mnn4 gene mutant reduces phosphoman- same methods, the N-linked oligosaccharides of the rAT nosylation in the cell-surface mannoproteins. We performed produced by the wild-type strain and the PNO1 disruptant

Fig. 4. Homology plot and amino acid sequence identity between P. pastoris PNO1p and S. cerevisiae Mnn4p. (A) Homology plot of P. pastoris PNO1p (left) and S. cerevisiae Mnn4p (top). (B) Highly homologous regions of P. pastoris PNO1p and S. cerevisiae Mnn4p. The amino acid sequence of the Pno1p is presented in the upper line and that of the Mnn4p in the lower line. M. Miura et al. / Gene 324 (2004) 129–137 135

Table 1 peaks was 20% in rAT from the wild-type strain RH101, Effect of PNO1 gene disruption and reversion on alcian blue binding but less than 1% in rAT from the PNO1 disruptant. These Strain Genotype Alcian blue assay Color results indicate that the PNO1 gene products must play an (OD620 –OD492) essential role in the phosphomannosylation of N-linked RH101 wild-type 0.62 F 0.04 blue oligosaccharides such as Man GlcNAc , the structure F 9–12 2 PNO1 disruptant Dpno1 0.56 0.04 blue of which is similar to that of the core oligosaccharides PNO1 revertant Dpno1DPNO1, 0.57 F 0.04 blue SUC2 Man8GlcNAc2. LB6-5D (S. cerevisiae) Dmnn4 ND white Each value represents average F standard deviation (n = 8). 3.3. The PNO1 gene reversion and sugar chain analysis of ND: not detected. secreted rAT were isolated and separated to neutral and acidic fractions, To confirm the function of the PNO1 gene, the plasmid and the ratios of acidic oligosaccharides were compared. pMM127 for the PNO1 gene reversion was introduced into One-charged acidic sugar chains were major acidic com- the chromosomal DNA of the PNO1 disruptant using the ponent in both strains and two-charged was hardly S. cerevisiae SUC2 gene as a selection marker. A PNO1 detectable in the PNO1 disruptant (data not shown). Size gene revertant (Fig. 2) was obtained and its properties distribution HPLC analysis of neutral and one-charged were investigated. The cell growth and rAT secretion levels acidic sugar chains using an amide adsorption column are from the PNO1 revertant were almost the same as in the shown in Fig. 6(A)–(D). The proportion of acidic to total PNO1 disruptant and the wild-type strain RH101 (data not oligosaccharides calculated from the dimensions of the shown). The level of alcian blue dye staining of the PNO1

Fig. 6. Size-distribution analysis of neutral and acidic PA-oligosaccharides using amide adsorption column. (A) Neutral sugar chain of RH101 (wild-type); (B) acidic sugar chain of RH101; (C) neutral sugar chain of the PNO1 disruptant; (D) acidic sugar chain of the PNO1 disruptant; (E) neutral sugar chain of the PNO1 revertant; (F) acidic sugar chain of the PNO1 revertant. The amount of neutral sugar chain applied was 1/2.5 compared with that of the acidic sugar chain. Peaks were estimated as follows through comparison with previous data (Hirose et al., 2002): Peak a, Man9GlcNAc2-PA; Peak b, Man10GlcNAc2-PA; Peak c, Man11GlcNAc2-PA; Peak d, Man12GlcNAc2-PA; Peak e, Man-P-Man9GlcNAc2-PA; Peak f, Man-P-Man10GlcNAc2-PA; Peak g, Man-P- Man11GlcNAc2-PA; Peak h, Man-P-Man12GlcNAc2-PA. Elution time was converted to dextrose unit (DU) standardized time using the retention time of authentic PA-glucose oligomers. 136 M. Miura et al. / Gene 324 (2004) 129–137 disruptant was also unchanged (Table 1). However, it On the other hand, the PNO1 disruptant in the present study should be noted that the phosphomannosylation ratio of led to significantly reduced phosphomannosylation of core N-linked oligosaccharides in the rAT from the PNO1 oligosaccharides without affecting the phosphomannosyla- revertant recovered to the same level as in the wild-type tion of outer sugar chains. These observations suggested strain (Fig. 6), and the major acidic sugar had one-charge that both genes were involved in phosphomannosylation, as well as wild-type derivative (data not shown). Since but that the PNO1 gene had a different function from the phosphomannosylation ability was fully restored by the MNN4 gene. PNO1 gene re-introduction, we concluded that the function of Pno1p is to promote phosphomannosylation of N-linked 4.2. Yeast host for expression of recombinant glycoprotein oligosaccharides. In the recombinant production of mammalian glycoproteins in yeast, it is necessary to pay attention to undesirable 4. Discussion changes in properties or activity caused by yeast-derived sugar chains attached to the recombinant proteins. In 4.1. Functions of the PNO1 gene particular, neo-antigenicity is a serious problem when yeast derived-recombinant glycoprotein is developed for pharma- We cloned the novel gene PNO1 and investigated its ceutical use. Three features of glycosylation in S. cerevi- function using an rAT-expressing PNO1 gene disruptant and siae—hypermannosylation in the outer chain, terminal a1– revertant. In wild-type P. pastoris, the phosphomannosyla- 3 mannosylation and phosphomannosylation—are not pres- tion ratio of N-linked oligosaccharides in the secreted rAT ent in mammalian secretory glycoproteins and should was 20%, but this fell to less than 1% following the PNO1 therefore be considered for potential neo-antigenicity in gene disruption. It was already known that the N-linked humans (Ballou, 1990). Phosphomannosylation is not ob- oligosaccharides attached to the rAT produced by P. pas- served in Kluyveromyces lactis but it contains GlcNAc toris consisted of mannose-type sugar chains and that the residue in the outer sugar chain and other yeasts have major structure was Man9–12GlcNAc2 (Hirose et al., 2002). various N-glycan structure different from that of mamma- Therefore, the PNO1 gene was thought to have the function lian (Gemmli and Trimble, 1999). Recently, genetically of leading the phosphomannosylation of oligosaccharides engineered P. pastoris strain was reported to synthesize such as Man9–12GlcNAc2, the structure of which is similar human-like hybrid N-glycosylation, but phosphomannosy- to that of the core sugar chain Man8GlcNAc2 synthesized in lation event was not mentioned (Choi et al., 2003).InP. the endoplasmic reticulum. However, the PNO1 disruptant pastoris, only a small amount of hypermannosylated forms as well as the wild-type strain stained positive with alcian is present in heterologous recombinant protein (Bretthauer blue, which is known to bind to the negatively charged and Castellino, 1999), and a1–3 mannosylation does not sugar chains of cell-surface mannans containing phospho- occur because of the deficiency of a1–3 mannosyltransfer- mannose, indicating that the PNO1 gene is not involved in ase (Trimble et al., 1991). These findings suggest that the phosphomannosylation of the outer chain of oligosac- suppression of phosphomannosylation would pave the charides. We separately obtained two P. pastoris mutants by way for the development of P. pastoris-derived recombinant treatment of the mutagen ethyl methanesulfonate. Unlike glycoproteins for pharmaceutical use. From this point of the PNO1 disruptant, these did not stain with alcian blue, view, we have demonstrated the possibility of using the indicating reduced phosphomannosylation of the outer PNO1 disruptant for the production of pharmaceutical chain of the mannoproteins (data not shown). Taking these recombinant glycoproteins. Moreover, the unchanged prop- findings together, we are sure that the PNO1 gene is erties of the PNO1 disruptant, such as cell growth and involved in the phosphomannosylation of only the core production ability, may be useful in the industrial produc- oligosaccharides and not of the outer sugar chains. tion of recombinant proteins. When we compared the amino acid sequence of Pno1p and Mnn4p, sequence homology was observed, at the level of 45%, only in a short region of Pno1p from position 450 to Acknowledgements 606. Both proteins have a transmembrane domain near the N-terminal region, but the total length of Pno1p is consid- We would like to thank Dr. Wataru Ohtani and Mr. erably shorter than that of Mnn4p, and the Lys-Glu repeat Kenmi Miyano for helpful discussions. sequence considered to play an important role in Mnn4p is not present in Pno1p. It has been reported that the ratio of acidic core oligosaccharides decreases to 30% or less by the References MNN4 disruption of S. cerevisiae (Odani et al., 1996), but this finding also shows that a considerable amount of acidic Ballou, C.E., 1990. Isolation, characterization, and properties of Saccha- sugar chains still remain, and suggests that there are other romyces cereviviae mnn mutants with nonconditional protein glycosy- pathways for positive regulation of phosphomannosylation. lation defects. Methods Enzymol. 185, 440–470. M. Miura et al. / Gene 324 (2004) 129–137 137

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