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 phosphomanno- sylation 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.