Cloning and characterization in Pichia pastoris of PNO1 gene required for phosphomannosylation of N-linked oligosaccharides.
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.
Miura
Masami
M
Protein Research Laboratory, Mitsubishi Pharma Corporation, Shodai-Ohtani, Hirakata, Osaka 573-1153, Japan. Miura.Masami@mk.m-pharma.co.jp
Hirose
Masaaki
M
Miwa
Taeko
T
Kuwae
Shinobu
S
Ohi
Hideyuki
H
D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008358:Mannose / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D005656:Fungal Proteins; D008565:Membrane Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000990:Antithrombin III; D008358:Mannose