title: Characterization of the Pichia pastoris protein-O-mannosyltransferase gene family.
abstract: The methylotrophic yeast, Pichiapastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of β-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P. pastoris, thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P. pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P. pastoris PMT family. Like Candida albicans, P. pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P. pastoris platform as a suitable system for the production of therapeutic glycoproteins.
authors.affiliation: Biologics Discovery-GlycoFi Inc., Merck Research Laboratories, Lebanon, New Hampshire, United States of America. juergen_nett@merck.com
authors.forename: Juergen H
authors.initials: JH
authors.lastname: Nett
authors.forename: W James
authors.initials: WJ
authors.lastname: Cook
authors.forename: Ming-Tang
authors.initials: MT
authors.lastname: Chen
authors.forename: Robert C
authors.initials: RC
authors.lastname: Davidson
authors.forename: Piotr
authors.initials: P
authors.lastname: Bobrowicz
authors.forename: Warren
authors.initials: W
authors.lastname: Kett
authors.forename: Elena
authors.initials: E
authors.lastname: Brevnova
authors.forename: Thomas I
authors.initials: TI
authors.lastname: Potgieter
authors.forename: Mark T
authors.initials: MT
authors.lastname: Mellon
authors.forename: Bianka
authors.initials: B
authors.lastname: Prinz
authors.forename: Byung-Kwon
authors.initials: BK
authors.lastname: Choi
authors.forename: Dongxing
authors.initials: D
authors.lastname: Zha
authors.forename: Irina
authors.initials: I
authors.lastname: Burnina
authors.forename: John T
authors.initials: JT
authors.lastname: Bukowski
authors.forename: Min
authors.initials: M
authors.lastname: Du
authors.forename: Stefan
authors.initials: S
authors.lastname: Wildt
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hamilton
mesh_terms: D002176:Candida albicans / Q000201:enzymology / Q000235:genetics; D055786:Gene Knockout Techniques; D005800:Genes, Fungal; D006031:Glycosylation; D008364:Mannosyltransferases / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D010843:Pichia / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase