title: PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris.
abstract: Protein mannosyltransferases (PMTs) catalyze the O-mannosylation of serine and threonine residues of proteins in the endoplasmic reticulum. The five PMT genes coding for protein mannosyltransferases, designated as PMT1, 2, 4, 5 and 6, were identified from Pichia pastoris genome based on the homology to PMT genes in Saccharomyces cerevisiae genome, which has seven PMT genes. The homologues of S. cerevisiae PMT 3 &7 genes are absent in P. pastoris genome. Approximately 5% of the recombinant insulin precursor expressed in P. pastoris is O-mannosylated. In this study, we attempted to prevent O-mannosylation of insulin precursor in vivo, through inactivation of the Pichia PMT genes. Since multiple PMTs are found to be expressed, it was important to understand which of these are involved in O-mannosylation of the insulin precursor. The genes encoding PMT1, 4, 5 and 6 were knocked out by insertional inactivation method. Inactivation of PMT genes 4, 5 and 6 showed ∼16-28% reductions in the O-mannosylation of insulin precursor. The PMT1 gene disrupted Pichia clone showed ∼60% decrease in O-mannosylated insulin precursor, establishing its role as an important enzyme for insulin precursor O-mannosylation.
authors.affiliation: Biocon Research Limited - SEZ unit, Plot Nos. 2 & 3, Phase IV - BIA, Bommasandra-Jigani Link Road, Bengaluru 560 099, India. Nagaraj.Govindappa@biocon.com
authors.forename: Nagaraj
authors.initials: N
authors.lastname: Govindappa
authors.forename: Manjunatha
authors.initials: M
authors.lastname: Hanumanthappa
authors.forename: Krishna
authors.initials: K
authors.lastname: Venkatarangaiah
authors.forename: Komal
authors.initials: K
authors.lastname: Kanojia
authors.forename: Krishnamurthy
authors.initials: K
authors.lastname: Venkatesan
authors.forename: Amarnath
authors.initials: A
authors.lastname: Chatterjee
authors.forename: Mutyalasetty
authors.initials: M
authors.lastname: Kusumanchi
authors.forename: Nitesh
authors.initials: N
authors.lastname: Dave
authors.forename: Partha
authors.initials: P
authors.lastname: Hazra
authors.forename: Sanjay
authors.initials: S
authors.lastname: Tiwari
authors.forename: Kedarnath
authors.initials: K
authors.lastname: Sastry
mesh_terms: D000818:Animals; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D055786:Gene Knockout Techniques; D016681:Genome, Fungal; D006031:Glycosylation; D007328:Insulin / Q000378:metabolism*; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
chemical_list: D007328:Insulin; D013912:Threonine; D012694:Serine; D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase