A Phenylalanine to Serine Substitution within an O-Protein Mannosyltransferase Led to Strong Resistance to PMT-Inhibitors in Pichia pastoris

Abstract

Protein O-mannosyltransferases (PMTs) catalyze the initial reaction of protein O-mannosylation by transferring the first mannose unit onto serine and threonine residues of a nascent polypeptide being synthesized in the endoplasmic reticulum (ER). The PMTs are well conserved in eukaryotic organisms, and in vivo defects of these enzymes result in cell death in yeast and congenital diseases in humans. A group of rhodanine-3-acetic acid derivatives (PMTi) specifically inhibits PMT activity both in vitro and in vivo. As such, these chemical compounds have been effectively used to minimize the extent of O-mannosylation on heterologously produced proteins from different yeast expression hosts. However, very little is known about how these PMT-inhibitors interact with the PMT enzyme, or what structural features of the PMTs are required for inhibitor-protein interactions. To better understand the inhibitor-enzyme interactions, and to gain potential insights for developing more effective PMT-inhibitors, we isolated PMTi-resistant mutants in Pichia pastoris. In this study, we report the identification and characterization of a point mutation within the PpPMT2 gene. We demonstrate that this F664S point mutation resulted in a near complete loss of PMTi sensitivity, both in terms of growth-inhibition and reduction in O-mannosylglycan site occupancy. Our results provide genetic evidence demonstrating that the F664 residue plays a critical role in mediating the inhibitory effects of these PMTi compounds. Our data also indicate that the main target of these PMT-inhibitors in P. pastoris is Pmt2p, and that the F664 residue most likely interacts directly with the PMTi-compounds.

Protein O-mannosylation is crucial for E-cadherin–mediated cell adhesion

“…To rule out bias that quantitative differences in Pomt transcript levels might introduce in our functional analyses of O-mannosylation during preimplantation development, we established a method to inhibit POMT enzymatic activity directly in early embryos. Rhodanine-3-acetic acid derivatives such as compound 5a (R3A-5a) are highly specific inhibitors of fungal protein O-mannosyltransferases (23)(24)(25). We found that the enzymatic activity of mammalian POMTs also is blocked selectively by R3A-5a, both in vitro and in vivo.…”

Evidence of recombination of vaccine strains of lumpy skin disease virus with field strains, causing disease

“…The isolate Chechnya/2015 harbored an additional A to T nucleotide change, resulting in an amino acid change from serine to phenylalanine (Fig 1). Similar interchanges between two amino acids were shown to have a profound effect on mutated proteins [41]. Due to limited knowledge on LSDV proteins and their functions [1], it is challenging to predict the altered roles of RPO30.…”

Characterization of the Pichia pastoris Protein-O-mannosyltransferase Gene Family

“…In a parallel report by our laboratory a more potent RAA derivative (PMTi-4) was assessed for antibody production in P . pastoris [39]. Like the Kuroda et al study, a dramatic increase in antibody titer was observed in the presence of RAA derivatives.…”

“…Like the Kuroda et al study, a dramatic increase in antibody titer was observed in the presence of RAA derivatives. Incidentally, unlike PMTi-3 which appears to target Pmt2p and Pmt5p, PMTi-4 preferentially inhibited Pmt2p [39]. …”