Characterization of the Pichia pastoris protein-O-mannosyltransferase gene family.

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

Inhibitor sensitivity of <i>PMT</i> knockouts.

<p>For dilution assays, yeast cells were grown overnight in YSD and then adjusted to an OD600 of 1.0 using YSD, diluted in 10-fold increments, and then 3 µl of each dilution were spotted onto solid YSD media with varying concentrations of PMTi-3. The picture of the plate without PMTi-3 was taken after 48 hours, and the picture of the plates containing PMTi-3 were taken after 96 hours of incubation at 25^°C. YGLY4140 is the parent strain of YGLY6001 and YGLY4329, YGLY733 is the parent strain of YGLY3773 and YGLY5968, and NRRL -Y11430 is the parent strain of YGLY35032.</p

Western blot analysis of an IgG1 expressed in the presence of PMTi-3.

<p>The <i>PMT</i> wild type strain YGLY4280 was grown in 96 well deep-well plates and induced in medium containing no PMTi-3 (C) or increasing concentrations of PMTi-3 (0.0187, 0.0375, 0.075, 0.15, 0.3, 0.625, 1.25, 2.5, 5, 10 µg/ml). After overnight induction, 7 µl of each supernatant was separated by polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto nitrocellulose membranes. IgG1 was detected using an anti-human IgG (heavy and light chain) antibody. Hyperglycosylated heavy-chain (indicated by an asterisk) is barely detectable at PMTi-3 concentrations of 0.15 µg/ml and higher, however strain growth and fitness (strain lysis) was still unaffected at PMTi-3 concentrations of up to 1.25 µg/ml. A representation of the PMTi-3 increase in concentration and the relative reduction in cell fitness are illustrated by <i>light</i> and <i>dark gray triangles</i> respectively.</p

<i>PMT</i> expression profile by microarray analysis.

<p>The relative levels of PMT mRNA levels were assessed by microarray analysis under glycerol (<i>shaded bars</i>) or methanol (<i>black bars</i>) growth conditions as described in the Materials and Methods Section. mRNA levels for <i>ACT1</i> and <i>GPD</i> have been added for reference.</p

Overview of <i>PMT</i> knockout experiment.

<p>(A) The PCR overlap and <i>PMT</i> knockout procedure. First a linear gene deletion allele was constructed the following way: a region 5’ of the <i>PMT</i> ORF was amplified using primers 1 and 2, the selectable marker was amplified using primers 3 and 4, and a region 3’ of the <i>PMT</i> ORF was amplified using primers 5 and 6. The primers in parentheses were added to the schematic to illustrate that primers 2 and 3, as well as primers 4 and 5, are complementary to each other (thereby creating the overlaps). The amplified products were then used as templates together with primers 1 and 6 in the overlap reaction. The linear deletion allele was then integrated into the <i>P</i><i>. pastoris</i> genome via homologous recombination, and knockouts were selected on plates supplemented with the appropriate antibiotic. (B) Western blot analysis of Kringle 1-3 protein (K1-3) expressed in wild type and <i>PMT</i> knockout strains. Strains JC53 (wild type), JC55 (<i>Δpmt1</i>), JC66 (<i>Δpmt2</i>), JC65 (<i>Δpmt4</i>), and JC51 (<i>Δpmt5/6</i>) were grown and induced in shake flasks. Seven µl of each supernatant was separated by polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto nitrocellulose membranes. K1-3 protein was detected using an anti-His antibody. O-glycan site occupancy (Occ.) was determined as described in Materials and Methods. The expected mass of the fully deglycosylated protein is 44.2 kDa, and the band at 36 kDa probably represents proteolytic cleavage.</p