Overexpression of cell-wall GPI-anchored proteins restores cell growth of N-glycosylation-defective och1 mutants in Schizosaccharomyces pombe

The glycoproteins of yeast contain a large outer chain on N-linked oligosaccharides; therefore, yeast is not suitable for producing therapeutic glycoproteins for human use. Using a deletion mutant strain of alpha 1,6-mannosyltransferase (och1 Delta), we previously produced humanized N-glycans in fission yeast; however, the Schizosaccharomyces pombe och1 Delta cells displayed a growth delay even during vegetative growth, resulting in reduced productivity of heterologous proteins. To overcome this problem, here we performed a genome-wide screen for genes that would suppress the growth defect of temperature-sensitive och1 Delta cells. Using a genomic library coupled with screening of 18,000 transformants, we identified two genes (pwp1(+), SPBC1E8.05), both encoding GPI-anchored proteins, that increased the growth rate of och1 Delta cells, lacking the outer chain. We further showed that a high copy number of the genes was needed to improve the growth rate. Mutational analysis of Pwp1p revealed that the GPI-anchored region of Pwp1p is important in attenuating the growth defect. Analysis of disruptants of pwp1(+) and SPBC1E8.05 showed that neither gene was essential for cell viability; however, both mutants were sensitive beta-glucanase, suggesting that Pwp1p and the protein encoded by SPBC1E8.05 non-enzymatically support beta-glucan on the cell-surface of S. pombe. Collectively, our work not only sheds light on the functional relationships between GPI-anchored proteins and N-linked oligosaccharides of glycoproteins in S. pombe, but also supports the application of S. pombe to the production of human glycoprotein.

Automated summary

Through a genome-wide screen of a deletion mutant strain in fission yeast, two genes (pwp1(+), SPBC1E8.05) encoding GPI-anchored proteins were identified to improve the growth rate of cells lacking the outer chain, thereby enhancing productivity of heterologous proteins. Mutational analysis revealed the importance of the GPI-anchored region of Pwp1p in attenuating the growth defect and supporting beta-glucan on the cell-surface of S. pombe non-enzymatically.