| Abstract [eng] |
Glycosylation is one of the most prevalent post-translational protein modifications in eukaryotes. Glycans attached to proteins are important for their activity, stability, and correct folding, also, glycoproteins perform regulatory, signaling, catalytic, and many other functions in cells. Dolichol kinase (DK) is an essential enzyme involved in glycosylation processes in the membrane of the endoplasmic reticulum and core steps of these processes are highly conserved from yeasts to higher eukaryotes. Defects in protein glycosylation pathway in yeasts have already been linked to changes in protein secretion efficiency, but more detailed studies are required for better understanding of these processes. The aim of this study was to evaluate the impact of protein glycosylation deficiencies, including defects caused by DK mutations, to the secretion efficiency of recombinant proteins in S. cerevisiae and to investigate the ability of homologous human and K. lactis DK to complement S. cerevisiae DK functions. The secretion efficiency of recombinant proteins in examined yeast mutants was dependent on protein glycosylation efficiency. The activity of DK was most affected by G420D and double G407S and L421S mutations, which was detected by decrease of carboxypeptidase Y glycosylation efficiency and changes in S. cerevisiae cell wall structure. Despite glycosylation defects, the mutants with DK carrying these mutations exhibited higher secretion efficiency of recombinant α-amylase, KEX2 peptidase and Pen-m4 protein. This improvement of protein secretion efficiency in S. cerevisiae might be related to a wide range of physiological changes caused by protein glycosylation defects leading to changes in lipid homeostasis, ET stress regulation, and cell wall structure. In this study for the first time, S. cerevisiae DK gene was replaced by human and K. lactis DK genes which, despite partial homology between protein sequences, were able to fully complement S. cerevisiae DK function. The constructed yeast strain with the human DK gene was also used to study the effects of human DK mutations on glycosylation processes in S. cerevisiae. |
