Effects of glycosylation on the secretion and enzyme activity of Mucor rennin, an aspartic proteinase of Mucor pusillus, produced by recombinant yeast.
The Mucor rennin gene encoding a prepro form of the fungal aspartic proteinase from Mucor pusillus was expressed under the control of the yeast GAL7 promoter in Saccharomyces cerevisiae. The mature M. pusillus rennin secreted efficiently by yeast was a highly glycosylated protein. Analysis by a combination of site-directed mutagenesis of each of the three possible glycosylation sites and treatment of the secreted M. pusillus rennins with endo-beta-N-acetylglucosaminidase H revealed that the mature yeast M. pusillus rennin contained two asparagine-linked glycosylation sites among the three possible glycosylation sites. A mutation of the 2 glycosylated asparagine residues of M. pusillus rennin resulted in significant decreases in the level of secretion by yeast cells. In addition, the extent of glycosylation of M. pusillus rennin was found to affect the enzyme properties such as milk-clotting and proteolytic activities.
Aikawa
J
J
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Yamashita
T
T
Nishiyama
M
M
Horinouchi
S
S
Beppu
T
T
D000118:Acetylglucosaminidase; D001483:Base Sequence; D012085:Chymosin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009089:Mucor / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D012085:Chymosin