Polymannosylation to asparagine-19 in hen egg white lysozyme in yeast.
Complementary DNA encoding hen egg white lysozyme (HEWL) was subjected to site-directed mutagenesis to have the N-glycosylation signal sequence (Asn19-Tyr20-Thr21) by substituting Arg with Thr at position 21. The mutant lysozyme (R21T) was expressed in Saccharomyces cerevisiae carrying the yeast expression plasmid inserting the mutant HEWL cDNA. The mutant lysozyme was expressed in the glycosylated forms which are mainly a polymannosyl form with a small amount of oligomannosyl form. The polymannosyl lysozyme was susceptible to Endo H cleavage of the carbohydrate chain. The length of the polymannose chain was predicted to be approximately 340 residues/mol of lysozyme from carbohydrate analysis. According to the estimation with low-angle laser light scattering combined with HPLC, the average molecular weight of polymannosyl lysozyme was 75 kDa, which is consistent with the value obtained from the carbohydrate analysis. The size of polymannosyl lysozyme R21T is similar or somewhat larger than that of G49N reported previously. Thus, it was confirmed that the unusually large polymannose chain was attached to heterologous mutant lysozyme, regardless of the N-linked position, in yeast.
Kato
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A
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
Takasaki
H
H
Ban
M
M
D000117:Acetylglucosamine / Q000032:analysis; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D008358:Mannose / Q000032:analysis / Q000737:chemistry*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D021382:Protein Sorting Signals; D012441:Saccharomyces cerevisiae / Q000235:genetics
D018076:DNA, Complementary; D021382:Protein Sorting Signals; D001216:Asparagine; D006596:Hexosaminidases; D009113:Muramidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008358:Mannose; D000117:Acetylglucosamine