Secretion in yeast of mutant human lysozymes with and without glutathione bound to cysteine 95.
A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.
Protein Engineering Research Institute, Osaka, Japan.
Y
Y
Taniyama
C
C
Seko
M
M
Kikuchi
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004220:Disulfides / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D009154:Mutation; D010434:Pepsin A; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D016339:Spectrometry, Mass, Fast Atom Bombardment
D004220:Disulfides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009113:Muramidase; D010434:Pepsin A; D003545:Cysteine