Behavior of cysteine mutants of human lysozyme in de novo synthesis and in vivo secretion.
To investigate the mechanism of disulfide-bond-coupled de novo folding of human lysozyme, we have constructed 23 mutant enzymes in which cysteine residue(s) were replaced by alanine(s). The mutant genes were translated in vitro in a system composed of rabbit reticulocyte lysate, canine pancreatic microsomal vesicles and oxidized glutathione. This system allows the formation of intramolecular disulfide bonds in translation products translocated into the microsomal lumen. The mobilities of the translation products were analyzed by SDS/PAGE in nonreducing conditions. Some mutant lysozymes were found to form a compact conformation with native-like mobility in the presence of SDS. The de novo formation of the SDS-resistant compact conformation of each mutant correlated well with its efficiency of secretion by Saccharomyces cerevisiae. Our results suggest that the de novo synthesized products reflect the conformational states in vivo to some extent, and that the formation of SDS-resistant compact conformation can be regarded as a necessary condition for allowing lysozyme to be secreted. In addition, the analysis of a mutant C116A (Cys116----Ala) under different oxidative conditions suggests two distinct pathways for the disulfide-bond-coupled formation of the compact conformation.
Omura
F
F
Protein Engineering Research Institute, Osaka, Japan.
Taniyama
Y
Y
Kikuchi
M
M
D000818:Animals; D001483:Base Sequence; D003545:Cysteine; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014158:Transcription, Genetic
D015345:Oligonucleotide Probes; D009113:Muramidase; D003545:Cysteine