Accelerated secretion of human lysozyme with a disulfide bond mutation.
The mutant human lysozyme, [Ala77, Ala95]lysozyme, in which the disulfide bond Cys77-Cys95 is eliminated, is known to exhibit increased secretion in yeast, compared to wild-type human lysozyme [Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M. & Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967]. To investigate this phenomenon, mammalian cells were used to analyze the secretion kinetics of [Ala77, Ala95]lysozyme and wild-type human lysozyme. The secretion rate of [Ala77, Ala95]lysozyme during the 150-min chase period was significantly accelerated [half-life (t1/2) = 29 min] compared to that of wild-type human lysozyme (t1/2 = 83 min), when expressed at the same levels within the cells. In contrast, after the 150-min chase, the rates of disappearance of both wild-type and mutant human lysozymes within the cells were similar, and considerably slower (t1/2 = 220 min), respectively. The remaining intracellular wild-type human lysozyme was localized mainly in the endoplasmic reticulum, whereas accelerated transport of the [Ala77, Ala95]lysozyme mutant protein from the endoplasmic reticulum to the Golgi apparatus was observed. Also in yeast cells, similar secretion kinetics and the differences in t1/2 for wild-type and mutant human lysozymes during the early chase period were observed. The two-phase kinetics of disappearance of intracellular human lysozymes suggest that only a proportion of the proteins becomes secretion competent soon after synthesis and is completely secreted during the early chase period, whereas others enter the distinct, slow pathways of intracellular transport and/or degradation. Increased secretion of [Ala77, Ala95]lysozyme is possibly due to enhanced competence for secretion acquired in the endoplasmic reticulum at the early stage of transport events, which is closely connected with the removal of a disulfide bond.
Omura
F
F
Protein Engineering Research Institute, Osaka, Japan.
Otsu
M
M
Kikuchi
M
M
D000230:Adenocarcinoma; D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004220:Disulfides; D015870:Gene Expression; D005813:Genes, Synthetic; D006801:Humans; D007700:Kinetics; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection; D014709:Vero Cells
D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D009113:Muramidase; D000409:Alanine