Effects of cysteine to serine substitutions in the two inter-chain disulfide bonds of insulin.
Using site-directed mutagenesis we deleted the two inter-chain disulfide bonds of insulin, separately or both, by substitution of the cysteine residues with serine. Deletion of A20-B19 or both of the two inter-chain disulfide bonds resulted in the complete loss of secretion of the mutant single-chain porcine insulin precursor (PIP) from Saccharomyces cerevisiae cells. Removal of the A7-B7 disulfide bond resulted in a large reduction of secretion, but we could obtain the mutant for analysis of its biological and some physico-chemical properties. The A7-B7 disulfide bond deleted insulin mutant retained only 0.1% receptor-binding activity compared with porcine insulin, and its in vivo biological potency measured by mouse convulsion assay was also very low. We also studied some physico-chemical properties of the mutant using circular dichroism, native polyacrylamide gel electrophoresis and reversed-phase HPLC, which revealed some structural changes of the mutant peptides compared to native insulin. The present study shows that the two inter-chain disulfide bonds are important for efficient in vivo folding/secretion of PIP from yeast, especially the A20-B19 disulfide bond, and that the A7-B7 disulfide bond is crucial for maintaining the native conformation and biological activity of insulin.
Guo
Z Y
ZY
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, People's Republic of China.
Feng
Y M
YM
D019943:Amino Acid Substitution; D000818:Animals; D002942:Circular Dichroism; D003545:Cysteine; D004220:Disulfides; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D016297:Mutagenesis, Site-Directed; D011384:Proinsulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D011972:Receptor, Insulin / Q000378:metabolism; D012694:Serine; D013552:Swine
D004220:Disulfides; D007328:Insulin; D012694:Serine; D011384:Proinsulin; D011972:Receptor, Insulin; D003545:Cysteine