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 bo nds of insulin, separately or both, by substitution of the cysteine residue s with serine. Deletion of A20-B19 or both of the two interchain disulfide bonds resulted in the complete toss of secretion of the mutant single-chain porcine insulin precursor (PIP) from Saccharomyces cerevisiae cells. Remov al 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 phys ico-chemical properties. The A7-B7 disulfide bond deleted insulin mutant re tained only 0.1% receptor-binding activity compared with porcine insulin, a nd its in vivo biological potency measured by mouse convulsion assay was al so very low. We also studied some physico-chemical properties of the mutant using circular dichroism, native polyacrylamide gel electrophoresis and re versed-phase HPLC, which revealed some structural changes of the mutant pep tides compared to native insulin. The present study shows that the two inte r-chain disulfide bonds are important for efficient in vivo folding/secreti on of PIP from yeast, especially the A20-B19 disulfide bond, and that the A 7-B7 disulfide bond is crucial for maintaining the native conformation and biological activity of insulin.