Secretion and processing of insulin precursors in yeast.

Abstract

A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor al leader sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system. The secreted peptides were purified from the fermentation broth and characterized by sequencing and amino acid analysis. Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors isolated from such constructions could be enzymatically converted into insulin by treatment with trypsin and carboxypeptidase B. The above results suggest that the C-peptide region of proinsulin serves to direct the trypsin-like converting enzyme to process at the two dibasic sequences. We propose that in hormone precursors in general the spacer peptides serve to expose dibasic sequences for processing.Human preproinsulin consists of a prepeptide of 24 amino acid residues followed by proinsulin containing 86 amino acid residues in the configuration: prepeptide-B-Arg-Arg-C-LysArg-A in which C is the C peptide of 31 amino acid residues (1), and A and B are the A and B chain of human proinsulin, respectively. The prepeptide is removed during transport of the nascent polypeptide into the endoplasmic reticulum. By the time it reaches the Golgi, the disulfide bridges of proinsulin (2)

Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site.

“…Kex2 functions late in the secretory pathway of Saccharomyces cerevisiae to cleave the polypeptide chains of prepro-killer toxin and prepro-a-factor at the paired amino acid sequences Lys-Arg and Arg-Arg. In addition to these natural functions, the Kex2 protease can mediate the processing of proinsulin and proalbumin expressed in yeast (21,22) and can properly cleave the propeptide of proalbumin in vitro (23). Finally, Kex2 can function in transfected murine cells to process proopiomelanocortin prohormone to product peptides normally found in vivo (11).…”

Insulin or insulin-like studies on unicellular organisms: a review

“…Greenfield et al (1988) have observed that the substance detected in this fungi, which somehow resembles mammalian insulin, exerts metabolic effect in mammalian adypocites. The insulin precursor expression and secretion, including proinsulin, was found in S. cerevisiae by Thim et al (1986). According to Lenard (1992) hormones and hormones binding proteins resembling those of vertebrates are widespread in fungi, yeast and bacteria, and functional responses of microbial cells to mamaliam hormone have been found.…”

“…The authors conceded that the fungi material obtained was similar to purified mammalian insulins in its solubility in acid-ethanol, elution profile, bioactivity in bioassay systems, and the neutralization of its bioactivity by anti-insulin antibody. The findings from Thim et al (1986) showed a series of dibasic insulin precursors including proinsulin expressed and secreted by the fungi Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor alpha leader sequence and an insulin precursor.…”

Primary structures of three fragments of proglucagon from the pancreatic islets of the daddy Sculpin (Cottus scorpius)

“…Similar pressure has acted to conserve the length and overall net charge of the C peptide of proinsulin rather than the amino acid sequence [26]. It has been speculated, therefore, that such spacer peptides may function as 'second signal peptides' ensuring that the prohormone adopts the correct conformation for processing at the appropriate dibasic residues [27].…”