In vitro evolution of amphioxus insulin-like peptide to mammalian insulin.
By site-directed mutagenesis, six insulin residues related to the insulin-receptor interaction were grafted, partially or fully, onto the corresponding position of a recombinant amphioxus insulin-like peptide (ILP) that contained the A- and B-domains of the deduced amphioxus ILP. After fermentation, purification, and enzymatic cleavage, six insulin-like double-chain ILP analogues were obtained: [A2Ile]ILP, [B12Val, B16Tyr]ILP, [B25Phe]ILP, [A2Ile, B12Val, B16Tyr, B25Phe]ILP (four-mutated ILP), [A2Ile, B12Val, B16Tyr, B24Phe, B25Phe]ILP (five-mutated ILP), and [A2Ile, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr]ILP (six-mutated ILP). Circular dichroism analysis showed that such replacement did not significantly affect their secondary and tertiary structure compared with that of the wild-type ILP. The insulin-receptor-binding activity of the four-, five-, and six-mutated ILP was 0.14%, 11%, and 11% of native insulin, respectively; the other three ILP analogues acquired none of the detectable insulin-receptor-binding potency. The growth-promoting activities of the five- and six-mutated ILP were both about 50% of native insulin, while that of the wild-type ILP was not detectable. By structure-function-based mutagenesis, the completely inactive amphioxus ILP was converted into a molecule with moderate mammalian insulin activity. These results indicated the following: first, the grafted as well as those inborn insulin-receptor-binding related residues can form an insulin-receptor-binding patch on the ILP analogues; second, the ILP can be used as a scaffold molecule to investigate the role of the insulin residues; third, the natural evolution of amphioxus ILP to mammalian insulin is a possible process and can be mimicked in the laboratory.
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Zhan-Yun
ZY
Guo
Lu
L
Shen
Wen
W
Gu
An-Zheng
AZ
Wu
Jian-Guo
JG
Ma
You-Min
YM
Feng
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002942:Circular Dichroism; D019020:Directed Molecular Evolution; D005057:Eukaryotic Cells; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008322:Mammals; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011972:Receptor, Insulin / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D007328:Insulin; C093255:amphioxus insulin-like peptide; D011972:Receptor, Insulin