Porcine IGF1 synonymous mutation alter gene expression and protein binding affinity with IGF1R

Context: Insulin like growth factor 1 (IGF1) is privotal in the regulation of animal growth and is a single-chain globular protein composed of B, C, A, and D regions, of which the C region is involved in maintaining high affinity binding to the IGF1 receptor (IGF1R). Purpose: In this study, significant expression differences between large pigs and miniature pigs were detected and only one synonymous SNP (c.258G>A) in the C region of the coding sequence of IGF1 gene was screened. The aim of this manuscript was to clear the function of the SNP and clarify the mechanism of its influnce. Methods: The expression vectors contained A allele and G allele were constructed, and the expression assays of the two groups were determined by qRT-PCR and western blotting, then the stability assays of the mRNA and protein were carried out under the inhibitation of actinomycin D and cycloheximide, respectively. At last, the binding affinity of IGF1-G and IGF1-A with IGF1R were indicated by co-immunoprecipitation and double immunofluorescence labeling methods, the conformation difference was detected by differential immunodetection. Results: The IGF1-G expressed higher than IGF1-A in both transcription and translation levels, and the mRNA and protein stabilities of IGF1-G were lower than IGF1-A (P < 0.05). Furthermore, the relative binding affinity of GG-genotype IGF1 with IGF1R was significantly higher than that of the AA-genotype IGF1 (P< 0.05), and there was a difference in the conformation of the IGF1 with two genotypes. Conclusion: Our findings indicated the synonymous mutation altered the IGF1 gene expression and confirmed the synonymous mutation affected the IGF1 folding and the interactions with the IGF1R preliminarily. (C) 2018 Published by Elsevier B.V.