Screen,Synthesis And Activity Detect Of Mimetic Peptides Of Insulin

Insulin is a kind of protein hormone secreted by pancreas isletβcells. It is well known that insulin maintains glucose homeostasis by adjusting glucose intake and oxidation and glycogen generation and store. The complex physiological action of insulin depends on its binding to insulin receptor. The insulin receptor is an intrinsic disulfide-linked dimer of heterodimeric disulfide-linked proteins of the form (αβ)2. The alpha subunits are entirely extracellular and contain the ligand-binding domain.Interactions of insulin with the other monomer are predominantly electrostatic, with no obvious hydrophobic components.Although much is know about the IR in terms of its mode of action and signaling pathways, there is a paucity of information regarding the molecular architecture of the insulin binding site, the contact sites for insulin binding and the mechanism by which insulin induces its biological effects. People have been devoted in researching the hot spots on IR and on insulin binding sites. They hope to clear these doubts and obtain the minimum while act as full binding action fragments. Insulin can not be taken orally or by nasal cavity, otherwise, will be decomposed. Mainline is the only way for diabetics, but it has its drawback that the high concentration above physiological one makes insulin polymerization and impede its effect. Therefore seeking and designing small analogs for making oral surrogate of insulin is of great importance. It is a right way to screen in a phage-display random peptide library.Our group takes the Chinese hamster ovary cells over-expressing insulin receptors(CHO-IR) as targets to screen CHO-IR binding peptides in a phage display hexapeptide library. After four round of panning, we obtain fifteen ELISA positive recombined clones whose Kds all range in nanomolar scale .Considering the Kd values as main indexes combining with the yield ratio would lead to more preferable results. The amino acid sequences of these peptides display distinct conservation with each other. None of the peptides identified in this study had an sequence homology with insulin, but there was a significant number of important hydrophobic residues found in the various motifs.The motif L~L is high frequency which coincide with the literature . Now there in no literatures indicate clearly that S~S and A P play a role in the binding of insulin and insulin receptor. We suppose that the motifs S~S and A P may analogue the spatial structure of insulin. Two peptides are synthesized by manual way. Purify by HPLC, determined by MS and lyophilized.HepG2 cell have many essence features of hepatocyte, So we choose HepG2 cell to construct kata-glucose model in vitro. The biology activity of peptide A and B is detection by this model. They didn't show apparent effect of glucose consumption and cell generation in low concentration, but in high concentration they can inhibit glucose consumption notably. The two peptides in 1μM concentration can synergism with insulin (<10μM concentration), P<0.01, have mostly significant deviation. The peptides in respective low concentration can synergism with insulin.The insulin receptor is an insulin-stimulated tyrosine-specific protein kinase. The tyrosine kinase catalyzes the very rapid autophosphorylation of its ownβ-subunit on multiple tyrosines. We detect the effect of peptide A on tyrosine phosphorylation of insulin receptor by Western-blot. It shows that peptide A have weak effect on tyrosine phosphorylation of insulin receptor, but it can inhibit the binding of insulin and insulin receptor, it shows apparente antagonistic activity.