Primary Exploration Of The Correlation Of Characters Of Two Positions In Scorpion Analgesic Peptide Ⅲ With Its Activity

The three-dimensional structure of analgesic peptide Ⅲ(BmK-AGP-SYPU Ⅲ),a polypeptide purified from the venom of scorpion Buthus martensii Karsch,was established by homology modeling.In order to obtain information about residues involved in the analgesic activity,BmK-AGP-SYPU Ⅲ was subjected to mutagenesis analysis at several sites belong to different structural domains.Based on the cDNA sequence encoding the mature peptide of BmK-AGP-SYPU Ⅲ,a series of mutagenic primers used to generate the desired mutations were designed and synthesized.Using pSYPU-1b-His-BmK-AGP-SYPU Ⅲ as a template along with T7 promoter primer and the mutagenic primers,seven mutant genes were obtained by PCR.All the genes were inserted into plasmid pSYPU-1b.The recombinant pSYPU-1b plasmids were used to transform competent E.coli BL21(λDE3)cells.E.coli BL21(λDE3)cells harboring each of the recombinant pSYPU-1b plasmids were grown in the Luria-Bertani medium and the expression of recombinant proteins was induced by addition of isopropy-β-D-thiogalactoside(IPTG).The recombinant proteins were purified to homogeneity through immobilized metal affinity chromatography and cation exchange chromatography.The mouse-twisting action model was used to test the analgesic effect of recombinant proteins,and both mutations at Asp53 and Ser54 of BmK-AGP-SYPU Ⅲ produced significant change in the analgesic activity.Asp53 in BmK-AGP-SYPU Ⅲ was replaced by various classes’ amino acids and four mutants were obtained.Except for the conserved substitution of negatively charged Glu for Asp53,the other three mutations at position 53 siginificantly decreased the analgesic activity.Modelling structures analysis showed the overall conformation of toxin was not changed by the mutations;the analgesic activity differences of different mutants should not be attributed to the local conformation change of N-terminal a-amino.The obtained results suggested that residue 53 was important for the analgesic activity of BmK-AGP-SYPU Ⅲ,its side-chain functional group exerts is effect by means of an intermolecular electrostatic interaction.BmK-AGP-SYPU Ⅲ was then subjected to a series of site-directed mutations at position 54 and a total of six mutants were sequentially obtained.The results of bioassay on mice showed that any substitution of Ser54 by various polar amino acids produced no significant change in the analgesic activity,while all substitutions of nonpolar amino acid for Ser54 led to a significant loss of analgesic activity.The modeling structure indicated that the side-chain of residue 54 is protruding from the molecular surface without any interaction with the rest of the molecule.The results of our present work show that residue 54 plays an important role in the analgesic activity of BmK-AGP-SYPU Ⅲ,its side-chain functional exerts is effect by means of an intermolecular hydrogen bond.