| Human placental ribonuclease inhibitor is an acidic protein of about 50kDa with an amino acid composition characterized by unusually high contents of leucine and cysteine. It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease. HRI has 32 cysteine residues, the formation of disulfide bonds from those cysteine residues is a rapid cooperative process that inactivates HRI. The most proximal cysteine residues in native HRI are two pairs that are adjacent in sequence, such as Cys328 and Cys329. A cystine from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight-membered ring, which would disrupt the structure of HRI and could facilitate further oxidation. Otherwise, HRI is constructed of 7 leucine-rich repeats. The repeat units must form a circle, a helix, or a line. Of these, a circular arrangement may bring the N and C termini into close proximity.In this present paper, two alanine residues substitute for cys328/cys329 by site-directed mutagenesis and N-termini 10 residues were deleted. The site-mutated HRI cDNA and 5'-termini of 30 bp deletion-mutated HRI cDNA were constructed into plasmid pPIC9K respectively, and then transformed into Pichia pastoris GS115 by electroporation. After screening the colonies, the bacteria were cultured and the products were purified with CNBr Activated Sepharose 4B affinity chromatography. The affinity of the recombinant human RI with double site mutation for RNase A and its anti-oxidative effect were examined. The results indicated that there is no much change of the affinity for RNase A (Ki=3.24(10-14mol/L) detected when compared with the wild type of RI (Ki=2.88(10-14mol/L). But the capacity of anti-oxidative effect was increased by 7(9 times. The enhance of the anti-oxidative effect may be the reason of preventing the formation of disulfide bond between cys328 and cys329, thereby the native three dimensional structure of the RI was maintained. The conclusion is that replacing adjacent cysteine residues (cys328/cys329) in this protein can confer oxidation resistance. Meanwhile, the affinity of the recombinant human RI with 30 bp deletion-mutation for RNase A and its capacity of inhibition to RNase A activity were examined. The results indicated that there is two-times decreases in the affinity for RNase A (Ki=5.76(10-14mol/L) when compared with the wild type of RI. N-termini residues 1-10 of HRI can be deleted without destroying its inhibitory activity.HRI has 32 cysteine residues in one molecule and at least 30 cysteine residues exist in reduced states. It is well known that reduced glutathione protects organisms from peroxidation by using its reduced thiol group. RI also has antioxidative functions. In this sense, it is predicted that Cys328Ala-Cys329Ala site-mutated HRI may enhance its capability of oxidation resistance, but N-termini 10 residues deleted HRI may has no much change on its capability of oxidation resistance. First, mutant HRI cDNA, 5'-termini of 30 bp deletion-mutated HRI cDNA and wild HRI cDNA were blunted and inserted into the reverse-transcript virus vector pLNCX, then positive clone was tranfected into C6 cell by liposome. Effects of wild HRI, Cys328Ala-Cys329Ala site-mutated HRI and 5'-termini of 30 bp deletion-mutated HRI on the rat glial cell line C6 injured with H2O2 were studied by the comparison between the transfected C6 cells with Cys328Ala-Cys329Ala site-mutated HRI cDNA, 5'-termini of 30 bp deletion-mutated HRI cDNA and wild HRI cDNA in which RI were overexpressed. The main experiment results were as following: the transfected C6-mutant-RI cells had higher viability, less MDA contents, but more CAT and SOD activities. This result showed that mutant HRI had more effective antioxidant capability to protect cells from peroxidative injuries. C6 with N-terminal deletion mutant–HRI had lower viability, more MDA contents, lower CAT and SOD activities than wild type HRI. This result showed that N-deletion mutant HRI had decreased its...
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