Structure and Function of the Phospholipase AィイD22ィエD2 Inhibitor Having Leucine-rich Repeats

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1. A large-scale purification of phospholipase AィイD22ィエD2 inhibitors from the serum of Chinese mamushi and the crystallization of the inhibitorsWe have purified three types of phospholipase AィイD22ィエD2 inhibitors (PLIα, PLIβ, and PLIγ) by the sequential chromatography on Blue-Sepharose FF, Q-Sepharose FF, Phenyl-Sepharose HP, and Superdex 200pg columns. Finally, 10 mg of the purified PLIα, 5 mg of the PLIβ, and 20 mg of the PLIγ could be obtained from 10 ml of the Chinese mamushi serum. We are determining the best crystallization conditions of these inhibitors by the standard technique of vapor diffusion with the hanging drop methods. By this time, we could not obtain the crystals suitable for X-ray crystallography.2. Establishment of the bacterial expression system for PLAィイD22ィエD2 inhibitors and elucidation of their structure function relationships by means of the site-directed mutagenesis analysisThe PLIα cDNA was subcloned into the expression vector pET-16b and used to transform Esc … More herichia coli. We could obtain the recombinant protein having the inhibition activity as strong as that of the natural PLIα. Alternatively, we have identified a PLIα-like protein (PLIα-LP) from the serum of the non-venomous striated snake and cloned its cDNA. The purified PLIα-LP was found to have no inhibitory activity toward any PLAィイD22sィエD2, although its amino acid sequence showed about 70% homology to that of Chinese mamushi PLIα. We have constructed the recombinant chimeric proteins between the PLIα-LP and Chinese mamushi PLIα and examined their inhibitory activity in order to specify the region responsible for the PLAィイD22ィエD2 inhibition. It was suggested that the CRD structure in the PLIα molecule played the basic structural roles such as the formation of trimeric structure and that the N-terminal and C-terminal regions of PLIα other than CRD were required for the interaction of PLIα to a PLAィイD22ィエD2 molecule.3. Purification of leucine-rich αィイD22ィエD2 glycoprotein (LRG) from the human serum and the examination of its PLAィイD22ィエD2 inhibitory activity.Using the synthesized peptide (LRG1-20) corresponding to the reported N-terminal 20 amino acid sequence of LRG as an antigen, we immunized a rabbit and obtained the anti-LRG1-20 antibody. The ELISA using this antibody was found to be a good tool for detecting LRG during the purification of LRG from human serum. Less