Identification And Gene Cloning Of The Proteinases That Convert Plasminogen To Angiostatin

Angiostatin, an endothelial cell-specific inhibitor of angiogenesis, plays an important role in inhibiting the growth and metastasis of tumor by blocking tumor angiogenesis. Further understanding the mechanism of the generation of angiostatin is necessary. Angiostatin is an internal proteolytic fragment of plasminogen. A number of proteinases have been found to cleave plasminogen to form angiostatin. Further investigation on these proteinases may help us insight into the regulatory mechanism of angiogenesis, meanwhile, it might provide a novel therapeutic strategy for anti-angiogenesis cancer treatment.In this study the proteolysis mechanism of angiostatin generation was explored through the identification of two proteinase derived from a bacterium and a tumor cell line respectively.A serine proteinase secreted by a strain of aeromonas hydrophila (A.h), which specifically converted plasminogen to angiostatin, was first identified and purified by Dr. Fuyang Li. Through protein mass-spectrum analysis andamino acid sequencing, the amino acid sequences of eight peptides of the protein were obtained. In this work, according to these amino acid sequences and the results of their homology analysis with other proteins, the probe was devised. Then the genomic library of A.h was constructed and was further screened by hybridization method. The DNA sequence of a fragment of the interested gene was obtained. Together with genomic walking method, the DNA sequence with an open reading frame was gained. The bioinformatics analysis of the amino acid sequence deduced from the open reading frame has been performed. The analysis showed that this sequence contained all the amino acid sequences of eight peptides of the interested proteinase. The analysis also indicated a signal peptide at the amino-terminal of the protein, which suggested it was a secretory protein. The two putative conserved domains, Peptidase_S8 domain and P_proprotein domain, in the protein, suggested that Esp30 belonged to a family of subtilisin-like proprotein convertase (SPC). Based on the above analysis, the novel gene was named esp30 (extracellular serine proteinase 30) . On the basis of the structure of SPC, Esp30 was predicated to be a precursor of a serine proteinase. To investigate its function, the recombinant expression of esp30 was performed in E.coli and in mammalian cells and the antibody against Esp30 was prepared. But till now, the enzymatic activity of the recombinant protein has not been detected. How to express biologically active Esp30 recombinant protein is to be investigated.In addition to the work on the proteolytic mechanism of bacterium-derived angiostatin, we explored the production mechanism of cancer-cell-mediated angiostatin generation. The specific cleavage of plasminogen when incubated with the culture supernatant of a lung cancer...