| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 88 === Angiogenesis plays an important role in variety of physiological processes such as embryonic development, wound healing, and tissue or organ regeneration. These processes are highly regulated and occur within a short period. Abnormal growth of new blood vessels can lead to the progression of many diseases such as diabetic retinopathy, rheumatoid arthritis and tumor growth. Direct experimental evidence shows that angiogenesis is necessary for tumor growth and metastases.
Angiostatin is an endogenous anti-angiogenic agent and initially isolated from urine and sera of mice bearing Lewis lung carcinoma. It was found to inhibit endothelial cell proliferation and to block basic FGF-elicited angiogenesis in corneal micropocket assay. Microsequence analysis of angiostatin revealed that it had greater than 98% identity to an internal fragment of plasminogen which includes the first four kringle domains.
Many studies have demonstrated that individual and multiple kringle fragments of angiostatin have different inhibitory activity of angiogenesis. Because each kringle contains the triple loop disulfide-linked structures, we want to understand the relationship between the structure and function of angiostatin and its related fragments. We selected three specific cleavage sites in kringle 4 and kringle 5, ie. 543, 555, 560 in kringle 5 and 454, 449, 437 in kringle 4 to generate various fragments of plasminogen. In addition, we also prepared two wild type fragments, ie. K5567(wt) and K4462(wt).
Various fragments were successfully expressed in Pichia Pastoris expression system and purified from the inducing medium by lysine affinity chromatography. In endothelial cell migration assay, we found that the fragments which contained denatured kringle 5 had more potent inhibitory activity than K5wt and K4wt. The same situation happened to the fragments which contained denatured kringle 4, implying that the kringle conformation may shield kringle domain from effectively interacting with endothelial cells.
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