sturctural and functional studies on angiostatin

• Main Authors: Chen, Wen-Yu, 陳汶妤
• Other Authors: Lin, Ming-Te
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/18006184663633615370

碩士 === 國立成功大學 === 生物化學研究所 === 89 === Angiogenesis plays a fundamental role in many physiological and pathological processes, such as the tissue or organ regeneration, embryonic development, tumor growth and metastastases. Angiostatin, a 38kD protein, is an inhibitor of angiogensis. It is also an internal fragment of plasminogen comprised of the first four triple disulfide-linked kringle structure. Since the first isolation of the angiostatin protein by Dr. Folkman’s group, numerous researches have been conducted on this topic. Many studies have demonstrated that angiostatin containing K1-4 fragment isn’t the most powerful inhibitor of angiogenesis. In endothelial cell migration assay, the reduced kringle 5 domain fragment achieves the highest inhibition; the kringle 1 domain or kringle 1-3 fragment has the lowest activity of inhibition. In endothelial cell proliferation assay, the kringle 5 domain alone demonstrates highest level of inhibition; the kringle 4 domain has lower level of inhibition. It has also been shown that when disulfide bond of plasmin kringle 5 is reduced, the more potent angiostatin can be generated by enzyme catalization. Therefore multiple kringle fragments of angiostatin have different inhibitory effect on the angiogenesis. In this thesis, we want to understand the effect of the different levels of disulfide bridge disruption on the anti-angiogenic activity. We selected three specific cleavage sites in kringle 3, kringle 4 and kringle 5, i.e. k3₃₁₆, k3₃₂₈, k3₃₃₃in kringle 3; k4₄₁₈, k4₄₃₀ and k4₄₃₅ in kringle 4; and, k5₅₂₄, k5₅₃₆ and k5₅₄₁ in kringle 5 to generate various fragments of plasminogen. In addition, we also prepare three fragments with intact kringle, i.e. K3_354(wt), K4_443(wt)and K5_548(wt). Currently, we have successfully expressed twelve different length fragments of angiostatin using the Pichia pastoris expression system and tested their anti-angiogenic activity by Calf Pulmonary Artery Endothelial Cell (CPAE). In endothelial cell migration assay, the plasminogen fragments with reduced disulfide bridge had higher inhibitory effect then that of fragments with intact kringle structure, suggesting that the breakage of disulfide linkage in kringle is the prerequisite for the generation of potent angiostatin. In the proliferation assay, we found that K4₄₁₈ and K4₄₃₀ had higher inhibitory activity then other fragments suggesting the potential role of K4. In HPLC analysis, one or two peaks (RT19 and RT22) were identified for each fragment.