纤溶酶原K5抗血管增生活性依赖其完整Kringle结构域

根据K5蛋白(Pro451—Ala541)的结构特征和二硫键分布特点，设计K5的两个缺失突变体K5 mut1(Cys461—Cys540，保留K5 kringle环3个完整二硫键但去除N端和C端多余氨基酸)和K5 mut2 (Cys482—Cys535，打开kringle环，只保留2个二硫键).以野生型人纤溶酶原K5 cDNA为模板，用PCR方法得到编码缺失突变体的DNA片段，定向克隆入pET22b(+)质粒载体，重组体转化进大肠杆菌BL21（DE3），诱导表达，产物经亲和层析和高浓度甘油透析纯化后进行鉴定和生物活性测定.K5 mut1蛋白特异性抑制人视网膜微血管内皮细胞增殖，且活性强度是完整的K5蛋白2倍；K5 mut2对人视网膜微血管内皮细胞无显著抑制作用.结果提示，完整的Kringle结构（包含3个二硫键）是维持人纤溶酶原K5抗血管增生活性的必需结构域，而K5分子中Kringle结构域外的N端和C端氨基酸臂则并非其活性所必需.

Anti-angiogenic Activity of Plasminogen Kringle 5 Depending on Its Intact Kringle Domain

The relationship between the kringle structure and the anti-angiogenic activity of human plasminogen kringle 5 was explored. Two deletion mutants of K5 were designed on the basis of the structure and disulfide bond distribution of K5 (Pro451-Ala541). The first mutant, named K5 mut1(Cys461-Cys540), retained the three disulfide bonds and deleted the amino acid residues of both N- and C- terminals outside kringle domain of K5. The second mutant, named K5 mut2(Cys482-Cys535), kept two disulfide bonds and the intact kringle domain of K5 was opened. The genes of mutants were amplified from the cDNA of plasminogen K5 by polymerase chain reaction(PCR) and cloned into EcoRⅠ/Hind Ⅲ sites of pET22b(+). The recombinants were transformed into E.coli BL21(DE3) and expressed with IPTG induction. The three soluble recombinant proteins were purified by affinity chromatography and glycerol dialysis.Reaching 92.6% purity for K5 intact,96.0% for K5 mutl and 90.1% for K5 mut2, respectively. K5 mut1 specifically inhibited proliferation of primary human retinal capillary endothelial cells (HRCEC) in dose-related manner with more potent activity than intact K5. Whereas K5 mut2 had little or no obvious inhibition effect on HRCEC and pericytes. The results showed that intact kringle domain is the prerequisite for the anti-angiogenic activity of human plasminogen K5 while the amino acids residues of both terminals outside kringle domain are not essential for the activity.