Achê David Collares, Gomes Mário Sérgio R, de Souza Dayane Lorena Naves, Silva Makswell Almeida, Brandeburgo Maria Inês Homsi, Yoneyama Kelly Aparecida Geraldo, Rodrigues Renata Santos, Borges Márcia Helena, Lopes Daiana Silva, Rodrigues Veridiana de Melo
Institute of Genetics and Biochemistry, Federal University of Uberlandia, UFU, 38400-902 Uberlandia, MG, Brazil.
Department of Chemical and Physical, State University of Southwest Bahia (UESB), 45506-210 Jequie, BA, Brazil.
Int J Biol Macromol. 2015 Jan;72:445-53. doi: 10.1016/j.ijbiomac.2014.08.050. Epub 2014 Sep 6.
In the present work, we demonstrate some biochemical and functional properties of a new PI snake venom metalloproteinase (SVMP) isolated from Bothrops pauloensis snake venom (BpMP-II), in addition we evaluated its capacity to inhibit endothelial cell adhesion and in vitro angiogenesis. BpMP-II was purified after a combination of three chromatography steps and showed molecular mass of 23,000 Da determined by MALDI-TOF, an isoelectric point of 6.1 and the sequence of some fragments obtained by MS/MS (MALDI TOF\TOF) presented high structural similarity with other PI-SVMPs. BpMP-II showed proteolytic activity against azocasein, was able to degrade bovine fibrinogen and was inhibited by EDTA, 1.10 phenantroline and β-mercaptoethanol. BpMP-II did not induce local hemorrhage in the dorsal region of mice even at high doses and did not affect plasma creatine kinase (CK) levels when administered intramuscularly into the gastrocnemius muscle of mice. Moreover, this metalloproteinase decreased tEnd cells viability at concentrations higher than 20 μg/mL. With sub-toxic doses this metalloproteinase affected tEnd cell adhesion and was also able to inhibit in vitro angiogenesis. BpMP-II showed very important functional properties suggesting considerable therapeutic potential for this class of protein.
在本研究中，我们展示了从保罗矛头蝮蛇毒中分离出的一种新型PI蛇毒金属蛋白酶（SVMP，即BpMP-II）的一些生化和功能特性，此外，我们还评估了其抑制内皮细胞黏附和体外血管生成的能力。通过三步色谱法组合纯化后，BpMP-II经基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）测定分子量为23,000 Da，等电点为6.1，通过串联质谱（MS/MS，MALDI TOF\TOF）获得的一些片段序列与其他PI-SVMPs具有高度结构相似性。BpMP-II对偶氮酪蛋白具有蛋白水解活性，能够降解牛纤维蛋白原，并受到乙二胺四乙酸（EDTA）、1,10-菲啰啉和β-巯基乙醇的抑制。即使高剂量给药，BpMP-II也不会在小鼠背部区域引起局部出血，肌肉注射到小鼠腓肠肌中时也不会影响血浆肌酸激酶（CK）水平。此外，当浓度高于20 μg/mL时，这种金属蛋白酶会降低tEnd细胞活力。在亚毒性剂量下，这种金属蛋白酶会影响tEnd细胞黏附，并且还能够抑制体外血管生成。BpMP-II显示出非常重要的功能特性，表明这类蛋白质具有相当大的治疗潜力。
