Crotalid Snake Venom Subproteomes Unraveled by the Antiophidic Protein DM43

Snake venoms are mixtures of proteins and peptides with different biological activities, many of which are very toxic. Several animals, including the opossum Didelphis aurita, are resistant to snake venoms due to the presence of neutralizing factors in their blood. An antihemorrhagic protein named DM43 was isolated from opossum serum. It inhibits snake venom metalloproteinases through noncovalent complex formation with these enzymes. In this study, we have used DM43 and proteomic techniques to explore snake venom subproteomes. Four crotalid venoms were chromatographed through an affinity column containing immobilized DM43. Bound fractions were analyzed by one- and two-dimensional gel electrophoresis, followed by identification by MALDI-TOF/TOF mass spectrometry. With this approach, we could easily visualize and compare the metalloproteinase compositions of Bothrops atrox, Bothrops jararaca, Bothrops insularis, and Crotalus atrox snake venoms. The important contribution of proteolytic processing to the complexity of this particular subproteome was demonstrated. Fractions not bound to DM43 column were similarly analyzed and were composed mainly of serine proteinases, C-type lectins, C-type lectin-like proteins, l-amino acid oxidases, nerve growth factor, cysteine-rich secretory protein, a few metalloproteinases (and their fragments), and some unidentified spots. Although very few toxin families were represented in the crotalid venoms analyzed, the number of protein spots detected was in the hundreds, indicating an important protein variability in these natural secretions. DM43 affinity chromatography and associated proteomic techniques proved to be useful tools to separate and identify proteins from snake venoms, contributing to a better comprehension of venom heterogeneity.

蛇毒是具有不同生物学活性的蛋白质和肽类的混合物，其中许多具有极高的毒性。由于血液中存在中和因子，多种动物，包括负鼠Didelphis aurita，对蛇毒具有抵抗力。一种名为DM43的抗出血蛋白从负鼠血清中分离出来。它通过与非共价复合物形成抑制蛇毒金属蛋白酶。在本研究中，我们利用DM43和蛋白质组学技术探讨了蛇毒亚蛋白质组。四种蝰蛇科蛇毒通过包含固定化DM43的亲和层析柱进行层析。结合的组分通过一维和二维凝胶电泳进行分析，随后通过MALDI-TOF/TOF质谱鉴定。采用此方法，我们可以轻松地可视化和比较Bothrops atrox、Bothrops jararaca、Bothrops insularis和Crotalus atrox蛇毒中的金属蛋白酶组成。蛋白质水解处理对于该特定亚蛋白质组复杂性的重要贡献得到了证实。未与DM43柱结合的组分也进行了类似分析，主要组成包括丝氨酸蛋白酶、C型凝集素、C型凝集素样蛋白、L-氨基酸氧化酶、神经生长因子、富含半胱氨酸的分泌蛋白、少量金属蛋白酶（及其片段）以及一些未知的斑点。尽管在分析的蝰蛇科蛇毒中仅代表极少数毒素家族，但检测到的蛋白质点数达数百个，表明这些天然分泌物中蛋白质的变异性非常重要。DM43亲和层析及其相关的蛋白质组学技术被证明是分离和鉴定蛇毒蛋白的有用工具，有助于更好地理解蛇毒的异质性。