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.

蛇毒是包含具有不同生物学活性的蛋白质和肽的混合物，其中许多具有极高的毒性。由于血液中存在中和因子，包括负鼠 Didelphis aurita 在内的多种动物对蛇毒具有抵抗力。一种名为 DM43 的抗出血蛋白已从负鼠血清中分离出来。该蛋白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，对蛇毒亚蛋白质组进行了探索。四种蝰蛇毒液通过含有固定化 DM43 的亲和色谱柱进行层析。结合组分通过一维和二维凝胶电泳进行分析，随后由MALDI-TOF/TOF质谱进行鉴定。采用此方法，我们能够轻松地可视化和比较 Bothrops atrox、Bothrops jararaca、Bothrops insularis 和 Crotalus atrox 蝰蛇毒液中的金属蛋白酶组成。蛋白质水解加工对这一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，主要由丝氨酸蛋白酶、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）以及一些未鉴定斑点组成。尽管在分析的蝰蛇毒液中只有极少数毒素家族得到表征，但检测到的蛋白质点数达到数百个，这表明这些天然分泌物中存在重要的蛋白质变异性。DM43 亲和色谱和相关蛋白质组学技术已被证明是分离和鉴定蛇毒蛋白的有用工具，有助于更深入地理解毒液的异质性。