Department of Science and Technology , Federal University of São Paulo (ICT-UNIFESP) , São José dos Campos , SP 12231-280 , Brazil.
Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS) , Instituto Butantan , São Paulo , SP 05503-000 , Brazil.
J Proteome Res. 2019 Sep 6;18(9):3419-3428. doi: 10.1021/acs.jproteome.9b00325. Epub 2019 Aug 8.
Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.
蛇咬伤是世界上许多地区的一个主要医学关注点，金属蛋白酶在蝰蛇科毒液的病理作用中起着重要作用，包括局部组织损伤、出血和凝血功能障碍。来自毒液的金属蛋白酶 3（HF3）诱导局部出血，并靶向细胞外基质（ECM）成分，包括胶原蛋白和蛋白聚糖以及血浆蛋白。然而，这种金属蛋白酶的完整底物谱尚不清楚。我们报告了定位蛋白质组学研究，鉴定出超过 2000 个 N 末端，包括小鼠胚胎成纤维细胞分泌蛋白中 HF3 切割位点的新 N 末端。末端胺同位素标记的底物（TAILS）分析确定了候选 HF3 底物（包括 ECM 和焦点粘连蛋白以及半胱氨酸蛋白酶抑制剂胱抑素 C）中 P1' 位的亮氨酸偏好。有趣的是，190 个独特的肽与 TopFIND N 末端数据库中注释的切割位点匹配，这表明这些切割发生在易于切割的位点，或者可能是在与 HF3 孵育时被其他激活的蛋白酶（包括 caspase-3 和 -7、组织蛋白酶 D 和 E、颗粒酶 B 以及 MMPs 2 和 9）生成的。使用切割位点特异性的蛋白质组学鉴定（PICS），我们使用源自 THP-1 单核细胞的胰蛋白酶文库作为 HF3 底物，用于鉴定肽中的蛋白酶切割位点和序列偏好。总共检测到 799 个独特的切割位点，与使用 MEF 细胞的天然分泌蛋白底物的 TAILS 分析一致，表明 P1' 位明显偏好亮氨酸。总之，这些结果大大扩展了 HF3 的已知底物降解组，并揭示了潜在的新靶标，这可能为更好地阐明蛇咬伤的复杂病理生理学提供基础。
