Jia L G, Wang X M, Shannon J D, Bjarnason J B, Fox J W
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
J Biol Chem. 1997 May 16;272(20):13094-102. doi: 10.1074/jbc.272.20.13094.
Snake venom hemorrhagic metalloproteinase toxins that have metalloproteinase, disintegrin-like and cysteine-rich domains are significantly more potent than toxins with only a metalloproteinase domain. The disintegrin-like domains of these toxins differ from the disintegrin peptides found in crotalid and viperid venoms by the nature of their different disulfide bond structure and, in lieu of the disintegrins' signature Arg-Gly-Asp (RGD) integrin binding sequence, there is an XXCD disulfide-bonded cysteinyl sequence in that region. Due to these apparent differences, the contribution to the overall function of the hemorrhagic metalloproteinases by the disintegrin-like domain has been unknown. In this investigation we have expressed in insect cells the disintegrin-like/cysteine-rich (DC) domains of the Crotalus atrox hemorrhagic metalloproteinase atrolysin A and demonstrated that the recombinant protein (A/DC) can inhibit collagen- and ADP-stimulated platelet aggregation. Using synthetic peptides, we have evidence that the region of the disintegrin-like domain that is positionally analogous to the RGD loop of the disintegrins is the site responsible for inhibition of platelet aggregation. For these synthetic peptides to have significant inhibitory activity, the -RSECD- cysteinyl residue must be constrained by participation in a disulfide bond with another cysteinyl residue. The two acidic amino acids adjacent to the middle cysteinyl residue in these peptides are also important for biological activity. These studies emphasize a functional role for the disintegrin-like domain in toxins and suggest structural possibilities for the design of antagonists of platelet aggregation.
具有金属蛋白酶、解整合素样和富含半胱氨酸结构域的蛇毒出血性金属蛋白酶毒素，其效力显著高于仅具有金属蛋白酶结构域的毒素。这些毒素的解整合素样结构域与蝰蛇科和蝰蛇科毒液中的解整合素肽不同，在于它们不同的二硫键结构性质，并且在该区域中，代替解整合素标志性的精氨酸-甘氨酸-天冬氨酸（RGD）整合素结合序列，存在一个XXCD二硫键连接的半胱氨酸序列。由于这些明显的差异，解整合素样结构域对出血性金属蛋白酶整体功能的贡献一直未知。在本研究中，我们在昆虫细胞中表达了锯鳞蝰蛇出血性金属蛋白酶阿曲溶素A 的解整合素样/富含半胱氨酸（DC）结构域，并证明重组蛋白（A/DC）可抑制胶原蛋白和ADP 刺激的血小板聚集。使用合成肽，我们有证据表明，解整合素样结构域中与解整合素的RGD 环位置相似的区域是负责抑制血小板聚集的位点。为使这些合成肽具有显著的抑制活性，-RSECD-半胱氨酸残基必须通过与另一个半胱氨酸残基形成二硫键来受限。这些肽中与中间半胱氨酸残基相邻的两个酸性氨基酸对生物活性也很重要。这些研究强调了解整合素样结构域在毒素中的功能作用，并为血小板聚集拮抗剂的设计提出了结构可能性。
