Laboratório de Hemostase e Venenos, Instituto de Bioquímica Médica Leopoldo de Meis, And Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (Inbeb) - Universidade Federal do Rio de Janeiro -UFRJ, RJ, Brazil.
Laboratório de Farmacologia Celular e Molecular, IBRAG, Universidade do Estado do Rio de Janeiro - UERJ, RJ, Brazil.
Toxicon. 2022 Oct 15;217:87-95. doi: 10.1016/j.toxicon.2022.08.010. Epub 2022 Aug 15.
Integrins are transmembrane heterodimeric glycoproteins, present in most cell types that act as mechanoreceptors, connecting extracellular matrix proteins to the cytoskeleton of the cell, mediating several physiological and pathological processes. The disintegrins are peptides capable of modulating the activity of integrins, such as αIIbβ3, responsible for the platelet aggregation and αvβ3, related to angiogenesis. The aim of this study was to produce the recombinant disintegrin jarastatin (rJast), to evaluate its secondary structure and biological activity. rJast was expressed in the yeast Komagataella phaffii (earlier Pichia pastoris) purified using molecular exclusion chromatography and the internal sequence and molecular mass were confirmed by mass spectrometry. The yield was approximately 40 mg/L of culture. rJast inhibited platelet aggregation induced by 2-4 μM ADP, 10 nM thrombin, and 1 μg/mL collagen (IC of 244.8 nM, 166.3 nM and 223.5 nM, respectively). It also blocked the adhesion of platelets to collagen under continuous flow in approximately 60% when used 1 μM. We also evaluated the effect of rJast on HMEC-1 cells. rJast significantly inhibited the adhesion of these cells to vitronectin, as well as cell migration (IC 1.77 μM) without changing the viability. Conclusions: rJast was successfully expressed with activity in human platelets aggregation identical to the native molecule. Also, rJast inhibits adhesion and migration of endothelial cells. Thus, being relevant for the development of anti-thrombotic and anti-angiogenic drugs.
整合素是一种跨膜异二聚体糖蛋白，存在于大多数细胞类型中，作为机械感受器，将细胞外基质蛋白与细胞的细胞骨架连接起来，介导多种生理和病理过程。解整合素是能够调节整合素活性的肽，如负责血小板聚集的αIIbβ3和与血管生成有关的αvβ3。本研究的目的是生产重组解整合素 jarastatin（rJast），并评估其二级结构和生物活性。rJast 在酿酒酵母 Komagataella phaffii（以前称为 Pichia pastoris）中表达，使用分子筛层析法纯化，通过质谱法确认内部序列和分子量。产量约为每升培养物 40 毫克。rJast 抑制由 2-4 μM ADP、10 nM 凝血酶和 1 μg/mL 胶原蛋白诱导的血小板聚集（IC 分别为 244.8、166.3 和 223.5 nM）。当使用 1 μM 时，它还可以阻止血小板在连续流动下与胶原蛋白的黏附，抑制率约为 60%。我们还评估了 rJast 对 HMEC-1 细胞的影响。rJast 显著抑制这些细胞与纤连蛋白的黏附以及细胞迁移（IC 为 1.77 μM），而不改变细胞活力。结论：rJast 成功表达，其在人血小板聚集中的活性与天然分子相同。此外，rJast 还抑制内皮细胞的黏附和迁移。因此，它与抗血栓和抗血管生成药物的开发相关。
