Li D, Scherfer C, Korayem A M, Zhao Z, Schmidt O, Theopold U
Department of Applied and Molecular Ecology, Adelaide University, Glen Osmond, SA 5064, Australia.
Insect Biochem Mol Biol. 2002 Aug;32(8):919-28. doi: 10.1016/s0965-1748(02)00030-9.
Here we describe a novel approach to isolate proteins involved in insect hemolymph coagulation. In order to avoid problems in purifying clot proteins after they had been crosslinked, we performed an in vitro coagulation reaction with cell-free hemolymph from the lepidopteran Galleria mellonella and used the resulting complexes to produce a specific antiserum. The antiserum reacted with a subset of hemolymph proteins as well as with granular cells, but not with other hemocyte types of Galleria. Screening expression libraries identified some positive clones, which turned out to code for some previously characterized components of immune cascades, as well as some novel candidates for clotting factors. Known components include members of both the coagulation system and the prophenol-activating cascade, lending support to the idea that both systems work together during the formation of a hemolymph clot. Novel candidates for insect clotting factors include a mucin-like protein, a glutathione-S-transferase, and a distant member of the alpha-crystallin/small heat shock protein family. Using assays measuring the activity of transglutaminase, a key enzyme in clotting reactions in both vertebrates and invertebrates, we found a partial overlap between transglutaminase substrates and proteins recognized by the antiserum against the in vitro-induced clot.
在此，我们描述了一种分离参与昆虫血淋巴凝固的蛋白质的新方法。为了避免在纯化已交联的凝血蛋白时出现问题，我们用鳞翅目昆虫黄粉虫的无细胞血淋巴进行了体外凝血反应，并利用所得复合物制备了特异性抗血清。该抗血清与一部分血淋巴蛋白以及颗粒细胞发生反应，但不与黄粉虫的其他血细胞类型发生反应。筛选表达文库鉴定出了一些阳性克隆，结果发现它们编码一些先前已表征的免疫级联反应成分，以及一些凝血因子的新候选物。已知成分包括凝血系统和前酚激活级联反应的成员，这支持了两个系统在血淋巴凝块形成过程中协同作用的观点。昆虫凝血因子的新候选物包括一种粘蛋白样蛋白、一种谷胱甘肽 - S - 转移酶，以及α - 晶体蛋白/小热休克蛋白家族的一个远亲成员。通过测定转谷氨酰胺酶（脊椎动物和无脊椎动物凝血反应中的关键酶）的活性，我们发现转谷氨酰胺酶底物与针对体外诱导凝块的抗血清所识别的蛋白质之间存在部分重叠。
