Research of the molecular mechanism in the wound-healing system of marine shellfish

扇贝血细胞含有谷氨酰胺转酶（TGase），其电泳与内收肌中的TGase不同。将血细胞匀浆与MDC （monodansylcadaverine）在10℃下孵育，MDC被掺入血细胞的230 kDa和100 kDa蛋白中。100 kDa蛋白仅在上清液中检测到，230 kDa蛋白不溶，210 kDa蛋白在两个组分中均检测到。在缺乏MDC的情况下，230 kDa、210 kDa和100 kDa蛋白通过内源性转谷氨酰胺酶交联。230 kDa的蛋白质在5分钟内就能快速交联并形成巨大的聚合物。该100 kDa蛋白的BrCN-cleaved片段的氨基酸序列与其他tgase的保守序列具有明显的同源性，表明该组分就是该酶本身。虽然对230 kDa蛋白的氨基末端区域和6个brcn切割片段进行了测序，但未检测到与已知蛋白的显著同源性；230 kDa的蛋白质被认为是一种新的蛋白质。210 kDa蛋白仅在brcn片段的氨基末端8个残基上测序。本研究首次使用微通道阵列流量分析仪（MC-FAN）对贝类血细胞的聚集进行分析。钙离子的存在刺激了这种聚集，EGTA抑制了这种聚集，但个体差异较大。咸淡水蛤含有至少3种含有海洋和淡水型酶的TGase同工酶，表明TGase可能在细胞外而不是细胞内具有活性。这些结果表明，如果扇贝组织受到损伤，血细胞转谷氨酰胺酶可能被激活，首先将不溶性的血细胞230 kDa蛋白交联，然后将210 kDa蛋白交联，形成血细胞片相互交联的交联蛋白基质，从而止血。

Scallop hemocytes contain a transglutaminase (TGase) that is electrophoretically different from the TGase in the adductor muscle. When hemocyte homogenate was incubated with monodansylcadaverine (MDC) at 10℃, MDC was incorporated into the 230 kDa and 100 kDa proteins of the hemocytes. The 100 kDa protein was only detected in the supernatant, the 230 kDa protein was insoluble, and the 210 kDa protein was detected in both fractions. In the absence of MDC, the 230 kDa, 210 kDa, and 100 kDa proteins were cross-linked by endogenous transglutaminase. The 230 kDa protein was most quickly cross-linked and formed huge polymers within five minutes. The amino acid sequence of a BrCN-cleaved fragment derived from the 100 kDa protein showed the obvious homology to the conservative sequences of the other TGases, suggesting this component was the enzyme itself. Although the amino terminal region and six BrCN-cleaved fragments of the 230 kDa protein were sequenced, no significant homology to the known proteins was detected ; the 230 kDa protein was supposed to be a novel protein. The 210 kDa protein was only sequenced at the amino terminal eight residues of a BrCN-fragment. In this research, micro channel array flow analyzer (MC-FAN) was used for the first time to analyze the aggregation of shellfish hemocytes. The aggregation was stimulated in the presence of calcium ion and suppressed by EGTA, though an individual difference was comparatively large. Brackish water clam contained at least three TGase isozymes containing marine and fresh water-type enzymes, suggesting TGase could be active extracellularly rather than intracellularly. These results suggest that if scallop tissues are injured, hemocyte transglutaminase may be activated, initially cross-linking the insoluble hemocyte 230 kDa protein, followed by the 210 kDa proteins, to form a cross-linked protein matrix with inter cross-linking of hemocyte sheets, to stop the bleeding.