Mueller S G, Paterson A J, Kudlow J E
Department of Clinical Biochemistry, University of Toronto, Banting and Best Diabetes Centre, Toronto General Hospital, Ontario, Canada.
Mol Cell Biol. 1990 Sep;10(9):4596-602. doi: 10.1128/mcb.10.9.4596-4602.1990.
Analysis of the transforming growth factor alpha (TGF alpha) cDNA predicts that the mature TGF alpha polypeptide is cleaved from the extracellular domain of its precursor, which is an integral membrane protein. Furthermore, the cleavage sites for the release of this mitogen are compatible with the participation of an elastaselike protease. We have immunohistochemically localized TGF alpha to the vascular smooth muscle cells in the arterioles. To investigate whether polymorphonuclear (PMN) leukocytic elastase, a blood-borne protease, could process the cell surface TGF alpha, NR6 cells were transfected with the rat TGF alpha cDNA. The cDNA encoded the entire open reading frame, and its expression was under the control of the mouse metallothionein I promoter. A cloned transfectant, termed 1B2, synthesized the TGF alpha precursor in a zinc-inducible manner, and the precursor was localized to the cell surface. Western blot (immunoblot) analysis indicated that treatment of the zinc-induced 1B2 cells with either PMN leukocytic or pancreatic elastase resulted in the release of the mature TGF alpha polypeptide. The released TGF alpha was bioactive, as it was capable of both competing with epidermal growth factor for binding to its receptor and stimulating [3H]thymidine incorporation in the mitogenic assay. Formaldehyde fixation of the 1B2 cells eliminated basal release of TGF alpha but allowed normal processing by both PMN leukocytic and pancreatic elastase to occur. However, human cathepsin G, bovine pancreatic alpha 1-chymotrypsin, collagenase, trypsin, subtilisin, and plasmin failed to release any detectable fragments of the TGF alpha precursor from the fixed cells. The location of TGF alpha in the arterioles and ability of PMN leukocytic elastase to process the membrane-bound TGF alpha precursor suggests a novel role for this elastase at the wound site.
对转化生长因子α（TGFα）cDNA的分析预测，成熟的TGFα多肽是从其前体的细胞外结构域切割而来，该前体是一种整合膜蛋白。此外，这种促有丝分裂原释放的切割位点与一种类弹性蛋白酶的参与是相符的。我们通过免疫组织化学方法将TGFα定位到小动脉的血管平滑肌细胞中。为了研究血液中的蛋白酶——多形核（PMN）白细胞弹性蛋白酶是否能够加工细胞表面的TGFα，用大鼠TGFα cDNA转染NR6细胞。该cDNA编码整个开放阅读框，其表达受小鼠金属硫蛋白I启动子的控制。一个克隆的转染细胞系，称为1B2，以锌诱导的方式合成TGFα前体，并且该前体定位于细胞表面。蛋白质印迹（免疫印迹）分析表明，用PMN白细胞弹性蛋白酶或胰弹性蛋白酶处理锌诱导的1B2细胞会导致成熟TGFα多肽的释放。释放的TGFα具有生物活性，因为它既能与表皮生长因子竞争结合其受体，又能在促有丝分裂试验中刺激[3H]胸腺嘧啶核苷掺入。1B2细胞的甲醛固定消除了TGFα的基础释放，但允许PMN白细胞弹性蛋白酶和胰弹性蛋白酶正常加工。然而，人组织蛋白酶G、牛胰α1-糜蛋白酶、胶原酶、胰蛋白酶、枯草杆菌蛋白酶和纤溶酶未能从固定细胞中释放出任何可检测到的TGFα前体片段。TGFα在小动脉中的定位以及PMN白细胞弹性蛋白酶加工膜结合TGFα前体的能力表明这种弹性蛋白酶在伤口部位具有新的作用。
