Diemer V, Hoyle M, Baglioni C, Millis A J
Department of Biological Sciences, University at Albany, State University of New York 12222.
J Biol Chem. 1992 Mar 15;267(8):5257-64.
Porcine smooth muscle cells (SMC) grown to a high density monolayer culture undergo a morphological transition in which the cells draw away from the substrate and form multicellular nodules. The cells within the nodule resemble SMC in the aortic media and in some atherosclerotic plaques. The process of nodule formation is associated with the enhanced production of a secreted 38-kDa glycoprotein. To characterize the 38-kDa protein and its expression, a cDNA clone (pc38K) was isolated by immunological screening of an expression library. The 1646-base pair cDNA contains a single open reading frame encoding 446 amino acids. This sequence shows 72% homology with the human complement cytolysis inhibitor (CLI), also called serum protein-40,40, and 68% identity with rat sulfated glycoprotein-2. Based on this homology, we refer to the protein encoded by pc38K as CLI. This polypeptide includes a potential signal sequence, seven glycosylation sites and 10 cysteines in two clusters of five each. Southern blot analysis reveals that a single copy gene encoding CLI is present in mammals and chicken. In Northern blot analysis of SMC RNA, pc38K hybridizes to a mRNA of about 1.9 kilobases that is preferentially expressed in nodular SMC. The steady state level of this mRNA increases as the cultures begin to form multilayered regions. High levels of the mRNA persist after the cells are trypsin-dissociated. Culture medium conditioned by nodular SMC also induces an increase of CLI mRNA. Analysis of RNA extracted from porcine tissues show the highest levels of CLI mRNA in brain and liver; lower levels are detected in other tissues, including the aorta. Possible functions for the CLI are discussed.
生长至高密度单层培养的猪平滑肌细胞（SMC）会经历一种形态转变，即细胞脱离底物并形成多细胞结节。结节内的细胞类似于主动脉中膜和一些动脉粥样硬化斑块中的SMC。结节形成过程与一种分泌型38 kDa糖蛋白的产生增加有关。为了表征这种38 kDa蛋白及其表达情况，通过对一个表达文库进行免疫筛选分离出了一个cDNA克隆（pc38K）。这个1646个碱基对的cDNA包含一个编码446个氨基酸的单一开放阅读框。该序列与人类补体溶细胞抑制因子（CLI，也称为血清蛋白-40）具有72%的同源性，与大鼠硫酸化糖蛋白-2具有68%的同一性。基于这种同源性，我们将pc38K编码的蛋白称为CLI。该多肽包括一个潜在的信号序列、七个糖基化位点以及两个每组五个的10个半胱氨酸簇。Southern印迹分析表明，编码CLI的单拷贝基因存在于哺乳动物和鸡中。在对SMC RNA进行Northern印迹分析时，pc38K与一个约1.9千碱基的mRNA杂交，该mRNA在结节状SMC中优先表达。随着培养物开始形成多层区域，这种mRNA的稳态水平会升高。细胞经胰蛋白酶解离后，该mRNA仍保持高水平。结节状SMC条件培养基也会诱导CLI mRNA增加。对从猪组织中提取的RNA的分析表明，CLI mRNA在脑和肝中的水平最高；在包括主动脉在内的其他组织中检测到的水平较低。文中讨论了CLI可能的功能。
