Creutz C E, Tomsig J L, Snyder S L, Gautier M C, Skouri F, Beisson J, Cohen J
Department of Pharmacology, University of Virginia, Charlottesville 22908, USA.
J Biol Chem. 1998 Jan 16;273(3):1393-402. doi: 10.1074/jbc.273.3.1393.
In an attempt to identify proteins that might underlie membrane trafficking processes in ciliates, calcium-dependent, phospholipid-binding proteins were isolated from extracts of Paramecium tetraurelia. The major protein obtained, named copine, had a mass of 55 kDa, bound phosphatidylserine but not phosphatidylcholine at micromolar levels of calcium but not magnesium, and promoted lipid vesicle aggregation. The sequence of a 920-base pair partial cDNA revealed that copine is a novel protein that contains a C2 domain likely to be responsible for its membrane active properties. Paramecium was found to have two closely related copine genes, CPN1 and CPN2. Current sequence data bases indicate the presence of multiple copine homologs in green plants, nematodes, and humans. The full-length sequences reveal that copines consist of two C2 domains at the N terminus followed by a domain similar to the A domain that mediates interactions between integrins and extracellular ligands. A human homolog, copine I, was expressed in bacteria as a fusion protein with glutathione S-transferase. This recombinant protein exhibited calcium-dependent phospholipid binding properties similar to those of Paramecium copine. An antiserum raised against a fragment of human copine I was used to identify chromobindin 17, a secretory vesicle-binding protein, as a copine. This association with secretory vesicles, as well the general ability of copines to bind phospholipid bilayers in a calcium-dependent manner, suggests that these proteins may function in membrane trafficking.
为了鉴定可能是纤毛虫膜运输过程基础的蛋白质，从四膜虫提取物中分离出了钙依赖性磷脂结合蛋白。得到的主要蛋白质名为“共结合蛋白”，质量为55 kDa，在微摩尔浓度的钙而非镁存在时结合磷脂酰丝氨酸而非磷脂酰胆碱，并促进脂质囊泡聚集。一段920个碱基对的部分cDNA序列显示，共结合蛋白是一种新型蛋白质，含有一个可能与其膜活性特性有关的C2结构域。发现四膜虫有两个密切相关的共结合蛋白基因，CPN1和CPN2。目前的序列数据库表明，绿色植物、线虫和人类中存在多种共结合蛋白同源物。全长序列显示，共结合蛋白在N端由两个C2结构域组成，后面跟着一个类似于介导整合素与细胞外配体相互作用的A结构域。一种人类同源物共结合蛋白I在细菌中作为与谷胱甘肽S-转移酶的融合蛋白表达。这种重组蛋白表现出与四膜虫共结合蛋白相似的钙依赖性磷脂结合特性。针对人类共结合蛋白I片段产生的抗血清被用于鉴定一种分泌囊泡结合蛋白chromobindin 17为共结合蛋白。这种与分泌囊泡的关联，以及共结合蛋白以钙依赖性方式结合磷脂双层的一般能力，表明这些蛋白质可能在膜运输中发挥作用。
