Briggs K K, Silvers A J, Johansen K M, Johansen J
Department of Zoology and Genetics, Iowa State University, Ames 50011.
J Cell Biol. 1995 Jun;129(5):1355-62. doi: 10.1083/jcb.129.5.1355.
The mAb lan3-6 recognizes a cytosolic antigen which is selectively expressed in the growth cones and axons of a small subset of peripheral sensory neurons fasciculating in a single tract common to all hirudinid leeches. We have used this antibody to clone a novel EF-hand calcium-binding protein, calsensin, by screening an expression vector library. A full-length clone of 1.1 kb identified by the antibody was isolated and sequenced. In situ hybridizations with calsensin probes and antibody staining using new polyclonal antisera generated against calsensin sequence demonstrate that calsensin indeed corresponds to the lan3-6 antigen. Calsensin consists of 83 residues with a calculated molecular mass of 9.1 kD that contains two helix-loop-helix domains. The calcium-binding domains are likely to be functional in vivo since a fusion protein derived from the calsensin clone binds 45Ca2+ in vitro. Immunoaffinity purification experiments with the lan3-6 antibody shows that a large 200,000 M(r) protein selectively copurifies with calsensin in two different leech species. These results suggest that calsensin may be functioning as a trigger protein which interacts with the larger protein. These data are consistent with the hypothesis that calsensin may mediate calcium-dependent signal transduction events in the growth cones and axons of this small group of sensory neurons which fasciculate in a single axon tract.
单克隆抗体lan3 - 6识别一种胞质抗原，该抗原选择性地表达于所有蛭类水蛭共有的单一神经束中聚集的一小部分外周感觉神经元的生长锥和轴突中。我们利用这种抗体通过筛选表达载体文库克隆了一种新型的EF手型钙结合蛋白——钙传感蛋白。通过该抗体鉴定出一个1.1 kb的全长克隆，并进行了测序。用钙传感蛋白探针进行原位杂交以及使用针对钙传感蛋白序列产生的新的多克隆抗血清进行抗体染色，结果表明钙传感蛋白确实对应于lan3 - 6抗原。钙传感蛋白由83个氨基酸残基组成，计算分子量为9.1 kD，包含两个螺旋 - 环 - 螺旋结构域。由于源自钙传感蛋白克隆的融合蛋白在体外能结合45Ca2 +，所以钙结合结构域在体内可能具有功能。用lan3 - 6抗体进行的免疫亲和纯化实验表明，在两种不同的水蛭物种中，一种分子量为200,000 M(r)的大蛋白能与钙传感蛋白选择性地共同纯化。这些结果表明钙传感蛋白可能作为一种触发蛋白，与较大的蛋白相互作用。这些数据与以下假设一致：钙传感蛋白可能在这一小群在单一轴突束中聚集的感觉神经元的生长锥和轴突中介导钙依赖性信号转导事件。
