Studies on Regulation of Cellular Function by Calpastatin

钙调蛋白是一种内源性抑制蛋白，特异性作用于细胞内钙依赖蛋白(Calain)。在本研究中，我们扩展了钙调蛋白的结构和功能分析，并研究了这种抑制物在动物细胞中的作用。主要研究结果如下：1.将人钙调蛋白基因定位于染色体5q14-22.2。四个内部重复结构域之间的高度保守区被基因组DNA中的内含子隔开。与外显子1B相对应的27个残基的合成寡肽具有抑制钙蛋白酶的活性，当加入到BIM细胞(神经母细胞瘤细胞系)的培养上清液中时，它能抑制TPA诱导的蛋白激酶C的下调。4.2维核磁共振研究表明，该寡肽在高度保守的序列中形成I型β-转角结构。制备了抗人calastatin的单抗，并建立了一种区分肌肉和红细胞两种calastatin分子的方法，与哺乳动物红细胞相比，鸡红细胞(有核细胞)保留了calastatin的氨基末端结构域，并具有肌型结构。研究发现，在HTLV-I感染的T细胞白血病细胞系中，m-calain和calastatin的mRNA和蛋白水平升高，而在HTLV-I转化成纤维细胞系中不表达。钙调蛋白除具有抑制钙蛋白酶的作用外，还具有L式钙通道激活作用。

Calpastatin is endogenous inhibitor protein acting specifically on intracellular calcium dependent proteinase(calpain). In the present study we extended structure-function analyses of calpastatin and examined roles of this inhibitor in animal cells. The major results obtained were as follows :1. Locus of human calpastatin gene was assigned to chromosome 5ql4-22.2. Highly conserved regions among the four internally repetitive domains were separated by introns in the genomic DNA.3. The 27-residue synthetic oligopeptide corresponding to'the Exon 1B was active as calpain inhibitor, and it suppressed TPA induced down-regulation of protein kinase C when added to culture medium of BIM cells(neuroblastoma cell line).4.2-dimensional NMR studies have revaeled that the oligopeptide forms type I beta-turn structure in the highly conserved sequence.5. Monoclonal antibodies were prepared for human calpastatin, and a method of distinguishing two types(muscle and erythrocyte)of calpastatin molecules were developed.In contrast to mammalian erythrocytes, chicken erythrocytes(nucleated cells)were suggested to retain amino-terminal domains of calpastatin and to have a muscle type structure.6. MRNA and protein levels of m-calpain and calpastatin were found to be elevated in HTLV-I infected T-cell leukemia cell lines, but not in HTLV-I tax-transformed fibroblast cell lines.7. Besides calpain inhibition, calpastatin was suggested to have L-type calcium channel activating function.