Watakabe A, Kobayashi R, Helfman D M
Cold Spring Harbor Laboratory, NY 11724, USA.
J Cell Sci. 1996 Sep;109 ( Pt 9):2299-310. doi: 10.1242/jcs.109.9.2299.
We have identified and characterized two proteins in rat brain that bind to the neuron-specific tropomyosin isoform, TMBr3. The two proteins were identified by blot overlay assay, in which the proteins immobilized on the membrane were probed by epitope-tagged TMBr3, followed by detection with anti-epitope antibody. We have purified these proteins using a TMBr3 affinity column. Peptide sequencing as well as immunoblotting showed that one of the two proteins is identical to tropomodulin, a tropomyosin-binding protein originally identified in erythrocytes. The cDNA for the other protein was cloned from an adult rat brain cDNA library using degenerate oligonucleotides that we designed based on the peptide sequences. Sequence analysis of the cDNA clone revealed this protein to be a novel isoform of tropomodulin which is the product of a distinct gene, and is herein referred to as N-tropomodulin. Recombinant N-tropomodulin bound to TMBr3 as well as to other low molecular mass tropomyosins (TM5a or TM5), but not to high molecular mass tropomyosins (TM2 or TMBr1). Northern blotting and RNase protection assays as well as immunoblotting showed that N-tropomodulin is expressed predominantly in brain. Furthermore, RNase protection assays revealed no alternatively spliced regions within the coding sequence. Developmentally, N-tropomodulin was detected in rat brain as early as embryonic day 14 and reaches the adult level before birth. Immunofluorescence of primary frontal cortex cell cultures showed that N-tropomodulin is specifically expressed in neurons. The neuron-specific expression of N-tropomodulin strongly suggests specialized roles of this TM-binding protein in neurons.
我们已经在大鼠脑中鉴定并表征了两种与神经元特异性原肌球蛋白异构体TMBr3结合的蛋白质。这两种蛋白质是通过印迹覆盖分析鉴定的，在该分析中，固定在膜上的蛋白质用表位标签化的TMBr3进行探测，然后用抗表位抗体进行检测。我们使用TMBr3亲和柱纯化了这些蛋白质。肽测序以及免疫印迹表明，这两种蛋白质中的一种与原肌球蛋白调节蛋白相同，原肌球蛋白调节蛋白是最初在红细胞中鉴定出的一种原肌球蛋白结合蛋白。另一种蛋白质的cDNA是使用我们根据肽序列设计的简并寡核苷酸从成年大鼠脑cDNA文库中克隆出来的。对该cDNA克隆的序列分析表明，这种蛋白质是原肌球蛋白调节蛋白的一种新型异构体，它是一个不同基因的产物，在此被称为N-原肌球蛋白调节蛋白。重组N-原肌球蛋白调节蛋白与TMBr3以及其他低分子量原肌球蛋白（TM5a或TM5）结合，但不与高分子量原肌球蛋白（TM2或TMBr1）结合。Northern印迹、RNase保护分析以及免疫印迹表明，N-原肌球蛋白调节蛋白主要在脑中表达。此外，RNase保护分析显示编码序列内没有可变剪接区域。在发育过程中，早在胚胎第14天就在大鼠脑中检测到了N-原肌球蛋白调节蛋白，并且在出生前达到成年水平。原代额叶皮质细胞培养物的免疫荧光显示，N-原肌球蛋白调节蛋白在神经元中特异性表达。N-原肌球蛋白调节蛋白的神经元特异性表达强烈表明这种TM结合蛋白在神经元中具有特殊作用。
