Cathepsin L Colocalizes with Chromogranin A in Chromaffin Vesicles to Generate Active Peptides
Cathepsin L Colocalizes with Chromogranin A in Chromaffin Vesicles to Generate Active Peptides
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DOI：
10.1210/en.2008-1613
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发表时间：
2009-08-01
期刊：
影响因子：
4.8
通讯作者：
Mahata, Sushil K.
中科院分区：
文献类型：
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作者：
Biswas, Nilima;Rodriguez-Flores, Juan L.;Mahata, Sushil K.
嗜铬粒蛋白A（CgA）是嗜铬粒中的主要可溶性蛋白质，其经蛋白水解加工产生生物活性肽，包括儿茶酚胺释放抑制肽catestatin。在这里，我们试图确定是否半胱氨酸蛋白酶组织蛋白酶L（CTSL），一种新的酶的神经肽的蛋白水解加工，像成熟的丝氨酸蛋白酶[激素原转化酶（PC）1/3或PC 2]产生catestatin通过蛋白水解加工的CgA。我们发现，内源性CTSL与CGA共定位在原代大鼠嗜铬细胞的分泌囊泡。用编码CTSL的表达质粒转染PC 12细胞，引导CTSL向分泌囊泡表达。去卷积荧光显微镜表明更大的共定位CTSL与CGA比溶酶体标志物LGP 110。CTSL在PC 12细胞中的过表达引起全长CgA的切割。CTSL还在体外以时间和剂量依赖性方式切割CgA，并且通过E64（硫醇试剂）抑制记录了该过程的特异性。对CTSL消化的重组CgA的质谱鉴定了对应于CgA（360-373）的catestatin区肽。由CgA的CTSL裂解产生的肽池抑制尼古丁诱导的PC 12细胞的儿茶酚胺分泌。CTSL加工在catestatin区域减少了天然存在的catestatin变体，特别是Pro370 Leu和Gly 364 Ser。在CTSL产生的肽中，一个子集与体内catestatin区域中发现的肽相匹配。这些研究结果表明，CGA可以是一个底物的半胱氨酸蛋白酶CTSL在体外和切拉内，和它们的共定位在切拉内的嗜铬颗粒表明在体内的酶/底物的关系的可能性。（内分泌学150：3547-3557，2009）
Chromogranin A (CgA), the major soluble protein in chromaffin granules, is proteolytically processed to generate biologically active peptides including the catecholamine release inhibitory peptide catestatin. Here we sought to determine whether cysteine protease cathepsin L (CTSL), a novel enzyme for proteolytic processing of neuropeptides, acts like the well-established serine proteases [prohormone convertase (PC) 1/3 or PC2] to generate catestatin by proteolytic processing of CgA. We found that endogenous CTSL colocalizes with CgA in the secretory vesicles of primary rat chromaffin cells. Transfection of PC12 cells with an expression plasmid encoding CTSL directed expression of CTSL toward secretory vesicles. Deconvolution fluorescence microscopy suggested greater colocalization of CTSL with CgA than the lysosomal marker LGP110. The overexpression of CTSL in PC12 cells caused cleavage of full-length CgA. CTSL also cleaved CgA in vitro, in time-and dose-dependent fashion, and specificity of the process was documented through E64(thiol reagent) inhibition. Mass spectrometry on CTSL-digested recombinant CgA identified a catestatin-region peptide, corresponding to CgA(360-373). The pool of peptides generated from the CTSL cleavage of CgA inhibited nicotine-induced catecholamine secretion from PC12 cells. CTSL processing in the catestatin region was diminished by naturally occurring catestatin variants, especially Pro370Leu and Gly364Ser. Among the CTSL-generated peptides, a subset matched those found in the catestatin region in vivo. These findings indicate that CgA can be a substrate for the cysteine protease CTSL both in vitro and in cella, and their colocalization within chromaffin granules in cella suggests the likelihood of an enzyme/substrate relationship in vivo. (Endocrinology 150: 3547-3557, 2009)