Hirayoshi K, Kudo H, Takechi H, Nakai A, Iwamatsu A, Yamada K M, Nagata K
Department of Cell Biology, Kyoto University, Japan.
Mol Cell Biol. 1991 Aug;11(8):4036-44. doi: 10.1128/mcb.11.8.4036-4044.1991.
We report the isolation and characterization of a cDNA clone encoding HSP47, a transformation-sensitive heat shock protein that binds to collagen. A cDNA library was prepared from total RNA isolated from heat-shocked chicken embryo fibroblasts and screened by using oligonucleotide mixtures prepared on the basis of the N-terminal amino acid sequence of biochemically purified HSP47. The cDNA insert contained 3,278 bp, which encoded a 15-amino-acid signal peptide and a mature protein coding region consisting of 390 amino acid residues; it also included part of the 5' noncoding region and a long 3' noncoding region. The deduced amino acid sequence revealed an RDEL sequence at the C terminus, which is a variant of the KDEL retention signal for retention of proteins in the endoplasmic reticulum. Northern (RNA) blot analyses and nuclear run-on assays established that the induction of HSP47 by heat shock and its suppression after transformation of chicken embryo fibroblasts by Rous sarcoma virus are regulated at the transcriptional level. A homology search revealed that this protein belongs to the serpin family, the superfamily of plasma serine protease inhibitors. Although structurally homologous to the serpins, HSP47 lacks the active site thought to be essential for the inhibition of proteases and does not appear to bind to intracellular proteases. HSP47 is the first heat shock protein found to be a member of the serpin superfamily. Conversely, it is the first serpin family member that is not secreted from cells, which could be explained by acquisition of the RDEL retention signal during evolution.
我们报道了编码HSP47的cDNA克隆的分离和特性分析，HSP47是一种与胶原蛋白结合的转化敏感型热休克蛋白。从热休克鸡胚成纤维细胞分离的总RNA制备cDNA文库，并使用基于生化纯化的HSP47的N端氨基酸序列制备的寡核苷酸混合物进行筛选。cDNA插入片段包含3278bp，编码一个15个氨基酸的信号肽和一个由390个氨基酸残基组成的成熟蛋白编码区；它还包括部分5'非编码区和一个长的3'非编码区。推导的氨基酸序列在C端显示一个RDEL序列，这是内质网中蛋白质保留的KDEL保留信号的变体。Northern（RNA）印迹分析和核转录分析表明，热休克诱导HSP47以及劳氏肉瘤病毒转化鸡胚成纤维细胞后对其的抑制是在转录水平上调控的。同源性搜索显示该蛋白属于丝氨酸蛋白酶抑制剂家族，即血浆丝氨酸蛋白酶抑制剂超家族。尽管HSP47在结构上与丝氨酸蛋白酶抑制剂同源，但它缺乏被认为对蛋白酶抑制至关重要的活性位点，并且似乎不与细胞内蛋白酶结合。HSP47是发现的第一个属于丝氨酸蛋白酶抑制剂超家族的热休克蛋白。相反，它是第一个不分泌到细胞外的丝氨酸蛋白酶抑制剂家族成员，这可以通过进化过程中获得RDEL保留信号来解释。
