Hosokawa N, Hohenadl C, Satoh M, Kühn K, Nagata K
Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8397, Japan.
J Biochem. 1998 Sep;124(3):654-62. doi: 10.1093/oxfordjournals.jbchem.a022162.
HSP47 is a stress protein (heat shock protein) which resides in the endoplasmic reticulum, and is postulated to function as a collagen-specific molecular chaperone. To elucidate the role of HSP47 in procollagen biosynthesis, we have established human embryonic kidney 293 cell lines, which were stably transfected with alpha1(III) procollagen chains with or without HSP47. 293 cells do not produce any extracellular matrix proteins including collagens, and the level of HSP47 expression is almost undetectable in this cell line. Recombinant type III procollagens in 293 cells form trypsin-resistant homotrimers, which are secreted into the medium as trimers in the presence or absence of recombinant mouse HSP47. The secretion of procollagen III was delayed in 293 cells stably transfected with proalpha1(III) collagen chains [293+proalpha1(III) cells] in comparison with human rhabdomyosarcoma cell line RD, which normally produces type III procollagens. In this study, we examined the rate of type III procollagen secretion in detail. In cells cotransfected with mouse HSP47 [293+proalpha1(III)+HSP47 cells], the rate of type III procollagen secretion was slower than in 293+proalpha1(III) cells. The binding of HSP47 with proalpha1(III) collagen chains was confirmed by immunoprecipitation using the chemical cross-linker, DSP. The electrophoretic mobility of proalpha1(III) collagen chains in 293+proalpha1(III) cells was slightly slower than that in RD cells, whereas the recombinant proalpha1(III) chains of 293+proalpha1(III)+HSP47 cells showed almost the same electrophoretic mobility as those of RD cells. The melting temperature (Tm) of type III procollagen in 293+proalpha1(III)+HSP47 cells was almost the same as that in RD cells, and the Tm in 293+proalpha1(III) cells was slightly higher than that in RD cells. These data suggest that the recombinant proalpha1(III) collagen chain is overmodified in 293+proalpha1(III) cells, but not in 293+proalpha1(III)+HSP47 cells.
热休克蛋白47（HSP47）是一种驻留在内质网的应激蛋白（热休克蛋白），被认为作为一种胶原特异性分子伴侣发挥作用。为了阐明HSP47在原胶原生物合成中的作用，我们建立了人胚肾293细胞系，该细胞系用含或不含HSP47的α1（III）原胶原链进行稳定转染。293细胞不产生任何细胞外基质蛋白，包括胶原，并且在该细胞系中几乎检测不到HSP47的表达水平。293细胞中的重组III型原胶原形成抗胰蛋白酶的同三聚体，无论有无重组小鼠HSP47，它们都以三聚体形式分泌到培养基中。与正常产生III型原胶原的人横纹肌肉瘤细胞系RD相比，稳定转染了原α1（III）胶原链的293细胞[293 +原α1（III）细胞]中III型原胶原的分泌延迟。在本研究中，我们详细检测了III型原胶原的分泌速率。在与小鼠HSP47共转染的细胞[293 +原α1（III）+ HSP47细胞]中，III型原胶原的分泌速率比293 +原α1（III）细胞慢。使用化学交联剂DSP通过免疫沉淀证实了HSP47与原α1（III）胶原链的结合。293 +原α1（III）细胞中原α1（III）胶原链的电泳迁移率比RD细胞中的略慢，而293 +原α1（III）+ HSP47细胞的重组原α1（III）链显示出与RD细胞几乎相同的电泳迁移率。293 +原α1（III）+ HSP47细胞中III型原胶原的解链温度（Tm）与RD细胞中的几乎相同，而293 +原α1（III）细胞中的Tm略高于RD细胞中的。这些数据表明，重组原α1（III）胶原链在293 +原α1（III）细胞中过度修饰，但在293 +原α1（III）+ HSP47细胞中没有。
