HSP47, a Collagen-Specific Molecular Chaperone, Delays the Secretion of Type III Procollagen Transfected in Human Embryonic Kidney Cell Line 293: A Possible Role for HSP47 in Collagen Modification

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Abstract
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 al(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 proal(III) collagen chains [293+proαl(III) cells] in comparison with human rhabdomyo-sarcoma 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+proαl(III) +HSP47 cells], the rate of type III procollagen secretion was slower than in 293+proαl(III) cells. The binding of HSP47 with proal(III) collagen chains was confirmed by immunoprecipitation using the chemical cross-linker, DSP. The electrophoretic mobility of proal(III) collagen chains in 293+proαl(III) cells was slightly slower than that in RD cells, whereas the recombinant proal(III) chains of 293+proαl(III)+ HSP47 cells showed almost the same electrophoretic mobility as those of RD cells. The melting temperature (Tm) of type III procollagen in 293+proαl(III)+HSP47 cells was almost the same as that in RD cells, and the Tm in 293+proαl(III) cells was slightly higher than that in RD cells. These data suggest that the recombinant proal(III) collagen chain is overmodified in 293+proα1(III) cells, but not in 293+proal(III)+HSP47 cells.

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