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

HSP47 is a stress protein (heat shock protein) which resides in the en doplasmic reticulum, and is postulated to function as a collagen-speci fic molecular chaperone. To elucidate the role of HSP47 in procollagen biosynthesis, we have established human embryonic kidney 293 cell lin es, which were stably transfected with alpha 1(III) procollagen chains with or without HSP47. 293 cells do not produce any extracellular mat rix proteins including collagens, and the level of HSP47 expression is almost undetectable in this cell line. Recombinant type III procollag ens in 293 cells form trypsin-resistant homotrimers, which are secrete d into the medium as trimers in the presence or absence of recombinant mouse HSP47. The secretion of procollagen III was delayed in 293 cell s stably transfected with pro alpha 1(III) collagen chains [293+pro al pha 1(III) cells] in comparison with human rhabdomyosarcoma cell line RD, which normally produces type III procollagens. In this study, we e xamined the rate of type In procollagen secretion in detail. In cells cotransfected with mouse HSP47 [293 + pro alpha 1(III) + HSP47 cells], the rate of type III procollagen secretion was slower than in 293 + p ro alpha 1(III) cells. The binding of HSP47 with pro alpha 1(III) coll agen chains was confirmed by immunoprecipitation using the chemical cr oss-linker, DSP, The electrophoretic mobility of pro alpha 1(HI) colla gen chains in 293 + pro alpha 1(III) cells was slightly slower than th at in RD cells, whereas the recombinant pro alpha 1(III) chains of 293 +pro alpha 1(III) + HSP47 cells showed almost the same electrophoretic mobility as those of RD cells. The melting temperature (T-m) of type III procollagen in 293+pro alpha 1 (III) + HSP47 cells was almost the same as that in RD cells, and the T-m in 293+pro alpha 1(III) cells wa s slightly higher than that in RD cells. These data suggest that the r ecombinant pro alpha 1(III) collagen chain is overmodified in 293 + pr o alpha 1(III) cells, but not in 293 + pro alpha 1(III) + HSP47 cells.