Effect of HSP47 on prolyl 4-hydroxylation of collagen model peptides

Prolyl 4-hydroxylation, the most important post-translational modification in collagen biosynthesis, is catalyzed by prolyl 4-hydroxylase, an endoplas mic reticulum-resident enzyme. HSP47 is a collagen-binding stress protein w hich also resides in the endoplasmic reticulum (Nagata, K. and Yamada, K.M. (1986) J. Biol. Chem., 261, 7531-7536). Both prolyl 4-hydroxylase and HSP4 7 interact with procollagen alpha-chains during their folding and/or modifi cation in the endoplasmic reticulum. Recent study has revealed that a simpl e collagen model peptide, (Pro-Pro-Gly)(n), is recognized by HSP47 as well as by prolyl 4-hydroxylase in vitro (Koide et al., manuscript submitted). I n the present study, we investigated the effect of HSP47 on the prolyl 4-hy droxylation of such collagen model peptides. To monitor the enzymatic hydro xylation of the peptides, we developed a non-RI assay system based on rever sed-phase HPLC. When HSP47 was added to the reaction mixture, substrate and less-hydroxylated materials accumulated. This effect depended on the pepti de-binding activity of HSP47, because a mutant HSP47 without collagen-bindi ng activity did not show any inhibitory effect on prolyl 4-hydroxylation. K inetic analysis and other biochemical analyses suggest that HSP47 retards t he enzymatic reaction competing for the substrate peptide.