Endoplasmic reticulum protein HSP47 binds specifically to the N-Terminal globular domain of the amino-propeptide of the procollagen I α1(I)-chain

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibroblasts has gelatin-binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47-procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with³⁵[S]-methionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Sepharose 4B beads. The gelatin-bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro α1 (l)-N-propeptide at 25 μg/ml compeletely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86–90 of the pro α1 (l)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro α1 (l)-N-propeptide domain were confirmed by examination of polysome-associated pro α chains. The nascent pro α1(l)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro α1 (l)-N-propeptide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion protein constructs of residues 23–108 (NP1), 23–151 (NP2), and 23–178 (NP3) within the pro α1 (l)-N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotranslation systems in which the complete pro α1(l)- and pro α2(l)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro α2(l)-chains and hsp47, whereas there was strong interaction between pro α1 (l)-chains and nascent hsp47. SP1.D8 could not precipitate pro α1 (l)-chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a “chaperone” role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the proα1 (l) chain, and the pro α1 (l) amino-propeptide region in particular. © 1995 Wiley-Liss, Inc.