ENDOPLASMIC-RETICULUM PROTEIN HSP47 BINDS SPECIFICALLY TO THE N-TERMINAL GLOBULAR DOMAIN OF THE AMINO-PROPEPTIDE OF THE PROCOLLAGEN-I ALPHA-1(I)-CHAIN

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibrobl asts, 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 c ollagens and collagen domain peptides were incubated with (35)[S]-meth ionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Se pharose 4B beads. The gelatin-bound proteins were collected and analyz ed by gel electrophoresis and autoradiography. Collagenase digested pr ocollagen I had the same effect as denatured intact procollagen, indic ating that the propeptides were the major interaction sites. The addit ion of intact pro alpha 1(1)-N-propeptide at 25 mu g/ml completely inh ibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide V PTDE, residues 86-90 of the pro alpha 1(I)-N-propeptide, inhibits hsp4 7-gelatin binding. These data implicating the pro alpha 1(I)-N-propept ide domain were confirmed by examination of polysome-associated pro al pha chains. The nascent pro alpha 1(I)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro alpha 1(I)-N-propept ide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion prot ein constructs of residues 23-108 (NP1), 23-151 (NP2), and 23-178 (NP3 ) within the pro alpha 1(I)-N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysat es. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotrans lation systems in which the complete pro alpha 1(I)- and pro alpha 2(I )-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro alpha>2( I)-chains and hsp47, whereas there was strong interaction between pro alpha 1(I)-chains and nascent hsp47. SP1.D8 could not precipitate pro alpha 1(I)-chains from the translation mix if nascent hsp47 was presen t. These data all suggest that if hsp47 has a ''chaperone'' role durin g procollagen chain processing and folding it performs this specific r ole via its preferential interaction with the pro (alpha 1(1) chain, a nd the pro alpha 1(I) amino-propeptide region in particular. (C) 1995 Wiley-Liss, Inc.