Sequence-specific Recognition of Collagen Triple Helices by the Collagen-specific Molecular Chaperone HSP47

Mohammed Tasab, Lynsey Jenkinson, Neil J. Bulleid
2002 Journal of Biological Chemistry  
HSP47 is a molecular chaperone that plays an unknown role during the assembly and transport of procollagen. Our previous studies showed that, unlike most chaperones, HSP47 interacts with a correctly folded substrate. We suggested that HSP47 either stabilizes the correctly folded collagen helix from heat denaturation or prevents lateral aggregation prior to its transport from the endoplasmic reticulum. In this study we have addressed the role of triple helix stability in the binding of HSP47 to
more » ... inding of HSP47 to procollagen by expressing procollagen molecules with differing thermal stabilities and analyzing their ability to interact with HSP47 within the endoplasmic reticulum. Our results show that HSP47 interacts with thermostable procollagen molecules, suggesting that helix stabilization is not the primary function of HSP47 and that the interaction of HSP47 with procollagen depends upon the presence of a minimum of one Gly-X-Arg triplet within the triple helical domain. Interestingly, procollagen chains containing high proportions of stabilizing triplets formed triple helices and interacted with HSP47 even in the absence of proline hydroxylation, demonstrating that recognition does not depend upon this modification. Our results support the view that HSP47 functions early in the secretory pathway by preventing the lateral aggregation of procollagen chains. The folding and assembly of procollagen molecules occurs within the lumen of the endoplasmic reticulum (ER) 1 following translation and translocation of procollagen chains on membrane-bound ribosomes. Post-translational modifications take place as the chains enter the lumen of the ER, and the assembly process is known to involve interactions with multiple ER resident chaperones (1). Each procollagen chain contains a collagenous domain of repeating Gly-X-Y triplets that determines the characteristic triple helical structure of collagens. The contribution of specific Gly-X-Y triplets to the thermal stability of the triple helical structure depends upon the identity of the X and Y residues (2). The C-propeptide regions associate to form a trimeric procollagen molecule (3). This association ensures that the triple helix nucleation regions of the adjacent chains (4) are in close proximity, allowing for helix nucleation to occur followed by propagation of the triple helix toward the N terminus (5). As well as carrying out post-translational modifications, proteins such as protein disulfide isomerase and prolyl 4-hydroxylase assist in the folding process either by preventing nonspecific association of the individual chains prior to trimerization and triple helix formation (6) or by helping to retain unfolded molecules within the ER (7) . HSP47 is an ER resident glycoprotein that plays a crucial role during the folding, maturation, and secretion of procollagen (8). Evidence to support this role comes from the fact that HSP47 is up-regulated in collagen-producing cells (9 -11) and binds specifically to collagenous peptides (12, 13). More convincingly, when the hsp47 gene was disrupted in mice, the resulting nullizygous animals were severely deficient in the mature processed form of collagen and did not survive past day 11.5 postcoitus (14). These results demonstrate that HSP47 is required to facilitate the secretion of stable and correctly processed collagen and that mice cannot develop without this specialized molecular chaperone. However, this work does not reveal the precise function of HSP47, whether HSP47 recognizes a specific sequence within the collagenous domain, or how binding of HSP47 to procollagen is regulated within the cell. One approach that has been taken to address the question of substrate recognition has been to study the binding of collagenlike peptides to HSP47 in vitro (13). These studies demonstrate that HSP47 binds with highest affinity to peptides that are capable of forming thermostable triple helices. The binding of peptides to HSP47 can be abolished by lowering the pH, leading to the suggestion that the regulation of binding in vivo could be due to changes in pH during transport through the secretory pathway (15). These in vitro binding studies have also shown that the affinity of HSP47 for peptides containing arginine residues within the Y position of the Gly-X-Y collagen triplet is much higher than when this amino acid is absent (16). However a recent report on binding of HSP47 to (PPG) 10 peptide demonstrated high affinity binding (15). Although there are conflicting results in the literature, these experiments do establish that HSP47 binds preferentially to procollagen peptides that adopt a triple helical conformation and contain the amino acid arginine. Our previous studies demonstrated that HSP47 binds to procollagen molecules once they have formed triple helical molecules and dissociates upon thermal denaturation of the triple helical structure (17). The approach taken was to analyze the interaction of HSP47 within newly synthesized procollagen molecules within a functionally and morphologically intact ER (18). Having established that HSP47 interacts with correctly folded procollagen, we hypothesized that this interaction could lead to a stabilization of the procollagen triple helix, in particular regions of the helix with lower thermal stability. An alternative explanation for the binding of HSP47 to correctly folded procollagen is to prevent the lateral association of the chains occurring within the ER. It is known that once the procollagen
doi:10.1074/jbc.m202782200 pmid:12114508 fatcat:chmloe7pu5fexmazp4ugxtjkjy