Substrate Recognition of Collagen-specific Molecular Chaperone HSP47

Abstract

Prior to secretion, procollagen molecules are correctly folded to triple helices in the endoplasmic reticulum (ER). HSP47 specifically associates with procollagen in the ER during its folding and/or modification processes and is thought to function as a collagen-specific molecular chaperone (Nagata, K. (1996) Trends Biochem. Sci. 21, 23-26). However, structural requirements for substrate recognition and regulation of the binding have not yet been elucidated. Here, we show that a typical collagen model sequence, (Pro-Pro-Gly) n , possesses sufficient structural information required for recognition by HSP47. A structure-activity relationship study using synthetic analogs of (Pro-Pro-Gly) n has revealed the requirements in both chain length and primary structure for the interaction. The substrate recognition of HSP47 has also been shown to be similar but distinct from that of prolyl 4-hydroxylase, an ER resident enzyme. Further, it has shown that the interaction of HSP47 with the substrate peptides is abolished by prolyl 4-hydroxylation of the second Pro residues in Pro-ProGly triplets and that the fully prolyl 4-hydroxylated peptide, (Pro-Hyp-Gly) n , does not interact with HSP47. We thus have proposed a model in which HSP47 dissociates from procollagen during the process of prolyl 4-hydroxylation in the ER.Collagen is the most abundant protein in vertebrate bodies and is folded as procollagen in the endoplasmic reticulum (ER). 1 The folding of procollagen in cells is unique, as is the final structure itself. In the folding of type I collagen, for instance, three ␣ chains (two ␣1(I) and one ␣2(I)) first associate at C-terminal propeptides, and then approximately 340 repeats of the Xaa-Yaa-Gly triplet form the 300-nm length triple helix (1). In the triple helix, three left-handed polyproline type II (PP-II) helices are supercoiled (2). The helix-forming process is believed to be coupled with prolyl 4-hydroxylation at Yaa positions (3). HSP47 transiently interacts with procollagen molecules in the ER in the process of folding and/or modification (4, 5). HSP47 is not secreted with procollagen from the cells, because it possesses the ER retention signal, Arg-Asp-Glu-Leu, at the carboxyl end (5, 6). The expression of HSP47 is well correlated with that of various types of collagen in cultured cells, in various tissues during development, and in pathophysiological conditions (7-12). Thus HSP47 is assumed to be a collagen-specific molecular chaperone (13). However, the binding sites or motif in the procollagen molecule have not been well elucidated. The molecular basis of HSP47 function, including the regulation of the substrate association and dissociation, also remains an enigma.Here, we studied substrate recognition of HSP47 using the recombinant chaperone protein and synthetic collagen model peptides and found that a very simple collagen model peptide mimics the native substrate of HSP47. The difference in substrate recognition between HSP47 and prolyl 4-hydroxylase (P4-H) was discussed in both primary and secondary s...

The Molecular Interactions of Heat Shock Protein 47 (Hsp47) and Their Implications for Collagen Biosynthesis

“…4a); this is much weaker than that previously recorded using a comparable fluorescence-based assay for Hsp47 binding to collagen (K d of 140 nM) (39). These results for collagen are in disagreement with Koide and coworkers (20) who have determined K d values (300 -1100 nM) for the interaction between mrmHsp47 and full-length type I-IV collagen using surface plasmon resonance. It seems more reasonable to compare our results with McDonald and Bä chinger (39) as our methodologies are identical.…”

“…Furthermore, recent work by Tasab and co-workers (16) has demonstrated that Hsp47 will bind to fully formed helical trimers in vivo, perhaps thereby stabilizing the triple helix and preventing chain dissociations. This functional hypothesis is supported by a recent demonstration that Hsp47 binds specifically to stretches of non-hydroxylated Pro-Gly-X repeats (20), regions of low structural stability within the full-length collagen triple helices. After binding there is clear evidence that triple helical procollagen-Hsp47 complexes are eventually transported from the endoplasmic reticulum to cis-Golgi (21), where Hsp47 is dissociated and recycled back to the endoplasmic reticulum (18).…”

“…Cation (MonoS) exchange chromatography was then used to separate monomeric Hsp47 from mrmHsp47c, providing a more rapid and convenient purification procedure. Previous work (20,44) with mrmHsp47c has always been conducted on the basis that mrmHsp47 was only monomer in character. Therefore, the existence of the trimeric state of mrmHsp47 is an important revelation.…”

Endothelial–mesenchymal transition in human atrial fibrillation

“…S100A4 [19], also known as fibroblast-specific protein 1 (FSP1) [20,21], is a member of the S100 superfamily of EF-hand calcium-binding proteins. P4HD is an essential enzyme for the biosynthesis of procollagen [22] and HSP47 is an ER-resident stress protein that is believed to function as a molecular chaperone specific to the biosynthesis of procollagen [23,24]. We identified the co-expressions of these profibrotic molecules within the atrial endocardial cells, suggesting transition to fibroblast phenotype.…”

Folding defects in fibrillar collagens

“…27 8C instead of 43 8C in the fully hydroxylated form. Type I procollagen molecules synthesized when the enzyme is inhibited by iron chelators are stored in the ER but can be hydroxylated and secreted if the Koide et al 1999). If correct, this provides one part of the solution to the problem of maintaining the long proa chains in a favourable conformation until the carboxyterminal propeptide, the locus of chain^chain interaction, is synthesized and released.…”