HSP47: Mechanisms and Interactions

Hsp47 is an ER-resident unique molecular chaperone crucially involved in the correct folding of procollagen ^{2, 3}. Procollagen represents the precursor molecule of collagen, the major component of the ECM. So far, 29 types of collagen have been identified and described ¹⁹⁰. Procollagen is synthesized in the ER, followed by its transition to the Golgi apparatus and subsequent release from the cell (Figure 6). The most abundant collagen in humans is type I collagen, forming typical collagen fibers that consist of two α₁-chains and one α₂-chain. The three polypeptide chains are synthesized as preprocollagen in the ribosomes along the rough endoplasmic reticulum (rER) and co-translationally inserted into the rER. The individual α-chains bear a triple helix-establishing domain of more than 300 repeats of the Gly-Xaa-Yaa triplet, in which X and Y are often occupied by proline and hydroxyproline, respectively. After cleavage of the presequence, the single chains (now termed pro-alpha-chains) associate at the C-propeptide region via inter-chain disulfide bridges followed by a zipper-like triple-helix formation from the C- to the N-terminus ^{191, 192}. Afterwards, hydroxylation of lysyl and prolyl residues takes place inside the ER lumen requiring the presence of three hydroxylases and ascorbic acid (vitamin C) as a cofactor ¹⁹³. Noteworthy, hydroxylation of proline residues at the third position in the Gly-Xaa-Yaa triplet represents an essential step in the formation of procollagen as it stabilizes the triple-helical conformation of the molecule. Additional post-translational modifications such as glycosylation and formation of disulfide bridges have been noted, on the one hand, to contribute to the correct folding and stability of procollagen and, on the other hand, to prevent aggregation of triple-helical procollagen ^{194, 195}. This triple-helical monomer of procollagen is subsequently shipped to the Golgi, packed and secreted via exocytosis. After extracellular cleavage of the C- and N-propeptides yielding tropocollagen (Figure 6), collagen fibrils and fibers are generated by covalent cross-linking (aldol reaction) of lysyl residues catalyzed by lysyl oxidase. Collagen fibers are attached to the cell membrane through various cell adhesion molecules such as fibronectin, integrin, and laminin (for a review see Olsen, 1981; Aigner et al., 2003) ^{196, 197}.

Figure 6. Cartoon illustrating procollagen folding in the rough ER. Newly synthesized preprocollagen is inserted co-translationally into the ER. Cleavage of the presequence yields the pro-alpha chains that associate at the C-propeptide region followed by a zipper-like triple-helix formation from the C- to the N-terminus. Hsp47 binds to triple-helical procollagen in the ER and dissociates in the cis-Golgi or ERGIC under low pH. Triple-helical procollagen is shipped subsequently to the Golgi, packed and secreted via exocytosis. After cleavage of the C- and N-propeptides yielding tropocolloagen, collagen fibrils and fibers are generated. For details see text.

The correct folding of the triple-helical procollagen molecules is a prerequisite for the formation of biologically active collagen. In this context, Hsp47 serves as a critical regulator in intracellular processing during the assembly of triple-helical procollagen molecules ¹⁹⁸. Hsp47 has been proposed to protect newly synthesized procollagen chains from being folded or aggregated in the ER ¹⁹⁹, to block the desintegration of procollagen in the ER prior to its transport to the Golgi ²⁰⁰, and to prevent the secretion of immature or misfolded procollagen molecules from the ER ¹⁵⁶. In addition, Hsp47 stabilizes the partially and correctly folded triple-helical procollagen intermediates in the ER that are unstable at body temperature ²⁰¹. Hsp47 was also reported to assist in packing procollagens in certain vesicles intended for the transport to the Golgi by interacting with the Src homology 3 (SH-3) domain of transmembrane protein transport and Golgi organisation 1 (TANGO-1) ²⁰².

Several investigations were conducted to determine the molecular nature of the Hsp47-procollagen interaction. There is a wealth of evidence to suggest that the Hsp47-procollagen complex is formed by two Hsp47 molecules that attach in a head-to-head fashion via β-sheet C to two strands of one procollagen trimer (Figure 2). The consensus motif for binding of Hsp47 to the procollagen triple-helix has been identified previously as being Gly – Xaa – Arg ¹⁴³, with Arg playing an important role in the Hsp47-procollagen interaction ^{25, 144, 145}. Widmer and colleagues additionally determined the Hsp47 residues interacting with Arg at the Yaa⁰ position and the Yaa⁻³ position of the Yaa⁻³ – Gly – Xaa – Arg sequence of procollagen as being Asp385 and Arg222, respectively ²⁴. Moreover, the critical amino acid residues Leu381 andTyr383 in Hsp47 have been confirmed to play a pivotal role in several hydrophobic interactions. The first structural investigation of the procollagen binding site in Hsp47 using NMR and mutational analysis proposed a possible contribution of Met223, Met363 and Tyr230 in the B/C β-barrel domain to procollagen binding ²⁵. Of note, Hsp47 has been shown to preferentially recognize the folded triple-helical conformation of procollagen ^144-149. In contrast to the critical Arg in the consensus binding motif Gly – Xaa – Arg, the identity of the residue in the Xaa position is negligible; Hsp47 also recognizes the amino acid in the Yaa⁻³ position in the sequence Yaa⁻³ – Gly – Xaa – Arg. Explicitly, Hsp47 predominantly recognizes Thr and Pro at Yaa⁻³, followed by Ser, Hyp, Val, and Ala, but it does not recognize Lys, Gln, or Glu ²³. Adapted from the characterization of Hsp47-binding motifs, the number of Hsp47-binding sites in native procollagens was estimated by the group of Kouki Kitagawa, revealing that the triple-helical region (1029 amino acid residues) of human type III procollagen harbors 29 putative Hsp47-binding sites ²³. In a more recent study using canine Hsp47 further important residues could be determined. Herein, His238 located to the interface of HSP47-procollagen binding, was described to form hydrogen bonds with Asp220 and Ser305. By forming a stable salt bridge to Arg222 and Tyr383, Asp220 then enables binding of Hsp47 to procollagen ²⁷.

The interaction of Hsp47 with procollagen has been found to be pH-dependent, as evidenced previously ²². Hsp47 binds to triple-helical procollagen in the ER under neutral pH and dissociates in the cis-Golgi or ERGIC under low pH ¹⁵⁶. Apo-(unbound-) Hsp47 returns to the ER and is released into the ER lumen; this retrograde transport is mediated by an interaction of the C-terminal RDEL motif in Hsp47 with the KDEL receptor that cycles between the Golgi and ER ²⁶. In this context, studies using various trafficking inhibitors verified the addiction of the Hsp47-procollagen interaction to pH. Inhibitors of the medial-Golgi or post-trans-Golgi transport, monensin or bafilomycin A1, as well as the ER-cis-Golgi transport inhibitor brefeldin A have been found to block procollagen exocytosis from cells ²⁶. As the Hsp47-procollagen interaction only occurred in brefeldin A-treated cells, one can assume that Hsp47 does not associate in the cis-Golgi or a more distal compartment in the secretory pathway; it rather dissociates from procollagen in the cis-Golgi or ERGIC due to the low pH in these compartments ²⁶. Growing evidence suggests that histidine residues contribute to the pH-dependent release of Hsp47 from procollagen. Several investigations identified His274 as being the central residue for the pH-dependent procollagen release, whereby the neighbouring His273 significantly affects the pKa of His274 ²⁷