Sequence-specific recognition of collagen triple helices by the collagen-specific molecular chaperone HSP47

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 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.     

Intracellular retention of procollagen within the endoplasmic reticulum is mediated by prolyl 4-hydroxylase.

The correct folding and assembly of proteins within the endoplasmic reticulum (ER) are prerequisites for subsequent transport from this organelle to the Golgi apparatus. The mechanisms underlying the ability of the cell to recognize and retain unassembled or malfolded proteins generally require binding to molecular chaperones within the ER. One classic example of this process occurs during the biosynthesis of procollagen. Here partially folded intermediates are retained and prevented from secretion, leading to a build up of unfolded chains within the cell. The accumulation of these partially folded intermediates occurs during vitamin C deficiency due to incomplete proline hydroxylation, as vitamin C is an essential co-factor of the enzyme prolyl 4-hydroxylase. In this report we show that this retention is tightly regulated with little or no secretion occurring under conditions preventing proline hydroxylation. We studied the molecular mechanism underlying retention by determining which proteins associate with partially folded procollagen intermediates within the ER. By using a combination of cross-linking and sucrose gradient analysis, we show that the major protein binding to procollagen during its biosynthesis is prolyl 4-hydroxylase, and no binding to other ER resident proteins including Hsp47 was detected. This binding is regulated by the folding status rather than the extent of hydroxylation of the chains demonstrating that this enzyme can recognize and retain unfolded procollagen chains and can release these chains for further transport once they have folded correctly.     

Roles of collagen fibers and its specific molecular chaperone: analysis using HSP47-knockout mice.

Collagen is the most abundant protein of mammals and produces highly organized ultrastructures in the extracellular matrix. There are at least 27 types of collagen in mammalian tissues. While fibrillar collagen (eg. types I, II, III, V and XI) assembles into large fibril structures in the extracellular matrix, type IV collagen produces meshwork-like structures in the basement membranes. As collagen has a distinct triple helix structure composed of Gly-X-Y repeats whose Y position is often hydroxyproline, its folding and maturation process differs considerably from globular proteins. Type I collagen is an assembly of two alpha-1 chains and one alpha-2 chain, and each of the alpha chains contain the N-terminal propeptide, C-terminal propeptide and central triple helical region. The 47-kDa heat shock protein (HSP47) is an endoplasmic reticulum (ER)-resident molecular chaperone that specifically recognizes the triple helical region of collagen and is required for productive folding and maturation of collagen molecules. Only in the presence of HSP47, collagen type I molecules can be assembled into the correctly folded triple helices in the ER of mouse embryos without producing misfolded or non-functionally aggregated molecules. HSP47-knockout embryos die just after 10.5 day due to the absence of functional collagen. Recent our data demonstrated that the non-fibrillar network-forming collagen type IV also requires HSP47 for productive folding and maturation. Here, we discuss the role of HSP47 in the folding and maturation of collagen type IV as well as type I.     

Substrate recognition of collagen-specific molecular chaperone HSP47. Structural requirements and binding regulation

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-Pro-Gly 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.     

Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis

Triple helix formation of procollagen after the assembly of three alpha-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of alpha1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.     

Procollagen triple helix assembly: an unconventional chaperone-assisted folding paradigm

Fibers composed of type I collagen triple helices form the organic scaffold of bone and many other tissues, yet the energetically preferred conformation of type I collagen at body temperature is a random coil. In fibers, the triple helix is stabilized by neighbors, but how does it fold? The observations reported here reveal surprising features that may represent a new paradigm for folding of marginally stable proteins. We find that human procollagen triple helix spontaneously folds into its native conformation at 30-34 degrees C but not at higher temperatures, even in an environment emulating Endoplasmic Reticulum (ER). ER-like molecular crowding by nonspecific proteins does not affect triple helix folding or aggregation of unfolded chains. Common ER chaperones may prevent aggregation and misfolding of procollagen C-propeptide in their traditional role of binding unfolded polypeptide chains. However, such binding only further destabilizes the triple helix. We argue that folding of the triple helix requires stabilization by preferential binding of chaperones to its folded, native conformation. Based on the triple helix folding temperature measured here and published binding constants, we deduce that HSP47 is likely to do just that. It takes over 20 HSP47 molecules to stabilize a single triple helix at body temperature. The required 50-200 microM concentration of free HSP47 is not unusual for heat-shock chaperones in ER, but it is 100 times higher than used in reported in vitro experiments, which did not reveal such stabilization.     

Insufficient folding of type IV collagen and formation of abnormal basement membrane-like structure in embryoid bodies derived from Hsp47-null embryonic stem cells

Hsp47 is a molecular chaperone that specifically recognizes procollagen in the endoplasmic reticulum. Hsp47-null mouse embryos produce immature type I collagen and form discontinuous basement membranes. We established Hsp47-/- embryonic stem cell lines and examined formation of basement membrane and production of type IV collagen in embryoid bodies, a model for postimplantation egg-cylinder stage embryos. The visceral endodermal cell layers surrounding Hsp47-/- embryoid bodies were often disorganized, a result that suggested abnormal function of the basement membrane under the visceral endoderm. Rate of type IV collagen secretion by Hsp47-/- cells was fourfold lower than that of Hsp47+/+ cells. Furthermore, type IV collagen secreted from Hsp47-/- cells was much more sensitive to protease digestion than was type IV collagen secreted from Hsp47+/+ cells, which suggested insufficient or incorrect triple helix formation in type IV collagen in the absence of Hsp47. These results indicate for the first time that Hsp47 is required for the molecular maturation of type IV collagen and suggest that misfolded type IV collagen causes abnormal morphology of embryoid bodies.     

Type I collagen in Hsp47-null cells is aggregated in endoplasmic reticulum and deficient in N-propeptide processing and fibrillogenesis

Heat-shock protein of 47 kDa (Hsp47) is a molecular chaperone that recognizes collagen triple helices in the endoplasmic reticulum (ER). Hsp47-knockout mouse embryos are deficient in the maturation of collagen types I and IV, and collagen triple helices formed in the absence of Hsp47 show increased susceptibility to protease digestion. We show here that the fibrils of type I collagen produced by Hsp47-/- cells are abnormally thin and frequently branched. Type I collagen was highly accumulated in the ER of Hsp47-/- cells, and its secretion rate was much slower than that of Hsp47+/+ cells, leading to accumulation of the insoluble aggregate of type I collagen within the cells. Transient expression of Hsp47 in the Hsp47-/- cells restored normal extracellular fibril formation and intracellular localization of type I collagen. Intriguingly, type I collagen with unprocessed N-terminal propeptide (N-propeptide) was secreted from Hsp47-/- cells and accumulated in the extracellular matrix. These results indicate that Hsp47 is required for correct folding and prevention of aggregation of type I collagen in the ER and that this function is indispensable for efficient secretion, processing, and fibril formation of collagen.     

Mechanisms of procollagen and HSP47 sorting during ER-to-Golgi trafficking

Efficient quality control and export of procollagen from the cell is crucial for extracellular matrix homeostasis, yet it is still incompletely understood. One of the debated questions is the role of a collagen-specific ER chaperone HSP47 in these processes. Most ER chaperones preferentially bind to unfolded polypeptide chains, enabling selective export of natively folded proteins from the ER after chaperone release. In contrast, HSP47 preferentially binds to the natively folded procollagen and is believed to be released only in the ER-Golgi intermediate compartment (ERGIC) or cis-Golgi. HSP47 colocalization with procollagen in punctate structures observed by immunofluorescence imaging of fixed cells has thus been interpreted as evidence for HSP47 export from the ER together with procollagen in transport vesicles destined for ERGIC or Golgi. To understand the mechanism of this co-trafficking and its physiological significance, we imaged the dynamics of fluorescently tagged type I procollagen and HSP47 punctate structures in live MC3T3 murine osteoblasts with up to 120 nm spatial and 500 ms time resolution. Contrary to the prevailing model, we discovered that most bona fide carriers delivering procollagen from ER exit sites (ERESs) to Golgi contained no HSP47, unless the RDEL signal for ER retention in HSP47 was deleted or mutated. These transport intermediates exhibited characteristic rapid, directional motion along microtubules, while puncta with colocalized HSP47 and procollagen similar to the ones described before had only limited, stochastic motion. Live cell imaging and fluorescence recovery after photobleaching revealed that the latter puncta (including the ones induced by ARF1 inhibition) were dilated regions of ER lumen, ERESs, or autophagic structures surrounded by lysosomal membranes. Procollagen was colocalized with HSP47 and ERGIC53 at ERESs. It was colocalized with ERGIC53 but not HSP47 in Golgi-bound transport intermediates. Our results suggest that procollagen and HSP47 sorting occurs at ERES before procollagen is exported from the ER in Golgi-bound transport intermediates, providing new insights into mechanisms of procollagen trafficking.     

Mapping Hsp47 binding site(s) using CNBr peptides derived from type I and type II collagen

As a crucial molecular chaperone in collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation.     

Evolution and function of diverse Hsp90 homologs and cochaperone proteins

Members of the Hsp90 molecular chaperone family are found in the cytosol, ER, mitochondria and chloroplasts of eukaryotic cells, as well as in bacteria. These diverse family members cooperate with other proteins, such as the molecular chaperone Hsp70, to mediate protein folding, activation and assembly into multiprotein complexes. All examined Hsp90 homologs exhibit similar ATPase rates and undergo similar conformational changes. One of the key differences is that cytosolic Hsp90 interacts with a large number of cochaperones that regulate the ATPase activity of Hsp90 or have other functions, such as targeting clients to Hsp90. Diverse Hsp90 homologs appear to chaperone different types of client proteins. This difference may reflect either the pool of clients requiring Hsp90 function or the requirement for cochaperones to target clients to Hsp90. This review discusses known functions, similarities and differences between Hsp90 family members and how cochaperones are known to affect these functions. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Direct in vitro and in vivo evidence for interaction between Hsp47 protein and collagen triple helix

Hsp47 (heat shock protein 47), a collagen-specific molecular chaperone, is essential for the maturation of various types of procollagens. Previous studies have suggested that Hsp47 may preferentially recognize the triple-helix form of procollagen rather than unfolded procollagen chains in the endoplasmic reticulum. However, the underlying mechanism has remained unclear because of limitations in the available methods for detecting in vitro and in vivo interactions between Hsp47 and collagen. In this study, we established novel methods for this purpose by adopting a time-resolved FRET technique in vitro and a bimolecular fluorescence complementation technique in vivo. Using these methods, we provide direct evidence that Hsp47 binds to collagen triple helices but not to the monomer form in vitro. We also demonstrate that Hsp47 binds a collagen model peptide in the trimer conformation in vivo. Hsp47 did not bind collagen peptides that had been modified to block their ability to form triple helices in vivo. These results conclusively indicate that Hsp47 recognizes the triple-helix form of procollagen in vitro and in vivo.     

Defective folding and stable association with protein disulfide isomerase/prolyl hydroxylase of type I procollagen with a deletion in the pro alpha 2(I) chain that preserves the Gly-X-Y repeat pattern.

We have studied the folding, processing, and association with two endoplasmic reticulum (ER) resident proteins of the abnormal type I procollagen molecules produced by a strain of fibroblasts harboring a 4.5 kilobase deletion in an allele of COL1A2 (Willing, M. C., Cohn, D.H., Starman, B. Holbrook, K.A., Greenberg, C.R., and Byers, P.H. (1988) J. Biol. Chem. 263, 8398-8404). By sequencing cDNA, we found that the mutant allele encodes pro alpha 2(I) chains that are shortened by 180 amino acids but retain the Gly-X-Y repeat pattern crucial for collagen triple helix formation. The type I procollagen molecules that incorporated the shortened chain were retained intracellularly and were stable. The triple helical domain in these molecules did not attain a normal conformation and remained accessible to posttranslational modifying enzymes amino-terminal to the deletion site for a prolonged period. The abnormal molecules folded into a triple helical conformation more slowly than the normal molecules, and the amino-terminal ends of the pro alpha 1(I) chains failed to become protease-resistant. While the abnormal procollagen molecules were not bound by the ER-resident protein BiP, they stably associated with protein disulfide isomerase, the beta-subunit of prolyl-4-hydroxylase. These results indicate that some mutations in type I collagen genes both transiently delay folding and permanently disrupt the structure of the triple helix and suggest that binding to prolyl-4-hydroxylase helps to retain certain abnormal procollagen molecules within the ER.     

Procollagen binds to both prolyl 4-hydroxylase/protein disulfide isomerase and HSP47 within the endoplasmic reticulum in the absence of ascorbate

In cells, only properly folded procollagen trimers are secreted from the endoplasmic reticulum (ER), while improperly folded abnormal procollagens are retained within the ER. Ascorbic acid is a co-factor in procollagen hydroxylation, which in turn is required for trimer formation. We examined chaperone proteins which bound to procollagen in the absence of ascorbic acid, a model which mimics the human disease scurvy at the cellular level. We found that both prolyl 4-hydroxylase (P4-H)/protein disulfide isomerase (PDI) and HSP47 bound to procollagen in the absence of ascorbic acid. However, the binding of PDI to procollagen decreased when HSP47 was co-transfected, suggesting that HSP47 and PDI compete for binding to procollagen. These data indicate that P4-H/PDI and HSP47 have cooperative but distinct chaperone functions during procollagen biosynthesis.     

The C-propeptide domain of procollagen can be replaced with a transmembrane domain without affecting trimer formation or collagen triple helix folding during biosynthesis.

The folding and assembly of procollagen occurs within the cell through a series of discrete steps leading to the formation of a stable trimer consisting of three distinct domains: the N-propeptide, the C-propeptide and the collagen triple helix flanked at either end by short telopeptides. We have established a semi-permeabilized cell system which allows us to study the initial stages in the folding and assembly of procollagen as they would occur in the intact cell. By studying the folding and assembly of the C-propeptide domain in isolation, and a procollagen molecule which lacks the C-propeptide, we have shown that this domain directs the initial association event and is required to allow triple helix formation. However, the essential function of this domain does not include triple helix nucleation or alignment, since this can occur when the C-propeptide is substituted with a single transmembrane domain. Also the telopeptide region is not involved in triple helix nucleation; however, a minimum of two hydroxyproline-containing Gly-X-Y triplets at the C-terminal end of the triple helix are required for nucleation to occur. Thus, the C-propeptide is required solely to ensure association of the monomeric chains; once these are brought together, the triple helix is able to nucleate and fold to form a correctly aligned triple helix.     

XPORT-dependent transport of TRP and rhodopsin

TRP channels have emerged as key biological sensors in vision, taste, olfaction, hearing, and touch. Despite their importance, virtually nothing is known about the folding and transport of TRP channels during biosynthesis. Here, we identify XPORT (exit protein of rhodopsin and TRP) as a critical chaperone for TRP and its G protein-coupled receptor (GPCR), rhodopsin (Rh1). XPORT is a resident ER and secretory pathway protein that interacts with TRP and Rh1, as well as with Hsp27 and Hsp90. XPORT promotes the targeting of TRP to the membrane in Drosophila S2 cells, a finding that provides a critical first step toward solving a longstanding problem in?the successful heterologous expression of TRP. Mutations in xport result in defective transport of TRP and Rh1, leading to retinal degeneration. Our results identify XPORT as a molecular chaperone and provide a mechanistic link between TRP channels and their GPCRs during biosynthesis and transport.     

Specific recognition of the collagen triple helix by chaperone HSP47: minimal structural requirement and spatial molecular orientation

The unique folding of procollagens in the endoplasmic reticulum is achieved with the assistance of procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed "flying capstan model" (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319).     

In vitro culture decreases the expression of TGF(beta), Hsp47 and type I procollagen and increases the expression of CTGF in avian tendon explants

Weight-bearing tendons in many species, including humans, chickens and horses, are prone to failure, in many cases without a discernible cause. The normal function of the tendon depends on the proper assembly of fibrils of type I collagen, the main structural component of the tendon. We studied the effect of in vitro culture, temperature (37 degrees C vs. 43 degrees C) and wounding on the expression of mRNAs for several collagen regulators, transforming growth factor beta (TGF(beta)), heat shock protein 47 (Hsp47) and connective tissue growth factor (CTGF), in chicken embryonic gastrocnemius tendon explants. The expression of mRNAs for TGF(beta) and Hsp47, a chaperone of collagen assembly, remained strong during the first day of in vitro culture, but then it decreased, slightly more at higher temperature. Additional injury in selected tendons had no significant effect on the levels of TGF(beta) and Hsp47 mRNAs. Likewise, the level of immunostained type I procollagen also decreased with the length of culture. The expression of CTGF gradually increased from 0 at the time of tendon removal with the duration of culture to strong after three days of culture when the expression of TGF(beta) and Hsp47 was low. We conclude that in vitro culture over the period of several days rather than an increase in temperature or additional wounding decreases the expression of TGF(beta), Hsp47 and type I procollagen and increases the expression of CTGF.     

Molecular Consequences of the SERPINH1/HSP47 Mutation in the Dachshund Natural Model of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a heritable connective tissue disease characterized by bone fragility and increased risk of fractures. Up to now, mutations in at least 18 genes have been associated with dominant and recessive forms of OI that affect the production or post-translational processing of procollagen or alter bone homeostasis. Among those, SERPINH1 encoding heat shock protein 47 (HSP47), a chaperone exclusive for collagen folding in the ER, was identified to cause a severe form of OI in dachshunds (L326P) as well as in humans (one single case with a L78P mutation). To elucidate the disease mechanism underlying OI in the dog model, we applied a range of biochemical assays to mutant and control skin fibroblasts as well as on bone samples. These experiments revealed that type I collagen synthesized by mutant cells had decreased electrophoretic mobility. Procollagen was retained intracellularly with concomitant dilation of ER cisternae and activation of the ER stress response markers GRP78 and phospho-eIF2α, thus suggesting a defect in procollagen processing. In line with the migration shift detected on SDS-PAGE of cell culture collagen, extracts of bone collagen from the OI dog showed a similar mobility shift, and on tandem mass spectrometry, the chains were post-translationally overmodified. The bone collagen had a higher content of pyridinoline than control dog bone. We conclude that the SERPINH1 mutation in this naturally occurring model of OI impairs how HSP47 acts as a chaperone in the ER. This results in abnormal post-translational modification and cross-linking of the bone collagen.