Specific Recognition of the Collagen Triple Helix by Chaperone HSP47

Abstract

The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa-3) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg0) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa-3 and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa-3 determined the binding affinity for HSP47. Maximal binding was observed when Yaa-3 was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr-3 and one Arg0. The results revealed that HSP47 recognizes the Yaa-3 and Arg0 residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47. The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa-3) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg0) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa-3 and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa-3 determined the binding affinity for HSP47. Maximal binding was observed when Yaa-3 was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr-3 and one Arg0. The results revealed that HSP47 recognizes the Yaa-3 and Arg0 residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47. Heat-shock protein 47 (HSP47) 3The abbreviations used are: HSP47, heat-shock protein 47; ER, endoplasmic reticulum; Hyp or O, 4-hydroxy-l-proline; Bpa, p-benzoyl-l-phenylalanine; MALDI-TOF-MS, matrix-assisted laser-desorption time-of-flight mass spectrometry; CBB, Coomassie Brilliant Blue R-250; SPR, surface plasmon resonance; GST, glutathione S-transferase; NHS, N-hydroxysuccinimide; C1q, complement 1q; MSR1, macrophage scavenger receptor 1; CL-L1/Collectin10, collectin liver 1; CL-P1/Collectin12, collectin placenta 1; MBL2, mannose-binding lectin 2; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Standard three-letter and one-letter abbreviations were used for the common amino acids. is an essential molecular chaperone for normal procollagen biosynthesis in mammals. Disruption of both alleles of the hsp47 gene in mice causes abnormal procollagen folding in the endoplasmic reticulum (ER), resulting in an embryonic lethal phenotype (1Nagai N. Hosokawa M. Itohara S. Adachi E. Matsushita T. Hosokawa N. Nagata K. J. Cell Biol. 2000; 150: 1499-1506Crossref PubMed Scopus (291) Google Scholar, 2Marutani T. Yamamoto A. Nagai N. Kubota H. Nagata K. J. Cell Sci. 2004; 117: 5913-5922Crossref PubMed Scopus (55) Google Scholar). Although the molecular function of HSP47 remains unclear, it has been suggested that HSP47 could stabilize correctly folded triple-helical intermediates of procollagen that are otherwise unstable at body temperature (3Tasab M. Batten M.R. Bulleid N.J. EMBO J. 2000; 19: 2204-2211Crossref PubMed Scopus (174) Google Scholar, 4Koide T. Aso A. Yorihuzi T. Nagata K. J. Biol. Chem. 2000; 275: 27957-27963Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). HSP47 has also been suggested to inhibit lateral associations that form insoluble aggregates in the ER (5Thomson C.A. Ananthanarayanan V.S. Biochem. J. 2000; 349: 877-883Crossref PubMed Scopus (52) Google Scholar). Efforts toward elucidating the collagen-recognition mechanism of HSP47 have been made by our group and others, and to date, Arg residues at Yaa positions in the collagenous Gly-Xaa-Yaa repeats have been shown to be necessary for interaction with HSP47 (6Koide T. Takahara Y. Asada S. Nagata K. J. Biol. Chem. 2002; 277: 6178-6182Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 7Tasab M. Jenkinson L. Bulleid N.J. J. Biol. Chem. 2002; 277: 35007-35012Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The importance of the triple-helical structure for HSP47 binding has also been strongly suggested, and it was finally proven in the preceding paper by using conformationally constrained collagenous peptides (8Koide T. Asada S. Takahara Y. Nishikawa Y. Nagata K. Kitagawa K. J. Biol. Chem. 2006; 281: 3432-3438Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The minimal number of Arg residues per triple helix required for the specific interaction was further determined to be one. In addition, the Yaa residue occupying position -3 (denoted as Yaa-3, and similar nomenclatures are used later), based on the essential Arg residue at a Yaa position (whose position is defined as position 0), has also been suggested to be important for the interaction, since replacement of the 4-hydoxy-l-proline (Hyp) residue at position -3 in (Pro-Hyp-Gly)2-Pro-Hyp-3-Gly-Pro-Arg0-Gly-(Pro-Hyp-Gly)4-Pro-Hyp-amide with p-benzoyl-l-phenylalanine (Bpa) abolished the binding, even though the peptide had a triple-helical structure and contained an Arg residue at a Yaa position. In the present study, we focused on the structure of Yaa-3, which was expected to be another structural determinant for HSP47 binding, together with the side chain structure of Arg0. A structure-activity relationship study was performed using collagen-like peptides with various Yaa-3 substitutions. Moreover, the three-dimensional arrangement of the Yaa-3 and Arg0 residues required for HSP47 binding was determined by utilizing heterotrimeric collagen-model peptides with different fixed chain arrangements. To date, HSP47 has been shown to interact with at least the major types of collagen, including types I to V (9Natsume T. Koide T. Yokota S.-i. Hirayoshi K. Nagata K. J. Biol. Chem. 1994; 269: 31224-31228Abstract Full Text PDF PubMed Google Scholar). However, it has not been elucidated whether all 27 types of mammalian collagens require the assistance of HSP47 during their folding processes. In addition, there is currently little available information regarding the interactions of HSP47 with other collagen-related proteins. In the final part of this article, we show the results of a motif search for HSP47-binding proteins against human protein data bases using an originally developed computational search program and predict possible clients of HSP47. Synthetic Peptides—Peptide chains were constructed manually based on the N-(9-fluorenyl)methoxycarbonyl-based solid-phase method on Rink-amide resins (Novabiochem, Darmstadt, Germany). Heterotrimerization of the peptides were conducted according to the procedure described elsewhere (Ref. 10Koide T. Nishikawa Y. Takahara Y. Bioorg. Med. Chem. Lett. 2004; 14: 125-128Crossref PubMed Scopus (29) Google Scholar and also see supplemental materials). The peptides were purified by reversed-phase high performance liquid chromatography and characterized by electron spray-ionization mass spectrometry or matrix-assisted laser-depsorption time-of-flight mass spectrometry (MALDI-TOF-MS). Prior to use, all peptide solutions were kept at 4 °C for at least 2 days to allow the formation of triple helices. CD Spectrometry—To characterize the conformational states of the synthetic collagen-like peptides, CD spectra of the refolded trimers (1 mg/ml in 20 mm bis-Tris-HCl (pH 7.4), 150 mm NaCl) were recorded on a Jasco J-820 spectropolarimeter as described previously (10Koide T. Nishikawa Y. Takahara Y. Bioorg. Med. Chem. Lett. 2004; 14: 125-128Crossref PubMed Scopus (29) Google Scholar). Thermal transition curves of the triple helices were determined by monitoring the changes in the molar ellipticity at 225 nm at a heating rate of 0.3 °C/min. All the peptides were triple-helical in the buffer used for the experiments at the temperature at which the binding assays were conducted. Solid-phase Peptide Binding (Peptide Pull-down) Assay—Solid-phase binding assays to examine HSP47 binding to peptide-immobilized Sepharose beads were performed as reported previously (6Koide T. Takahara Y. Asada S. Nagata K. J. Biol. Chem. 2002; 277: 6178-6182Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Briefly, Escherichia coli lysates containing either recombinant mouse (11Koide T. Asada S. Nagata K. J. Biol. Chem. 1999; 274: 34523-34526Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) or human (12Yokota S.-i. Kubota H. Matsuoka Y. Naitoh M. Hirata D. Minota S. Takahashi H. Fujii N. Nagata K. Biochem. Biophys. Res. Commun. 2003; 303: 413-418Crossref PubMed Scopus (37) Google Scholar) HSP47 protein (70 μl) were mixed with 130 μl of binding buffer (50 mm HEPES-Na (pH 7.5), 150 mm NaCl, 1 mm EDTA) and 25-μl bed of the affinity beads. The binding was carried out at 4 °C for 1 h with gentle rotation. After washing, the proteins retained on the beads were eluted with 2× Laemmli's SDS sample buffer (50 mm Tris-HCl (pH 6.7), 2% SDS, 10% glycerol, 0.002% bromphenol blue) and separated by SDS-PAGE in 12% gels. The gels were stained with Coomassie Brilliant Blue R-250 (CBB). Purification of HSP47—Recombinant mouse HSP47 was purified from E. coli lysates by gelatin-affinity chromatography essentially as described previously (8Koide T. Asada S. Takahara Y. Nishikawa Y. Nagata K. Kitagawa K. J. Biol. Chem. 2006; 281: 3432-3438Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13Saga S. Nagata K. Chen W.T. Yamada K.M. J. Cell Biol. 1987; 105: 517-527Crossref PubMed Scopus (146) Google Scholar). Competition Assay—Competitive binding assays were conducted at 25 °C on a surface plasmon resonance (SPR) The peptide (10Koide T. Nishikawa Y. Takahara Y. Bioorg. Med. Chem. Lett. 2004; 14: 125-128Crossref PubMed Scopus (29) Google Scholar) was on the surface of a to resonance using at according to the The binding was performed in mm bis-Tris-HCl (pH containing NaCl, 1 mm and HSP47 was with various of the peptides for at 25 was the at a rate of The to changes in the using a surface was from the binding each to the specific The surface was by with μl of mm HSP47-binding were in and on an on a of amino acid from and were from the of and to the The of H. M. and in each database were and Briefly, HSP47-binding proteins were from the database by of proteins containing at least repeats with in its or Yaa of proteins was with repeats of or repeats of resulting in the of collagenous and of proteins with in their triple-helical to HSP47-binding proteins. The binding were is or affinity is or and affinity is or according to the results of the binding assays supplemental materials). Cell and were obtained from The human was as described previously K. Y. S. T. T. H. Y. A. T. H. T. M. N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were for 2 days with their containing Binding was obtained from one of the of this Cell lysates were obtained by of buffer (50 mm Tris-HCl (pH 150 mm NaCl, 1 mm by the of insoluble by at for of or μl of human was mixed with a bed of or beads After the beads with buffer (50 mm Tris-HCl (pH 150 mm NaCl, 1 mm proteins retained on the beads were separated by SDS-PAGE using gels and to The were with in and with scavenger receptor K. Y. S. T. T. H. Y. A. T. H. T. M. N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google or or was used and the were with a or an as Yaa-3 and Arg0 in the for the with has been shown to interact with is amino in triple-helical regions of collagen (6Koide T. Takahara Y. Asada S. Nagata K. J. Biol. Chem. 2002; 277: 6178-6182Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 7Tasab M. Jenkinson L. Bulleid N.J. J. Biol. Chem. 2002; 277: 35007-35012Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). shown in binding replacement of the residue of (Pro-Hyp-Gly)2-Pro-Hyp-3-Gly-Pro-Arg0-Gly-(Pro-Hyp-Gly)4-Pro-Hyp-amide with a residue results in of HSP47 binding, similar of other positions have little on the binding (8Koide T. Asada S. Takahara Y. Nishikawa Y. Nagata K. Kitagawa K. J. Biol. Chem. 2006; 281: 3432-3438Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). that the amino acid Yaa-3 is another key residue for by HSP47. Based on this finding, the relationship between the structure of the Yaa-3 residue and HSP47 binding was analyzed using with various in the Yaa-3 The peptides and used are shown in and which are in Yaa positions of collagens J. Biol. PubMed Scopus Google were All the peptides triple-helical at 25 °C in the buffer used for the as by CD not The peptides were and their interactions with recombinant HSP47 were examined by The peptide was from these assays its group with the shown in the of HSP47 retained on the beads to on the structure of the Yaa-3 The with and were the by with and and were at the Yaa-3 the protein to HSP47 was on the they all contained Arg residues at of HSP47 with collagen-like peptides with various Yaa-3 binding of HSP47 to to Sepharose beads. E. coli containing HSP47 was mixed with affinity beads on which the peptides were After washing, the proteins retained on the beads were separated by 12% SDS-PAGE and by The molecular are shown in binding assays on HSP47 binding was as described The data for only of the peptides are shown of the are The relative HSP47 binding of these peptides were further determined by assays on an HSP47 protein was mixed with of the peptides and a on which an (10Koide T. Nishikawa Y. Takahara Y. Bioorg. Med. Chem. Lett. 2004; 14: 125-128Crossref PubMed Scopus (29) Google was In these the peptide was as to between the Arg residues at positions and Yaa-3 by a residue at position 1 and The curves obtained by the HSP47 were used to the The results were with obtained by the peptide The were to from the for and to the Yaa-3 and amino were by HSP47, amino with or side chains were The of Thr-3 and Arg0 was revealed to as the structure for HSP47. A residue at the Yaa-3 position was also to to the high affinity However, it be that in the folding of of the residues in Yaa positions are to Hyp to the triple helix In addition, between the and the of the triple helices were observed and the obtained from per was the temperature at which a of a triple helix is the obtained from per was the temperature at which a of a triple helix is in a of the for HSP47 The using collagen-like peptides indicated that the Yaa-3 residue and the Arg residue at form an HSP47-binding epitope in the collagen triple The collagen triple helix is a of helices with a relative to each other J. M. 1994; PubMed Scopus Google Scholar). different spatial for the of these key residues are possible we which arrangement is recognized by HSP47 by a utilizing heterotrimeric collagen-like peptides. peptides were to fixed of the important residues we used to the peptide since were reported to be with the collagen triple helix and a between the chains be J. L. J. Chem. 1999; Scopus Google Scholar). one of the amino was to Sepharose beads in a fixed molecular In these the position of the Arg residue was fixed to a and one and residues were at the in different In these peptides, and residues were as of the and amino for HSP47 binding, 2 and heterotrimeric peptides were triple-helical at the similar to one another on CD The for the triple helices of these peptides were also similar to one another After to Sepharose the interactions of these peptides with HSP47 were examined by solid-phase shown in HSP47 only with in which the residue and Arg residue are present in the same peptide strand. the structure required for HSP47 binding is in the primary structure. Based on the results shown in HSP47-binding were determined as shown in and for and according to their in in a HSP47-binding in of HSP47-binding us to the number and of HSP47-binding in the triple-helical regions of In a (6Koide T. Takahara Y. Asada S. Nagata K. J. Biol. Chem. 2002; 277: 6178-6182Abstract Full Text Full Text PDF PubMed Scopus (85) Google we that procollagen HSP47-binding based on the that form these binding binding were predicted to be present in human and Ananthanarayanan C.A. Ananthanarayanan V.S. Sci. 2003; PubMed Scopus Google Scholar) out the that we had the number of HSP47-binding based on their results for HSP47 binding assays using collagen The of HSP47-binding were in the present study by taking the of the amino at Yaa-3 positions The triple-helical amino acid of human procollagen contains a of possible HSP47-binding that all the residues at are to Hyp residues to triple helix high affinity binding the motif are The of the and affinity are to be and of HSP47 on HSP47 have been carried out by only at types of as types and the of HSP47 in the biosynthesis of collagens or collagen-related proteins has been The of HSP47-binding further us to search for proteins that could interact with HSP47. using a developed computational search we candidate HSP47-binding proteins from and mouse The search results from the human data are shown in collagenous proteins at least repeats of the proteins contained the HSP47-binding All the of the of all types of collagen I to were revealed to HSP47-binding of the based on their and in the of were the collagen results that all collagen are clients of HSP47. In to the collagen a number of collagen-related as proteins and macrophage scavenger and and proteins and and were also as possible proteins the HSP47-binding of the residues at Yaa positions be to Hyp to triple helix The of a HSP47-binding motif is not or are available in a a of the residues at Yaa positions be to Hyp to triple helix formation The of a HSP47-binding motif is not or proteins are available To this collagen-related proteins were examined their binding to to Sepharose beads. C1q, which has one affinity and one affinity HSP47-binding motif and in its was shown to interact with scavenger receptor 1 with affinity collectin liver 1 with one affinity and collectin placenta 1 with affinity also specific binding to MSR1, and the other lectin which has HSP47-binding not not show binding results obtained by the in binding the of our in the biosynthesis of a collagen-related protein the assistance of HSP47, its HSP47 binding is expected to be the HSP47 binding of and 1 and C1q, and protein were not to be the in and their folding are not to require the chaperone function of HSP47. also be that a protein during its folding with the chaperone. is a protein collagen-like is to the ER A. M. H. S. H. H. H. H. Y. T. Takahashi K. D. T. Sci. S. A. PubMed Scopus Google and the HSP47 proteins are in the same the relationship between the folding of the collagen-related proteins and the function of HSP47 are since HSP47 was reported to be in protein A T. J. S. H. Y. M. Y. S. 2000; PubMed Scopus Google there be a relationship between the folding of proteins and HSP47 Although was shown to interact with HSP47 in it is in the K. Y. S. T. T. H. T. H. Y. A. T. N. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). that is to be a for the HSP47. also was shown to interact with HSP47 in the major of was reported to be M. J. Google Scholar) ER contains little HSP47. In addition, collagen-like as MBL2, receptor with collagenous and not HSP47-binding not was revealed that not all of collagen-related proteins require the assistance of HSP47. To the molecular of the function of a it is important to the structural of its the in which family proteins were revealed to in to a of their molecular J. PubMed Scopus Google Scholar, S. Full Text PDF PubMed Scopus Google Scholar, S. L. J. EMBO J. PubMed Scopus Google Scholar). other HSP47 with correctly that is clients (8Koide T. Asada S. Takahara Y. Nishikawa Y. Nagata K. Kitagawa K. J. Biol. Chem. 2006; 281: 3432-3438Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In the present we elucidated that the side chain structure of the Yaa-3 residue is a structural determinant for specific interaction with HSP47, in to the previously reported Arg residue at (6Koide T. Takahara Y. Asada S. Nagata K. J. Biol. Chem. 2002; 277: 6178-6182Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 7Tasab M. Jenkinson L. Bulleid N.J. J. Biol. Chem. 2002; 277: 35007-35012Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). was also shown that HSP47 recognizes Yaa-3 residues with and that the structure of Yaa-3 the affinity for HSP47 Moreover, binding assays heterotrimeric triple-helical peptides with fixed chain in of the structural epitope in the triple In this the side chains of Arg0 and Yaa-3 out from the same the structural of this side chain arrangement the HSP47-binding the possible chain to be the with the determined molecular of HSP47 in (8Koide T. Asada S. Takahara Y. Nishikawa Y. Nagata K. Kitagawa K. J. Biol. Chem. 2006; 281: 3432-3438Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). HSP47 is to the of procollagen triple helices by of folded triple-helical intermediates (3Tasab M. Batten M.R. Bulleid N.J. EMBO J. 2000; 19: 2204-2211Crossref PubMed Scopus (174) Google Scholar, 4Koide T. Aso A. Yorihuzi T. Nagata K. J. Biol. Chem. 2000; 275: 27957-27963Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). was by a that the of I collagen is body temperature E. N. S. Sci. S. A. 2002; PubMed Scopus Google the of this is J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). HSP47 was suggested to function as an of formation by in the ER, based on in experiments (5Thomson C.A. Ananthanarayanan V.S. Biochem. J. 2000; 349: 877-883Crossref PubMed Scopus (52) Google Scholar). the as to which function the major role of HSP47 in results to the we the In the present study, collagens were shown to a number of HSP47-binding with on the of their Yaa-3 residues 4 and the the role of HSP47 as a folding of require triple helix an could be by an in the of HSP47 in the The binding of HSP47 to affinity not normal could this has been an important whether all collagens and other proteins containing collagenous triple helices require the assistance of HSP47 during their biosynthesis. HSP47-binding that are in the primary and database a number of possible clients for HSP47. In all types of collagen are predicted to interact with HSP47, and the HSP47 binding were in their mouse and not is strongly suggested that all types of collagen are clients of HSP47 they in has been little information available regarding the interactions of HSP47 with other collagen-related proteins. the binding of HSP47, not all collagen were by the search for the HSP47 and of a protein with that of the chaperone. The relationship between and the biosynthesis of candidate clients be of collagen by HSP47 function is expected to be a mechanism for the of since the of in various and has been shown to be with in HSP47 H. M. Hirayoshi K. Nagata K. J. 1994; PubMed Scopus Google Scholar, T. H. T. H. K. Fujii T. H. M. J. S. J. 2004; PubMed Scopus Google Scholar, T. PubMed Scopus (37) Google Scholar). To date, the of against HSP47 was reported to the of collagen in M. K. N. H. Nagata K. T. T. J. PubMed Scopus Google Scholar) and M. T. K. A. T. S. 2003; PubMed Scopus Google Scholar). molecular were also to inhibit HSP47 function in C.A. Ananthanarayanan V.S. J. Med. Chem. PubMed Scopus Google Scholar). In the present study, we that an HSP47-binding containing the of a at Yaa-3 and an Arg at the binding to HSP47. The structural of the interactions elucidated structural information for the of structure-activity relationship study including amino acid residues a structure of an HSP47 are to of for the with