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Hsp47: a collagen-specific molecular chaperone
REVIEWS
TIBS 21 - JANUARY 1996
Hsp47: a collagen-specific molecular chaperone Kazuhiro Nagata Hsp47 is a novel stress protein in the endoplasmic reticulum that binds specifically to various types of collagens and procollagens. Hsp47 transiently associates with procollagen and is involved in collagen processing and/or secretion under normal conditions. Under conditions of stress, Hsp47 is part of the quality control system for procollagen, including the prevention of the secretion of procollagen with abnormal conformation. In addition to its role as a molecular chaperone, Hsp47 synthesis always parallels that of collagen in developing tissues and various cell lines, and in collagen-related pathological conditions such as fibrosis. THE ORIGINAL EXPERIMENTS demonstrating that urea-denatured ribonuclease spontaneously refolds after the denaturing buffer is diluted prompted Anfinsen to suggest that the information contained within the protein sequence is sufficient for its correct foldingL Although this paradigm is still essentially correct, it is now recognized that, in the crowded contents of the cell, molecular chaperones are required for proteins to fold correctly. Proteins destined for secretion, or for incorporation into membranes, enter the endoplasmic reticulum (ER) cotranslationally, where they fold and assemble into their active conformations. Within the ER are enzymes that form disulfide bonds, such as protein disulfide isomerase (PDI) and peptidylprolyl isomerase (PPlase); and chaperone proteins, including the glucose-regulated proteins GRP94 and BiP/GRP78, calnexin (an ER-membrane glycoprotein) and calreticulin (located in the ER lumen). Hsp47 is another resident ER protein, first identified as the major collagen-binding heat-inducible glycoprotein in fibroblasts 2,3. It is characterized by its substrate specificity for collagen, and plays a major role in collagen processing and quality control under stress conditions, by preventing the secretion of procotlagen with abnormal conformation.
K. Nagatais at the Departmentof Cell Biology,ChestDiseaseResearchInstitute, KyotoUniversity,Sakyo-ku,Kyoto606-01, Japan. 9 1996,ElsevierScienceLtd
Molecular characterization of Hsp47
immunofluorescence and immunogoldelectron microscopy9.
Hsp47 binds transiently to procollagen The binding of Hsp47 to collagen has been examined in vitro using the optical sensor system BIAcoreTM, which is based on detecting surface plasmon resonance. The association and dissociation rate constants, and the dissociation constant have been determined using purified collagens (types l-V) and recombinant mouse Hsp47 (rmHsp47) ~~ Dissociation constants of rmHsp47 with type I-V collagens are 10-6-10 -7 M, which is relatively low for molecular interactions and can be accounted for by the rapid dissociation rate constant (Kd > 10-2s-1) and considerably high association rate constant (Ka~2• -is-i). The binding of Hsp47 with collagen is pH sensitive both in vitro and in vivo; indeed, when the pH of the buffer is lowered to around 6.3, no binding is observed ~~ Transient binding of Hsp47 with procollagen is also observed in vivo. Immunoprecipitation of labeled cell extract with anti-Hsp47 or anti-collagen antibody reveals that both Hsp47 and procollagen co-precipitate with either antibody, implying an association between Hsp47 and procollagen within the cells tt. In addition, BiP/GRP78 and PDI are also co-precipitated with the Hsp47-procollagen complex. Cyclophilin B, but not yet calnexin, has been identified as a component of the complex ~2. Although the biological implications of this complex are not clear, a functionally active complex consisting of several chaperones might facilitate the processing and/or secretion of procollagen.
Hsp47, after its initial identification, was demonstrated to be transformation sensitive 2. Two other gelatin-binding proteins, colligin 4 and gp46 (Ref. 5), were also discovered in the mouse teratocarcinoma cell-line F9 and rat L6 myoblasts, respectively, and were later established to be identical to Hsp47. As Hsp47 binds to collagen, it can easily be purified by gelatin-Sepharose or type-I-collagen-coupled Sepharose beads from both cultured cells and chick embryos. Hsp47 has a molecular mass of approximately 47 kDa and its isoelectric point is around pH9. Hsp47 cDNA has been cloned independently by Wang et al. 6 from F9 cells after differentiation induced by retinoic acid, and by Hirayoshi et al. 7, who isolated a clone encoding Hsp47 from a cDNA library of heat-shocked chick embryo flbro- Associations in the ER The dynamic relationship between blasts. Both cDNAs, called J6 (Ref. 6) and MH47a (Ref. 7), encode a protein Hsp47 and procollagen has been carebelonging to the serpin (serine protease fully examined by pulse--chase experiinhibitor) superlamily, which also in- ments combined with crosslinking and cludes afantitrypsin, aFantichymotryp- immunoprecipitation analysis u. Under sin, antithrombin Ill and ovalbumin. normal conditions, Hsp47 dissociates Some members of the family, including from procollagen within 10-15 min after the J6 and MH47a gene products, have pulse labeling, while the amount of total no protease inhibitor activity. Hsp47 procollagen that is precipitated with an has a signal sequence at its amino anti-collagen antibody decreases after terminal and an ER-retention signal 20-30 min. However, under heat shock sequence, RDEL (Arg-Asp-Glu-Leu) at stress or when the cells are treated with its carboxy terminus 7. Hsp47 also has an iron chelater, such as ~,ed-dipyridyl two sites that can be glycosylated with (which inhibits triple helix formation of high-mannose-type sugar moieties 8. The procollagen; see Fig. 1), there is no presence of these signal sequences dissociation of Hsp47 from procollagen and glycosylation sites suggest that within the two hour chase period of the Hsp47 localizes to the ER; this has experiment. Confocal microscopy, using also been directly demonstrated by cells labeled with both anti-Hsp47 and
23
REVIEWS
TIBS 21 - JANUARY 1 9 9 6
Endoplasmic reticulum
Golgi stack cis
Translation
e
medial
trans
c
N
ccc s s
0
Procollagen NN
N
cqo~'-dipyridyl - - ~ Secretion
NI
Brefeldin A
Monensin
cis-Golgi network
Bafiromycin A1
trans-Golgi network
Rgure 1 Hsp47 binds to the nascent procollagen polypeptides immediately after they are co-translated into the endoplasmic reticulum (ER). The triple helix of type I procollagen is then formed: two (~1(I) and one c~2(I)chains of procollagen make contact at their carboxyltermini initially by forming inter-chain disulfide bonds. The formation of the triple helix then proceeds specifically from the carboxylto the amino terminus29, which provides the appropriate alignment and orientation to ensure correct helix formation. The triple helix then proceeds through the Golgi complex, where it dissociates from the Hsp47 in the cis compartment, before continuing on through the trans-Golgi network to be secreted at the cell surface. Hsp47 molecules are cycled back to the ER when bound to receptors in the cis compartment that recognize the RDEL peptide. The sites of action of inhibitors used to study the passage of procollagen are indicated.
anti-collagen antibodies, also indicates the co-localization of Hsp47 and procollagen in the ER under these conditions (M. Satoh et al., unpublished). Similar observations have been reported for BiP13 and calnexin 14. In fact, when the correct folding of newly synthesized polypeptides is inhibited, either by preventing glycosylation or by introducing folding-incompetent mutant molecules, these unfolded or misfolded proteins are bound with BiP or calnexin for much longer periods resulting in their retention in the ER15. Thus, Hsp47 might also be involved in a quality control mechanism similarly to BiP and calnexin.
unpublished). Confocal microscopy clearly indicates that both Hsp47 and procollagen co-localize in the intermediate compartment. When the cells are treated with monensin or bafiromycin A1 (which are reported to inhibit the secretion at the post-cis-Golgi or posttrans-Golgi compartments, respectively; see Fig. 1), there is no association of procollagen with Hsp47, and the accumulation of procollagen within the cell is clearly shown to be in a compartment distinct from that of Hsp47 (M. Satoh et al., unpublished). This series of experiments shows that Hsp47 dissociates from procollagen in the cisGolgi network (Fig. 1).
Associations in the Golgi Hsp47 and procollagen interactions Triple helix formation and Hsp47 Hsp47 was shown to bind to a further along the secretory pathway have also been studied. If transport nascent polypeptide of ctl(1) procolfrom the ER to the Golgi is inhibited lagen by immunoprecipitating a polywith brefeldin A or by low temperature some fraction with anti-Hsp47 antibody (15~ procollagen is not dissociated and by short pulse-chase experiments from Hsp47, which is revealed by (Ref. 16; M. Satoh et al., unpublished). pulse--chase experiments (M. Satoh et aL, However, Hsp47 also binds to mature
24
triple-helix procollagen as well as mature collagen without propeptides in vitro and in vivo (Refs 3, 9; M. Satoh et al., unpublished). By incorporating these observations, Fig. 1 shows a schematic representation of the interaction of Hsp47 during folding and assembly of procollagen. Procollagen chains must be unfolded until the whole chain has been translated and has entered the ER before the carboxyl termini make contact and triple-helix formation begins. Hsp47 could, therefore, prevent the incorrect folding and aggregation of these nascent polypeptide chains until the correct carboxy-terminal associations have been made. Although there are no data as to how Hsp47 facilitates procollagen triple helix formation, it is certainly true that Hsp47 remains bound to helical procollagen in the ER. The low affinity of Hsp47 for collagen (10-6-10-7 M) allows rapid association and dissociation during this process. If forms of stress, such as heat shock or the exposure to a,a'-dipyridyl,
TIBS 21 - JANUARY 1996
REVIEWS
Hsp47 gene reveals a well-conserved expression of Hsp4 7 was examined in an heat-shock element consisting of three in vivo pathological situation. Liver tandem repeats of five base pair units fibrosis is frequently observed in chronic (nGAAn, where n is any nucleotide) at liver diseases such as cirrhosis, where the promoter region (around -80 from there is an accumulation and disorder the transcription start site) 2~ This sug- of fibrous connective tissue. In a rat gests that under stressful conditions, liver fibrosis model, prepared by oral Hsp47 is induced via a heat-shock- administration of carbon tetrachloride, element-heat-shock-factor system as the mPd~lA levels of genes encoding with other stress proteins, although it Hsp47 and types I and lII collagens are cannot be ruled out that the parallel dramatically upregulated during the expression of Hsp47 with collagen is progression of fibrosis, whereas the regulated by other cis-elements. One levels of these mPd~lAs are barely candidate for such a regulatory mecha- detectable in normal rat liver. It is innism is the GATAfamily of transcription teresting to note that only cells around factors, which are reported to function the collagen fibrils actively synthesize during the differentiation of F9 cells. Hsp47 mRj~IA,which again suggests the The Hsp47 gene has GATA-binding functional involvement of Hsp47 in motifs in the promoter region and collagen accumulation. Coexpression of these elements are occupied by GATA-4 the genes encoding both Hsp47 and protein after the differentiation of F9 collagen is also reported in the develop ceils with retinoic acid 2~,22. However, ment of murine femurs and molars 23 whether collagen induction during F9 and various chick tissues 24. differentiation is also regulated by the same factors, and also whether this Heat shock causes the Hsp47geneto be parallel induction of Hsp47 and col- alternatively spliced ?mother interesting aspect in the lagen is regulated by the same mechaSynthesis of Hsp47 parallels that nism in other cell lines or systems, is expression of Hsp47 is the alternative of collagen splicing caused by heat shock. The It is well known that the synthesis not yet clear. Does this close relationship of Hsp47 Hsp47 cDNAs, J6 ~ef. 6) isolated by of type I collagen, a major collagen secreted by fibroblasts, decreases after with collagens extend to collagen func- subtractive cloning of F9 ceils before malignant transformation of these cells, tion as well as to their regulation? To and after differentiation (and renamed and that this decrease in synthesis is investigate this question further, the by Takechi, H. et al. 25 as MH47b and concomitant with that of other extracellular matrix proteins, including fibroTable I. Correlation of Hsp47 synthesis to collagen synthesis nectin and laminin. When fibroblasts are transformed with Rous sarcoma Collagen (type) virus, simian virus 40 or the oncogenic protein c-Ha-Ras, synthesis of both type I Cell/tissue type Cell line/type Hsp47 I II Ill IV collagen and Hsp47 decreases by the Fibroblast CEF +++ +++ same extent 2,18. However, both type IV RSV-transformed CEF + + collagen and Hsp47 (colligin) are draBALB/3T3 +++ +++ SV40-transformed 3T3 + + matically induced during the differentiNIH3T3 ++ ++ ation of F9 cells by either retinoic acid c-Ha4Ras-transformed 3T3 + + alone, or retinoic acid in combination with dibutyryl cAMP4. Interestingly, in 3T3&1 +++ +++ + Adipocyte Dif~rentiated 3T3-L1 ++ ++ + cells that do not contain detectable levels of any type of collagen, for example, Myelocyte M1 mouse myeloid leukemia cell line M1 or mouse pheochromocytoma cell line Neurocyte PC12 PC12, do not synthesize Hsp47 either. F9 Jr 4Embryonic Clarke et aL [9 reported the parallel carcinoma F9a ++ ++ induction of Hsp47 with collagens after F9b +++ +++ treatment of the myoblastic cell lines Hepatocyte . . . . . with transforming growth factor [~-1 Hepatic tissue Itch cells ++ ++ ~GF[3-1), suggesting that the expression Fibrosis in rat liver +++ +++ +++ of Hsp47 is always accompanied by that Bone Osteoblasts +++ +++ of collagen (Table I). The exception to this rule is when Cartilage Chondrocytes +++ +++ Hsp47 is induced by heat shock or Teeth Odontoblasts ++ ++ other forms of stress, after which Hsp47 is rapidly induced while collagen synaTreated with retinoic acid. thesis is either unaffected or even de~Treated with retinoic acid and dibutyryl cAMP. creases 7. Closer analysis of the cloned
cause the inhibition of procollagen triple-helix formation, Hsp47 must therefore act as a quality control step to prevent the secretion of abnormal collagen from the cells. Consistent with this, Hsp47 has a higher affinity for heatdenatured collagen than for mature collagen (T. Natsume et al., unpublished). Recently, Hsp47 has been reported to prevent the degradation of procollagen in the ER17. Future experiments should determine whether the inhibition of procollagen degradation is due to the serpin activity of Hsp47 ~efs 6, 7). It is likely that Hsp47 recycles back to the ER via the Golgi. Hsp47 probably gets trapped by receptors in the Golgi membrane that recognize the RDEL p e p tide ~erhaps KJ)ELreceptors), accounting for the low concentration of free Hsp47 in the Golgi compartment, which in turn allows the rapid dissociation of Hsp47 from procollagen in the cis-Golgi compartment. Hsp47 is then free to shuttle back to the ER.
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REVIEWS (a)
TIBS 2 1 - J A N U A R Y 1 9 9 6
Normal temperature MH47b
II
MH47c
~
III
However, it remains to be seen whether the alternative splicing caused by heat shock can be generalized to other stress proteins or even to other cellular proteins.
Can the concept for substratespecific molecular chaperone be generalized?
Hsp47 can be thought of as a substrate-specific molecular chaperone that only interacts with certain proIll collagens within the cells. II l However, both BiP and calI! nexin were also reported to bind specific substrates MH47a at first (BiP was shown to bind to immunoglobulin Figure 2 heavy chain, and calnexin Alternative splicing of Hsp47 5' non-coding region under to class I major histo(a) normal, and (b) heat shock conditions. Non-coding compatibility molecules14), exons are colored green and coding exons are colored but these chaperones have red (three additional exons are present downstream since been found to bind a of the transcript portion shown). (a) Under normal wide spectrum of secretory conditions, the transcript MH47b (comprising 5' nonand membrane proteins in coding regions la and III) predominates in the cell, although small amounts of MH47c (comprising 5' nonthe ER. Although Hsp47 coding regions la, II and III) are detectable. MH47a is was reported to bind fetuin not present. (b) After heat shock, the MH47c tranin vitro, immunoprecipiscript disappears and the MH47a transcript (consisttation shows that the major ing of regions la, Ib and III) accumulates. secretory protein co-precipitated with Hsp47 is procolMH47c), and MH47a ~ef. 25) cloned lagen in the fibroblasts 3,u. A more from a cDNA library of heat-shocked detailed examination of whether Hsp47 mouse fibroblasts, are identical except binds to other secretory or membrane for the 5' non-coding region ~ig. 2). proteins in the ER is still required. Under normal conditions, transcripts Other substrate-specific molecular consist of MH47b as the major compo- chaperones have also been reported. For nent and MH47c as the minor compo- example, the TCP-1 complex (a heteronent, while MH47a is not detected at all. oligomeric chaperonin containing tHowever, MH47a and MH47b become complex polypeptide 1), is reported to the major transcripts after heat shock be specific for actin and tubulin as a at 42~ or at 45~ while MH47c disap- molecular chaperone 27. Also, a 39kDa pears 2S. While MH47a is induced after receptor-associated protein has been heat shock, it is induced neither by reported to be an ER-resident molecular treatment with other stress-causing chaperone specific for low-density reagents such as arsenite and azetidine, lipoprotein-receptor-related proteins 28. nor after differentiation of F9 cells. These findings suggest that substrateMH47a, which has a long 5' non-coding specific molecular chaperones might region, is shown to be more efficiently play important roles in a variety of celtranslated at high temperature (42~ or lular events, particularly in folding and 45~ 2s. Thus, the induction of Hsp47 assembly of multimeric and heteroafter heat shock is regulated mainly at oligomeric proteins, such as collagen, the transcriptional level, but also at the actin and tubulin. Thus, it seems likely translational level. This was, essentially, that even more substrate-specific the first evidence that exogenous molecular chaperones will be found stimuli such as heat shock can induce in the future. alternative splicing. It will also be important to examVery recently, we have found that the ine whether these substrate-specific gene encoding mouse HspI05 is also chaperones work with other general alternatively spliced after heat shock, chaperones, such as BiP and calnexin. resulting in the formation of different Hsp47 is reported to associate with mPd~lAs at 42~ and at 45~ ~ef. 26). BiP, PDI and cyclophilin B11,12,but it is (b) Heat shock
26
not clear whether this association has biological implications for its chaperone function.
References 1 Anfinsen, C. B. (1973) Science 181, 223-230 2 Nagata, K. and Yamada, K. M. (1986) J. Biol. Chem. 261, 7531-7536 3 Nagata, K., Saga, S. and Yamada, K. M. (1986) J. Cell Biol. 103, 223-229 4 Kurkinen, M., Taylor,A., Garrels, G. I. and Hogan, B. L. M. (1984) J. Biol. Chem. 259, 5915-5922 5 Cates, G. A., Nandan, D., Brickenden, A. M. and Sanwal, B. D. (1987) Biochem. Cell Biol. 65, 767-775 6 Wang, S. Y. and Gudas, L. J. (1990) J. Biol. Chem. 265, 15818-15822 7 Hirayoshi, K. et al. (1991) Mol. Cell. Biol. 11, 4036--4044 8 Hughes, R. C., Taylor, A., Sage, H. and Hogan, B. L. M. (1987) Eur. J. Biochem. 163, 57-65 9 Saga, S., Nagata, K., Chen, W-T. and Yamada, K. M. (1987) J. Cell Biol. 105, 517-527 10 Natsume, T. et al. (1994) J. Biol. Chem. 269, 31224-31228 11 Nakai, A., Satoh, M., Hirayoshi, K. and Nagata, K. (1992) J. Cell Biol. 117, 903-914 12 Smith, T. et al. (1995) J. Biol. Chem. 270, 18323-18328 13 Gething, M-J. and Sambrook, J. F. (1992) Nature 355, 33-45 14 Bergeron, J. J. M., Brenner, M. B., Thomas, D. Y. and Williams, D. B. (1994) Trends Biochem. Sci. 19, 124-128 15 Helenius, A., Marquart, T. and Braakman, I. (1992) Trends Cell Biol. 2, 227-231 16 Sauk, J. J., Smith, T., Norris, K. and Ferreira, L. (1994) J. Biol. Chem. 269, 3941-3946 17 Jain, N., Brickenden, A., Ball, E. H. and Sanwal, B. D. (1994) Arch. Biochem. Biophys. 314, 23-30 18 Nakai, A., Hirayoshi, K. and Nagata, K. (1990) J. Biol. Chem. 265, 992-999 19 Clarke, E. P. et al. (1993) J. Cell Biol. 121, 193-199 20 Hosokawa, N. et al. (1993) Gene 126, 187-193 21 Bielinska, M. and Wilson D. B. (1995) Biochem. J. 307,183-189 22 Wang, S. Y. (1994) J. Biol. Chem. 269, 607-613 23 Shroff, B. et al. (1993) Connect. Tissue Res. 29, 273-286 24 Miyaishi, 0., Sakata, K., Matsuyama, M. and Saga, S. (1992) J. Histochem. Cytochem. 40, 1021-1029 25 Takechi, H., Hosokawa, N., Hirayoshi, K. and Nagata, K. (1994) Mol. Cell. Biol. 14, 567-575 26 Yasuda, K., Nakai, A., Hatayama, T. and Nagata, K. J. Biol. Chem. (in press) 27 Willison, K. R. and Kubota, H. (1994) in The Biology of Heat Shock Proteins and Molecular Chaperones, pp. 299-312, Cold Spring Harbor
Press 28 Bu, G., Geuze, H. J., Strous, G. J. and Schwartz, A. L. (1995) EMBO J. 14, 2269-2280 29 Mayne, R. and Burgeson, R. E. (1987) Structure and Function of Collagen Types, Academic Press
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TIBS 21 - JANUARY 1996
Hsp47: a collagen-specific molecular chaperone Kazuhiro Nagata Hsp47 is a novel stress protein in the endoplasmic reticulum that binds specifically to various types of collagens and procollagens. Hsp47 transiently associates with procollagen and is involved in collagen processing and/or secretion under normal conditions. Under conditions of stress, Hsp47 is part of the quality control system for procollagen, including the prevention of the secretion of procollagen with abnormal conformation. In addition to its role as a molecular chaperone, Hsp47 synthesis always parallels that of collagen in developing tissues and various cell lines, and in collagen-related pathological conditions such as fibrosis. THE ORIGINAL EXPERIMENTS demonstrating that urea-denatured ribonuclease spontaneously refolds after the denaturing buffer is diluted prompted Anfinsen to suggest that the information contained within the protein sequence is sufficient for its correct foldingL Although this paradigm is still essentially correct, it is now recognized that, in the crowded contents of the cell, molecular chaperones are required for proteins to fold correctly. Proteins destined for secretion, or for incorporation into membranes, enter the endoplasmic reticulum (ER) cotranslationally, where they fold and assemble into their active conformations. Within the ER are enzymes that form disulfide bonds, such as protein disulfide isomerase (PDI) and peptidylprolyl isomerase (PPlase); and chaperone proteins, including the glucose-regulated proteins GRP94 and BiP/GRP78, calnexin (an ER-membrane glycoprotein) and calreticulin (located in the ER lumen). Hsp47 is another resident ER protein, first identified as the major collagen-binding heat-inducible glycoprotein in fibroblasts 2,3. It is characterized by its substrate specificity for collagen, and plays a major role in collagen processing and quality control under stress conditions, by preventing the secretion of procotlagen with abnormal conformation.
K. Nagatais at the Departmentof Cell Biology,ChestDiseaseResearchInstitute, KyotoUniversity,Sakyo-ku,Kyoto606-01, Japan. 9 1996,ElsevierScienceLtd
Molecular characterization of Hsp47
immunofluorescence and immunogoldelectron microscopy9.
Hsp47 binds transiently to procollagen The binding of Hsp47 to collagen has been examined in vitro using the optical sensor system BIAcoreTM, which is based on detecting surface plasmon resonance. The association and dissociation rate constants, and the dissociation constant have been determined using purified collagens (types l-V) and recombinant mouse Hsp47 (rmHsp47) ~~ Dissociation constants of rmHsp47 with type I-V collagens are 10-6-10 -7 M, which is relatively low for molecular interactions and can be accounted for by the rapid dissociation rate constant (Kd > 10-2s-1) and considerably high association rate constant (Ka~2• -is-i). The binding of Hsp47 with collagen is pH sensitive both in vitro and in vivo; indeed, when the pH of the buffer is lowered to around 6.3, no binding is observed ~~ Transient binding of Hsp47 with procollagen is also observed in vivo. Immunoprecipitation of labeled cell extract with anti-Hsp47 or anti-collagen antibody reveals that both Hsp47 and procollagen co-precipitate with either antibody, implying an association between Hsp47 and procollagen within the cells tt. In addition, BiP/GRP78 and PDI are also co-precipitated with the Hsp47-procollagen complex. Cyclophilin B, but not yet calnexin, has been identified as a component of the complex ~2. Although the biological implications of this complex are not clear, a functionally active complex consisting of several chaperones might facilitate the processing and/or secretion of procollagen.
Hsp47, after its initial identification, was demonstrated to be transformation sensitive 2. Two other gelatin-binding proteins, colligin 4 and gp46 (Ref. 5), were also discovered in the mouse teratocarcinoma cell-line F9 and rat L6 myoblasts, respectively, and were later established to be identical to Hsp47. As Hsp47 binds to collagen, it can easily be purified by gelatin-Sepharose or type-I-collagen-coupled Sepharose beads from both cultured cells and chick embryos. Hsp47 has a molecular mass of approximately 47 kDa and its isoelectric point is around pH9. Hsp47 cDNA has been cloned independently by Wang et al. 6 from F9 cells after differentiation induced by retinoic acid, and by Hirayoshi et al. 7, who isolated a clone encoding Hsp47 from a cDNA library of heat-shocked chick embryo flbro- Associations in the ER The dynamic relationship between blasts. Both cDNAs, called J6 (Ref. 6) and MH47a (Ref. 7), encode a protein Hsp47 and procollagen has been carebelonging to the serpin (serine protease fully examined by pulse--chase experiinhibitor) superlamily, which also in- ments combined with crosslinking and cludes afantitrypsin, aFantichymotryp- immunoprecipitation analysis u. Under sin, antithrombin Ill and ovalbumin. normal conditions, Hsp47 dissociates Some members of the family, including from procollagen within 10-15 min after the J6 and MH47a gene products, have pulse labeling, while the amount of total no protease inhibitor activity. Hsp47 procollagen that is precipitated with an has a signal sequence at its amino anti-collagen antibody decreases after terminal and an ER-retention signal 20-30 min. However, under heat shock sequence, RDEL (Arg-Asp-Glu-Leu) at stress or when the cells are treated with its carboxy terminus 7. Hsp47 also has an iron chelater, such as ~,ed-dipyridyl two sites that can be glycosylated with (which inhibits triple helix formation of high-mannose-type sugar moieties 8. The procollagen; see Fig. 1), there is no presence of these signal sequences dissociation of Hsp47 from procollagen and glycosylation sites suggest that within the two hour chase period of the Hsp47 localizes to the ER; this has experiment. Confocal microscopy, using also been directly demonstrated by cells labeled with both anti-Hsp47 and
23
REVIEWS
TIBS 21 - JANUARY 1 9 9 6
Endoplasmic reticulum
Golgi stack cis
Translation
e
medial
trans
c
N
ccc s s
0
Procollagen NN
N
cqo~'-dipyridyl - - ~ Secretion
NI
Brefeldin A
Monensin
cis-Golgi network
Bafiromycin A1
trans-Golgi network
Rgure 1 Hsp47 binds to the nascent procollagen polypeptides immediately after they are co-translated into the endoplasmic reticulum (ER). The triple helix of type I procollagen is then formed: two (~1(I) and one c~2(I)chains of procollagen make contact at their carboxyltermini initially by forming inter-chain disulfide bonds. The formation of the triple helix then proceeds specifically from the carboxylto the amino terminus29, which provides the appropriate alignment and orientation to ensure correct helix formation. The triple helix then proceeds through the Golgi complex, where it dissociates from the Hsp47 in the cis compartment, before continuing on through the trans-Golgi network to be secreted at the cell surface. Hsp47 molecules are cycled back to the ER when bound to receptors in the cis compartment that recognize the RDEL peptide. The sites of action of inhibitors used to study the passage of procollagen are indicated.
anti-collagen antibodies, also indicates the co-localization of Hsp47 and procollagen in the ER under these conditions (M. Satoh et al., unpublished). Similar observations have been reported for BiP13 and calnexin 14. In fact, when the correct folding of newly synthesized polypeptides is inhibited, either by preventing glycosylation or by introducing folding-incompetent mutant molecules, these unfolded or misfolded proteins are bound with BiP or calnexin for much longer periods resulting in their retention in the ER15. Thus, Hsp47 might also be involved in a quality control mechanism similarly to BiP and calnexin.
unpublished). Confocal microscopy clearly indicates that both Hsp47 and procollagen co-localize in the intermediate compartment. When the cells are treated with monensin or bafiromycin A1 (which are reported to inhibit the secretion at the post-cis-Golgi or posttrans-Golgi compartments, respectively; see Fig. 1), there is no association of procollagen with Hsp47, and the accumulation of procollagen within the cell is clearly shown to be in a compartment distinct from that of Hsp47 (M. Satoh et al., unpublished). This series of experiments shows that Hsp47 dissociates from procollagen in the cisGolgi network (Fig. 1).
Associations in the Golgi Hsp47 and procollagen interactions Triple helix formation and Hsp47 Hsp47 was shown to bind to a further along the secretory pathway have also been studied. If transport nascent polypeptide of ctl(1) procolfrom the ER to the Golgi is inhibited lagen by immunoprecipitating a polywith brefeldin A or by low temperature some fraction with anti-Hsp47 antibody (15~ procollagen is not dissociated and by short pulse-chase experiments from Hsp47, which is revealed by (Ref. 16; M. Satoh et al., unpublished). pulse--chase experiments (M. Satoh et aL, However, Hsp47 also binds to mature
24
triple-helix procollagen as well as mature collagen without propeptides in vitro and in vivo (Refs 3, 9; M. Satoh et al., unpublished). By incorporating these observations, Fig. 1 shows a schematic representation of the interaction of Hsp47 during folding and assembly of procollagen. Procollagen chains must be unfolded until the whole chain has been translated and has entered the ER before the carboxyl termini make contact and triple-helix formation begins. Hsp47 could, therefore, prevent the incorrect folding and aggregation of these nascent polypeptide chains until the correct carboxy-terminal associations have been made. Although there are no data as to how Hsp47 facilitates procollagen triple helix formation, it is certainly true that Hsp47 remains bound to helical procollagen in the ER. The low affinity of Hsp47 for collagen (10-6-10-7 M) allows rapid association and dissociation during this process. If forms of stress, such as heat shock or the exposure to a,a'-dipyridyl,
TIBS 21 - JANUARY 1996
REVIEWS
Hsp47 gene reveals a well-conserved expression of Hsp4 7 was examined in an heat-shock element consisting of three in vivo pathological situation. Liver tandem repeats of five base pair units fibrosis is frequently observed in chronic (nGAAn, where n is any nucleotide) at liver diseases such as cirrhosis, where the promoter region (around -80 from there is an accumulation and disorder the transcription start site) 2~ This sug- of fibrous connective tissue. In a rat gests that under stressful conditions, liver fibrosis model, prepared by oral Hsp47 is induced via a heat-shock- administration of carbon tetrachloride, element-heat-shock-factor system as the mPd~lA levels of genes encoding with other stress proteins, although it Hsp47 and types I and lII collagens are cannot be ruled out that the parallel dramatically upregulated during the expression of Hsp47 with collagen is progression of fibrosis, whereas the regulated by other cis-elements. One levels of these mPd~lAs are barely candidate for such a regulatory mecha- detectable in normal rat liver. It is innism is the GATAfamily of transcription teresting to note that only cells around factors, which are reported to function the collagen fibrils actively synthesize during the differentiation of F9 cells. Hsp47 mRj~IA,which again suggests the The Hsp47 gene has GATA-binding functional involvement of Hsp47 in motifs in the promoter region and collagen accumulation. Coexpression of these elements are occupied by GATA-4 the genes encoding both Hsp47 and protein after the differentiation of F9 collagen is also reported in the develop ceils with retinoic acid 2~,22. However, ment of murine femurs and molars 23 whether collagen induction during F9 and various chick tissues 24. differentiation is also regulated by the same factors, and also whether this Heat shock causes the Hsp47geneto be parallel induction of Hsp47 and col- alternatively spliced ?mother interesting aspect in the lagen is regulated by the same mechaSynthesis of Hsp47 parallels that nism in other cell lines or systems, is expression of Hsp47 is the alternative of collagen splicing caused by heat shock. The It is well known that the synthesis not yet clear. Does this close relationship of Hsp47 Hsp47 cDNAs, J6 ~ef. 6) isolated by of type I collagen, a major collagen secreted by fibroblasts, decreases after with collagens extend to collagen func- subtractive cloning of F9 ceils before malignant transformation of these cells, tion as well as to their regulation? To and after differentiation (and renamed and that this decrease in synthesis is investigate this question further, the by Takechi, H. et al. 25 as MH47b and concomitant with that of other extracellular matrix proteins, including fibroTable I. Correlation of Hsp47 synthesis to collagen synthesis nectin and laminin. When fibroblasts are transformed with Rous sarcoma Collagen (type) virus, simian virus 40 or the oncogenic protein c-Ha-Ras, synthesis of both type I Cell/tissue type Cell line/type Hsp47 I II Ill IV collagen and Hsp47 decreases by the Fibroblast CEF +++ +++ same extent 2,18. However, both type IV RSV-transformed CEF + + collagen and Hsp47 (colligin) are draBALB/3T3 +++ +++ SV40-transformed 3T3 + + matically induced during the differentiNIH3T3 ++ ++ ation of F9 cells by either retinoic acid c-Ha4Ras-transformed 3T3 + + alone, or retinoic acid in combination with dibutyryl cAMP4. Interestingly, in 3T3&1 +++ +++ + Adipocyte Dif~rentiated 3T3-L1 ++ ++ + cells that do not contain detectable levels of any type of collagen, for example, Myelocyte M1 mouse myeloid leukemia cell line M1 or mouse pheochromocytoma cell line Neurocyte PC12 PC12, do not synthesize Hsp47 either. F9 Jr 4Embryonic Clarke et aL [9 reported the parallel carcinoma F9a ++ ++ induction of Hsp47 with collagens after F9b +++ +++ treatment of the myoblastic cell lines Hepatocyte . . . . . with transforming growth factor [~-1 Hepatic tissue Itch cells ++ ++ ~GF[3-1), suggesting that the expression Fibrosis in rat liver +++ +++ +++ of Hsp47 is always accompanied by that Bone Osteoblasts +++ +++ of collagen (Table I). The exception to this rule is when Cartilage Chondrocytes +++ +++ Hsp47 is induced by heat shock or Teeth Odontoblasts ++ ++ other forms of stress, after which Hsp47 is rapidly induced while collagen synaTreated with retinoic acid. thesis is either unaffected or even de~Treated with retinoic acid and dibutyryl cAMP. creases 7. Closer analysis of the cloned
cause the inhibition of procollagen triple-helix formation, Hsp47 must therefore act as a quality control step to prevent the secretion of abnormal collagen from the cells. Consistent with this, Hsp47 has a higher affinity for heatdenatured collagen than for mature collagen (T. Natsume et al., unpublished). Recently, Hsp47 has been reported to prevent the degradation of procollagen in the ER17. Future experiments should determine whether the inhibition of procollagen degradation is due to the serpin activity of Hsp47 ~efs 6, 7). It is likely that Hsp47 recycles back to the ER via the Golgi. Hsp47 probably gets trapped by receptors in the Golgi membrane that recognize the RDEL p e p tide ~erhaps KJ)ELreceptors), accounting for the low concentration of free Hsp47 in the Golgi compartment, which in turn allows the rapid dissociation of Hsp47 from procollagen in the cis-Golgi compartment. Hsp47 is then free to shuttle back to the ER.
25
REVIEWS (a)
TIBS 2 1 - J A N U A R Y 1 9 9 6
Normal temperature MH47b
II
MH47c
~
III
However, it remains to be seen whether the alternative splicing caused by heat shock can be generalized to other stress proteins or even to other cellular proteins.
Can the concept for substratespecific molecular chaperone be generalized?
Hsp47 can be thought of as a substrate-specific molecular chaperone that only interacts with certain proIll collagens within the cells. II l However, both BiP and calI! nexin were also reported to bind specific substrates MH47a at first (BiP was shown to bind to immunoglobulin Figure 2 heavy chain, and calnexin Alternative splicing of Hsp47 5' non-coding region under to class I major histo(a) normal, and (b) heat shock conditions. Non-coding compatibility molecules14), exons are colored green and coding exons are colored but these chaperones have red (three additional exons are present downstream since been found to bind a of the transcript portion shown). (a) Under normal wide spectrum of secretory conditions, the transcript MH47b (comprising 5' nonand membrane proteins in coding regions la and III) predominates in the cell, although small amounts of MH47c (comprising 5' nonthe ER. Although Hsp47 coding regions la, II and III) are detectable. MH47a is was reported to bind fetuin not present. (b) After heat shock, the MH47c tranin vitro, immunoprecipiscript disappears and the MH47a transcript (consisttation shows that the major ing of regions la, Ib and III) accumulates. secretory protein co-precipitated with Hsp47 is procolMH47c), and MH47a ~ef. 25) cloned lagen in the fibroblasts 3,u. A more from a cDNA library of heat-shocked detailed examination of whether Hsp47 mouse fibroblasts, are identical except binds to other secretory or membrane for the 5' non-coding region ~ig. 2). proteins in the ER is still required. Under normal conditions, transcripts Other substrate-specific molecular consist of MH47b as the major compo- chaperones have also been reported. For nent and MH47c as the minor compo- example, the TCP-1 complex (a heteronent, while MH47a is not detected at all. oligomeric chaperonin containing tHowever, MH47a and MH47b become complex polypeptide 1), is reported to the major transcripts after heat shock be specific for actin and tubulin as a at 42~ or at 45~ while MH47c disap- molecular chaperone 27. Also, a 39kDa pears 2S. While MH47a is induced after receptor-associated protein has been heat shock, it is induced neither by reported to be an ER-resident molecular treatment with other stress-causing chaperone specific for low-density reagents such as arsenite and azetidine, lipoprotein-receptor-related proteins 28. nor after differentiation of F9 cells. These findings suggest that substrateMH47a, which has a long 5' non-coding specific molecular chaperones might region, is shown to be more efficiently play important roles in a variety of celtranslated at high temperature (42~ or lular events, particularly in folding and 45~ 2s. Thus, the induction of Hsp47 assembly of multimeric and heteroafter heat shock is regulated mainly at oligomeric proteins, such as collagen, the transcriptional level, but also at the actin and tubulin. Thus, it seems likely translational level. This was, essentially, that even more substrate-specific the first evidence that exogenous molecular chaperones will be found stimuli such as heat shock can induce in the future. alternative splicing. It will also be important to examVery recently, we have found that the ine whether these substrate-specific gene encoding mouse HspI05 is also chaperones work with other general alternatively spliced after heat shock, chaperones, such as BiP and calnexin. resulting in the formation of different Hsp47 is reported to associate with mPd~lAs at 42~ and at 45~ ~ef. 26). BiP, PDI and cyclophilin B11,12,but it is (b) Heat shock
26
not clear whether this association has biological implications for its chaperone function.
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