Aberrant binding of mutant HSP47 affects posttranslational modification of type I collagen and leads to osteogenesis imperfecta

Autoři: Delfien Syx aff001;  Yoshihiro Ishikawa aff002;  Jan Gebauer aff004;  Sergei P. Boudko aff002;  Brecht Guillemyn aff001;  Tim Van Damme aff001;  Sanne D’hondt aff001;  Sofie Symoens aff001;  Sheela Nampoothiri aff005;  Douglas B. Gould aff003;  Ulrich Baumann aff004;  Hans Peter Bächinger aff002;  Fransiska Malfait aff001
Působiště autorů: Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium aff001;  Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon, United States of America aff002;  Department of Ophthalmology, UCSF School of Medicine, San Francisco, California, United States of America aff003;  Institute of Biochemistry, University of Cologne, Cologne, Germany aff004;  Amrita Institute of Medical Sciences and Research Center, Cochin, Kerala, India aff005;  Department of Anatomy, Institute for Human Genetics, UCSF School of Medicine, San Francisco, California, United States of America aff006
Vyšlo v časopise: Aberrant binding of mutant HSP47 affects posttranslational modification of type I collagen and leads to osteogenesis imperfecta. PLoS Genet 17(2): e1009339. doi:10.1371/journal.pgen.1009339
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1009339


Heat shock protein 47 (HSP47), encoded by the SERPINH1 gene, is a molecular chaperone essential for correct folding of collagens. We report a homozygous p.(R222S) substitution in HSP47 in a child with severe osteogenesis imperfecta leading to early demise. p.R222 is a highly conserved residue located within the collagen interacting surface of HSP47. Binding assays show a significantly reduced affinity of HSP47-R222S for type I collagen. This altered interaction leads to posttranslational overmodification of type I procollagen produced by dermal fibroblasts, with increased glycosylation and/or hydroxylation of lysine and proline residues as shown by mass spectrometry. Since we also observed a normal intracellular folding and secretion rate of type I procollagen, this overmodification cannot be explained by prolonged exposure of the procollagen molecules to the modifying hydroxyl- and glycosyltransferases, as is commonly observed in other types of OI. We found significant upregulation of several molecular chaperones and enzymes involved in procollagen modification and folding on Western blot and RT-qPCR. In addition, we showed that an imbalance in binding of HSP47-R222S to unfolded type I collagen chains in a gelatin sepharose pulldown assay results in increased binding of other chaperones and modifying enzymes. The elevated expression and binding of this molecular ensemble to type I procollagen suggests a compensatory mechanism for the aberrant binding of HSP47-R222S, eventually leading to overmodification of type I procollagen chains. Together, these results illustrate the importance of HSP47 for proper posttranslational modification and provide insights into the molecular pathomechanisms of the p.(R222S) alteration in HSP47, which leads to a severe OI phenotype.

Klíčová slova:

osteogenesis imperfecta – Amino acid analysis – Binding analysis – Collagens – Fibroblasts – Gelatin – Heat shock response – Secretion


1. Birk DE, Bruckner P. Collagens, Suprastructures, and Collagen Fibril Assembly. The Extracellular Matrix: an Overview. Berlin, Heidelberg: Springer Berlin Heidelberg; 2010. pp. 77–115. doi: 10.1007/978-3-642-16555-9_3

2. Ishikawa Y, Bächinger HP. A molecular ensemble in the rER for procollagen maturation. Biochim Biophys Acta. 2013;1833: 2479–2491. doi: 10.1016/j.bbamcr.2013.04.008 23602968

3. Nagata K, Saga S, Yamada KM. A major collagen-binding protein of chick embryo fibroblasts is a novel heat shock protein. J Cell Biol. The Rockefeller University Press; 1986;103: 223–229.

4. Nagata K. Hsp47: a collagen-specific molecular chaperone. Trends Biochem Sci. 1996;21: 22–26. doi: 10.1016/0968-0004(96)80881-4 8848834

5. Nagata K. Expression and function of heat shock protein 47: a collagen-specific molecular chaperone in the endoplasmic reticulum. Matrix Biol. 1998;16: 379–386. doi: 10.1016/s0945-053x(98)90011-7 9524358

6. Ono T, Miyazaki T, Ishida Y, Uehata M, Nagata K. Direct in vitro and in vivo evidence for interaction between Hsp47 protein and collagen triple helix. J Biol Chem. American Society for Biochemistry and Molecular Biology; 2012;287: 6810–6818. doi: 10.1074/jbc.M111.280248 22235129

7. Koide T, Nishikawa Y, Asada S, Yamazaki CM, Takahara Y, Homma DL, et al. Specific recognition of the collagen triple helix by chaperone HSP47. II. The HSP47-binding structural motif in collagens and related proteins. J Biol Chem. American Society for Biochemistry and Molecular Biology; 2006;281: 11177–11185. doi: 10.1074/jbc.M601369200 16484215

8. Widmer C, Gebauer JM, Brunstein E, Rosenbaum S, Zaucke F, Drögemüller C, et al. Molecular basis for the action of the collagen-specific chaperone Hsp47/SERPINH1 and its structure-specific client recognition. Proc Natl Acad Sci USA. National Acad Sciences; 2012;109: 13243–13247. doi: 10.1073/pnas.1208072109 22847422

9. Makareeva E, Leikin S. Procollagen triple helix assembly: an unconventional chaperone-assisted folding paradigm. Lu J, editor. PLoS ONE. Public Library of Science; 2007;2: e1029. doi: 10.1371/journal.pone.0001029 17925877

10. Bonfanti L, Mironov AA, Martínez-Menárguez JA, Martella O, Fusella A, Baldassarre M, et al. Procollagen traverses the Golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation. Cell. 1998;95: 993–1003. doi: 10.1016/s0092-8674(00)81723-7 9875853

11. Ishikawa Y, Ito S, Nagata K, Sakai LY, Bächinger HP. Intracellular mechanisms of molecular recognition and sorting for transport of large extracellular matrix molecules. Proc Natl Acad Sci USA. National Acad Sciences; 2016;113: E6036–E6044. doi: 10.1073/pnas.1609571113 27679847

12. Satoh M, Hirayoshi K, Yokota S, Hosokawa N, Nagata K. Intracellular interaction of collagen-specific stress protein HSP47 with newly synthesized procollagen. J Cell Biol. 1996;133: 469–483. doi: 10.1083/jcb.133.2.469 8609177

13. Ishida Y, Nagata K. Hsp47 as a collagen-specific molecular chaperone. Meth Enzymol. Elsevier; 2011;499: 167–182. doi: 10.1016/B978-0-12-386471-0.00009-2 21683254

14. Nagai N, Hosokawa M, Itohara S, Adachi E, Matsushita T, Hosokawa N, et al. Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. J Cell Biol. 2000;150: 1499–1506. doi: 10.1083/jcb.150.6.1499 10995453

15. Drögemüller C, Becker D, Brunner A, Haase B, Kircher P, Seeliger F, et al. A missense mutation in the SERPINH1 gene in Dachshunds with osteogenesis imperfecta. Barsh GS, editor. PLoS Genet. Public Library of Science; 2009;5: e1000579. doi: 10.1371/journal.pgen.1000579 19629171

16. Christiansen HE, Schwarze U, Pyott SM, AlSwaid A, Balwi Al M, Alrasheed S, et al. Homozygosity for a missense mutation in SERPINH1, which encodes the collagen chaperone protein HSP47, results in severe recessive osteogenesis imperfecta. Am J Hum Genet. 2010;86: 389–398. doi: 10.1016/j.ajhg.2010.01.034 20188343

17. Duran I, Nevarez L, Sarukhanov A, Wu S, Lee K, Krejci P, et al. HSP47 and FKBP65 cooperate in the synthesis of type I procollagen. Hum Mol Genet. Oxford University Press; 2014;24: 1918–1928. doi: 10.1093/hmg/ddu608 25510505

18. Essawi O, Symoens S, Fannana M, Darwish M, Farraj M, Willaert A, et al. Genetic analysis of osteogenesis imperfecta in the Palestinian population: molecular screening of 49 affected families. Mol Genet Genomic Med. 2017;86: 551. doi: 10.1002/mgg3.331 29150909

19. Marshall C, Lopez J, Crookes L, Pollitt RC, Balasubramanian M. A novel homozygous variant in SERPINH1 associated with a severe, lethal presentation of osteogenesis imperfecta with hydranencephaly. Gene. 2016;595: 49–52. doi: 10.1016/j.gene.2016.09.035 27677223

20. Song Y, Zhao D, Xu X, Lv F, Li L, Jiang Y, et al. Novel compound heterozygous mutations in SERPINH1 cause rare autosomal recessive osteogenesis imperfecta type X. Osteoporos Int. Springer London; 2018;387: 1657–8. doi: 10.1007/s00198-018-4448-2 29520608

21. Schwarze U, Cundy T, Liu YJ, Hofman PL, Byers PH. Compound heterozygosity for a frameshift mutation and an upstream deletion that reduces expression of SERPINH1 in siblings with a moderate form of osteogenesis imperfecta. Am J Med Genet A. John Wiley & Sons, Ltd; 2019;260: 1734. doi: 10.1002/ajmg.a.61170 31179625

22. Barnes AM, Chang W, Morello R, Cabral WA, Weis M, Eyre DR, et al. Deficiency of Cartilage-Associated Protein in Recessive Lethal Osteogenesis Imperfecta. N Engl J Med.; 2006;355: 2757–64. doi: 10.1056/NEJMoa063804 17192541

23. Cabral WA, Chang W, Barnes AM, Weis M, Scott MA, Leikin S, et al. Prolyl 3-hydroxylase 1 Deficiency Causes a Recessive Metabolic Bone Disorder Resembling Lethal/Severe Osteogenesis Imperfecta. Nat Genet. 2007;39: 359–65. doi: 10.1038/ng1968 17277775

24. Yamauchi M, Sricholpech M. Lysine post-translational modifications of collagen. Essays Biochem. Portland Press Limited; 2012;52: 113–133. doi: 10.1042/bse0520113 22708567

25. Lindert U, Weis MA, Rai J, Seeliger F, Hausser I, Leeb T, et al. Molecular Consequences of Defective SERPINH1/HSP47 in the Dachshund Natural Model of Osteogenesis Imperfecta. J Biol Chem. American Society for Biochemistry and Molecular Biology; 2015; doi: 10.1074/jbc.M115.661025 26004778

26. Koide T, Takahara Y, Asada S, Nagata K. Xaa-Arg-Gly triplets in the collagen triple helix are dominant binding sites for the molecular chaperone HSP47. J Biol Chem. American Society for Biochemistry and Molecular Biology; 2002;277: 6178–6182. doi: 10.1074/jbc.M106497200 11751879

27. Koide T, Asada S, Takahara Y, Nishikawa Y, Nagata K, Kitagawa K. Specific recognition of the collagen triple helix by chaperone HSP47: minimal structural requirement and spatial molecular orientation. J Biol Chem. American Society for Biochemistry and Molecular Biology; 2006;281: 3432–3438. doi: 10.1074/jbc.M509707200 16326708

28. Ito S, Nagata K. Mutants of collagen-specific molecular chaperone Hsp47 causing osteogenesis imperfecta are structurally unstable with weak binding affinity to collagen. Biochem Biophys Res Commun. 2015;469: 437–442 doi: 10.1016/j.bbrc.2015.12.028 26692483

29. Marini JC, Forlino A, Bächinger HP, Bishop NJ, Byers PH, Paepe AD, et al. Osteogenesis imperfecta. Nat Rev Dis Primers. Nature Publishing Group; 2017;3: 17052. doi: 10.1038/nrdp.2017.52 28820180

30. Pokidysheva E, Mizuno K, Bächinger HP. The Collagen Folding Machinery: Biosynthesis and Post-Translational Modifications of Collagens. Osteogenesis Imperfecta. 2014. pp. 57–70.

31. Ishikawa Y, Holden P, Bächinger HP. Heat Shock Protein 47 and 65-kDa FK506-binding Protein Weakly but Synergistically Interact During Collagen Folding in the Endoplasmic Reticulum. J Biol Chem. 2017;292: 17216–17224. doi: 10.1074/jbc.M117.802298 28860186

32. Asada S, Koide T, Yasui H, Nagata K. Effect of HSP47 on Prolyl 4-hydroxylation of Collagen Model Peptides. Cell Struct Funct. 1999;24: 187–96. doi: 10.1247/csf.24.187 10532353

33. Duran I, Martin JH, Weis M, Krejci P, Konik P, Li B, et al. A Chaperone Complex Formed by HSP47, FKBP65, and BiP Modulates Telopeptide Lysyl Hydroxylation of Type I Procollagen. J Bone Miner Res. 2017 Jun;32(6):1309–1319. doi: 10.1002/jbmr.3095 28177155

34. Marutani T, Yamamoto A, Nagai N, Kubota H, Nagata K. Accumulation of type IV collagen in dilated ER leads to apoptosis in Hsp47-knockout mouse embryos via induction of CHOP. J Cell Sci. The Company of Biologists Ltd; 2004;117: 5913–5922. doi: 10.1242/jcs.01514 15522896

35. Ishida Y, Yamamoto A, Kitamura A, Lamandé SR, Yoshimori T, Bateman JF, et al. Autophagic elimination of misfolded procollagen aggregates in the endoplasmic reticulum as a means of cell protection. Mol Biol Cell. American Society for Cell Biology; 2009;20: 2744–2754. doi: 10.1091/mbc.e08-11-1092 19357194

36. Ishida Y, Kubota H, Yamamoto A, Kitamura A, Bächinger HP, Nagata K. Type I collagen in Hsp47-null cells is aggregated in endoplasmic reticulum and deficient in N-propeptide processing and fibrillogenesis. Mol Biol Cell. American Society for Cell Biology; 2006;17: 2346–2355. doi: 10.1091/mbc.e05-11-1065 16525016

37. Forlino A, Cabral WA, Barnes AM, Marini JC. New perspectives on osteogenesis imperfecta. Nat Rev Endocrinol. 2011;7: 540–557. doi: 10.1038/nrendo.2011.81 21670757

38. Bettica P, Baylink DJ, Moro L. Galactosyl Hydroxylysine and Deoxypyridinoline: A Methodological Comparison. Eur J Clin Chem Clin Biochem. 1993;31: 459–65. doi: 10.1515/cclm.1993.31.7.459 8399787

39. De Leeneer K, Hellemans J, Steyaert W, Lefever S, Vereecke I, Debals E, et al. Flexible, Scalable and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice. Hum Mutat. 2014;36: 379–387. doi: 10.1002/humu.22739 25504618

40. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Springer Nature; 2015. pp. 405–424. doi: 10.1038/gim.2015.30 25741868

41. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9: 676–682. doi: 10.1038/nmeth.2019 22743772

42. Zheng L., Baumann U., Reymond J.L. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Research, 2004;32: e115. doi: 10.1093/nar/gnh110 15304544

43. Ericsson U.B., Hallberg B.M., Detitta G.T., Dekker N., Nordlund P. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Analytical Biochemistry; 2006;357: 289–298. doi: 10.1016/j.ab.2006.07.027 16962548

44. Oecal S, Socher E, Uthoff M, Ernst C, Zaucke F, Sticht H, et al. The pH-Dependent Client Release from the Collagen-Specific Chaperone HSP47 is Triggered by a Tandem Histidine Pair. J Biol Chem. American Society for Biochemistry and Molecular Biology; 2016;291: 12612–12626. doi: 10.1074/jbc.M115.706069 27129216

45. Findlay JWA, Dillard RF. Appropriate calibration curve fitting in ligand binding assays. AAPS J. Springer-Verlag; 2007;9: E260–7. doi: 10.1208/aapsj0902029 17907767

46. Syx D, De Wandele I, Symoens S, De Rycke R, Hougrand O, Voermans N, et al. Bi-allelic AEBP1 mutations in two patients with Ehlers-Danlos syndrome. Hum Mol Genet. 2019;28: 1853–1864. doi: 10.1093/hmg/ddz024 30668708

47. Pace JM, Kuslich CD, Willing MC, Byers PH. Disruption of one intra-chain disulphide bond in the carboxyl-terminal propeptide of the proalpha1(I) chain of type I procollagen permits slow assembly and secretion of overmodified, but stable procollagen trimers and results in mild osteogenesis imperfecta. 2001;38: 443–449.

48. Vranka JA, Pokidysheva E, Hayashi L, Zientek K, Mizuno K, Ishikawa Y, et al. Prolyl 3-hydroxylase 1 null mice display abnormalities in fibrillar collagen-rich tissues such as tendons, skin, and bones. J Biol Chem. American Society for Biochemistry and Molecular Biology; 2010;285: 17253–17262. doi: 10.1074/jbc.M110.102228 20363744

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