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

Abstract

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 collagen 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 collagen, this overmodification cannot be explained by prolonged exposure of the collagen 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 collagen suggests a compensatory mechanism for the aberrant binding of HSP47-R222S, eventually leading to overmodification of type I collagen 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.

Collagen’s primary structure determines collagen:HSP47 complex stoichiometry

“…Proper triple-helix formation as well as exit from the ER of procollagen molecules depends critically on HSP47, an ER-resident chaperone belonging to the serpin family. Ablation of HSP47 leads to early embryonic death in mice ( 6 ), and several missense mutations in humans and dogs give rise to Osteogenesis imperfecta ( 7 , 8 , 9 , 10 ). The exact mechanism by which HSP47 chaperones collagen folding and secretion is not entirely clear, yet.…”

Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes

“…Defects of the complex members affected this modification either by enhancing (defect in Hsp47 and Bip) or diminishing (defect in FKBP65) LH2 activity 34 . In contrast, Syx et al has stated that a mutant Hsp47, which showed a reduced binding to type I collagen, resulted in decreased LH2 44 . These inconsistent data suggest that Hsp47 may act as a positive or negative regulator of LH2 in a context-dependent manner.…”

Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes

“…Defects of the complex members affected this modi cation either by enhancing (defect in Hsp47 and Bip) or diminishing (defect in Fkbp65) LH2 activity 34 . In contrast, Syx et al has stated that a mutant Hsp47, which showed a reduced binding to type I collagen, resulted in decreased LH2 44 . These inconsistent data suggest that Hsp47 may act as a positive or negative regulator of LH2 in a context-dependent manner.…”