A Chaperone Complex Formed by HSP47, FKBP65, and BiP Modulates Telopeptide Lysyl Hydroxylation of Type I Procollagen

Abstract

Lysine hydroxylation of type I collagen telopeptides varies from tissue to tissue, and these distinct hydroxylation patterns modulate collagen cross-linking to generate a unique extracellular matrix. Abnormalities in these patterns contribute to pathologies that include osteogenesis imperfecta (OI), fibrosis, and cancer. Telopeptide procollagen modifications are carried out by lysyl hydroxylase 2 (LH2); however, little is known regarding how this enzyme regulates hydroxylation patterns. We identified an ER complex of resident chaperones that includes HSP47, FKBP65, and BiP regulating the activity of LH2. Our findings show that FKBP65 and HSP47 modulate the activity of LH2 to either favor or repress its activity. BiP was also identified as a member of the complex, playing a role in enhancing the formation of the complex. This newly identified ER chaperone complex contributes to our understanding of how LH2 regulates lysyl hydroxylation of type I collagen C-telopeptides to affect the quality of connective tissues. © 2017 American Society for Bone and Mineral Research.

Fig. 1

LH2, FKBP65, HSP47 and BiP interact. A-C. Protein levels of LH2 were altered in cells with mutations in FKBP10 and SERPINH1. BiP levels were decreased in HSP47 defective cells. D-E. Rescue experiment using FKBP10^-/- cells transfected with FKBP10 tagged vector. LH2 levels were rescued by expression of wildtype FKBP65 protein. F. Co-immunoprecipitation of endogenous LH2, FKBP65, HSP47 and BiP in human osteoblasts (OB) with specific antibodies. LH2 immunoprecipitated HSP47 and BiP. G. Co-immunoprecipitation of tagged FKBP65 and LH2 in OB cells. LH2 and FKBP65 immunoprecipitated each other, including both LH2 isoforms, respectively.

Fig. 2. In situ

interaction of FKBP65 and LH2 by proximity ligation assay. A. FKBP65 and LH2 interacted in WT cells (red): B. FKBP10 mutant cells did not show interaction between LH2 and FKBP65. C-D. Interaction was increased in HSP47 defective cells.

Fig. 3

Cellular localization of LH2 and BiP. Intracellular immunolocalization of LH2 and BiP in human fibroblasts. A-F: LH2 is shown in red and FKBP65 in green. G-I: BiP is shown in green and HSP47 in red. control cells (A, D and G); FKBP10^-/- cells (B, E and H); and HSP47^M237T/M237T cells (C, F and I). LH2 colocalized with FKBP65 in the ER (D-F), including within abnormal vesicles in mutant cells (arrows). BiP colocalized partially with HSP47 in the ER but not in either the Golgi or abnormal vesicles (arrow). Nuclei were stained with DAPI (in blue). Green arrows identify the Golgi compartment.

Fig. 4

Lysyl Hydroxylation of type I collagen C-telopeptides. Percentage of hydroxylation of lysine 1208 in secreted type I collagen by fibroblasts treated with ascorbic acid. The control represents the average of three different fibroblast cell lines from unaffected persons. Control siRNA represents same control fibroblasts transfected with non-target siRNA. FKBP10^-/- and HSP47^M237T/M237T are patient fibroblast. PLOD2 and HSPA5 siRNA are knockdown fibroblast by use of validated iRNA sequences. FKBP10^-/-, and PLOD2 knockdown cells showed a significant decrease in hydroxylation. HSP47^M237T/M237T and HSPA5 knockdown cells showed a significant increase in hydroxylation.

Fig. 5

Interaction analysis by surface plasmon resonance. A. Type I procollagen, FKBP65, HSP47 proteins were exposed to immobilized LH2. Both FKBP65 and HSP47 bound to the LH2-procollagen mix but the association was altered when both chaperones were presented simultaneously. B. Same experiment for binding to LH2 and type I procollagen, but including BiP protein in addition. The association curve was observed during addition of proteins during the initial 180 seconds, with a positive slope followed by a peak of affinity around this time and the dissociation curve afterward. Presence of BiP increased the affinity of HSP47 and FKBP65 for Procollagen and LH2 without altering their competition.

Fig. 6

The chaperone complex modulates LH2 activity. A. Without stimulus favoring collagen crosslinking, HSP47 binds to the hydroxylation site, supported by BiP, and impedes LH2 function, resulting in lack of hydroxylation. B. In the presence of a hydroxylation stimulus, FKBP65 supports the binding and activity of LH2 to promote lysyl hydroxylation. C. In cells with loss of FKBP65, LH2 activity cannot be induced, resulting in decreased hydroxylation. D. In cells with altered HSP47, the defective HSP47 cannot compete with FKBP65 and LH2 has unlimited access to the hydroxylation site. E. In the absence of BiP, complex stability is affected, limiting HSP47 access to the hydroxylation site and allowing over hydroxylation by LH2.