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. 2017 Oct 20;292(42):17216-17224.
doi: 10.1074/jbc.M117.802298. Epub 2017 Aug 31.

Heat shock protein 47 and 65-kDa FK506-binding protein weakly but synergistically interact during collagen folding in the endoplasmic reticulum

Yoshihiro Ishikawa  1 , Paul Holden  1 , Hans Peter Bächinger  2
Affiliations

Heat shock protein 47 and 65-kDa FK506-binding protein weakly but synergistically interact during collagen folding in the endoplasmic reticulum

Yoshihiro Ishikawa et al. J Biol Chem. .

Abstract

Collagen is the most abundant protein in the extracellular matrix in humans and is critical to the integrity and function of many musculoskeletal tissues. A molecular ensemble comprising more than 20 molecules is involved in collagen biosynthesis in the rough endoplasmic reticulum. Two proteins, heat shock protein 47 (Hsp47/SERPINH1) and 65-kDa FK506-binding protein (FKBP65/FKBP10), have been shown to play important roles in this ensemble. In humans, autosomal recessive mutations in both genes cause similar osteogenesis imperfecta phenotypes. Whereas it has been proposed that Hsp47 and FKBP65 interact in the rough endoplasmic reticulum, there is neither clear evidence for this interaction nor any data regarding their binding affinities for each other. In this study using purified endogenous proteins, we examined the interaction between Hsp47, FKBP65, and collagen and also determined their binding affinities and functions in vitro Hsp47 and FKBP65 show a direct but weak interaction, and FKBP65 prefers to interact with Hsp47 rather than type I collagen. Our results suggest that a weak interaction between Hsp47 and FKBP65 confers mutual molecular stability and also allows for a synergistic effect during collagen folding. We also propose that Hsp47 likely acts as a hub molecule during collagen folding and secretion by directing other molecules to reach their target sites on collagens. Our findings may explain why osteogenesis imperfecta-causing mutations in both genes result in similar phenotypes.

Keywords: collagen; endoplasmic reticulum (ER); molecular chaperone; protein folding; protein-protein interaction.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Western blot analysis using an anti-FKBP65 antibody on proteins immunoprecipitated from chicken rER extracts by an Hsp47 antibody. Proteins immunoprecipitated from chicken rER extracts using a monoclonal Hsp47 antibody were electrophoresed on a Novex NuPAGE Bis-Tris 4–12% gel under reducing conditions. Proteins were transferred to a PVDF membrane and subsequently analyzed by Western blotting using a polyclonal FKBP65 antibody. Lane 1, chicken rER extract (immunoprecipitate input); lane 2, purified chicken FKBP65; lane 3, immunoprecipitate using protein G-Sepharose alone; lane 4, immunoprecipitate using protein G-Sepharose plus Hsp47 mAb. The arrow highlights the position of FKBP65. * and ** are protein G and preincubated BSA bands from the protein G-Sepharose. The black line spacing denotes irrelevant lanes that were eliminated from the image.
Figure 2.
Figure 2.
Determination of a direct interaction between Hsp47 and FKBP65. A, purified endogenous chick Hsp47 and FKBP65 were electrophoresed on a Bolt 4–12% Bis-Tris Plus Gel and stained with GelCode Blue Stain Reagent. B, CD spectra of 0.6 μm chick Hsp47 (open circles) and 0.6 μm chick FKBP65 (closed circles). Red and blue curves indicate the theoretical signal derived from addition of individual Hsp47 and FKBP65 signals, and the experimental signal from a mixture of Hsp47 and FKBP65, respectively. C, direct binding kinetics were measured by SPR analysis using a BIAcore X instrument. Various concentrations of Hsp47 were run over the FKBP65 chip. The following binding curves are shown: 0.5 μm (green), 1.0 μm (red), and 2.0 μm (blue) Hsp47. An antibody against FKBP65 (magenta line) was used as a positive control.
Figure 3.
Figure 3.
Thermal stability of Hsp47 and FKBP65. The thermal stabilities of Hsp47 and FKBP65 and a mixture of Hsp47 and FKBP65 were measured using circular dichroism and absorbance measurements. The final concentrations of Hsp47 and FKBP65 were 0.6 μm and 0.5 μm for CD and absorbance measurements, respectively. A, pictures of the protein solutions were taken at the beginning (4 °C) and the end (80 °C) of the thermal transition experiment. B, images showing aggregates of Hsp47 that are magnified views of areas a and b from A. The arrows highlight aggregates of Hsp47. C, the thermal transition of Hsp47 (green), FKBP65 (magenta), and an experimental mixture of Hsp47 and FKBP65 (cyan) was monitored as a function of temperature by circular dichroism at 220 nm. The yellow curve indicates the theoretical signal derived from addition of individual Hsp47 and FKBP65 signals. D, the protein aggregation and/or unfolding transition of Hsp47 (green), FKBP65 (magenta), and an experimental mixture of Hsp47 and FKBP65 (cyan) was monitored as a function of temperature by absorbance (turbidity) at 230 nm. All curves were averaged from a minimum of three measurements, and error bars indicating S.D. are shown at each 5 °C interval.
Figure 4.
Figure 4.
Molecular stability of FKBP65. Purified FKBP65, indicated by the arrowhead, was incubated at room temperature A, in the absence (−CaCl2) or presence (+CaCl2) of calcium, or B, in the presence of Hsp47 (+Hsp47) or CypB (+CypB). Aliquots were removed at 12-h intervals and electrophoresed on NuPAGE Novex Bis-Tris 4–12% gels under reducing conditions followed by staining with GelCode Blue Stain Reagent. The black line spacing denotes irrelevant lanes that were eliminated from the image. These results indicate that the interaction between Hsp47 and FKBP65 leads to an improvement of their individual molecular stabilities.
Figure 5.
Figure 5.
The effect of FKBP65 on collagen folding in the presence of Hsp47. A, kinetics of the refolding of full-length type III collagen in the presence of Hsp47 and/or FKBP65 monitored by CD at 220 nm is shown. The concentration of type III collagen, FKBP65, and Hsp47 were all 0.2 μm. The refolding curves shown are in the absence (black) and presence of Hsp47 (red) and FKBP65 (blue). The mixture of Hsp47 and FKBP65 with (green) and without (magenta) type III collagen are also shown. B, SPR analysis was carried out using a BIAcore X instrument to determine the binding orientation of Hsp47 and FKBP65 to the collagen chip. The open and closed circles are Hsp47 (0.05 μm) and FKBP65 (0.2 μm), respectively. Red and blue curves indicate the theoretical signal derived from the addition of individual Hsp47 and FKBP65 curves and the experimental curve from a mixture of both Hsp47 and FKBP65, respectively. C, fibril formation of type I collagen. A stock solution of type I collagen in 50 mm acetic acid was diluted to a final concentration of 0.1 μm. Fibril formation in the absence (black) and presence of 0.2 μm Hsp47 (red) and 0.2 μm FKBP65 (blue) is shown. 0.2 μm decorin was used as a positive control (green). The squares and circles indicate the theoretical signal derived from the addition of individual FKBP65 and Hsp47 signals, and the experimental signal from a mixture of FKBP65 and Hsp47, respectively. D, direct binding kinetics were measured by SPR analysis using a BIAcore X instrument. Various concentrations of pepsin-treated type I collagen were run over the FKBP65 chip. The following binding curves are shown: 0.1 μm (green), 0.2 μm (red), 0.3 (blue) and 0.4 μm (magenta) Hsp47. All curves in A–D are averaged by a minimum of three measurements. These results indicate that FKBP65 preferentially interacts with Hsp47 rather than type I collagen, and the interaction between Hsp47 and FKBP65 creates a synergistic function for collagen folding.
Figure 6.
Figure 6.
Schematic diagram of the interactions between collagen, Hsp47, and FKBP65 and a proposed model of collagen folding in the rER. A, illustration of the interactions between three proteins; collagen, Hsp47, and FKBP65. The length of the arrows indicates the strength of the association (green) and dissociation (red) between molecules. B, graphical representation of the combined effects of Hsp47 and FKBP65 on collagen folding. I, triple helix formation is initiated from the carboxyl-terminal end; II, Hsp47 binds to the newly formed triple helix; III, FKBP65 is recruited to Hsp47 at the triple helix propagation site and accelerates the rate of folding; IV, FKBP65 is replaced by the SH3 domain of TANGO1 at the ER exit site for the loading of collagen into a special COPII vesicle.
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References

    1. Araki K., and Nagata K. (2011) Protein folding and quality control in the ER. Cold Spring Harb. Perspect. Biol. 3, a007526. - PMC - PubMed
    1. Gidalevitz T., Stevens F., and Argon Y. (2013) Orchestration of secretory protein folding by ER chaperones. Biochim. Biophys. Acta 1833, 2410–2424 - PMC - PubMed
    1. Bächinger H. P., Mizuno K., Vranka J., and Boudko S. (2010) Collagen Formation and Structure. In Comprehensive Natural Products II: Chemistry and Biology (Mander L., and Liu H.-W. eds.) pp. 469–530, Elsevier Science Publishing Co., Inc., New York
    1. Bateman J. F., Boot-Handford R. P., and Lamandé S. R. (2009) Genetic diseases of connective tissues: cellular and extracellular effects of ECM mutations. Nat. Rev. Genet. 10, 173–183 - PubMed
    1. Bella J., and Hulmes D. J. (2017) Fibrillar Collagens. Subcell. Biochem. 82, 457–490 - PubMed

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