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Detection of substrate binding of a collagen-specific molecular chaperone HSP47 in solution using fluorescence correlation spectroscopy

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Title: Detection of substrate binding of a collagen-specific molecular chaperone HSP47 in solution using fluorescence correlation spectroscopy
Authors: Kitamura, Akira Browse this author →KAKEN DB
Ishida, Yoshihito Browse this author
Kubota, Hiroshi Browse this author →KAKEN DB
Pack, Chan-Gi Browse this author
Homma, Takayuki Browse this author
Ito, Shinya Browse this author
Araki, Kazutaka Browse this author
Kinjo, Masataka Browse this author →KAKEN DB
Nagata, Kazuhiro Browse this author →KAKEN DB
Keywords: Molecular chaperone
HSP47
Collagen
Fluorescence correlation spectroscopy
Issue Date: 10-Feb-2018
Publisher: Elsevier
Journal Title: Biochemical and Biophysical Research Communications
Volume: 497
Issue: 1
Start Page: 279
End Page: 284
Publisher DOI: 10.1016/j.bbrc.2018.02.069
PMID: 29438711
Abstract: Heat shock protein 47 kDa (HSP47), an ER-resident and collagen-specific molecular chaperone, recognizes collagenous hydrophobic amino acid sequences (Gly-Pro-Hyp) and assists in secretion of correctly folded collagen. Elevated collagen production is correlated with HSP47 expression in various diseases, including fibrosis and keloid. HSP47 knockdown ameliorates liver fibrosis by inhibiting collagen secretion, and inhibition of the interaction of HSP47 with procollagen also prevents collagen secretion. Therefore, a high-throughput system for screening of drugs capable of inhibiting the interaction between HSP47 and collagen would aid the development of novel therapies for fibrotic diseases. In this study, we established a straightforward method for rapidly and quantitatively measuring the interaction between HSP47 and collagen in solution using fluorescence correlation spectroscopy (FCS). The diffusion rate of HSP47 labeled with Alexa Fluor 488 (HSP47-AF), a green fluorescent dye, decreased upon addition of type I or III collagen, whereas that of dye-labeled protein disulfide isomerase (PDI) or bovine serum albumin (BSA) did not, indicating that specific binding of HSP47 to collagen could be detected using FCS. Using this method, we calculated the dissociation constant of the interaction between HSP47 and collagen. The binding ratio between HSP47-AF and collagen did not change in the presence of sodium chloride, confirming that the interaction was hydrophobic in nature. In addition, we observed dissociation of collagen from HSP47 at low pH and re-association after recovery to neutral pH. These observations indicate that this system is appropriate for detecting the interaction between HSP47 and collagen, and could be applied to high-throughput screening for drugs capable of suppressing and/or curing fibrosis.
Rights: ©2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/
http://creativecommons.org/licenses/by-nc-nd/4.0/
Type: article (author version)
URI: http://hdl.handle.net/2115/72752
Appears in Collections:生命科学院・先端生命科学研究院 (Graduate School of Life Science / Faculty of Advanced Life Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 北村 朗

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