ADAMTS4 (aggrecanase-1) interaction with the C-terminal domain of fibronectin inhibits proteolysis of aggrecan

Gakuji Hashimoto, Masayuki Shimoda, Yasunori Okada

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

ADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including ΔSp (ΔArg693-Lys 837, lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC50 = 110 nM), whereas no inhibition was observed with ΔSp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC50 = 170 nM). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage.

Original languageEnglish
Pages (from-to)32483-32491
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number31
DOIs
Publication statusPublished - 2004 Jul 30

Fingerprint

Proteolysis
Aggrecans
Fibronectins
Two-Hybrid System Techniques
Cartilage
Yeast
Inhibitory Concentration 50
Assays
Clone Cells
Thrombospondins
Disintegrins
Degradation
aggrecanase
ADAMTS4 Protein
Confocal microscopy
Metalloproteases
Proteoglycans
Chondrocytes
Hybrid systems
Gene Library

ASJC Scopus subject areas

  • Biochemistry

Cite this

ADAMTS4 (aggrecanase-1) interaction with the C-terminal domain of fibronectin inhibits proteolysis of aggrecan. / Hashimoto, Gakuji; Shimoda, Masayuki; Okada, Yasunori.

In: Journal of Biological Chemistry, Vol. 279, No. 31, 30.07.2004, p. 32483-32491.

Research output: Contribution to journalArticle

@article{cadf6d4b5e7f465eb16fa114b1bc1390,
title = "ADAMTS4 (aggrecanase-1) interaction with the C-terminal domain of fibronectin inhibits proteolysis of aggrecan",
abstract = "ADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including ΔSp (ΔArg693-Lys 837, lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC50 = 110 nM), whereas no inhibition was observed with ΔSp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC50 = 170 nM). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage.",
author = "Gakuji Hashimoto and Masayuki Shimoda and Yasunori Okada",
year = "2004",
month = "7",
day = "30",
doi = "10.1074/jbc.M314216200",
language = "English",
volume = "279",
pages = "32483--32491",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "31",

}

TY - JOUR

T1 - ADAMTS4 (aggrecanase-1) interaction with the C-terminal domain of fibronectin inhibits proteolysis of aggrecan

AU - Hashimoto, Gakuji

AU - Shimoda, Masayuki

AU - Okada, Yasunori

PY - 2004/7/30

Y1 - 2004/7/30

N2 - ADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including ΔSp (ΔArg693-Lys 837, lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC50 = 110 nM), whereas no inhibition was observed with ΔSp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC50 = 170 nM). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage.

AB - ADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including ΔSp (ΔArg693-Lys 837, lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC50 = 110 nM), whereas no inhibition was observed with ΔSp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC50 = 170 nM). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage.

UR - http://www.scopus.com/inward/record.url?scp=3543004686&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=3543004686&partnerID=8YFLogxK

U2 - 10.1074/jbc.M314216200

DO - 10.1074/jbc.M314216200

M3 - Article

C2 - 15161923

AN - SCOPUS:3543004686

VL - 279

SP - 32483

EP - 32491

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 31

ER -