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 language | English |
|---|---|
| Pages (from-to) | 32483-32491 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 279 |
| Issue number | 31 |
| DOIs | |
| Publication status | Published - 2004 Jul 30 |
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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 journal › Article
}
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 -