Fibronectin fragments active in chondrocytic chondrolysis can be chemically cross-linked to the alpha5 integrin receptor subunit

Osteoarthritis Cartilage. 2002 Dec;10(12):938-49. doi: 10.1053/joca.2002.0854.


Objective: To determine whether fibronectin fragments (Fn-f) known to enhance cartilage matrix degradation and to alter chondrocyte metabolism, bind on the chondrocyte cell surface close enough to the alpha(5)beta(1) fibronectin (Fn) receptor to be chemically cross-linked to it.

Design: Biotinylated Fn-fs were added to chondrocytes, followed by cross-linking with dithiobissulfosuccinimidyl propionate, and the resultant alpha(5) complexes trapped on to antialpha(5)-agarose. Adherent material was analysed by probing with avidin-HRP. In a more specific approach in which only proximal targets could be cross-linked, photoaffinity labeled Fn-fs or Fn were added to cells, the derivatives activated and the cross-linked material analysed. Interaction of biotinylated Fn-fs and Fn with insolubilized alpha(5)beta(1) receptor was also visualized and quantified.

Results: Biotinylated Fn-fs and Fn, but not a control of BSA, were cross-linked to alpha(5) protein in the presence of the propionate. Photoaffinity label Fn-f and Fn, but not BSA, were cross-linked to alpha(5) protein as well. Interaction was decreased by addition of an excess of unlabeled Fn-f or Fn. Fn-fs bound to alpha(5)beta(1)-agarose, although the affinity was 30-fold weaker and the stoichiometry 20-fold greater when the smallest Fn-f was compared to native Fn.

Conclusions: These data are consistent with a role for the alpha(5) subunit in Fn-f activities and suggest that the Fn-fs bind proximal or directly to alpha(5) receptors. The weaker, higher stoichiometry interaction of Fn-fs with receptor suggests that fragmentation has allowed de novo interactions not possible in native Fn.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Biotinylation
  • Blotting, Western
  • Cartilage, Articular / metabolism*
  • Cattle
  • Cells, Cultured
  • Chondrocytes / metabolism*
  • Cross-Linking Reagents / metabolism
  • Fibronectins / metabolism*
  • Integrin alpha5beta1 / metabolism*
  • Microscopy, Fluorescence
  • Microscopy, Phase-Contrast
  • Photoaffinity Labels
  • Succinimides / metabolism


  • Cross-Linking Reagents
  • Fibronectins
  • Integrin alpha5beta1
  • Photoaffinity Labels
  • Succinimides
  • dithiobis(succinimidylpropionate)