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


METHODS
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.
Homandberg
G A
GA
Department of Biochemistry, Rush Medical College at Rush-Presbyterian-St Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612-3864, USA. ghomandb@medicine.nodak.edu
Costa
V
V
Wen
C
C
D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D019920:Photoaffinity Labels; D013388:Succinimides / Q000378:metabolism
D003432:Cross-Linking Reagents; D005353:Fibronectins; D039081:Integrin alpha5beta1; D019920:Photoaffinity Labels; D013388:Succinimides; C011240:dithiobis(succinimidylpropionate)