TRANSGLUTAMINASE-CATALYZED MATRIX CROSS-LINKING IN DIFFERENTIATING CARTILAGE - IDENTIFICATION OF OSTEONECTIN AS A MAJOR GLUTAMINYL SUBSTRATE

The expression of tissue transglutaminase in skeletal tissues is stric tly regulated and correlates with chondrocyte differentiation and cart ilage calcification in endochondral bone formation and in maturation o f tracheal cartilage (Aeschlimann, D., A, Wetterwald, H. Fleisch, and M. Paulsson. 1993, J. Cell Biol. 120:1461-1470). We now demonstrate th e transglutaminase reaction product, the gamma-glutamyl-epsilon-lysine cross-link, in the matrix of hypertrophic cartilage using a novel cro ss-link specific antibody. Incorporation of the synthetic transglutami nase substrate monodansylcadaverine (amine donor) in cultured tracheal explants reveals enzyme activity in the pericellular matrix of hypert rophic chondrocytes in the central, calcifying areas of the horseshoe- shaped cartilages. One predominant glutaminyl substrate (amine accepto r) in the chondrocyte matrix is osteonectin as revealed by incorporati on of the dansyl label in culture. Indeed, nonreducible osteonectin-co ntaining complexes of similar to 65, 90, and 175 kD can be extracted f rom mature tracheal cartilage. In vitro cross-linking of osteonectin b y tissue transglutaminase gives similar products of similar to 90 and 175 kD, indicating that the complexes in cartilage represent osteonect in oligomers. The demonstration of extracellular transglutaminase acti vity in differentiating cartilage, i.e., cross-linking of osteonectin in situ, shows that tissue transglutaminase-catalyzed cross-linking is a physiological mechanism for cartilage matrix stabilization.