The addition of TGF-beta1 to bovine articular chondrocytes resulted in increased synthesis and secretion of two anionic glycoproteins. including, a previously Studied but unidentified high molecular weight anionic glycoconjugate (HMW-AG). Sequencing by mass spectroscopy identified these anionic glycoproteins as fibronectin. Western blot analysis confirmed the identity of these two overexpressed glycoproteins as fibronectin. In the presence and absence of TGF-beta1 both V- and V- isoforms of fibronectin, which are EDA Lind EDB-, are synthesized. Dual labeling experiments suggest that the HMW-AG, the larger of the two overexpressed glycoproteins (apparent molecular weight of monomer approximately 260,000 Da), is more heavily glycosylated than the lower molecular weight anionic glycoprotein. Since fibronectin proteolytic fragments appear to enhance matrix metalloproteinase synthesis. TGF-beta1-mediated hyperglycosylation of fibronectin Could regulate cartilage metabolism by providing protection of fibronectin from proteolysis. a mechanism that Would also favor articular cartilage health. (C) 2004 Elsevier Inc. All rights reserved.