Keratan sulfate is thought to influence the cleavage of aggrecan by metalloenzymes. We have therefore produced a recombinant substrate, substituted with keratan sulfate, suitable for the study of aggrecanolysis in vitro. Recombinant human G1-G2 was produced in primary bovine keratocytes using a vaccinia virus expression system. Following purification and digestion with specific hydrolases, fluorophore-assisted carbohydrate electrophoresis was used to confirm the presence of the monosulfated Gal-GlcNAc6S and GlcNAc6s-Gal disaccharides and the disulfated Gal6S-GlcNAc6S disaccharides of keratan sulfate. Negligible amounts of fucose or sialic acid were detected, and the level of unsulfated disaccharides was minimal. Treatment with keratanases reduced the size of the recombinant G1-G2 by ϳ5 kDa on SDS-PAGE. Treatment with N-glycosidase F also reduced the size of G1-G2 by ϳ5 kDa and substantially reduced G1-G2 immunoreactivity with monoclonal antibody 5-D-4, indicating that keratan sulfate on the recombinant protein is Nlinked. Cleavage of G1-G2 by aggrecanase was markedly reduced when keratan sulfate chains were removed by treatment with keratanase, keratanase II, endo--galactosidase, or N-glycosidase F. These results indicate that modification of oligosaccharides in the aggrecan interglobular domain with keratan sulfate, most likely at asparagine residue 368, potentiates aggrecanase activity in this part of the core protein.Aggrecan is a major structural component of cartilage and together with type II collagen it enables this tissue to bear load and resist compression. Aggrecan has three globular domains, G1 and G2 at the N terminus and G3 at the C terminus. An extended sequence between the G2 and G3 domains is heavily substituted with chondroitin sulfate and keratan sulfate chains, organized into distinct chondroitin sulfate-1, chondroitin sulfate-2, and keratan sulfate-rich regions. An interglobular domain (IGD) 1 of ϳ150 amino acids separates G1 from G2 and is substituted with keratan sulfate chains as well as O-linked and N-linked oligosaccharides.The IGD is highly sensitive to proteinases. Cleavage in the IGD releases the entire chondroitin sulfate and keratan sulfate-rich regions essential for the biomechanical properties of aggrecan and is therefore the most detrimental for cartilage function. In pathology, proteolysis is driven by aggrecanases and, to a lesser extent, by matrix metalloproteinases. Aggrecanase was first identified as a novel activity that cleaved the aggrecan core protein at the E 373 2 374 A bond in the IGD (1-3); the products of this cleavage were found in synovial fluids from patients with osteoarthritis, joint injury, and inflammatory joint disease (4, 5). Subsequently, cartilage enzymes with aggrecanase activity were revealed as members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family (6, 7), and ADAMTS5 has recently been identified as the major aggrecanase in mouse cartilage (8,9).Proteolysis by matrix metalloproteinases at the N 341 2 342 F bond i...