N-Linked Keratan Sulfate in the Aggrecan Interglobular Domain Potentiates Aggrecanase Activity

Abstract

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...

TIMP-3 Inhibition of ADAMTS-4 (Aggrecanase-1) Is Modulated by Interactions between Aggrecan and the C-terminal Domain of ADAMTS-4

“…In contrast, chondroitin 6-sulfate (the major GAG component of human aggrecan) (24) was able to enhance TIMP-3 potency to a level much more similar to that of aggrecan when added at equivalent concentrations of GAGs. Interestingly, keratan sulfate, which has been shown to potentiate aggrecanase activity and which is the predominant GAG found near the Glu 373 -Ala 374 cleavage site (25), had no effect at all on TIMP-3 interactions. These results therefore suggest that the enhanced potency observed with aggrecan is mediated primarily through GAG-binding interactions, potentially through the FIGURE 6.…”

“…However, it should also be noted that, in this study, we investigated only a single form of keratan sulfate, which has been reported previously to undergo both age-related changes in chain length, sulfation, fucosylation, and sialic acid capping (26) and to vary in structure between the keratan sulfate-rich region and interglobular domain of aggrecan (25). Therefore, it remains a possibility that alternative forms of keratan sulfate may demonstrate properties different from those observed in this study.…”

Low molecular weight isoforms of the aggrecanases are responsible for the cytokine‐induced proteolysis of aggrecan in a porcine chondrocyte culture system

“…Experiments using rHuADAMTS-4 have suggested that lower molecular weight enzyme isoforms (55-30 kd) cleave at the IGD-aggrecanase site, while higher molecular weight isoforms cleave aggrecan within its C-terminal chondroitin sulfate attachment regions (13,15). The apparent absence of low molecular weight isoforms of ADAMTS-4 and ADAMTS-5 isolated from extracts of agarose plugs may result from masking of the epitopes through binding of the enzyme isoforms to aggrecan mediated through interaction with keratan sulfate (26). Following cleavage of the aggrecan substrate within the extracellular matrix and subsequent release of aggrecan catabolites to the culture media, dissociation of the low molecular weight ADAMTS-4 and ADAMTS-5 isoforms may occur.…”

The accumulation of intracellular ITEGE and DIPEN neoepitopes in bovine articular chondrocytes is mediated by CD44 internalization of hyaluronan

“…The dialyzed extracts were applied to an HA-Sepharose column equilibrated in PBS, and the G1-NITEGE fragments were eluted with buffered 4M GuHCl as described previously for G1-G2 (19,31). Purified G1-ITEGE (1.776 mg) was digested with 100 g/ml trypsin-activated MMP-13 for 22 hours at 37°C to generate G1-DIPEN.…”