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J. Biol. Chem., Vol. 280, Issue 25, 23615-23621, June 24, 2005

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

Christopher J. Poon, Anna H. Plaas¶, Doug R. Keene||, David J. McQuillan**, Karena Last, and Amanda J. Fosang

From the Department of Paediatrics, University of Melbourne and Murdoch Childrens Research Institute, Arthritis Research Group, Royal Children's Hospital, Parkville, Victoria 3052, Australia, ^¶Department of Internal Medicine, University of South Florida, Tampa, Florida 33602, ^||Shriners Hospital for Children, Portland, Oregon 97239, and ^**LifeCell Corporation, Branchburg, New Jersey 08876

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 5 kDa on SDS-PAGE. Treatment with N-glycosidase F also reduced the size of G1-G2 by 5 kDa and substantially reduced G1-G2 immunoreactivity with monoclonal antibody 5-D-4, indicating that keratan sulfate on the recombinant protein is N-linked. 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.

Received for publication, October 27, 2004 , and in revised form, April 12, 2005.

^* This work was supported in part by funds from the National Health and Medical Research Council (Australia) and the Mizutani Foundation for Glycoscience, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by Eileen Urquhart and Frank G. Spurway Scholarships from the Arthritis Foundation of Australia.

To whom correspondence should be addressed: Dept. of Paediatrics, University of Melbourne and Murdoch Childrens Research Institute, Arthritis Research Group, Royal Children's Hospital, Flemminton Rd. Parkville, Victoria 3052, Australia. Tel.: 61-3-9345-6628; Fax: 61-3-9345-7997; E-mail: amanda.fosang{at}mcri.edu.au.

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