The cleavage of biglycan by aggrecanases

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General Information Osteoarthritis and Cartilage

Presentation transcript:

The cleavage of biglycan by aggrecanases L.I. Melching, Ph.D., W.D. Fisher, M.D., E.R. Lee, Ph.D., J.S. Mort, Ph.D., P.J. Roughley, Ph.D. Osteoarthritis and Cartilage Volume 14, Issue 11, Pages 1147-1154 (November 2006) DOI: 10.1016/j.joca.2006.05.014 Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 Effect of aggrecanase-1 on bovine decorin and biglycan. The action of aggrecanase-1 on bovine decorin and biglycan was studied by SDS/PAGE and immunoblotting. (A) Lane 1, intact decorin; lane 2, intact decorin following chondroitinase treatment; lane 3, aggrecanase-treated decorin following chondroitinase treatment. (B) Lane 1, intact biglycan; lane 2, aggrecanase-treated biglycan; lane 3, intact biglycan following chondroitinase treatment; lane 4, aggrecanase-treated biglycan following chondroitinase treatment; lane 5, biglycan incubated with aggrecanase in the presence of o-phenanthroline; lane 6, biglycan incubated with aggrecanase in the presence of o-phenanthroline and subsequent chondroitinase treatment. Osteoarthritis and Cartilage 2006 14, 1147-1154DOI: (10.1016/j.joca.2006.05.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Effect of aggrecanases on human biglycan. Aggrecanase-1 or aggrecanase-2 was incubated with human biglycan at either pH 8.5 or pH 7.5, and the products analyzed by SDS/PAGE and immunoblotting. All digestion mixtures were treated with chondroitinase prior to analysis. Lane 1, biglycan treated with aggrecanase-1 at pH 8.5; lane 2, biglycan treated with aggrecanase-1 at pH 7.5; lane 3, biglycan treated with aggrecanase-2 at pH 8.5; lane 4, biglycan treated with aggrecanase-2 at pH 7.5. Osteoarthritis and Cartilage 2006 14, 1147-1154DOI: (10.1016/j.joca.2006.05.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 Effect of aggrecanases on aggrecan. Aggrecanase-1 or aggrecanase-2 was incubated with bovine aggrecan at pH 7.5, and the products analyzed by SDS/PAGE and immunoblotting using an antibody to the aggrecan G3 domain. The digestion mixtures were treated with keratanase and chondroitinase prior to analysis. Lane 1, intact aggrecan; lane 2, aggrecan treated with aggrecanase-1; lane 3, aggrecan treated with aggrecanase-2. Osteoarthritis and Cartilage 2006 14, 1147-1154DOI: (10.1016/j.joca.2006.05.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 Analysis of aggrecanase composition. The aggrecanase preparations were analyzed by SDS/PAGE and immunoblotting, using antibodies specific for each proteinase. (A) Aggrecanase-1; (B) aggrecanase-2. Osteoarthritis and Cartilage 2006 14, 1147-1154DOI: (10.1016/j.joca.2006.05.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 Analysis of biglycan in normal and arthritic human cartilage. Extracts of human articular cartilage were analyzed by SDS/PAGE and immunoblotting using an antibody to biglycan. All extracts were treated with chondroitinase prior to analysis. (A) Samples were from normal individuals aged: (1) 7 months, (2) 6 years, (3) 22 years, (4) 39 years, and (5) 61 years. (B) Samples were from individuals with arthritic joints aged: (1) 73 years, (2) 75 years, (3) 74 years, (4) 50 years, (5) 77 years and (6) 62 years. Samples 1–3 were from OA cartilage, and samples 4–6 were from RA cartilage. Arrow indicates the position of the biglycan cleavage product. Osteoarthritis and Cartilage 2006 14, 1147-1154DOI: (10.1016/j.joca.2006.05.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 Schematic representation of biglycan and its aggrecanase cleavage site. Biglycan is depicted as a line representing its mature core protein, with the location of the 10 leucine-rich repeats (filled boxes) and the flanking disulfide-bonded domains (open boxes) being indicated. The location of the aggrecanase cleavage site and its surrounding amino acid sequence are also indicated. Osteoarthritis and Cartilage 2006 14, 1147-1154DOI: (10.1016/j.joca.2006.05.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions