Characterization of an ADAMTS-5-mediated cleavage site in aggrecan in OSM- stimulated bovine cartilage M. Durigova, M.Sc., P. Soucy, B.Sc., K. Fushimi,

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Characterization of an ADAMTS-5-mediated cleavage site in aggrecan in OSM- stimulated bovine cartilage M. Durigova, M.Sc., P. Soucy, B.Sc., K. Fushimi, Ph.D., Dr., H. Nagase, Ph.D., Dr., J.S. Mort, Ph.D., Dr., P.J. Roughley, Ph.D., Dr. Osteoarthritis and Cartilage Volume 16, Issue 10, Pages 1245-1252 (October 2008) DOI: 10.1016/j.joca.2008.02.013 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Aggrecanase cleavage sites in bovine aggrecan. (A) Schematic representation of the bovine aggrecan core protein and its constituent domains: G1, IGD, G2 and G3, and the GAG-substituted domains: KS and CS-1 and CS-2 domains. Previously described aggrecanase cleavage sites are indicated by numbered arrows (1–5). Position of the additional aggrecanase-mediated cleavage site described in the present work is indicated by the dashed arrow (6). (B) Sites cleaved by aggrecanases. Cleavage site is indicated by an arrow and the position of the P1 glutamic acid residue in the bovine aggrecan sequence is also indicated. Osteoarthritis and Cartilage 2008 16, 1245-1252DOI: (10.1016/j.joca.2008.02.013) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Release of G3-containing fragments from bovine cartilage explant cultures. Bovine cartilage explants were cultured in the presence of IL-1β and OSM over a 4-day period. Culture medium was collected at day 2 and day 4, treated with keratanase II and chondroitinase ABC, analyzed by SDS/PAGE and immunoblotting using an antibody recognizing the G3 domain of aggrecan. Material released from equal amounts of cartilage explants was loaded onto the gel. Lane 1, control day 2; lane 2, IL-1β+OSM day 2; lane 3, IL-1β+OSM day 4. Migration positions of molecular weight markers are indicated on the left. Migration position of the additional G3-bearing fragment is indicated by an arrow. Osteoarthritis and Cartilage 2008 16, 1245-1252DOI: (10.1016/j.joca.2008.02.013) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Purification of G3-containing aggrecan fragments by ion-exchange chromatography. (A) Culture media from day 2 and day 4 of IL-1β/OSM-treated explant cultures were pooled and loaded onto a DEAE-Sepharose column pre-equilibrated in 50-mM Tris, 0.15-M NaCl, pH 7.5. Bound aggrecan material was retrieved by step elution with 0.3-M and 1-M NaCl. Protein content in the collected 1-ml fractions was estimated by measuring absorbance at 280nm. (B) Aliquots of the fractions were deglycosylated and analyzed by SDS/PAGE and western blotting using anti-G3 antibody. Migration positions of molecular weight markers are indicated on the left. Migration position of the G3-bearing fragment of interest is indicated by an arrow. Osteoarthritis and Cartilage 2008 16, 1245-1252DOI: (10.1016/j.joca.2008.02.013) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Amino acid sequence analysis of G3-containing aggrecan fragment. Fraction number 98 [see Fig. 3(B)] containing the G3 product of interest was treated with keratanase II and chondroitinase ABC. The sample was run on a 4–12% Bis–Tris Novex gels and transferred to a PVDF membrane. The Coomassie Blue-stained band of interest (marked by an arrow) was excised and analyzed by automated Edman degradation sequencing. The determined N-terminal sequence is indicated. Osteoarthritis and Cartilage 2008 16, 1245-1252DOI: (10.1016/j.joca.2008.02.013) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Digestion of fetal bovine aggrecan with aggrecanases. Purified fetal bovine aggrecan (2mg/ml) was digested with recombinant full-length human ADAMTS-4 or ADAMTS-5 (5nM) for 24h at 37°C. The samples were then treated with keratanase II and chondroitinase ABC, then analyzed by SDS/PAGE and immunoblotting using either an anti-aggrecan G3 antibody (A) or an anti-neoepitope antibody directed against the new N-terminus of the G3 fragment (B). Lane 1, undigested aggrecan; lane 2, aggrecan digested with ADAMTS-4; lane 3, aggrecan digested with ADAMTS-5; lane 4, culture media from IL-1β/OSM-treated bovine explants collected at day 4. Migration positions of molecular weight markers are indicated on the left. Migration position of the G3-bearing fragment of interest is indicated by an arrow. Osteoarthritis and Cartilage 2008 16, 1245-1252DOI: (10.1016/j.joca.2008.02.013) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Digestion of fetal bovine aggrecan by ADAMTS-5: a time-course study. Purified aggrecan (2mg/ml) was digested with recombinant human ADAMTS-5 (1nM) for 1, 3, 6 or 18h at 37°C. The samples were then treated with keratanase II and chondroitinase ABC, and analyzed by SDS/PAGE and immunoblotting using an anti-aggrecan G3 antibody. Lane 1, undigested aggrecan; lane 2, aggrecan digested with ADAMTS-5 for 1h; lane 3, aggrecan digested with ADAMTS-5 for 3h; lane 4, aggrecan digested with ADAMTS-5 for 6h; lane 5, aggrecan digested with ADAMTS-5 for 18h. Migration positions of molecular weight markers are indicated on the left. Migration position of the G3-bearing fragment of interest is indicated by an arrow. Osteoarthritis and Cartilage 2008 16, 1245-1252DOI: (10.1016/j.joca.2008.02.013) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Analysis of CS and KS presence of G3-containing aggrecan fragment. ADAMTS-5 digests of fetal bovine aggrecan were treated with either keratanase II or chondroitinase ABC, or both enzymes. Sample were then analyzed by SDS/PAGE on 4–12% Bis–Tris Novex gels and immunoblotting using either an anti-aggrecan G3 antibody or an anti-neoepitope antibody directed against the new N-terminus of the G3 fragment. Migration positions of molecular weight markers are indicated on the left. Migration position of the G3-bearing fragment of interest is indicated by an arrow. Osteoarthritis and Cartilage 2008 16, 1245-1252DOI: (10.1016/j.joca.2008.02.013) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 8 Sequence comparison between different species at the C-terminal aggrecanase cleavage site. Bovine, human, dog, mouse and rat aggrecan amino acid sequences at the cleavage site were aligned. P1 indicates the first amino acid upstream of the scissile bond; P1′ represents the first amino acid downstream of the scissile bond. Amino acids are represented by their respective single letter code. Osteoarthritis and Cartilage 2008 16, 1245-1252DOI: (10.1016/j.joca.2008.02.013) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions