Human osteoarthritis synovial fluid and joint cartilage contain both aggrecanase- and matrix metalloproteinase-generated aggrecan fragments A. Struglics,

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Human osteoarthritis synovial fluid and joint cartilage contain both aggrecanase- and matrix metalloproteinase-generated aggrecan fragments A. Struglics, Ph.D., S. Larsson, B.Sc., M.A. Pratta, M.Sc., S. Kumar, Ph.D., M.W. Lark, Ph.D., L.S. Lohmander, M.D., Ph.D. Osteoarthritis and Cartilage Volume 14, Issue 2, Pages 101-113 (February 2006) DOI: 10.1016/j.joca.2005.07.018 Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 Schematic illustration of human aggrecan with cleavage sites of aggrecanases (above) and MMP (below). G1, G2 and G3 – globular domains 1–3; IGD – interglobular domain; KS – keratan sulfate enriched region; CS1 and CS2 – chondroitin sulfate enriched region one and two. Amino acid numberings are based on mature human aggrecan starting with the N-terminal sequence 1VETS- (NCBI accession nr P16112). The cleavage sites represented by arrows were summarized from several publications (see text). Osteoarthritis and Cartilage 2006 14, 101-113DOI: (10.1016/j.joca.2005.07.018) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Western immuno-blot peptide blocking experiment against in vitro digested cartilage aggrecan. Human cartilage aggrecan (A1D1) digested in vitro with ADAMTS-4 (A, B, C, E lanes 1 and 3) or with MMP-3 (D, E lanes 2 and 4) was SDS-PAGE separated (3–8% gels) and analyzed by Western immuno-blot in the presence or absence of synthetic peptides. (A) Anti-ARGS Western with 1.5μg GAG per lane, ±10-μM neoepitope ARGSVILTVKGGC-peptide and ±10-μM spanning EGEARGSVILTVKPIF-peptide. (B) Anti-SELE Western with 1.25μg GAG per lane, ±2-μM neoepitope CASTASELE-peptide and ±2-μM spanning EVVTASTASELEGRGT-peptide. (C) Anti-KEEE Western with 3.6μg GAG per lane, ±2-μM neoepitope CEVAPTTFKEEE-peptide and ±2-μM spanning VAPTTFKEEEGLGS-peptide. (D) Anti-FFGV Western with 4.2μg GAG/lane, ±10-μM neoepitope FFGVGGEEDITVC-peptide and ±10-μM spanning DIPENFFGVGGEEDC-peptide. (E) Anti-G1 Western with 4.2μg GAG per lane, ±2-μM immunization CATEGQVRVNSIYQDKVSL-peptide. Mean values of Mw of aggrecan fragments are given as kDa, and were calculated based on electrophoretic migration. The anti-G1A band, marked with # (E) was identified as the deglycosylation enzyme chondroitinase ABC (results not shown). Osteoarthritis and Cartilage 2006 14, 101-113DOI: (10.1016/j.joca.2005.07.018) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 Aggrecan fragments detected in MMP-3 and ADAMTS-4 in vitro digested A1D1-cartilage, and in OA synovial fluid (D1-fraction) by Western immuno-blot. The samples were SDS-PAGE separated (3–8% gel) and analyzed by Western blot with different antibodies as shown. Different amounts of GAG per lane were loaded to facilitate the comparison between the samples. The positions of Mw (in KDa) are indicated. Osteoarthritis and Cartilage 2006 14, 101-113DOI: (10.1016/j.joca.2005.07.018) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 Detection of high Mw aggrecan fragments in OA synovial fluid. Human OA synovial fluid fraction D1 was SDS-PAGE separated (3–8% gel) and analyzed by Western immuno-blot with the following antibodies: (A) anti-G1, anti-SELE and anti-ARGS; (B) anti-KEEE, anti-FFGV and anti-SELE. Different amounts of GAG per lane were loaded to facilitate comparisons. Mean values of Mw (in kDa) of aggrecan fragments were calculated based on electrophoretic migration. Osteoarthritis and Cartilage 2006 14, 101-113DOI: (10.1016/j.joca.2005.07.018) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 Detection of aggrecan fragments G1-TEGE and G1-IPEN by Western immuno-blot. The human samples were SDS-PAGE separated (4–12% gel) and analyzed by Western immuno-blot using different antibodies as shown. Amount of protein loaded per lane is shown. Mean values of Mw of aggrecan fragments were calculated based on electrophoretic migration. Osteoarthritis and Cartilage 2006 14, 101-113DOI: (10.1016/j.joca.2005.07.018) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 High Mw aggrecan fragments detected in OA cartilage by Western immuno-blot. Human OA aggrecan cartilage-A1D1 was SDS-PAGE separated (3–8% gel) and analyzed by Western immuno-blot with different antibodies as shown. Amount of GAG loaded per lane, the positions of Mw markers and the aggrecan fragments detected are shown. Osteoarthritis and Cartilage 2006 14, 101-113DOI: (10.1016/j.joca.2005.07.018) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 7 The main distribution of aggrecan fragments detected by Western immuno-blot in OA cartilage (A1D1 and A1D3 samples) and OA synovial fluid (D1 and A1 samples). Fragments shown are generated by pathways initiated by aggrecanase action (A) and MMP-action (B) as shown in Fig. 8. Osteoarthritis and Cartilage 2006 14, 101-113DOI: (10.1016/j.joca.2005.07.018) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 8 A hypothetical model of the major proteolysis of cartilage aggrecan in OA joints. ARGS- and FFGV-fragments are generated by two different pathways (I and II). In the pericellular area, full length aggrecan monomers (G1-G2-G3) are expressed and aggregate with HA and link protein; cross-linking of the G3-domain with, e.g., fibulin-2 may also occur. In pathway I, aggrecan turnover in the pericellular area starts by MMP cleavage within the IGD producing G1-IPEN and FFGV-G3 fragments. The HA-bound G1-IPEN fragments are endocytosed by the chondrocytes. The FFGV-G3 fragments are further processed in the CS2 region by aggrecanases, producing FFGV-fragments with various C-terminal sequences (FFGV-G2—), which are partly retained in the tissue by sequence interactions (⊥). In pathway II in the interterritorial area, aggrecan monomers are first cleaved in the CS2 region by aggrecanases (AGNASE) producing HA-bound G1-fragments with various C-terminals. Then the G1-fragments are cleaved by aggrecanase within the IGD, generating HA-bound G1-TEGE fragments and unbound ARGS-fragments having various C-terminals (ARGS-G2—). Finally, in the interterritorial matrix compartment the aggrecan fragments are further processed by aggrecanases and other proteases producing fragments that are released into the synovial cavity. Osteoarthritis and Cartilage 2006 14, 101-113DOI: (10.1016/j.joca.2005.07.018) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions