Monoclonal antibodies that specifically recognize neoepitope sequences generated by ‘aggrecanase’ and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro

Abstract

Monoclonal antibodies have been prepared that react specifically with the neoepitopes present on proteoglycan degradation products generated from the proteolytic cleavage of aggrecan in the interglobular domain. Antibody BC-3 recognizes the new N-terminus (ARGSV...) on aggrecan degradation products produced by the action of the as yet uncharacterized proteolytic activity, 'aggrecanase', and antibody BC-4 recognizes the new C-terminus (...DIPEN) generated by the proteolytic action of matrix metalloproteinases. Specificity for these neoepitope sequences was determined in competitive e.l.i.s.a. using synthetic peptide antigens as inhibitors. Antibody BC-3 was used in the detection of aggrecan degradation products in the culture medium obtained from two different in vitro culture systems: bovine cartilage explants treated with either retinoic acid or interleukin-1, and secondly, rat chondrosarcoma cells treated with retinoic acid. Both interleukin-1 and retinoic acid treatment caused an increase in aggrecan catabolism resulting in an increased release to the medium of specific aggrecan degradation products containing the BC-3 neoepitope generated by the action of 'aggrecanase'. However, several additional aggrecan catabolites were present that were not immunoreactive with antibody BC-3. In addition, under control conditions, in the bovine cartilage cultures the BC-3 epitope was found on some of these aggrecan catabolites. In contrast, no immune-reactive material was found in the aggrecan degradation products present in control media of rat chondrosarcoma cells cultured in the absence of retinoic acid. Collectively, these results demonstrate that 'aggrecanase' activity is not a constitutive event in all cartilage culture systems and also suggest that proteolytic agents other than 'aggrecanase' are involved in aggrecan catabolism in normal turnover compared with pathological conditions. Antibody BC-4 was used to demonstrate the identity of the G1 domain of aggrecan following proteolytic cleavage of a purified G1-G2 preparation with collagenase, gelatinase A or stromelysin. The G2 product of this cleavage did not react with antibody BC-3, indicating that, under the experimental conditions used, none of these enzymes exhibited 'aggrecanase' activity. It is expected that both of these antibodies will play a pivotal role in detailed studies elucidating molecular mechanisms of aggrecan degradation and they will be particularly useful for the sensitive monitoring of aggrecan degradation products in tissue extracts and body fluids.

Aggrecan degradation in human intervertebral disc and articular cartilage

“…Monoclonal anti-neoepitope antibodies have recently been developed to permit the specific detection of the cleavage products of proteolytic action [21,22]. However, it has since been shown that polyclonal antisera with similar specificity can be prepared [23,24].…”

Degradation of cartilage aggrecan by collagenase‐3 (MMP‐13)

“…Samples electrophoresed on 5% SDS gels [30] or agarose/acrylamide composite gels [31] were transferred onto Immobilon membrane and analysed for AF-28 epitope [17] or BC-3 epitope [32]. After blocking with 5% skim milk powder in PBS, the membranes were incubated with AF-28 antibody (1 : 1,000 dilution) or BC-3 antibody (1 : 1,000) in 0.5% skim milk powder in PBS for 1 h at room temperature then washed six times in buffer containing 0.1% tween-20 in PBS.…”

“…Prior to immunodetection with BC-3 antibody, glycosaminoglycan chains present on whole aggrecan (but not G1-G2) were removed by digesting the membranes with 0.01 U/ml chondroitin ABC lyase, 0.01 U/ml keratanase and 0.001 U/ml keratanase 11 in 50 mM Tris/Acetate pH 7.4 for 2 h at room temperature [32].…”

Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro

“…Immunoblotting of membranes and incubations with primary and secondary antibodies was performed as previously described [27] using monoclonal antibodies (MAbs) or polyclonal antiserum as follows : MAb BC-13 (1 : 250) and MAb BC-3 (1 : 2000 ; [11]) recognizing the aggrecanase generated C-and N-terminal IGD neoepitopes ...ITEGE and ARGSV … respectively ; polyclonal anti-DIPEN (1 : 250 ; [13,19]) and MAb BC-14 (1 : 1000 ; [11,28]) recognizing the MMP-generated C-and N-terminal IGD neoepitopes … DIPEN and FFGVG … respectively ; and MAb 2-B-6 (1 : 10 000) recognizing a chondroitinase-generated chondroitin-4-sulphate disaccharide epitope. All Western blots were scanned and differences in staining intensity of digitized bands were assessed using NIH Image (version 1.55) software.…”

“…The study of aggrecan IGD catabolism by aggrecanase and MMPs has been greatly facilitated by the development of antibodies which specifically recognize the new N-and C-terminal amino acid sequences (' catabolic neoepitopes ') generated by cleavage at Glu$($-Ala$(% by aggrecanase (anti-ARGSV and anti-ITEGE) or at Asn$%"-Phe$%# by MMPs (anti-FFGVG and anti-DIPEN) [5,[10][11][12][13]. Studies utilizing antibodies to the C-terminal neoepitopes on aggrecan catabolites retained within the cartilage matrix have confirmed the presence of G1-bearing fragments resulting from cleavage at both the aggrecanase and MMP sites [14][15][16][17][18][19].…”