Elsevier

Biochimie

Volume 88, Issue 7, July 2006, Pages 767-773
Biochimie

Autoantibodies against aggrecan in systemic rheumatic diseases

https://doi.org/10.1016/j.biochi.2006.01.004Get rights and content

Abstract

Objective. – This study was undertaken to investigate the presence of autoantibodies against the main cartilage proteoglycan, aggrecan, in systemic rheumatic disease sera, and to identify substructure(s) responsible for the autoimmune response.

Methods. – Sera were obtained from 86 patients with various systemic rheumatic diseases, 14 with osteoarthritis (OA), 18 with cancer and 40 healthy individuals. The presence of autoantibodies against aggrecan was examined by a solid phase assay and by Western blotting, using proteoglycan aggregates treated with proteolytic enzymes. The positive bands were subjected to nanohigh performance liquid chromatography (nanoHPLC)–MS, in order to identify the aggrecan substructures involved in the autoimmune response.

Results. – Autoantibodies against aggrecan were identified in all systemic rheumatic disease sera at a high titre, almost three times that observed in healthy controls. OA and cancer sera produced a reaction equal to that of the healthy. Western blotting analysis of aggrecan proteolytic fragments revealed the presence of a triple band, reacting with the patients' sera, of about 37 kDa, which also reacted with a polyclonal antibody against hyaluronan-binding region. NanoHPLC–MS analysis suggested that this band belonged to the G2 domain of aggrecan.

Conclusion. – At least a part of the autoimmune reaction to aggrecan, displayed by the systemic disease sera, involves the G2 domain. The significant difference observed between these sera and those from other diseases, especially cancer, may suggest a possible discriminatory role of anti-aggrecan antibodies. This may help in the differential diagnosis in complicated clinical cases. However, for this to be confirmed, studies in larger cohorts of patients should be performed.

Introduction

Aggrecan, the predominant cartilage proteoglycan, contains a 230 kDa core protein, consisting of three globular domains G1, G2 and G3, from the N- to the C-terminus, separated by long segments, hosting the carbohydrates [1], [2], [3]. Through the G1 domain, it interacts strongly with hyaluronan, forming aggregates of enormous molecular mass, maintaining aggrecan within the collagen network. Stabilization of these aggregates is achieved by the link protein (~48 kDa), which binds to the protein core and hyaluronan [2], [3]. Mild trypsin treatment of aggrecan generates a protein fragment containing the G1 and G2 domains, which retains hyaluronan-binding capacity and is termed hyaluronan-binding region (HABR) [4]. The G3 domain includes substructures responsible for additional biologic functions.

Almost 90% of aggrecan mass is carbohydrate, mainly consisting of about 100 chains of chondroitin sulfate (CS) and about 50 chains of keratan sulfate (KS) and of N- and O-linked oligosaccharides. Due to the high glycosaminoglycan content, aggrecan possesses a very high negative charge, enabling cartilage to resist compressive forces.

Degradation of aggrecan occurs during normal cartilage metabolism, and the fragments obtained, corresponding to less than 5% of total aggrecan, are easily taken up by chondrocytes. Increased degradation in various arthritides results in entrance of these fragments into the circulation [5], [6], [7], [8], [9], [10]. Aggrecan negative charge density is decreased, along with its capacity to form aggregates [11], [12].

The development of several experimental models of arthritis contributed tremendously to our understanding of the pathogenesis in humans. In the aggrecan-induced arthritis model, an autoimmune response against the G1 domain of aggrecan was observed, amplified when purified G1, which did not contain KS, was injected [13]. In a similar model, absence of autoantibodies against the glycosaminoglycan structures was observed, suggesting that the immunogenic regions of aggrecan must be located within its protein core and especially in a part close to the CS-binding region [14]. Autoantibodies against aggrecan, with titres correlating with disease activity, have been detected in sera of experimental models [13], [15], and also in synovial fluid of patients with various joint diseases [16]. Interestingly, there are no clear data on serum levels of anti-aggrecan antibodies in patients with inflammatory and other arthritides. More precisely, Karopoulos et al. [16] failed to identify such antibodies in rheumatoid arthritis (RA) sera, whereas Polgar et al. [17] have only incidentally mentioned in a Table of their study the presence of such antibodies, without elaborating in the matter.

The present study aimed at investigating the presence of autoantibodies against aggrecan in the sera of patients with various systemic and joint diseases in comparison with healthy donors and cancer patients and, a preliminary characterization of the immunogenic part(s) of aggrecan.

Section snippets

Materials

V8 protease (Staphylococcus aureus), chondroitinase ACII, keratanase and polyvalent anti-mouse IgG, anti-rabbit IgG and anti-human IgG conjugated with peroxidase were from Sigma (USA). Proteoglycan aggregates (A1 fraction from ultracentrifugation), aggrecan, link protein and HABR of aggrecan were isolated from bovine laryngeal cartilage, as described in [4], [18], since they exhibit high homology to the respective human molecules.

The monoclonal antibodies 3-B-3 (against the CS stubs of aggrecan

Determination of aggrecan autontibodies in human sera

Polystyrene ELISA plate wells were activated by the sequential reaction of glutaraldehyde and spremine, followed by aggrecan immobilization as described previously in [26]. Human sera at 1:1000 dilution in 0.15 M NaCl, 0.01 M sodium phosphate pH 7.4 (PBS) was used for the interaction of the antibodies present with the immobilized aggrecan, which were then detected by anti-human IgG conjugated with peroxidase, followed by addition of o-phenylenediamine and hydrogen peroxide solution.

Control

Serum autoantibodies against aggrecan

The presence of autoantibodies against aggrecan was examined in activated plate wells, to which aggrecan was immobilized electrostatically. Systemic rheumatic disease sera had significantly higher titres than healthy sera (Fig. 1). The mean value of patients' titres was almost three times that of healthy controls (0.397 ± 0.254 versus 0.123 ± 0.76). More precisely, the mean values of titres were 0.389 ± 0.209 for RA, 0.437 ± 0.251 for SLE, 0.446 ± 0.308 for SS and 0.317 ± 0.148 for AS. In 50% of the

Discussion

In the present study, a simple, rapid and sensitive technique for quantitative determination of autoantibodies against aggrecan in human serum was introduced, together with a preliminary characterization of the peptide(s) region(s) of aggrecan that may contribute to its immunoreactivity. For the quantitation of autoantibodies, aggrecan was immobilized onto polystyrene plate wells through electrostatic interactions with spermine, covalently attached to glutaraldehyde bound to the wells. The

Acknowledgements

The financial support of the Research Committee of the University of Patras (K. Karatheodori program) to I. Tsagaraki and that of the Greek Ministry of Education and the European Community (program EPEAEK II––HERAKLITOS) to M. Assouti are acknowledged.

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