Chondroadherin Fragmentation as a Biochemical Marker for Early Stage Disk Degeneration

Introduction Intervertebral disk (IVD) degeneration has been strongly associated with and named a major cause of back pain. At present, little is known about the molecular mechanisms involved in the degeneration of IVD and how these may differ from normal turnover of the tissue. As a result of this, a biomarker for disk degeneration has not yet been identified and we propose chondroadherin (CHAD) fragmentation as a potential option. CHAD, a protein of the leucine rich repeat (LRR) family, is one of the proteins predominantly expressed in the extracellular matrix of cartilaginous tissue, including that of the IVD. This restricted distribution is unusual among LRR proteins, which commonly show a wide tissue distribution. CHAD is primarily found close to the cells, where it can interact with collagen fibrils of the ECM and molecules at the cell surface, providing a mechanism for regulating cell metabolism and ECM structure. These interactions may also aid in promoting matrix homeostasis, and variation in CHAD abundance or structure might therefore lead to pathological changes in the tissue over time. The aims of this study were to determine whether CHAD fragmentation occurs and is unique to disk degeneration, and to characterize the cleavage site within CHAD at which fragmentation occurs. Materials and Methods Healthy and degenerate lumbar IVDs were obtained through organ donations via Transplant Quebec. IVDs from patients with degenerative disk disease and from patients with scoliosis were obtained at the time of surgery. Punches of 4 mm were taken and disk tissue then extracted using 15 volumes of extraction buffer (4 M GuCl, 10 mM EDTA, COMPLETE, 50 mM NaAc, pH 5.8) on a wet weight per volume basis. Extracted proteins were ethanol precipitated, and CHAD fragmentation was studied using SDS-PAGE and western blotting in combination with specific antibodies. To characterize the CHAD cleavage site, a degenerate disk extract was subjected to CsCl density gradient centrifugation to remove proteoglycans. CHAD was purified from the protein fraction by chromatography through carboxymethyl 52. The CHAD-containing samples were then fractionated on an SDS-PAGE gel and stained with Coomassie Blue. Gel portions containing the CHAD fragment were excised, then lyophilized, reduced and alkylated, and subjected to trypsin digestion. Peptides were then identified by liquid chromatography mass spectrometry. Antineoepitope antibodies specifically recognizing the fragmented CHAD were generated by immunizing rabbits with synthetic peptides conjugated to KLH. The peptides represented the terminal amino acid sequences at the site of CHAD fragmentation. Results Proteolytic fragmentation of CHAD was observed in IVDs from patients with DDD and in some individuals with adolescent idiopathic scoliosis (AIS). Its presence appeared to be related to the degree of degeneration in both cases (Fig. 1). This phenomenon was found to be specific to disk degeneration, as CHAD fragmentation was not observed in healthy adolescent and adult disks from organ donors. Within the degenerate disk, fragmentation was evident in tissue with signs of degeneration but not in tissue that had no signs of degeneration. This same trend was also seen when comparing normal and degenerate disk tissue from different levels of the spine from the same donor. Upon analysis with the antineoepitope antibody, it was apparent that CHAD fragmentation occurred at the same site in degenerate disks from Transplant Quebec donors, surgical samples from adults with DDD, and surgical samples from adolescents with AIS. Normal tissue samples showed no antineoepitope antibody binding, confirming that CHAD fragmentation at this site was not present in the healthy disk. Conclusion CHAD fragmentation is associated with disk degeneration present both in the adult with DDD and the adolescent with scoliosis, and fragmentation is created by cleavage at the same site within both disorders. Fragmentation is not, however, present in normal disk tissue. Thus, CHAD fragmentation may distinguish catabolic processes leading to disk degeneration from those associated with normal turnover of the tissue, and as such serve as a marker of disk degeneration. It is necessary to recognize the biochemical processes that specifically contribute to disk degeneration, if degeneration is to be prevented or retarded, and if novel treatments initiating disk regeneration are to be developed. The mechanism leading to fragmentation and the proteinase involved are currently under investigation. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

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