Chondroadherin Fragmentation as a Biochemical Marker for Early Stage Disk Degeneration

Abstract

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...

Peptide Location Fingerprinting Reveals Tissue Region-specific Differences in Protein Structures in an Ageing Human Organ

“…Consequently, ECM assemblies residing in the IVD (such as collagen I in the AF [16] and aggrecan in the NP [17]) are susceptible to the progressive accumulation of damage to both their molecular structures and networks over time, leading to loss of functions of many diverse ECM components. The potential causative mechanisms of this remodelling include the chronic exposure of ECM proteins to elevated protease mediated cleavage [18], cross-linking by sugars leading to advanced glycation end products (AGEs) [19], mechanical fragmentation [20] and oxidation by reactive oxygen species (ROS) [21]. These processes may lead to a spectrum of modifications to protein higher order structures that are challenging to detect.…”