Degradome of the Intervertebral Disc

Abstract

Introduction: The intervertebral disc (IVD) is a complex structure consisting of the nucleus pulposus (NP), annulus fibrosus (AF) and cartilage endplates (EP). These components must work collectively for proper disc function. During aging and degeneration, changes to the IVD result in changes to the biochemical and mechanical function of the IVD – potentially leading to degeneration, of which the aetiology is still not fully understood. In human IVD, cell populations are sparse (relative to other species such as the mouse) of which the cells are surrounded by matrix. These cells are responsible for the production of matrix, of which its upkeep is important for disc maintenance and function. However, the processes that are associated with cell maintenance in the non-degenerated condition, versus the events that lead to degeneration of the IVD are not known. Moreover, the presence of many reported enzymes in the IVD are likely to contribute to changes in the disc matrix by cleaving proteins and potentially altering protein function, and subsequently disc function. By studying the degradome of the IVD, this may lead to the identification of fragmented proteins and the enzymes that degrade these proteins and elucidate events that occur in IVD maintenance versus IVD degeneration, of which cannot be identified by conventional mass spectrometry techniques. In this study, we examined the degradome of non-degenerated and degenerated IVD using Terminal Amine Isotopic Labelling of Substrates (TAILS) which facilitates the enrichment of N-termini peptides of degraded protein fragments. Material and Methods: NP and AF were isolated from three non-degenerated IVD and three degenerated IVD. Samples were snap frozen and subsequently pulverized in liquid nitrogen. Samples were extracted with 1% SDS with protease inhibitor and subsequently quanitified. N-terminal peptides were then enriched using the TAILS method. In brief, each sample was individually labelled with a Tandem Mass Tag (TMT) and then mixed together at equal ratios. The pooled sampe was digested with trypsin, and the peptides containing free amines were depleted by polymerization. Samples were cleaned and subsequently analysed by mass spectrometry on a Bruker Daltonics Impact II QTOF, and analysis was conducted using Scaffold Software. Results: Sixty-one peptides (corresponding to 61 protein fragments) were identified in the NP samples. In degenerate NP, there were more cleaved matrix proteins, including fibronectin, COMP, and lubricin. In non-degenerated NP, the cleaved proteins included collagen II and lysozyme. Ninety-one peptides (corresponding to 91 protein fragments) were identified in the AF samples. In degenerated AF, there were more cleaved matrix proteins including COMP and fibronectin, in addition to CILP and HAPLN1. Non-degenerate AF had more cleaved collagen I, II, and VI protein. Conclusion: In degenerate AF and NP, the presence of more cleaved matrix proteins, particularly those that are associated with stability, can potentially lead to alterations in the mechanical properties and function of the IVD. On the other hand, the cleaved proteins in the non- degenerate tissue could indicate turnover of proteins that is associated with maintaining a non-degenerate disc environment (such as collagen II in NP, and collagens I and II in AF). Understanding the degenerative process and maintenance of the disc via analysis of the degradome can highlight events that are occurring in health and in degeneration, in addition to the identification of enzymes that cleave these proteins, of which are currently in progress.