Detection of O-Linked-N-Acetylglucosamine Modification and Its Associated Enzymes in Human Degenerated Intervertebral Discs.

Abstract

Human herniated discs were obtained from discectomy specimens for the immunohistochemical detection of O-GlcNAc and O-GlcNAcase (OGA)/O-GlcNAc transferase (OGT). This study aimed to quantify the extent of O-GlcNAcylation and its associated enzymes (OGT/OGA) in human degenerated intervertebral discs. The O-GlcNAcylation of nuclear, cytoplasmic, and mitochondrial proteins as well as the effects of such post-translational modifications are currently the focus of extensive research. O-GlcNAcylation is believed to contribute to the etiology of chronic illnesses by acting as a nutrient and stress sensor in the cellular environment. Mature intervertebral disc cells are chondrocyte-like cells, and O-GlcNAc has been shown to promote chondrocyte apoptosis <i>in vitro</i>. We believe that O-GlcNAcylation is a key regulator of disc degeneration. Fifty-six specimens were fixed for 24 hours in a 10% solution of neutral-buffered formaldehyde, dehydrated, and embedded in paraffin. Tissue slices (4-µm-thick) were used for hematoxylin-eosin staining and immunohistochemistry. We found that O-GlcNAcylation of cytoplasmic proteins was less than that of nuclear proteins in both single cells and cell clusters. Cytoplasmic O-GlcNAcylation occurs subsequent to nuclear O-GlcNAcylation and is directly proportional to disc degeneration. OGT and O-GlcNAc expression levels were identical in all specimens examined. O-GlcNAc and OGA/OGT expression is shown to correlate for the first time with intervertebral disc cell degeneration. Increasing disc degeneration is associated with increasing O-GlcNAcylation in both nuclear and cytoplasmic proteins in human disc cells.