Background/Purpose: O-linked N-acetylglucosamine (O-GlcNAc) post-translational modifications have been implicated in the control of different signaling cascades and in the development and progression of degenerative and age-related diseases. We previously showed that accumulation of O-GlcNAc-modified proteins alters chondrocyte gene expression, leading to increased expression of chondrogenic markers in vitro and increased chondrogenic differentiation in vivo (Andrés-Bergós J, JBC 2012). We also showed that the total amount of O-GlcNAcylated proteins was significantly increased in human osteoarthritic (OA) cartilage compared to that of healthy cartilage (Tardío L, Arthritis Rheum, submitted). Here, we aimed to better profile and identify the O-GlcNAc-modified proteins in human OA cartilage.

Methods: Human OA cartilage was isolated from patients undergoing total knee replacement surgery (n=6), while healthy cartilage was obtained from age- and gender-matched donors (n=6). Wheat germ agglutinin (WGA) chromatography was used to selectively pull-down O-GlcNAc-modified proteins. To this end, equal amounts of proteins isolated from OA and healthy cartilage were loaded on WGA-affinity columns. The eluted fractions containing O-GlcNAc-modified proteins were processed by nano-LC-M/MS, using a 5500QTRAP mass spectrometer (AB Sciex), and subsequently analyzed using GeneCodis software, which grouped proteins based on predefined biological processes.

Results: Healthy and OA human cartilage shared a number of O-GlcNAc-modified proteins; however, our WGA-based analysis identified subsets of proteins O-GlcNAc-modified differentially in health and disease.  Around a 26% of healthy cartilage specific proteins were involved in cytoskeleton organization, and more than 16% play an important role in cell adhesion. None of these biological processes were found in the analysis of OA differential proteins suggesting that these protein modifications may have an important role in preserving cell structural integrity in a healthy state. Interestingly, O-GlcNAc modifications in proteins involved in growth plate cartilage development, cartilage condensation, or inhibition of angiogenesis were absent in OA samples and prominent in healthy cartilage. More importantly, O-GlcNAc modifications of proteins implicated in extracellular matrix degradation, cell growth, or complement activation were higher in OA cartilage than in non-OA/healthy cartilage, clearly separating healthy and damaged tissue based upon biological processes and subsets of O-GlcNAc-modified proteins. 

Conclusion: We report here for the first time subsets of differentially O-GlcNAc-modified proteins in healthy and OA human articular cartilage. Biological processes involving O-GlcNAc proteins are different in OA cartilage in comparison with the healthy one. Our results further highlight the contribution of this means of protein control to the development and progression of OA and, more importantly, may contribute to the identification of potential biomarkers and/or therapeutic targets in OA.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/o-linked-n-acetylglucosamine-modified-proteome-of-human-osteoarthritic-cartilage-biological-significance/