Characterization of modifications and incomplete elongation in the chondroitin sulfate linkage region on aggrecan in response to interleukin-1beta treatment

In contrast to transcription and translation, glycosylation is not a template driven process. Although many of the enzymes and substrates involved are known, regulation of glycosaminoglycan elongation is poorly understood. Aggrecan is a prominent proteoglycan in cartilage, and the abundance of chondroitin sulfate chains on aggrecan gives the tissue many of its resilient properties. The approximately one hundred chains of chondroitin sulfate are attached to the aggrecan core protein through a tetrasaccharide linkage structure consisting of xylose-galactose-galactose-glucuronic acid. This linkage region has been shown to contain galactosyl sulfations and xylosyl phosphorylations that can affect the activity of the enzymes involved in CS chain polymerization. Using chondrocytes extracted from human donor tali cartilage, alginate beads were cultured over the course of two weeks in 5% fetal bovine serum infused media in controlled conditions and interleukin-1beta treated. Unelongated and modified linkage structures were detected using a novel quantitative mass spectrometry-based method to look for targeted structures in controlled conditions and in cell cultures exposed to interleukin-1beta. Our findings show a presence of a variety of unelongated linkage structures, but the unmodified unelongated xylose residue is by far the most prevalent linkage region structure, constituting between 6--12% of the theoretical molar quantity of chondroitin sulfate chains. There was a presence of the di and trisaccharide linkage structures including phospho and sulfo modified structures, but the populations of each constituted less than 1% of molar equivalent of CS chains and therefore less than one chain per aggrecan molecule. Additionally, in comparing the control to IL-1beta treated cells, the amount of unelongated xylose per aggrecan core protein was shown to be significantly less (p-value = 0.03) in cytokine-treated chondrocytes. In conclusion, the application of quantitative mass spectrometry provides a new method for analyzing small glycosylations and the presence of unelongated linkage region is a novel finding in and of itself, especially that the predominant structure is unelongated xylose.