Abstract
Structural characterization of glycosaminoglycans remains a challenge but is essential for determining structure-function relationships between glycosaminoglycans and the biomolecules with which they interact and for gaining insight into the biosynthesis of glycosaminoglycans. We have recently reported that xyloside-primed chondroitin/dermatan sulfate derived from a human breast carcinoma cell line, HCC70, has cytotoxic effects and shown that it differs in disaccharide composition from nontoxic chondroitin/dermatan sulfate derived from a human breast fibroblast cell line, CCD-1095Sk. To further investigate the structural requirements for the cytotoxic effect, we developed a novel LC-MS/MS approach based on reversed-phase dibutylamine ion-pairing chromatography and negative-mode higher-energy collision dissociation and used it in combination with cell growth studies and disaccharide fingerprinting. This strategy enabled detailed structural characterization of linkage regions, internal oligosaccharides, and nonreducing ends, revealing not only differences between xyloside-primed chondroitin/dermatan sulfate from HCC70 cells and CCD-1095Sk cells, but also sialylation of the linkage region and previously undescribed methylation and sulfation of the nonreducing ends. Although the xyloside-primed chondroitin/dermatan sulfate from HCC70 cells was less complex in terms of presence and distribution of iduronic acid than that from CCD-1095Sk cells, both glucuronic acid and iduronic acid appeared to be essential for the cytotoxic effect. Our data have moved us one step closer to understanding the structure of the cytotoxic chondroitin/dermatan sulfate from HCC70 cells primed on xylosides and demonstrate the suitability of the LC-MS/MS approach for structural characterization of glycosaminoglycans.
Highlights
The ratio of m/z 282.03 to m/z 300.04 was roughly constant for the linkage region variants from HCC70 cells; m/z 282.03 was consistently dominating, suggesting that the GalNAc was primarily sulfated at position 6
We have recently shown a cytotoxic effect of CS/DS from a human breast carcinoma cell line, HCC70, primed on XylNap or XylNapOH, and linked the effect to the GAG structure, as the disaccharide composition differed from that of nontoxic CS/DS from a human breast fibroblast cell line, CCD-1095Sk, primed on the same xylosides [34]
By using a novel LC–MS/MS approach, we have in detail characterized the linkage region structures, internal saccharides, and NREs of the CS/DS from HCC70 cells and CCD-1095Sk cells primed on XylNap
Summary
To investigate whether the cytotoxic effect of XylNapprimed CS/DS from HCC70 cells could be linked to GlcUA- or IdoUA-containing structures of the CS/DS, XylNap-primed GAGs were isolated from culture media of HCC70 cells after treatment with 100 M XylNap and degraded enzymatically. The XylNap-primed GAGs degraded with a mixture of heparinase II and III and chondroitinase ABC were found to lack a distinct effect on the cell growth, indicating that the disaccharides and linkage region-containing structures obtained after this degradation were nontoxic. The XylNap-primed GAGs degraded both with the mixture of heparinase II and III and chondroitinase AC-I and -II, and the mixture of heparinase II and III and chondroitinase B, in contrast, had growth-reducing effects on the HCC70 cells, corresponding to IC50 values of ϳ9 g/ml in each case (Fig. 2) This suggests that both GlcUAand IdoUA-containing structures are important for the cytotoxic effect of the XylNap-primed CS/DS from HCC70 cells. The XylNap- and XylNap-d7primed GAGs were degraded using heparinase II and III, chon-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
