LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications

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.

The dual role of the glycosaminoglycan chondroitin‐6‐sulfate in the development, progression and metastasis of cancer

“…Evidence supporting the suggestion that the 6‐ O ‐sulfation of CS can strongly influence the behavior of a tumor cells (Fig. ) has come from the observation that xyloside‐primed CS/DS that was produced by a breast carcinoma cell line demonstrated a strong cytotoxic activity toward both normal and tumor cells . This effect, which involved the induction of apoptosis, was neutralized by cancer‐cell‐synthesized heparan sulfate .…”

“…Interestingly, CS/DS with these cytotoxic properties had a very high proportion of the 6-O-sulfated disaccharides in relation to the 4-O-sulfated ones (82-63% versus 15-30%) compared to the cytotoxically inactive GAG from normal breast fibroblasts, which contained~60% of the 4-O-sulfated disaccharides and~34% of the 6-O-sulfated ones [167,168]. Moreover, the cytotoxic CS/DS also differed from the GAG that was produced by normal cells in respect to a significantly lower content of the IdoA residues (~10% versus~40%, respectively) and to the occurrence of 4,6-O-disulfated disaccharides (E units) [167,168]. However, despite their known biological potential, the presence of E units cannot be the only structural determinant that is responsible for the cytotoxic effect of the xyloside-primed CS/DS because another CS/DS that was used in the research and that had a similar level of these disaccharides did not demonstrate any cytotoxic activity [167].…”

Glycosaminoglycan Domain Mapping of Cellular Chondroitin/Dermatan Sulfates

“…Thereafter, we subjected the samples to nanoflow reversed-phase ion-pairing (RPIP) chromatography using an in-house packed C18 column and dibutylamine (DBA) as an ion-pairing agent. RPIP was selected due to its high chromatographic resolution potential 21,[24][25][26] , and DBA was selected since it is relatively volatile, tends to result in shorter retention times and reduces the number of overlapping charge states compared to other ion-pairing agents 24 . The chromatographic system was directly coupled to an LTQ Orbitrap Elite mass spectrometer operating in negative ionization mode.…”

“…For initial evaluation of the approach, we used disaccharide standards and compared the results to our recently reported microscale LC-MS/MS setup (Fig. S1) 21 . GAGDoMa facilitated a ~300 times more sensitive detection of precursor ions than the previous approach, was more robust in terms of precursor ion intensity (Fig.…”

“…The MS analysis was performed by nanoflow reversed-phase dibutylamine ion-pairing chromatography with negative mode higher-energy collision dissociation (HCD) MS/MS, enabling fragment ion characterization, compositional profiling and semi-quantification of precursor ions. To challenge GAGDoMa, CS/DS primed on xylosides from two types of human cell lines were used as models as they were predicted to cover a wide range of structures 21,22 . Using this technology, we have identified over 200 structures and characterized more than 60 of these in depth.…”