Chromatographic analysis of polysialic acid released from neural adhesion molecule presented on glioma cell membranes

Polysialic acid (PolySia) is a cell-membrane carbohydrate polymer, composed of a-2,8 linked sialic acid monomers (SA). In mammals, PolySia is mainly presented on the neural adhesion molecule (NCAM) glycoprotein. Two closely related polysialyltransferases are responsible for polysialylation of NCAM, ST8Sia IV and ST8Sia II. It was reported that PolySia is expressed in a number of highly malignant tumours, where it was established that PolySia is associated with their progression. The critical role of PolySia in malignancy has focused our attention towards the development of convenient, rapid, and efficient chromatographic methods for the assessment of its level on the surfaces of tumour cells. Such development was undertaken using a rat C6 glioma cell line transfected with a human ST8Sia II gene (C6- ST8Sia II), which is over expressing PolySia, as a model, and C6- wild type cell line (C6-WT), with low expression of PolySia, as a control. After the detection of polysialylated NCAM at 250 kDa in C6-ST8Sia II cells by western blotting, endo-β-galactosidase and neuraminidases were utilised to cleave PolySia from C6- ST8Sia II cell membranes in order to analyse the released polymers, using chromatographic analysis. However, cellular Enzyme Linked-Immunosorbent Assay (ELISA) indicated the lack of such PolySia release. Therefore, a new strategy was then devised towards the digestion of PolySia from membranes of C6-ST8Sia II cells by endoneuraminidase (endo-N) enzymes, followed by a chromatographic measurement of totally hydrolysed PolySia polymers (as SA monomers). Although PolySia digestion by endo-N was successfully detected by western blotting, there was no differential chromatographic measurement in total SA observed between C6- ST8Sia II and C6-WT. We suggest that this could be due to the high background concentrations of SA monomers released from within both cell lines.