Glycosidic intermediates identified in ¹H MR spectra of intact tumour cells may contribute to the clarification of aspects of glycosylation pathways

Abstract

The glycosylation process, through the addition of carbohydrates, is a major post-translational modification of proteins and glycolipids. Proteins may be glycosylated in either the secretory pathway leading to N-linked or O-linked glycoproteins or as nucleocytoplasmic glycosylation that targets only single proteins involving a single β-linked N-acetylglucosamine. In both cases, the key precursors are the uridine diphospho-N-acetylhexosamines synthesised by the hexosamine biosynthetic pathway. Furthermore, uridine diphospho-N-acetylglucosamine participates in the biosynthesis of sialic acid. In this work, we propose MRS for the detection of uridine diphospho-N-acetylhexosamines visible in high-resolution MR spectra of intact cells from different human tumours. Signals from the nucleotide and amino sugar moieties, including amide signals observed for the first time in whole cells, are assigned, also taking advantage of spectral changes that follow cell treatment with ammonium chloride. Finally, parallel changes in uridine diphospho-N-acetylhexosamines and glutamine pools, observed after pH changes induced by ammonium chloride in the different tumour cell lines, may provide more details on the glycosylation processes.

1H‐MRS can detect aberrant glycosylation in tumour cells: a study of the HeLa cell line

“…Spectra of resonance at 2.08 ppm. In agreement with previous literature (17,27), the intensity of the doublet centred at 8.36 ppm in UDP-GlcNAc spectra (labelled as G NH in Fig. 3a,a' ,a''), originating from the NH of sugar moiety, decreased when the solution acidity was neutralized (Fig.…”

“…Similarly, resonance at 2.08 ppm, labelled G CH3 in Fig. 2b, was previously attributed to the CH 3 signal of UDP-GlcNAc (17).…”

“…In addition to the signals from uridine 5'-diphospho-Nacetylglucosamine (UDP-GlcNAc) labelled G NH , U 5 and U 6 , detected and assigned in a previous study (17), a doublet, labelled Gal NH in Fig. 2a, resonated in the low-field region of 1D spectra of HeLa cells, namely at 8.02 ppm.…”

“…A previous observation of sugar intermediates in the low field region of intact tumour cells spectra had recently raised some interest about the possibility of observing new signals, such as peaks from glycosylation precursors (17). Furthermore, the 2D technique is able to identify …”

“…The examination of low-field regions has recently raised some interest about the possibility of observing new signals from glycosylation precursors (15,16). In a previous study (17), we identified some of these signals in the low-field region of intact tumour cells spectra, taking advantage of the use of a very high resolution 1-mm microprobe and of two-dimensional (2D) COSY spectroscopy. In particular, the use of 2D experiments improves metabolite specificity and can solve most one-dimensional (1D) co-resonating signals while keeping good sensitivity, especially in association with high resolution coils.…”