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

Abstract

Glycosylation is the most abundant and diverse form of post-translational modification of proteins. Two types of glycans exist in glycoproteins: N-glycans and O-glycans often coexisting in the same protein. O-glycosylation is frequently found on secreted or membrane-bound mucins whose overexpression and structure alterations are associated with many types of cancer. Mucins have several cancer-associated structures, including high levels of Lewis antigens characterized by the presence of terminal fucose. The present study deals with the identification of MR signals from N-acetylgalactosamine and from fucose in HeLa cells by detecting a low-field signal in one-dimensional (1D) spectra assigned to the NH of N-acetylgalactosamine and some cross peaks assigned to fucose in two-dimensional (2D) spectra. The increase of Golgi pH by treatment with ammonium chloride allowed the N-acetylgalactosamine signal assignment to be confirmed. Behaviour of MR peak during cell growth and comparison with studies from literature taken together made it possible to have more insight into the relationship between aberrantly processed mucin and the presence of non-processed N-acetylgalactosamine residues in HeLa cells. Fucose signals, tentatively ascribed to residues bound to galactose and to N-acetylglucosamine, are visible in both intact cell and perchloric acid spectra. Signals assigned to fucose bound to galactose are more evident in ammonium chloride-treated cells where structural changes of mucin-related Lewis antigens are expected as a result of the higher Golgi pH. A common origin for the N-acetylgalactosamine and fucose resonances attributing them to aberrantly processed mucin can be inferred from the present results.

1H NMR spectroscopy of glioblastoma stem‐like cells identifies alpha‐aminoadipate as a marker of tumor aggressiveness

“…The measurement of cell samples lasted approximately 210 min (60 min for the 1D experiment and 150 min for the 2D experiment). Cell viability, tested by the trypan blue exclusion method, was greater than 90% at the beginning of the preparation and greater than 80% at the end of the NMR measurements, in agreement with previous works . NMR parameters were obtained in at least three independent experiments and data are expressed as mean ± standard deviation (SD) values.…”

“…An aliquot (volume 15 µl) of the suspension was inserted in a 1 mm NMR tube and then centrifuged to obtain the final packed cell volume; all cell samples were within the sensible NMR coil volume. Perchloric acid (PCA) extracts were prepared as described in a previous work . 10 mM L2‐aminoadipic acid pure compound was dissolved in H 2 O with 2 mM TMSP and 10% D 2 O, at pH 7.…”

“…NMR measurements and spectral analysis were performed as previously described . In particular, 1D 1 H NMR spectra were acquired with a 90° RF pulse.…”

“…This peak was considered representative of the cellular mass according to Palma et al . and references therein. αAAD being derived from Lys, the intensity ratio αAAD/(αAAD + Lys), which gives the percentage of catabolized Lys, was monitored when groups of different αAAD intensity were considered, as indicated in the legend of Figure (b).…”

1H NMR detects different metabolic profiles in glioblastoma stem‐like cells

“…Typically, 1000 scans were sufficient to obtain a good signal‐to‐noise ratio for intact cells, while 4000 scans were used for perchloric acid (PCA) extract spectra. PCA extracts were prepared as previously described .…”