Low Abundant <i>N</i>‑linked Glycosylation in Hen Egg White Lysozyme Is Localized at Nonconsensus Sites

Although wild-type hen egg white lysozyme (HEL) is lacking the consensus sequence motif NX­(S/T), in 1995 Trudel et al. (<i>Biochem. Cell Biol.</i> <b>1995</b>, <i>73</i>, 307–309) proposed the existence of a low abundant <i>N</i>-glycosylated form of HEL; however, the identity of active glycosylation sites in HEL remained a matter of speculation. For the first time since Trudel’s initial work, we report here a comprehensive characterization by means of mass spectrometry of <i>N</i>-glycosylation in wild-type HEL. Our analytical approach comprised ZIC-HILIC enrichment of <i>N</i>-glycopeptides from HEL trypsin digest, deglycosylation by ¹⁸O/PNGase F as well as by various endoglycosidases, and LC–MS/MS analysis of both intact and deglycosylated <i>N</i>-glycopeptides engaging multiple techniques of ionization and fragmentation. A novel data interpretation workflow based on MS/MS spectra classification and glycan database searching enabled the straightforward identification of the asparagine-rich <i>N</i>-glycopeptide [34–45] FESNFNTQATNR and allowed for compositional profiling of its modifying <i>N</i>-glycans. The overall heterogeneity profile of <i>N</i>-glycans in HEL comprised at least 26 different compositions. Results obtained from deglycosylation experiments provided clear evidence of asparagine residues N44 and N39 representing active glycosylation sites in HEL. Both of these sites do not fall into any known <i>N</i>-glycosylation-specific sequence motif but are localized in rarely observed nonconsensus sequons (NXN, NXQ)

α-Lactalbumin Forms with Oleic Acid a High Molecular Weight Complex Displaying Cytotoxic Activity

α-Lactalbumin (LA) forms with oleic acid (OA) a complex which has been reported to induce the selective death of tumor cells. However, the mechanism by which this complex kills a wide range of tumor cell lines is as yet largely unknown. The difficulty in rationalizing the cytotoxic effects of the LA/OA complex can be due to the fact that the molecular aspects of the interaction between the protein and the fatty acid are still poorly understood, in particular regarding the oligomeric state of the protein and the actual molar ratio of OA over protein in the complex. Here, the effect of LA addition to an OA aqueous solution has been examined by dynamic light scattering measurements and transmission electron microscopy. Upon protein addition, the aggregation state of the rather insoluble OA is dramatically changed, and more water-soluble and smaller aggregates of the fatty acid are formed. A mixture of LA and an excess of OA forms a high molecular weight complex that can be isolated by size-exclusion chromatography and that displays cellular toxicity toward Jurkat cells. On the basis of gel filtration data, cross-linking experiments with glutaraldehyde, and OA titration, we evaluated that the isolated LA/OA complex is given by 4−5 protein molecules that bind 68−85 OA molecules. The protein in the complex adopts a molten globule-like conformation, and it interacts with the fatty acid mostly through its α-helical domain, as indicated by circular dichroism measurements and limited proteolysis experiments. Overall, we interpret our and previous data as indicating that the cellular toxicity of a LA/OA complex is due to the effect of a protein moiety in significantly enhancing the water solubility of the cytotoxic OA and, therefore, that the protein/OA complex can serve mainly as a carrier of the toxic fatty acid in a physiological milieu

Exploring the Chemical Space of G‑Quadruplex Binders: Discovery of a Novel Chemotype Targeting the Human Telomeric Sequence

Recent findings have unambiguously demonstrated that DNA G-rich sequences can adopt a G-quadruplex folding in living cells, thus further validating them as crucial targets for anticancer therapy. Herein, to identify new potent G4 binders as antitumor drug candidates, we have targeted a 24-nt G4-forming telomeric sequence employing a receptor-based virtual screening approach. Among the best candidates, <i>in vitro</i> binding experiments allowed identification of three novel G4 ligands. Among them, the best compound features an unprecedented binding selectivity for the human telomeric DNA G-quadruplex with no detectable binding for other G4-forming sequences present at different genomic sites. This behavior correlates with the detected ability to generate DNA damage response in tumor cells at the telomeric level and efficient antiproliferative effect on different tumor cell lines at low micromolar concentrations