Chemoenzymatic Labeling Pathogens Containing Terminal N-Acetylneuraminic Acid−α(2–3)-Galactose Glycans

Abstract

The N-acetylneuraminic acid−α(2–3)-galactose epitope is often located at the nonreducing terminal ends of glycans on the envelopes of many pathogens, and it is believed that this structure mimics a host’s oligosaccharide so as to circumvent and/or counteract the host’s immune responses. A chemoenzymatic method for the rapid and sensitive detection of N-acetylneuraminic acid−α(2–3)-galactose has been built, so we planned to examine whether the chemoenzymatic method could be applied on the detection of N-acetylneuraminic acid−α(2–3)-galactose on pathogens. Our results revealed that the chemoenzymatic method was rapid and sensitive for labeling live or dead Gram-positive Streptococcus agalactiae A909 and Gram-negative Campylobacter jejuni MK104 with N-acetylneuraminic acid−α(2–3)-galactose. This study suggested that the chemoenzymatic method was a new strategy for labeling pathogens and had potential for the diagnosis of or therapeutics for pathogenic infection.

Protocol Considerations for In Vitro Metabolic Glycoengineering of Non‐Natural Glycans

“…When preparing adherent cells for flow cytometry analysis, take care to use the minimal amount of trypsin and shortest exposure time needed to detach the cells to avoid damaging cell-surface components bearing the metabolically glycoengineered glycans. Alternatively, metabolic incorporation of MGE analogs into adherent cells can be monitored by fluorescence microscopy or microplate reader assays (Smirnov et al, 2020;Wen et al, 2019;Wu et al, 2022).…”

“…Examples are described elsewhere and include three basic approaches. In one, cells are solubilized after labeling, releasing fluorophores into suspension where they can be quantitatively measured using a fluorescence microplate reader (Smirnov et al, 2020;Wen et al, 2019;Wu et al, 2022). In the second, metabolic incorporation of MGE analogs can be visualized by fluorescence microscopy, which provides rich detail on the subcellular distribution of the non-natural monosaccharide.…”

Cell-Surface Glycan Labeling and Sensing

“…Chen's group also summarized the development and application of glycosyltransferases until 2021 [53]. A few new glycosyltransferases [66][67][68] relevant to cell labeling have been reported in the last two years, with more attention being paid to improving performance [69] and expanding substrates [70] and applications [71][72][73][74][75][76]. The technique faces several challenges: glycosyltransferases that can tolerate unnatural monosaccharide donors are difficult to find; unnatural monosaccharide derivatives are more difficult to synthesize than the unnatural sugars of MGL; and chemoenzymatic labeling techniques are only suitable for reporting steady-state glycosylation and not for monitoring dynamic glycosylation.…”