Glycan specificity of neuraminidases determined in microarray format

Abstract

Neuraminidases hydrolytically remove sialic acids from glycoconjugates. Neuraminidases are produced by both humans and their pathogens, and function in normal physiology and in pathological events. Identification of neuraminidase substrates is needed to reveal their mechanism of action, but high-throughput methods to determine glycan specificity of neuraminidases do not exist. Here we use two glycan labeling reactions to monitor neuraminidase activity toward glycan substrates. While both periodate oxidation and aniline-catalyzed oxime ligation (PAL) and galactose oxidase and aniline-catalyzed oxime ligation (GAL) can be used to monitor neuraminidase activity toward glycans in microtiter plates, only GAL accurately measured neuraminidase activity toward glycans displayed on a commercial glass slide microarray. Using GAL, we confirm known linkage specificities of three pneumococcal neuraminidases and obtain new information about underlying glycan specificity.

Inhibition of sialidase activity as a therapeutic approach

“…This was the case with characterized substrate specificity of pneumococcal sialidases NanA, NanB, and NanC. 31 Zanamivir and oseltamivir display either weak, as in case of zanamivir, or medium, as in case of oseltamivir, inhibitory effect on pneumococcal neuraminidase. The first report of efficient neuraminidase-inhibiting activity reducing bacterial adherence to pulmonary epithelial cells and hampering biofilm formation and bacterial growth and viability was published in 2015.…”

Prevotellaare major contributors of sialidases in the human vaginal microbiome

“…Though we did not observe inhibition of GvNanH3 by Zanamivir in our experimental setup, it is possible that this enzyme can be partially inhibited as was previously demonstrated 31 . In an accompanying paper substrates such as human vaginal epithelial cell surface glycans 33 , glycan arrays 34 , or in more complex or natural settings 35,18 .…”

“…It is possible that Gv NanH4 acts intracellularly on an as-yet-uncharacterized substrate given its lack of a signal peptide, or is inactive, similar to the Group B Streptococcus sialidase NonA 28 . Future work should examine sialidase activity towards additional substrates such as human vaginal epithelial cell surface glycans 33 , glycan arrays 34 , or in more complex or natural settings 35,18 .…”

Carbohydrate Microarrays Containing Glycosylated Fluorescent Probes for Assessment of Glycosidase Activities

“…Recently, we showed that carbohydrate microarrays were useful for discovering functional glycans that elicited cellular responses . In spite of this high level of activity, applications of carbohydrate microarrays in profiling glycosidase activities have been rare . Below, we describe a new method to characterize glycosidase activities, which utilizes carbohydrate microarrays containing glycosylated fluorescent probes whose fluorescence signals increase upon glycosidase-promoted cleavage of glycosidic bonds.…”