Detection of Bacterial α-l-Fucosidases with an Ortho-Quinone Methide-Based Probe and Mapping of the Probe-Protein Adducts.

Yvette M C A Luijkx,Esther M Van ‘T Veld,Koen C A P Giesbers,Gerlof P Bosman,Albert J R Heck,Dario A T Cramer,Karli R Reiding,Tom Wennekes,Geert-Jan Boons,Karin Strijbis,Anniek J Henselijn

doi:10.3390/molecules27051615

Yvette M C A Luijkx, Esther M Van ‘T Veld + Show 9 more

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https://doi.org/10.3390/molecules27051615

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Abstract

Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria.

Highlights

Introduction αL-Fucosidases are enzymes capable of catalyzing the hydrolytic removal of terminalL-fucose residues from glycoconjugates
It is known that an abnormal increase in α-Lfucosidase activity in humans is associated with several pathological conditions [1–4]
The quinone methide generated by this probe mainly labels on specific tryptophan residues outside of the active site of the TmFuc fucosidase enzyme, with single protein–probe adducts dominating

Summary

Introduction

Introduction αL-Fucosidases are enzymes capable of catalyzing the hydrolytic removal of terminalL-fucose residues from glycoconjugates. L-Fucosidases are enzymes capable of catalyzing the hydrolytic removal of terminal. It is known that an abnormal increase in α-Lfucosidase activity in humans is associated with several pathological conditions [1–4]. Bacterial α-L-fucosidases are key enzymes for the degradation and metabolism of intestinal mucin O-glycans by gut microbes. This crucial family of enzymes thereby contributes to the composition of the gut microbiota and influences our health and disease [5]. We and others recently reported that fucosidases of two Bacteroides species from the human gut microbiota induced upregulation of growth and invasive properties of pathogenic Campylobacter jejuni strains [6,7]. A growing number of studies are implicating bacterial fucosidases in the host–microbe interplay in the intestine, microbiota cross-feeding, and colonization resistance [3,5,8–10]

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