Endo-β-N-acetylglucosaminidases from infant gut-associated bifidobacteria release complex N-glycans from human milk glycoproteins

Abstract

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-β-N-acetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man(3)GlcNAc(2)(α1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies.

Fig. 1.

Endoglycosidase activity found in Bifidobacterium isolates. A, deglycosylation of RNaseB by B. infantis ATCC 15697 over time. B–D, overnight incubation with RNaseB was evaluated for other isolates of B. longum (B), B. infantis (C), or B. breve (D). E, phylogenetic representation of predicted endoglycosidase protein sequences found in bifidobacterial isolates. C: control reactions.

Fig. 2.

Properties of the recombinant endoglycosidases in bifidobacteria. A, heat tolerance of EndoBI-1, EndoBI-2, or EndoBB evaluated in SDS-PAGE gels, as evaluated by RNaseB deglycosylation with each enzyme incubated at 95 °C for the times indicated. B, coincubations of bLF (upper panel) and hLF (lower panel) with EndoBI-1 (lane 1), EndoBI-2 (lane 2), EndoBB (lane 3), or PNGase F (lane 4). Control (lanes C) nondigested reactions were included in both experiments.

Fig. 3.

MALDI FT-ICR MS analysis of glycans released after coincubation of EndoBI-1 with bLF (A), hLF (B, positive mode), and IgA (C, positive mode). Green circles, mannose; blue squares, GlcNAc; red triangles, fucose; yellow circles, galactose; pink diamonds, NeuAc.

Fig. 4.

Properties of EndoBI-1 D184N. A, glycan array analysis of the mutant enzyme binding to mammalian glycans (x axis). The bars represent S.D. of sextuplicates. Glycans bound are provided, and the legend is as in Fig. 3. B, binding of EndoBI-1 D184N to coated glycoproteins, as detected by a FITC-anti-His antibody. The error bars represent S.D. from triplicate experiments, and the values of BSA binding to glycoproteins were withdrawn from EndoBI-1 D184N. The asterisks represent samples with p < 0.05 compared with BSA.

Fig. 5.

EndoBI-1 activity in breast milk. A, SDS-PAGE gel of overnight incubation of human milk (lane 1, control) with EndoBI-1 (lane 2), EndoBI-1 D184N (lane 3), or PNGase F (lane 4). Protein identities were deduced from Ref. . B, amount of N-glycosylation (proportional to α-mannose) in samples from A. The error bars represent S.D. from triplicate experiments. The asterisks represent samples with p < 0.05 compared with control.

Fig. 6.

Fold changes in gene expression for B. infantis ATCC 15697 genes during time coincubation with bLF or hLF, as indicated in the figure legend. Numbers in the x-axis represent B. infantis ATCC 15697 locus tags (Blon_). The error bars represent S.D. from three biological replicates. A, genes associated to GlcNAc metabolism and located close to EndoBI-1. B, genes previously described to be associated or induced by HMO.