Cooperation of β-galactosidase and β-N-acetylhexosaminidase from bifidobacteria in assimilation of human milk oligosaccharides with type 2 structure

Abstract

Bifidobacteria are predominant in the intestines of breast-fed infants and offer health benefits to the host. Human milk oligosaccharides (HMOs) are considered to be one of the most important growth factors for intestinal bifidobacteria. HMOs contain two major structures of core tetrasaccharide: lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc; type 1 chain) and lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc; type 2 chain). We previously identified the unique metabolic pathway for lacto-N-tetraose in Bifidobacterium bifidum. Here, we clarified the degradation pathway for lacto-N-neotetraose in the same bifidobacteria. We cloned one β-galactosidase (BbgIII) and two β-N-acetylhexosaminidases (BbhI and BbhII), all of which are extracellular membrane-bound enzymes. The recombinant BbgIII hydrolyzed lacto-N-neotetraose into Gal and lacto-N-triose II, and furthermore the recombinant BbhI, but not BbhII, catalyzed the hydrolysis of lacto-N-triose II to GlcNAc and lactose. Since BbgIII and BbhI were highly specific for lacto-N-neotetraose and lacto-N-triose II, respectively, they may play essential roles in degrading the type 2 oligosaccharides in HMOs.

Genome analysis of Bifidobacterium bifidum PRL2010 reveals metabolic pathways for host-derived glycan foraging

“…Additional glycosyl hydrolases involved in the breakdown of mucin-derived oligosaccharides were detected in the predicted proteome of B. bifidum PRL2010, including four putative N-acetyl-β-hexosaminidases (BBPR_0026, BBPR_1018, BBPR_1514, and BBPR_1529), four predicted β-galactosidases (BBPR_150, BBPR_0482, BBPR_1355, and BBPR_ 1460), and two putative exo-α-sialidases (BBPR_1793 and BBPR_ 1794) scattered across the PRL2010 chromosome. Interestingly, homologs of two putative N-acetyl-β-hexosaminidases (BBPR_1018 and BBPR_1529), as well as a predicted β-galactosidase (BBPR_ 0482) of B. bifidum JCM1254, are involved in the degradation of lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc) present in the core structures of HMOs as well as in core 2 mucin-type Oglycans (36) (36), the galactosidase (BBPR_0482) and both N-acetyl-β-hexosaminidases (BBPR_1018 and BBPR_1529) possess an Nterminal signal sequence and a C-terminal transmembrane region, suggesting that they act as extracellular membrane-bound enzymes. In contrast, the two other putative N-acetyl-β-hexosaminidases (BBPR_0026 and BBPR_1514) are predicted to represent intracellular proteins.…”

Carbohydrate metabolism in Bifidobacteria

“…Oral administration of galactooligosaccharides, produced by different b-glucosidases isolated from B. bifidum NCIMB41171 [140], significantly increases bifidobacterial numbers in the stool of healthy individuals, suggesting that these carbohydrates have bifidogenic properties and can successfully be used as prebiotic [21]. In addition, the characterized b-galactosidase from B. bifidum NCIMB 41171 was shown to degrade human milk oligosaccharides lacto-N-tetraose (LNT) and lacto-N-neotetraose more rapidly than lactose [79].…”

Unique Sugar Metabolic Pathways of Bifidobacteria

“…59) They are one GH2 -galactosidase (BbgIII, EC 3.2.1.23) and two GH20 HexNAcases (BbhI and BbhII), and two of these are responsible for thedegradation of type II oligosaccharides. BbgIII consists of 1,935 amino acids and contains an N-terminal signal peptide, a GH2 catalytic domain, two Ig-like domains, two CBM32 domains, three FIVAR domains, and a C-terminal membrane anchor.…”