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      1,6-α-L-Fucosidases from Bifidobacterium longum subsp. infantis ATCC 15697 Involved in the Degradation of Core-fucosylated N -Glycan

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          Abstract

          Bifidobacterium longum subsp. infantis ATCC 15697 possesses five α-L-fucosidases, which have been previously characterized toward fucosylated human milk oligosaccharides containing α1,2/3/4-linked fucose [Sela et al.: Appl. Environ. Microbiol., 78, 795-803 (2012)]. In this study, two glycoside hydrolase family 29 α-L-fucosidases out of five (Blon_0426 and Blon_0248) were found to be 1,6-α-L-fucosidases acting on core α1,6-fucose on the N-glycan of glycoproteins. These enzymes readily hydrolyzed p-nitrophenyl-α-L-fucoside and Fucα1-6GlcNAc, but hardly hydrolyzed Fucα1-6(GlcNAcβ1-4)GlcNAc, suggesting that they de-fucosylate Fucα1-6GlcNAcβ1-Asn-peptides/proteins generated by the action of endo-β- N-acetylglucosaminidase. We demonstrated that Blon_0426 can de-fucosylate Fucα1-6GlcNAc-IgG prepared from Rituximab using Endo-CoM from Cordyceps militaris. To generate homogenous non-fucosylated N-glycan-containing IgG with high antibody-dependent cellular cytotoxicity (ADCC) activity, the resulting GlcNAc-IgG has a potential to be a good acceptor substrate for the glycosynthase mutant of Endo-M from Mucor hiemalis. Collectively, our results strongly suggest that Blon_0426 and Blon_0248 are useful for glycoprotein glycan remodeling.

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          Most cited references 36

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          Nursing our microbiota: molecular linkages between bifidobacteria and milk oligosaccharides.

          As the sole nutrition provided to infants, bioactive molecules dissolved in milk influence the development of our gut microbiota. Accordingly, human milk oligosaccharides (HMOs) are minimally digested by the infant and persist to negatively and positively regulate gut microbiota. Infant-type bifidobacteria utilize these soluble carbohydrate oligomers by convergent mechanisms. Bifidobacterium longum subsp. infantis efficiently consumes several small mass HMOs and possesses a large gene cluster and other loci dedicated to HMO metabolism. In contrast, adult-associated bifidobacteria such as the closely related B. longum subsp. longum are deficient for HMO utilization, although they retain the capacity to ferment plant oligosaccharides and constituent pentose sugars. Thus, the ability to subsist on HMO could demark infant-associated ecotypes potentially adapted to colonize the nursing infant. Copyright 2010 Elsevier Ltd. All rights reserved.
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            Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria.

            The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.
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              Bifidobacterium longum subsp. infantis ATCC 15697 α-fucosidases are active on fucosylated human milk oligosaccharides.

              Bifidobacterium longum subsp. infantis ATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect. Specifically, among preferred HMOs, tetraose was favored early in fermentation, with other oligosaccharides consumed slightly later. In order to utilize fucosylated oligosaccharides, ATCC 15697 possesses several fucosidases, implicating GH29 and GH95 α-L-fucosidases in a gene cluster dedicated to HMO metabolism. Evaluation of the biochemical kinetics demonstrated that ATCC 15697 expresses three fucosidases with a high turnover rate. Moreover, several ATCC 15697 fucosidases are active on the linkages inherent to the HMO molecule. Finally, the HMO cluster GH29 α-L-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.
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                Author and article information

                Journal
                J Appl Glycosci (1999)
                J Appl Glycosci (1999)
                jag
                Journal of Applied Glycoscience
                The Japanese Society of Applied Glycoscience (Tokyo, JAPAN )
                1344-7882
                1880-7291
                20 February 2020
                2020
                : 67
                : 1
                : 23-29
                Affiliations
                1 Faculty of Biology-Oriented Science and Technology, Kindai University
                2 Graduate School of Biostudies, Kyoto University
                3 Faculty of Agriculture, Kyushu University
                4 Fushimi Pharmaceutical Co., Ltd.
                Author notes
                Corresponding author (Tel. +81–736–3888 , Fax. +81–736–77–4754 , E-mail: ashida@ 123456waka.kindai.ac.jp )
                Article
                jag.JAG-2019_0016
                10.5458/jag.jag.JAG-2019_0016
                8367633
                34429696
                2020 by The Japanese Society of Applied Glycoscience

                This is an open-access paper distributed under the terms of the Creative Commons Attribution Non-Commercial (by-nc) License (CC-BY-NC4.0: https://creativecommons.org/licenses/by-nc/4.0/).

                Categories
                Regular Paper

                core fucose, fucosidase, gh29, gut bacteria, probiotics

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