GH20 and GH84 β-N-acetylglucosaminidases with different linkage specificities underpin mucin O-glycan breakdown capability of Bifidobacterium bifidum

1. Johansson, M. E. V., Phillipson, M., Petersson, J., Velcich, A., Holm, L.,

and Hansson, G. C. (2008) The inner of the two Muc2 mucin-dependent

mucus layers in colon is devoid of bacteria. Proc. Natl. Acad. Sci. U. S. A.

105, 15064–15069

2. Asker, N., Axelsson, M. A. B., Olofsson, S. O., and Hansson, G. C. (1998)

Dimerization of the human MUC2 mucin in the endoplasmic reticulum is

followed by a N-glycosylation-dependent transfer of the mono- and dimers to the Golgi apparatus. J. Biol. Chem. 273, 18857–18863

3. Robbe, C., Capon, C., Coddeville, B., and Michalski, J.-C. (2004) Structural diversity and specific distribution of O-glycans in normal human

mucins along the intestinal tract. Biochem. J. 384, 307–316

4. Johansson, M. E. V., Larsson, J. M. H., and Hansson, G. C. (2011) The two

mucus layers of colon are organized by the MUC2 mucin, whereas the

outer layer is a legislator of host-microbial interactions. Proc. Natl. Acad.

Sci. U. S. A. 108, 4659–4665

5. Guzman-Aranguez, A., and Argüeso, P. (2010) Structure and biological

roles of mucin-type O-glycans at the ocular surface. Ocul. Surf. 8, 8–17

6. Yao, Y., Kim, G., Shafer, S., Chen, Z., Kubo, S., Ji, Y., et al. (2022) Mucus

sialylation determines intestinal host-commensal homeostasis. Cell. 185,

1172–1188.e28

7. Sheng, Y. H., and Hasnain, S. Z. (2022) Mucus and mucins: the underappreciated host defence system. Front. Cell Infect. Microbiol. 12, 856962

8. Lindén, S. K., Sheng, Y. H., Every, A. L., Miles, K. M., Skoog, E. C., Florin,

T. H. J., et al. (2009) MUC1 limits Helicobacter pylori infection both by

steric hindrance and by acting as a releasable decoy. PLoS Pathog. https://

doi.org/10.1371/journal.ppat.1000617

9. Wheeler, K. M., Cárcamo-Oyarce, G., Turner, B. S., Dellos-Nolan, S., Co,

J. Y., Lehoux, S., et al. (2019) Mucin glycans attenuate the virulence of

Pseudomonas aeruginosa in infection. Nat. Microbiol. 4, 2146–2154

10. Takagi, J., Aoki, K., Turner, B. S., Lamont, S., Lehoux, S., Kavanaugh, N.,

et al. (2022) Mucin O-glycans are natural inhibitors of Candida albicans

pathogenicity. Nat. Chem. Biol. 18, 762–773

11. Kudelka, M. R., Hinrichs, B. H., Darby, T., Moreno, C. S., Nishio, H.,

Cutler, C. E., et al. (2016) Cosmc is an X-linked inflammatory bowel

disease risk gene that spatially regulates gut microbiota and contributes to

sex-specific risk. Proc. Natl. Acad. Sci. U. S.A. 113, 14787–14792

12. Derrien, M., Vaughan, E. E., Plugge, C. M., and de Vos, W. M. (2004)

Akkermansia municiphila gen. nov., sp. nov., a human intestinal mucindegrading bacterium. Int. J. Syst. Evol. Microbiol. 54, 1469–1476

13. Koropatkin, N. M., Cameron, E. A., and Martens, E. C. (2012) How glycan

metabolism shapes the human gut microbiota. Nat. Rev. Microbiol. 10,

323–335

14. Martens, E. C., Chiang, H. C., and Gordon, J. I. (2008) Mucosal glycan

foraging enhances fitness and transmission of a saccharolytic human gut

bacterial symbiont. Cell Host Microbe 4, 447–457

15. Desai, M. S., Seekatz, A. M., Koropatkin, N. M., Kamada, N., Hickey, C.

A., Wolter, M., et al. (2016) A dietary fiber-deprived gut microbiota

degrades the colonic mucus barrier and enhances pathogen susceptibility.

Cell. 167, 1339–1353.e21

16. Sonnenburg, J. L., Xu, J., Leip, D. D., Chen, C.-H., Westover, B. P.,

Weatherford, J., et al. (2005) Glycan foraging in vivo by an intestineadapted bacterial symbiont. Science 307, 1955–1959

17. Birchenough, G., Schroeder, B. O., Bäckhed, F., and Hansson, G. C.

(2019) Dietary destabilisation of the balance between the microbiota and

the colonic mucus barrier. Gut Microbes 10, 246–250

18. Milani, C., Mangifesta, M., Mancabelli, L., Lugli, G. A., James, K.,

Duranti, S., et al. (2017) Unveiling bifidobacterial biogeography across the

mammalian branch of the tree of life. ISME J. 11, 2834–2847

19. Katoh, T., Yamada, C., Wallace, M. D., Yoshida, A., Gotoh, A., Arai, M.,

et al. (2023) A bacterial sulfoglycosidase highlights mucin O-glycan

breakdown in the gut ecosystem. Nat. Chem. Biol. https://doi.org/10.

1038/s41589-023-01272-y

20. Katoh, T., Ojima, M. N., Sakanaka, M., Ashida, H., Gotoh, A., and

Katayama, T. (2020) Enzymatic adaptation of Bifidobacterium bifidum to

host glycans, viewed from glycoside hydrolyases and carbohydratebinding modules. Microorganisms 8, 1–18

21. Katayama, T., Sakuma, A., Kimura, T., Makimura, Y., Hiratake, J., Sakata,

K., et al. (2004) Molecular cloning and characterization of Bifidobacterium bifidum 1,2-α-L-fucosidase (AfcA), a novel inverting glycosidase

(glycoside hydrolase family 95). J. Bacteriol. 186, 4885–4893

22. Nagae, M., Tsuchiya, A., Katayama, T., Yamamoto, K., Wakatsuki, S., and

Kato, R. (2007) Structural basis of the catalytic reaction mechanism of

novel 1,2-α-L-fucosidase from Bifidobacterium bifidum. J. Biol. Chem.

282, 18497–18509

23. Ashida, H., Miyake, A., Kiyohara, M., Wada, J., Yoshida, E., Kumagai, H.,

et al. (2009) Two distinct α-L-fucosidases from Bifidobacterium bifidum

are essential for the utilization of fucosylated milk oligosaccharides and

glycoconjugates. Glycobiology 19, 1010–1017

24. Sakurama, H., Fushinobu, S., Hidaka, M., Yoshida, E., Honda, Y., Ashida,

H., et al. (2012) 1,3–1,4-α-L-Fucosynthase that specifically introduces

Lewis a/x antigens into type-1/2 chains. J. Biol. Chem. 287, 16709–16719

25. Ashida, H., Tanigawa, K., Kiyohara, M., Katoh, T., Katayama, T., and

Yamamoto, K. (2018) Bifunctional properties and characterization of a

novel sialidase with esterase activity from Bifidobacterium bifidum. Biosci.

Biotechnol. Biochem. 82, 2030–2039

26. Kiyohara, M., Tanigawa, K., Chaiwangsri, T., Katayama, T., Ashida, H.,

and Yamamoto, K. (2011) An exo - sialidase from bifidobacteria involved

in the degradation of sialyloligosaccharides in human milk and intestinal

glycoconjugates. Glycobiology 21, 437–447

27. Wada, J., Ando, T., Kiyohara, M., Ashida, H., Kitaoka, M., Yamaguchi, M.,

et al. (2008) Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme

for the degradation of human milk oligosaccharides with a type 1

structure. Appl. Environ. Microbiol. 74, 3996–4004

28. Ito, T., Katayama, T., Hattie, M., Sakurama, H., Wada, J., Suzuki, R., et al.

(2013) Crystal structures of a glycoside hydrolase family 20 lacto-Nbiosidase from Bifidobacterium bifidum. J. Biol. Chem. 288, 11795–11806

29. Miwa, M., Horimoto, T., Kiyohara, M., Katayama, T., Kitaoka, M.,

Ashida, H., et al. (2010) Cooperation of β-galactosidase and β-N-acetylhexosaminidase from bifidobacteria in assimilation of human milk oligosaccharides with type 2 structure. Glycobiology 20, 1402–1409

30. Katoh, T., Maeshibu, T., Kikkawa, K., ichi, Gotoh, A., Tomabechi, Y.,

et al. (2017) Identification and characterization of a sulfoglycosidase from

Bifidobacterium bifidum implicated in mucin glycan utilization. Biosci.

Biotechnol. Biochem. 81, 2018–2027

31. Wakinaka, T., Kiyohara, M., Kurihara, S., Hirata, A., Chaiwangsri, T.,

Ohnuma, T., et al. (2013) Bifidobacterial a-galactosidase with unique

carbohydrate-binding module specifically acts on blood group B antigen.

Glycobiology 23, 232–240

32. Shimada, Y., Watanabe, Y., Wakinaka, T., Funeno, Y., Kubota, M.,

Chaiwangsri, T., et al. (2015) α-N-Acetylglucosaminidase from Bifidobacterium bifidum specifically hydrolyzes α-linked N-acetylglucosamine

at nonreducing terminus of O-glycan on gastric mucin. Appl. Microbiol.

Biotechnol. 99, 3941–3948

33. Turroni, F., Bottacini, F., Foroni, E., Mulder, I., Kim, J. H., Zomer, A.,

et al. (2010) Genome analysis of Bifidobacterium bifidum PRL2010 reveals metabolic pathways for host-derived glycan foraging. Proc. Natl.

Acad. Sci. U. S. A. 107, 19514–19519

J. Biol. Chem. (2023) 299(6) 104781

17

Mucin O-glycan breakdown by bacterial GH20 and GH84 enzymes

34. Pluvinage, B., Massel, P. M., Burak, K., and Boraston, A. B. (2020)

Structural and functional analysis of four family 84 glycoside hydrolases

from the opportunistic pathogen Clostridium perfringens. Glycobiology

30, 58–68

35. Garrido, D., Ruiz-Moyano, S., and Mills, D. A. (2012) Release and utilization of N-acetyl-d-glucosamine from human milk oligosaccharides by

Bifidobacterium longum subsp. infantis. Anaerobe 18, 430–435

36. Macauley, M. S., Whitworth, G. E., Debowski, A. W., Chin, D., and

Vocadlo, D. J. (2005) O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired

inhibitors. J. Biol. Chem. 280, 25313–25322

37. Zachara, N. E., and Hart, G. W. (2004) O-GlcNAc a sensor of cellular

state: The role of nucleocytoplasmic glycosylation in modulating cellular

function in response to nutrition and stress. Biochim. Biophys. Acta 1673,

13–28

38. Drula, E., Garron, M.-L., Dogan, S., Lombard, V., Henrissat, B., and

Terrapon, N. (2022) The carbohydrate-active enzyme database: functions

and literature. Nucl. Acids Res. 50, D571–D577

39. Hirokawa, T., Boon-Chieng, S., and Mitaku, S. (1998) SOSUI: classification and secondary structure prediction system for membrane proteins.

Bioinformatics 14, 378–379

40. Ojima, M. N., Jiang, L., Arzamasov, A. A., Yoshida, K., Odamaki, T., Xiao,

J., et al. (2022) Priority effects shape the structure of infant-type Bifidobacterium communities on human milk oligosaccharides. ISME J. https://

doi.org/10.1038/s41396-022-01270-3

41. Greig, I. R., Zahariev, F., and Withers, S. G. (2008) Elucidating the nature

of the Streptomyces plicatus beta-hexosaminidase-bound intermediate

using ab initio molecular dynamics simulations. J. Am. Chem. Soc. 130,

17620–17628

42. Cheng, P.-F., Snovida, S., Ho, M.-Y., Cheng, C.-W., Wu, A. M., and

Khoo, K.-H. (2013) Increasing the depth of mass spectrometry-based

glycomic coverage by additional dimensions of sulfoglycomics and

target analysis of permethylated glycans. Anal. Bioanal. Chem. 405,

6683–6695

43. Jin, C., Harvey, D. J., Struwe, W. B., and Karlsson, N. G. (2019) Separation

of isomeric O-glycans by ion mobility and liquid chromatography-mass

spectrometry. Anal. Chem. 91, 10604–10613

44. Gotoh, A., Katoh, T., Sakanaka, M., Ling, Y., Yamada, C., Asakuma, S.,

et al. (2018) Sharing of human milk oligosaccharides degradants within

bifidobacterial communities in faecal cultures supplemented with Bifidobacterium bifidum. Sci. Rep. https://doi.org/10.1038/s41598-01832080-3

45. Chiku, K., Nishimoto, M., and Kitaoka, M. (2010) Thermal decomposition of beta-D-galactopyranosyl-(1–>3)-2-acetamido-2-deoxy-D-hexopyranoses under neutral conditions. Carbohydr. Res. 345, 1901–1908

46. Yoshida, E., Sakurama, H., Kiyohara, M., Nakajima, M., Kitaoka, M.,

Ashida, H., et al. (2012) Bifidobacterium longum subsp. infantis uses two

different β-galactosidases for selectively degrading type-1 and type-2

human milk oligosaccharides. Glycobiology 22, 361–368

47. Klein, N. C., and Cunha, B. A. (1995) Tetracyclines. Med. Clin. North Am.

79, 789–801

48. Nishiyama, K., Nagai, A., Uribayashi, K., Yamamoto, Y., Mukai, T., and

Okada, N. (2018) Two extracellular sialidases from Bifidobacterium

bifidum promote the degradation of sialyl-oligosaccharides and support

the growth of Bifidobacterium breve. Anaerobe 52, 22–28

49. Parche, S., Beleut, M., Rezzonico, E., Jacobs, D., Arigoni, F., Titgemeyer,

F., et al. (2006) Lactose-over-glucose preference in Bifidobacterium longum NCC2705: glcP, encoding a glucose transporter, is subject to lactose

repression. J. Bacteriol. 188, 1260–1265

50. Sheldon, W. L., Macauley, M. S., Taylor, E. J., Robinson, C. E.,

Charnock, S. J., Davies, G. J., et al. (2006) Functional analysis of a

group A streptococcal glycoside hydrolase Spy1600 from family 84

reveals it is a β-N-acetylglucosaminidase and not a hyaluronidase.

Biochem. J. 399, 241–247

51. Ostrowski, A., Gundogdu, M., Ferenbach, A. T., Lebedev, A. A., and van

Aalten, D. M. F. (2015) Evidence for a functional O-linked N-acetylglucosamine (O-GlcNAc) system in the thermophilic bacterium Thermobaculum terrenum. J. Biol. Chem. 290, 30291–30305

18 J. Biol. Chem. (2023) 299(6) 104781

52. Navarro, M. A., Li, J., McClane, B. A., Morrell, E., and Beingesser, J.

(2018) NanI sialidase is an important contributor to Clostridium perfringens Type F Strain F4969 intestinal colonization in mice. Infect.

Immun. 86, e00462-18

53. Kim, M. J., Ku, S., Kim, S. Y., Lee, H. H., Jin, H., Kang, S., et al. (2018)

Safety evaluations of Bifidobacterium bifidum BGN4 and Bifidobacterium

longum BORI. Int. J. Mol. Sci. https://doi.org/10.3390/ijms19051422

54. Turroni, F., Foroni, E., Montanini, B., Viappiani, A., Strati, F., Duranti, S.,

et al. (2011) Global genome transcription profiling of Bifidobacterium

bifidum PRL2010 under in vitro conditions and identification of reference

genes for quantitative real-time PCR. Appl. Environ. Microbiol. 77,

8578–8587

55. Asakuma, S., Hatakeyama, E., Urashima, T., Yoshida, E., Katayama, T.,

Yamamoto, K., et al. (2011) Physiology of consumption of human milk

oligosaccharides by infant gut-associated bifidobacteria. J. Biol. Chem.

286, 34583–34592

56. Arzamasov, A. A., Nakajima, A., Sakanaka, M., Ojima, M. N., Katayama,

T., Rodionov, D. A., et al. (2022) Human milk oligosaccharide utilization

in intestinal bifidobacteria is governed by a global transcriptional regulator NagR. mSystems 7, e00343-22

57. Dennis, R. J., Taylor, E. J., Macauley, M. S., Stubbs, K. A., Turkenburg, J.

P., Hart, S. J., et al. (2006) Structure and mechanism of a bacterial βglucosaminidase having O-GlcNAcase activity. Nat. Struct. Mol. Biol. 13,

365–371

58. He, X., Gao, J., Peng, L., Hu, T., Wan, Y., Zhou, M., et al. (2021) Bacterial

O-GlcNAcase genes abundance decreases in ulcerative colitis patients

and its administration ameliorates colitis in mice. Gut 70, 1872–1883

59. Anderson, K., Li, S. C., and Li, Y. T. (2000) Diphenylamine-anilinephosphoric acid reagent, a versatile spray reagent for revealing glycoconjugates on thin-layer chromatography plates. Anal. Biochem. 287,

337–339

60. Takada, H., Katoh, T., and Katayama, T. (2020) Sialylated O-glycans from

hen egg white ovomucin are decomposed by mucin-degrading gut microbes. J. Appl. Glycosci. 67, 31–39

61. Kumagai, T., Katoh, T., Nix, D. B., Tiemeyer, M., and Aoki, K. (2013) Ingel β-elimination and aqueous - organic partition for improved O- and

sulfoglycomics. Anal. Chem. 85, 8692–8699

62. Cooper, C. A., Gasteiger, E., and Packer, N. H. (2001) GlycoMod - a

software tool for determining glycosylation compositions from mass

spectrometric data. Proteomics 1, 340–349

63. Yasui, K., Kano, Y., Tanaka, K., Watanabe, K., Shimizu-Kadota, M.,

Yoshikawa, H., et al. (2009) Improvement of bacterial transformation

efficiency using plasmid artificial modification. Nucl. Acids Res. 37, 2–8

64. Ruiz, L., Motherway, M. O., Lanigan, N., and van Sinderen, D. (2013)

Transposon mutagenesis in Bifidobacterium breve: construction and

characterization of a Tn5 transposon mutant library for Bifidobacterium

breve UCC2003. PLoS One 8, e64699

65. Odamaki, T., Bottacini, F., Kato, K., Mitsuyama, E., Yoshida, K., Horigome, A., et al. (2018) Genomic diversity and distribution of Bifidobacterium longum subsp. longum across the human lifespan. Sci. Rep. 8,

1–12

66. Horinouchi, S., and Weisblum, B. (1982) Nucleotide sequence and

functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150, 815–825

67. Sakanaka, M., Tamai, S., Hirayama, Y., Onodera, A., Koguchi, H., Kano,

Y., et al. (2014) Functional analysis of bifidobacterial promoters in Bifidobacterium longum and Escherichia coli using the α-galactosidase gene

as a reporter. J. Biosci. Bioeng. 118, 489–495

68. Park, M. J., Park, M. S., and Ji, G. E. (2019) Improvement of

electroporation-mediated transformation efficiency for a Bifidobacterium strain to a reproducibly high level. J. Microbiol. Methods 159,

112–119

69. Sakanaka, M., Hansen, M. E., Gotoh, A., Katoh, T., Yoshida, K., Odamaki,

T., et al. (2019) Evolutionary adaptation in fucosyllactose uptake systems

supports bifidobacteria-infant symbiosis. Sci. Adv. 5, 1–16

70. Finn, R. D., Tate, J., Mistry, J., Coggill, P. C., Sammut, S. J., Hotz, H. R.,

et al. (2008) The Pfam protein families database. Nucl. Acids Res. 36,

281–288

Mucin O-glycan breakdown by bacterial GH20 and GH84 enzymes

71. Madeira, F., Park, Y. M., Lee, J., Buso, N., Gur, T., Madhusoodanan, N.,

et al. (2019) The EMBL-EBI search and sequence analysis tools APIs in

2019. Nucl. Acids Res. 47, W636–W641

72. Neelamegham, S., Aoki-Kinoshita, K., Bolton, E., Frank, M., Lisacek, F.,

Lütteke, T., et al. (2019) Updates to the symbol nomenclature for glycans

guidelines. Glycobiology 29, 620–624

73. Krogh, A., Larsson, B., and Von Heijne, G. (2001) Predicting transmembrane protein topology with a hidden Markov model: application to

complete genomes. J. Mol. Biol. 305, 567–580

74. Benjamini, Y., and Hochberg, Y. (1995) Controlling the false discovery

rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc.

Ser. B 57, 289–300

J. Biol. Chem. (2023) 299(6) 104781

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