Bifidobacterium bifidum Lacto- N -Biosidase, a Critical Enzyme for the Degradation of Human Milk Oligosaccharides with a Type 1 Structure (cid:1)

Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto- N -biosidase that liberates lacto- N -biose I (Gal (cid:1) 1,3GlcNAc; type 1 chain) from lacto- N -tetraose (Gal (cid:1) 1,3GlcNAc (cid:1) 1,3Gal (cid:1) 1,4Glc), which is a major compo-nent of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB , was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified (cid:1) -linked lacto- N biose I structure. Lacto- N -biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto- N -biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type 1 chain. B. bifidum JCM1254 and a pair of primers (cid:4) (cid:4) and (cid:4) (cid:4) ) were designed based on the amino acid se- quence of LnbB from Streptomyces sp. strain (GenBank accession in order to the upstream region, inverse PCR carried out BamHI-digested, circularized genomic DNA a and primer pair (5 (cid:4) -GCTTGCCGACAACTCAGGCCGGAAGGATC-3 (cid:4) and 5 (cid:4) (cid:4) 3 (cid:4) colony hybridization PCR an PCR vitro cloning oligonucleotide (cid:4) -G forward primer reverse primer separately isolated in silico. a fragment the entire lnbB gene, PCR involving KOD polymerase performed using the genomic DNA as a template and a primer pair (5 (cid:4) -CTCTCCCCGCTGATGTAGGT-3 (cid:4) and 5 (cid:4) -AAGC TGACCGGCGTACTCTC-3 (cid:4) amplified into the of a low-copy-number plasmid, pMW118 Gene, and

[1]  R. Suzuki,et al.  Structural and Thermodynamic Analyses of Solute-binding Protein from Bifidobacterium longum Specific for Core 1 Disaccharide and Lacto-N-biose I* , 2008, Journal of Biological Chemistry.

[2]  Tadashi Nakamura,et al.  Variation of major neutral oligosaccharides levels in human colostrum , 2008, European Journal of Clinical Nutrition.

[3]  M. Nishimoto,et al.  Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124 , 2008, Applied Microbiology and Biotechnology.

[4]  C. Lebrilla,et al.  Glycoprofiling of bifidobacterial consumption of human milk oligosaccharides demonstrates strain specific, preferential consumption of small chain glycans secreted in early human lactation. , 2007, Journal of agricultural and food chemistry.

[5]  Hiroshi Mori,et al.  Comparative Metagenomics Revealed Commonly Enriched Gene Sets in Human Gut Microbiomes , 2007, DNA research : an international journal for rapid publication of reports on genes and genomes.

[6]  R. Suzuki,et al.  Purification, crystallization and preliminary X-ray analysis of the galacto-N-biose-/lacto-N-biose I-binding protein (GL-BP) of the ABC transporter from Bifidobacterium longum JCM1217. , 2007, Acta crystallographica. Section F, Structural biology and crystallization communications.

[7]  M. Nishimoto,et al.  Identification of N-Acetylhexosamine 1-Kinase in the Complete Lacto-N-Biose I/Galacto-N-Biose Metabolic Pathway in Bifidobacterium longum , 2007, Applied and Environmental Microbiology.

[8]  M. Nishimoto,et al.  Practical Preparation of Lacto-N-biose I, a Candidate for the Bifidus Factor in Human Milk , 2007, Bioscience, biotechnology, and biochemistry.

[9]  N. Iwabuchi,et al.  In Vitro Th1 Cytokine‐Independent Th2 Suppressive Effects of Bifidobacteria , 2007, Microbiology and immunology.

[10]  M. Nishimoto,et al.  Identification of the Putative Proton Donor Residue of Lacto-N-biose Phosphorylase (EC 2.4.1.211) , 2007, Bioscience, biotechnology, and biochemistry.

[11]  Kenji Yamamoto,et al.  Structural Basis of the Catalytic Reaction Mechanism of Novel 1,2-α-L-Fucosidase from Bifidobacterium bifidum* , 2007, Journal of Biological Chemistry.

[12]  A. Boraston,et al.  The Interaction of a Carbohydrate-binding Module from a Clostridium perfringens N-Acetyl-β-hexosaminidase with Its Carbohydrate Receptor* , 2006, Journal of Biological Chemistry.

[13]  A. Alexopoulos,et al.  Occurrence of Bifidobacterium in the intestine of newborns by fluorescence in situ hybridization. , 2006, Comparative immunology, microbiology and infectious diseases.

[14]  P. A. van den Brandt,et al.  Factors Influencing the Composition of the Intestinal Microbiota in Early Infancy , 2006, Pediatrics.

[15]  U. Wahn,et al.  A mixture of prebiotic oligosaccharides reduces the incidence of atopic dermatitis during the first six months of age , 2006, Archives of Disease in Childhood.

[16]  C. Lebrilla,et al.  In Vitro Fermentation of Breast Milk Oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri , 2006, Applied and Environmental Microbiology.

[17]  D. Jacobs,et al.  Lactose-over-Glucose Preference in Bifidobacterium longum NCC2705: glcP, Encoding a Glucose Transporter, Is Subject to Lactose Repression , 2006, Journal of bacteriology.

[18]  Robert D. Finn,et al.  Pfam: clans, web tools and services , 2005, Nucleic Acids Res..

[19]  K. Fujita,et al.  Identification and Molecular Cloning of a Novel Glycoside Hydrolase Family of Core 1 Type O-Glycan-specific Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum* , 2005, Journal of Biological Chemistry.

[20]  M. Nishimoto,et al.  Novel Putative Galactose Operon Involving Lacto-N-Biose Phosphorylase in Bifidobacterium longum , 2005, Applied and Environmental Microbiology.

[21]  J. Knol,et al.  Quantitative Real-Time PCR Assays To Identify and Quantify Fecal Bifidobacterium Species in Infants Receiving a Prebiotic Infant Formula , 2005, Applied and Environmental Microbiology.

[22]  K. Fujita,et al.  Novel bifidobacterial glycosidases acting on sugar chains of mucin glycoproteins. , 2005, Journal of bioscience and bioengineering.

[23]  J. Knol,et al.  Colon Microflora in Infants Fed Formula with Galacto- and Fructo-Oligosaccharides: More Like Breast-Fed Infants , 2005, Journal of pediatric gastroenterology and nutrition.

[24]  H. Kumagai,et al.  Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95) , 2004, Journal of bacteriology.

[25]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[26]  D. Harty,et al.  Characterisation of a novel homodimeric N-acetyl-beta-D-glucosaminidase from Streptococcus gordonii. , 2004, Biochemical and biophysical research communications.

[27]  I. Fliss,et al.  In vitro inhibition of Escherichia coli O157:H7 by bifidobacterial strains of human origin. , 2004, International journal of food microbiology.

[28]  Spencer J. Williams,et al.  Aspartate 313 in the Streptomyces plicatusHexosaminidase Plays a Critical Role in Substrate-assisted Catalysis by Orienting the 2-Acetamido Group and Stabilizing the Transition State* , 2002, The Journal of Biological Chemistry.

[29]  Peer Bork,et al.  The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Tadashi Nakamura,et al.  Oligosaccharides of milk and colostrum in non-human mammals , 2001, Glycoconjugate Journal.

[31]  S. Madsen,et al.  Intra- and Extracellular β-Galactosidases fromBifidobacterium bifidum and B. infantis: Molecular Cloning, Heterologous Expression, and Comparative Characterization , 2001, Applied and Environmental Microbiology.

[32]  H. Kumagai,et al.  Enzymatic syntheses of T antigen-containing glycolipid mimicry using the transglycosylation activity of endo-alpha-N-acetylgalactosaminidase. , 2001, Carbohydrate research.

[33]  W. T. Malone,et al.  Variability of human milk neutral oligosaccharides in a diverse population. , 2000, Journal of pediatric gastroenterology and nutrition.

[34]  Takeomi Murata,et al.  Facile enzymatic conversion of lactose into lacto-N-tetraose and lacto-N-neotetraose , 1999, Glycoconjugate Journal.

[35]  C. Brassart,et al.  β‐1,3‐Galactosyl‐N‐acetylhexosamine phosphorylase from Bifidobacterium bifidum DSM 20082: characterization, partial purification and relation to mucin degradation , 1999, Biotechnology and Applied Biochemistry.

[36]  M. James,et al.  Structural and Functional Characterization of Streptomyces plicatus β-N-Acetylhexosaminidase by Comparative Molecular Modeling and Site-directed Mutagenesis* , 1998, The Journal of Biological Chemistry.

[37]  J. Taylor‐Papadimitriou,et al.  Comparison of O-Linked Carbohydrate Chains in MUC-1 Mucin from Normal Breast Epithelial Cell Lines and Breast Carcinoma Cell Lines: , 1996, The Journal of Biological Chemistry.

[38]  F. Gavini,et al.  Characterization of the Lactose Transport System in the Strain Bifidobacterium bifidum DSM 20082 , 1996, Current Microbiology.

[39]  I. Kato,et al.  Purification and characterization of an enzyme releasing lacto-N-biose from oligosaccharides with type 1 chain. , 1993, The Journal of biological chemistry.

[40]  J. Wieruszeski,et al.  Primary structure of human milk octa-, dodeca- and tridecasaccharides determined by a combination of 1H-NMR spectroscopy and fast-atom-bombardment mass spectrometry. Evidence for a new core structure, the para-lacto-N-octaose. , 1993, European journal of biochemistry.

[41]  B Henrissat,et al.  A classification of glycosyl hydrolases based on amino acid sequence similarities. , 1991, The Biochemical journal.

[42]  M. Kanehisa,et al.  Expert system for predicting protein localization sites in gram‐negative bacteria , 1991, Proteins.

[43]  D. Podolsky,et al.  Oligosaccharide structures of human colonic mucin. , 1985, The Journal of biological chemistry.

[44]  K. Fujita,et al.  Development and differences of intestinal flora in the neonatal period in breast-fed and bottle-fed infants. , 1983, Pediatrics.

[45]  T. Ikenaka,et al.  Structure analyses of oligosaccharides by tagging of the reducing end sugars with a fluorescent compound. , 1978, Biochemical and biophysical research communications.

[46]  A. Kobata,et al.  Oligosaccharides of human milk. 3. Isolation and characterization of a new hexasaccharide, lacto-N-hexaose. , 1972, The Journal of biological chemistry.

[47]  F. Hytten,et al.  Oligosaccharides of Human Milk , 1957, Nature.

[48]  K. Fujita,et al.  Functions of novel glycosidases isolated from bifidobacteria , 2008 .

[49]  J. Vincken,et al.  Bifidobacterium carbohydrases-their role in breakdown and synthesis of (potential) prebiotics. , 2008, Molecular nutrition & food research.

[50]  C. Hoyen.  Factors Influencing the Composition of the Intestinal Microbiota in Early Infancy , 2007 .

[51]  T. Matsuzaki,et al.  Immunomodulatory function of lactic acid bacteria , 2004, Antonie van Leeuwenhoek.

[52]  Kenji Yamamoto,et al.  Chemo-enzymatic synthesis of bioactive glycopeptide using microbial endoglycosidase. , 2001, Journal of bioscience and bioengineering.

[53]  H. Harmsen,et al.  Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. , 2000, Journal of pediatric gastroenterology and nutrition.

[54]  N Klein,et al.  Oligosaccharides in human milk: structural, functional, and metabolic aspects. , 2000, Annual review of nutrition.

[55]  J. O'brien,et al.  Separation of glycoprotein-derived oligosaccharides by thin-layer chromatography. , 1979, Analytical biochemistry.

[56]  E. J. Bourne,et al.  Colour reactions given by sugars and diphenylamine-aniline spray reagents on paper chromatograms , 1960 .

[57]  R. Norris,et al.  Bifidus factor. I. A variant of Lactobacillus bifidus requiring a special growth factor. , 1954, Archives of biochemistry and biophysics.

[58]  F. Zilliken,et al.  Bifidus factor. II. Its occurrence in milk from different species and in other natural products. , 1954, Archives of biochemistry and biophysics.