Characterization of a sialate-O-acetylesterase (NanS) from the oral pathogen Tannerella forsythia that enhances sialic acid release by NanH, its cognate sialidase.

Tannerella forsythia, a Gram-negative member of the Bacteroidetes has evolved to harvest and utilize sialic acid. The most common sialic acid in humans is a mono-N-acetylated version termed Neu5Ac (5-N-acetyl-neuraminic acid). Many bacteria are known to access sialic acid using sialidase enzymes. However, in humans a high proportion of sialic acid contains a second acetyl group attached via an O-group, i.e. chiefly O-acetylated Neu5,9Ac2 or Neu5,4Ac2. This diacetylated sialic acid is not cleaved efficiently by many sialidases and in order to access diacetylated sialic acid, some organisms produce sialate-O-acetylesterases that catalyse the removal of the second acetyl group. In the present study, we performed bioinformatic and biochemical characterization of a putative sialate-O-acetylesterase from T. forsythia (NanS), which contains two putative SGNH-hydrolase domains related to sialate-O-acetylesterases from a range of organisms. Purification of recombinant NanS revealed an esterase that has activity against Neu5,9Ac2 and its glycolyl form Neu5Gc,9Ac. Importantly, the enzyme did not remove acetyl groups positioned at the 4-O position (Neu5,4Ac2). In addition NanS can act upon complex N-glycans released from a glycoprotein [erythropoietin (EPO)], bovine submaxillary mucin and oral epithelial cell-bound glycans. When incubated with its cognate sialidase, NanS increased sialic acid release from mucin and oral epithelial cell surfaces, implying that this esterase improves sialic acid harvesting for this pathogen and potentially other members of the oral microbiome. In summary, we have characterized a novel sialate-O-acetylesterase that contributes to the sialobiology of this important human pathogen and has potential applications in the analysis of sialic acid diacetylation of biologics in the pharmaceutical industry.

[1]  F. Dewhirst,et al.  Draft Genome Sequence of Tannerella forsythia Type Strain ATCC 43037 , 2015, Genome Announcements.

[2]  J. Potempa,et al.  The Link Between Periodontal Disease and Rheumatoid Arthritis: An Updated Review , 2014, Current Rheumatology Reports.

[3]  Xiangshan Zhou,et al.  Highly sialylated recombinant human erythropoietin production in large‐scale perfusion bioreactor utilizing CHO‐gmt4 (JW152) with restored GnT I function , 2014, Biotechnology journal.

[4]  P. Wright,et al.  Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species , 2013, The Biochemical journal.

[5]  Pedro M. Coutinho,et al.  The carbohydrate-active enzymes database (CAZy) in 2013 , 2013, Nucleic Acids Res..

[6]  Ashu Sharma,et al.  Protein-linked glycans in periodontal bacteria: prevalence and role at the immune interface , 2013, Front. Microbiol..

[7]  N. Hashii,et al.  Mass spectrometric glycoform profiling of the innovator and biosimilar erythropoietin and darbepoetin by LC/ESI-MS. , 2013, Journal of pharmaceutical and biomedical analysis.

[8]  G. Herrler,et al.  Sialic Acid Binding Properties of Soluble Coronavirus Spike (S1) Proteins: Differences between Infectious Bronchitis Virus and Transmissible Gastroenteritis Virus , 2013, Viruses.

[9]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[10]  Ashu Sharma,et al.  Sialic acid, periodontal pathogens and Tannerella forsythia: stick around and enjoy the feast! , 2012, Molecular oral microbiology.

[11]  Ashu Sharma,et al.  Role of sialidase in glycoprotein utilization by Tannerella forsythia , 2011, Microbiology.

[12]  J. Schrag,et al.  Structural and enzymatic characterization of NanS (YjhS), a 9‐O‐Acetyl N‐acetylneuraminic acid esterase from Escherichia coli O157:H7 , 2011, Protein science : a publication of the Protein Society.

[13]  J. L. Abrahams,et al.  Erythropoietin produced in a human cell line (Dynepo) has significant differences in glycosylation compared with erythropoietins produced in CHO cell lines. , 2011, Molecular pharmaceutics.

[14]  Ashu Sharma,et al.  Role of Tannerella forsythia NanH Sialidase in Epithelial Cell Attachment , 2010, Infection and Immunity.

[15]  C. Douglas,et al.  A Novel Sialic Acid Utilization and Uptake System in the Periodontal Pathogen Tannerella forsythia , 2010, Journal of bacteriology.

[16]  S. Pillai,et al.  Esterases and autoimmunity: the sialic acid acetylesterase pathway and the regulation of peripheral B cell tolerance. , 2009, Trends in immunology.

[17]  E. Vimr,et al.  YjhS (NanS) Is Required for Escherichia coli To Grow on 9-O-Acetylated N-Acetylneuraminic Acid , 2009, Journal of bacteriology.

[18]  Eric C. Martens,et al.  Complex Glycan Catabolism by the Human Gut Microbiota: The Bacteroidetes Sus-like Paradigm , 2009, The Journal of Biological Chemistry.

[19]  A. Hosie,et al.  An Orthologue of Bacteroides fragilis NanH Is the Principal Sialidase in Tannerella forsythia , 2009, Journal of bacteriology.

[20]  R. Wilson,et al.  Evolution of Symbiotic Bacteria in the Distal Human Intestine , 2007, PLoS biology.

[21]  Kankana Mukherjee,et al.  O-acetylation of sialic acids is required for the survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL) , 2006, Glycoconjugate Journal.

[22]  Pauline M Rudd,et al.  An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins. , 2002, Analytical biochemistry.

[23]  W. Wurzer,et al.  The sialate-4-O-acetylesterases of coronaviruses related to mouse hepatitis virus: a proposal to reorganize group 2 Coronaviridae. , 2002, The Journal of general virology.

[24]  M. Chatterjee,et al.  Identification and purification of cytolytic antibodies directed against O-acetylated sialic acid in childhood acute lymphoblastic leukemia. , 2000, Glycobiology.

[25]  S. Kauppinen,et al.  Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases. , 2000, Structure.

[26]  S. Vineeta,et al.  Identification of antibodies directed against O-acetylated sialic acids in visceral leishmaniasis: its diagnostic and prognostic role , 1998, Glycoconjugate Journal.

[27]  K. Kobayashi,et al.  Gingival crevicular pH in experimental gingivitis and occlusal trauma in man. , 1998, Journal of periodontology.

[28]  S. Socransky,et al.  Microbial complexes in subgingival plaque. , 1998, Journal of clinical periodontology.

[29]  S. Alonso,et al.  Sialic acid measurement by a modified Aminoff method: a time-saving reduction in 2-thiobarbituric acid concentration. , 1997, Journal of biochemical and biophysical methods.

[30]  H. Klenk,et al.  The catalytic triad of the influenza C virus glycoprotein HEF esterase: characterization by site-directed mutagenesis and functional analysis. , 1995, The Journal of general virology.

[31]  R. Parekh,et al.  Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. , 1995, Analytical biochemistry.

[32]  J. Vliegenthart,et al.  Structural analysis of the sialylated N- and O-linked carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. Sialylation patterns and branch location of dimeric N-acetyllactosamine units. , 1995, European journal of biochemistry.

[33]  T Corfield,et al.  Bacterial sialidases--roles in pathogenicity and nutrition. , 1992, Glycobiology.

[34]  A. Corfield,et al.  Mucin degradation in the human colon: production of sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria , 1992, Infection and immunity.

[35]  A. García-Sastre,et al.  Increased influenza A virus sialidase activity with N-acetyl-9-O-acetylneuraminic acid-containing substrates resulting from influenza C virus O-acetylesterase action☆ , 1992, Virus Research.

[36]  M. Fukuda,et al.  Leukosialin, a major O-glycan-containing sialoglycoprotein defining leukocyte differentiation and malignancy. , 1991, Glycobiology.

[37]  A. García-Sastre,et al.  Activity of influenza C virus O-acetylesterase with O-acetyl-containing compounds. , 1991, The Biochemical journal.

[38]  A. Varki,et al.  O-acetylation and de-O-acetylation of sialic acids. Purification, characterization, and properties of a glycosylated rat liver esterase specific for 9-O-acetylated sialic acids. , 1989, The Journal of biological chemistry.

[39]  F. Corpet.  Multiple sequence alignment with hierarchical clustering. , 1988, Nucleic acids research.

[40]  H. Klenk,et al.  Influenza C virus uses 9-O-acetyl-N-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells. , 1986, The Journal of biological chemistry.

[41]  A. Varki,et al.  A neuraminidase from Streptococcus sanguis that can release O-acetylated sialic acids. , 1983, The Journal of biological chemistry.

[42]  D AMINOFF,et al.  Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. , 1961, The Biochemical journal.

[43]  C. Douglas,et al.  Physiological adaptations of key oral bacteria. , 2014, Advances in microbial physiology.

[44]  R. D. de Groot.  Structure, function and evolution of the hemagglutinin-esterase proteins of corona- and toroviruses , 2006, Glycoconjugate Journal.

[45]  P. Roussel,et al.  O-acetylation of sialic acids. , 1998, Biochimie.

[46]  S. Sundar,et al.  Identification of antibodies directed against O-acetylated sialic acids in visceral leishmaniasis: its diagnostic and prognostic role. , 1998, Glycoconjugate journal.

[47]  R. Schauer.  Chemistry, metabolism, and biological functions of sialic acids. , 1982, Advances in carbohydrate chemistry and biochemistry.