Scholarly article on topic 'Sialidases from gut bacteria: a mini-review'

Sialidases from gut bacteria: a mini-review Academic research paper on "Biological sciences"

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Academic research paper on topic "Sialidases from gut bacteria: a mini-review"

Sialidases from gut bacteria: a mini-review

Nathalie Juge*1, Louise Tailford* and C David Owenf

'Institute of Food Research, The Gut Health and Food Safety Institute Strategic Programme, Norwich Research Park, Norwich NR4 7UA, U.K. fBlomolecular Sciences Building, University of St Andrews, St Andrews, Fife, KY16 9ST, UK

Abstract

Sialidases are a large group of enzymes, the majority of which catalyses the cleavage of terminal sialic acids from complex carbohydrates on glycoproteins or glycolipids. In the gastrointestinal (GI) tract, sialic acid residues are mostly found in terminal location of mucins via a2-3/6 glycosidic linkages. Many enteric commensal and pathogenic bacteria can utilize sialic acids as a nutrient source, but not all express the sialidases that are required to release free sialic acid. Sialidases encoded by gut bacteria vary in terms of their substrate specificity and their enzymatic reaction. Most are hydrolytic sialidases, which release free sialic acid from sialylated substrates. However, there are also examples with transglycosylation activities. Recently, a third class of sialidases, intramolecular frons-sialidase (IT-sialidase), has been discovered in gut microbiota, releasing (2,7-anhydro-Neu5Ac) 2,7-anydro-N-acetylneuraminic acid instead of sialic acid. Reaction specificity varies, with hydrolytic sialidases demonstrating broad activity against a2,3-, a2,6- and a2,8-linked substrates, whereas IT-sialidases tend to be specific for a2,3-linked substrates. In this minireview, we summarize the current knowledge on the structural and biochemical properties of sialidases involved in the interaction between gut bacteria and epithelial surfaces.

Sialic acid metabolism in the gut

In the gastrointestinal (GI) tract, sialic acid [N-acetylneuraminic acid (Neu5Ac)] is commonly found in terminal location of mucins [1,2]. Mucins are large glycoproteins, which can be broadly grouped as membrane-bound or secreted [3]. Membrane-bound mucins are essential contributors of the glycocalyx of mucosal surfaces where they play important biological roles in cell interactions and signalling [4]. Secreted mucins are the main structural components of the mucus gel covering the epithelium and essential to the maintenance of a homoeostatic relationship with our gut microbiota [1]. Mucins are characterized by a proline-threonine-serine (PTS) domain which is the site of extensive O-glycosylation with carbohydrates accounting for up to 80 % of the total mucin mass. The synthesis of mucin oligosaccharides starts with the transfer of N-acetyl-galactosamine (GalNAc) to serine and threonine residues of the mucin backbone to form mucin O-glycan core structures [5]. These core structures can be further elongated with galactose (Gal), N-acetyl-glucosamine (GlcNAc), GalNAc and frequently modified by terminal fucose or sialic acid residues via a1-2/3/4 and a2-3/6 linkages, respectively (Figure 1). The proportion of the major mucin glycan

Key words: sialidase, gut bacteria, gut microbiota, glycoside hydrolase, sialic acid, mucin degradation.

Abbreviations: 2,7-anydro-Neu5Ac, 2,7-anydro-N-acetylneuraminic acid; 4MU-Neu5Ac, 2'-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid; CBM, carbohydrate-binding module; Gal, galactose; GalNAc, N-acetyl-galactosamine; GH, glycoside hydrolase; GH33, GH family 33; GI, gastrointestinal; GlcNAc, N-acetyl-glucosamine; IT-sialidase, intramolecular frons-sialidase; MU, 4-Methylumbelliferonemethylumbelliferone; Neu5Ac, N-acetylneuraminic acid; Neu5Ac2en, N-acetyl-2,3-didehydro-2-deoxyneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; PNP-Neu5Ac, 2-0-(p-Nitrophenyl)-a-D-N-acetylneuraminic acid; PTS, proline-threonine-serine. 1 To whom correspondence should be addressed (email nathalie.juge@ifr.ac.uk).

epitopes, sialic acid and fucose, varies along the GI tract with a decreasing gradient of fucose and an increasing gradient of sialic acid from the ileum to the rectum in humans [6] and a reverse gradient in mice [7]. The Neu5Ac a2-6 N-acetylgalactosaminitol epitopes and Sda/Cad antigens found in humans [6,8] are absent or rare in mice where as the Neu5Ac-GlcNAc epitope and disialylated epitopes are more common along the murine GI tract [7].

The GI tract is heavily colonized with bacteria. Most species belong to the phyla Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia. The microbiota composition varies longitudinally along the GI tract but also transversally from the mucosa to the lumen [9,10]. The terminal mucin O-glycans have been proposed to serve as metabolic substrates, providing a nutritional advantage to bacteria which have adapted to the GI mucosal environment [11,12]. The release of sialic acid from non-reducing ends by sialidases is an initial step in the sequential degradation of mucins, since the terminal location of sialic acid residues in the mucin oligosaccharide chains may prevent the action of other glycoside hydrolases (GHs). In bacteria, the genes involved in sialic acid metabolism are usually found clustered together forming what is denominated as a Nan cluster. The canonical nanA/K/E cluster was first described in Escherichia coli [13] and an alternative pathway defined by the nanLET cluster was later discovered in Bacteroides fragilis [14]. The majority of the bacteria that harbour a Nan cluster colonize mucus regions of the human body, such as the gut where sialic acid is highly abundant and can serve as a source of energy, carbon and nitrogen [15]. As described below, a number of gut bacteria employ sialidases in the release of host sialic acids, including

Biochem. Soc. Trans. (2016) 44, 166-175; doi:10.1042/BST20150226

Figure 1 Sialylated terminal glycan structures in the gut

Structures shown are representative and not exhaustive. These glycans are appended to the core mucin structures. In mice the fucose (Fuc) residues are more commonly linked to Gal rather GlcNAc, ''structures common in human and rare in mice.

Monosialylated core-3*

|31-4 |31-3

a2-3 |i-3 |1 Sda/Cad antigen a2-6 a Sialyl Lewisa Q—Q—Q type 1 & 2

AnS t^O-OSer/Thr Ta1-4 chains*

|1-4 |1-3/4 |1-3

a2-3 |1-3 a Sialyl Lewisx

(y-CHJ-O—Ser/Thr

Sialyl-T-antigen

multiple species of Clostridia [16], Bacteroides [17], certain subspecies/serovars/strains of Bifidobacterium longum [18], Vibrio cholerae [19], Ruminococcus gnavus and Akkermansia muciniphila [20]. However, some bacteria appear to have incomplete packages of enzymes for utilizing host sialic acids. For example, Bacteroides thetaiotaomicron VPI-5482 encodes a sialidase and can release free sialic acid, but lacks the Nan operon required to consume the liberated monosaccharide and does not appear capable of consuming free sialic acid [21]. On the other hand Clostridium difficile strain 630 encodes the Nan operon but lacks the sialidase [22] and thus relies on other sialidase-producing organisms to acquire this potential nutrient source from the mucosal environment [23,24]. In contrast, some bacteria appear to possess the complete pathway of sialic acid catabolism including a predicted sialidase gene e.g. B. fragilis strains [14,25]. A recent study reported that mice monoassociated with B. thetaiotaomicron exhibited a significantly higher concentration of free Neu5Ac compared with germ-free mice, consistent with the ability of B. thetaiotaomicron to liberate but not consume the monosaccharide, whereas colonization of mice with B. fragilis, which is able to catabolize Neu5Ac, did not result in increased free sialic acid [24].

In contrast with gut commensals, which appear to use sialidases primarily for nutrient acquisition, some pathogens of the GI tract such as V cholerae or Clostridium perfringens strains also use sialidases to decrypt adhesin or toxin-binding sites [26]. All toxigenic strains of V cholerae have a sialidase encoded within a pathogenicity island in their genomes [27]. However it is also worth noting that, within particular pathogenic or commensal species, the presence or absence

of sialidase-encoding genes in bacterial genomes is often strain-specific. For example R. gnavus ATCC 29149 but not E1 expresses a sialidase [20,28], several strains of E. coli, e.g. enteropathogenic E. coli O127 strain (EPEC) [29] or probiotic strain Nissle 1917 [30] possess a sialidase-encoding gene whereas commensal E. coli strains such as E. coli strain EHV2 lack a sialidase [31]. However E. coli sialidases remain to be biochemically characterized. Similarly, not all Salmonella enterica strains encode a putative sialidase and only one sialidase has been functionally-characterized from S. enterica serovar typhimurium although it appears to have been acquired by horizontal transfer [32,33].

Sialic acid catabolism in the gut is important as increased free sialic acid levels in the intestinal mucosal compartment, e.g. post-antibiotic treatment, will favour outgrowth of some bacterial pathogenic strains of S. Typhimurium and C. difficile [24] or the outgrowth of E. coli during inflammation [31]. Such cross-feeding activity has also been reported between commensal bacteria, e.g. Bifidobacterium breve UCC2003 (containing a functional Nan cluster for sialic utilization) can utilize sialic acid released by the sialidase activity of Bifidobacterium bifidum PRL201048 [34]. The gut symbiont, R. gnavus ATCC 29149, is different from the above as it possesses the complete Nan cluster and an intramolecular trans-sialidase (IT-sialidase), thus producing (2,7-anhydro-Neu5Ac) 2,7-anydro-N-acetylneuraminic acid instead of free Neu5Ac, suggesting a novel mechanism of adaptation to the mucosal environment [20]. The biological role of bacterial sialidases produced by human gut commensal and pathogenic bacteria has been reviewed previously [15,26,35]. Here we focus on the structural and biochemical properties

Figure 2 Mechanism of action hydrolytic/frans/IT-sialidases sialidases act via a two-step double-displacement mechanism so that the «-configuration of the glycosidic bond is retained

The glycosylation step is the same for all three classes of sialidase, but for the deglycosylation step the Incoming molecule can be water, another sugar or the internal oxygen atom, as indicated. Figure adapted from www.cazypedia.org.

of characterized sialidases involved in the interaction between gut bacteria and epithelial surfaces.

Sialidases from gut bacteria: structure and mechanism of action

General features

Sialidases (also commonly referred to as neuraminidases) are a large group of enzymes, the majority of which are exo-sialidases catalysing the cleavage of terminal sialic acids from complex carbohydrates on glycoproteins or glycolipids. Based on amino acid sequence similarities, bacterial exo-sialidases are classified in the GH family 33 (GH33) of the CAZy classification (www.cazy.org) [36]. Hydrolysis occurs via an acid/base-catalysed double-displacement mechanism involving a covalent sialyl-enzyme intermediate, resulting in overall retention of configuration at the anomeric centre

[37,38] (Figure 2). Unusually the catalytic nucleophile is a tyrosine residue activated by a proximal glutamic acid, due to the charge on sialic acid itself as shown biochemically [39] and confirmed structurally [40] (Figure 2). Sialidases from the retaining sialidase families GH34 and GH83 are both restricted to viruses and examples of GH58 inverting endo-sialidases are found in some E. coli strains (www.cazy.org).

Based on their substrate specificity and catalytic mechanism, exo-sialidases can be separated into three classes: hydrolytic, trans-sialidases and IT-sialidase. Hydrolytic-sialidases cleave the glycosidic bond of terminal sialic acids and release free sialic acid, whereas trans-sialidases transfer the cleaved sialic acid to other glycoconjugates; according to the Enzyme Commission both classes belong to exo-a-sialidases (EC 3.2.1.18). Hydrolytic-sialidases usually have wide substrate specificity and cleave a2-3-, a2-6- and a2-8-linked terminal sialic acids. Trans-sialidase activity with specificity for a2-3-linked substrates was first discovered for the Trypanosoma

Figure 3 Structural features of sialidases and IT-sialidases from gut bacteria

(A) Cartoon representation of the GH33 catalytic domain from S. typhimurium NanH/STSA sialidase (PDB: 1DIL). The canonical six-bladed p-propeller fold is highlighted with alternate colouring of the propeller blades. (B) R. gnavus RgNanH IT-sialidase GH33 catalytic domain (cyan) with inserted domain (orange; PDB: 4X4A). 2,7-anhydro-Neu5Ac is shown bound into the active site. (C) The active site of R. gnavus RgNanH (cyan) with 2,7-anhydro-Neu5Ac bound (yellow). Selected hydrogen bonds are highlighted with black dashed lines. Two characteristic features of the IT-sialidase active site are highlighted with a semi-transparent surface: the hydrophobic stack responsible for «2-3 linkage specificity (grey) and the threonine residue responsible for sterically hindering the ligand glycerol group (cyan). The S. typhimurium NanH/STSA active site (orange) has been superimposed, demonstrating that the majority of active site features are conserved across the hydrolytic and IT-sialidase classes. Residue numbering refers to RgNanH.

cruzi sialidase TcTS [41]. Trans-sialidases with activity against a2-6- and a2-8-linked sialic acid substrates have been discovered in the intervening years [42,43]. The third class is the IT-sialidase (EC 4.2.2.15). Currently, the discovered and characterized IT-sialidases are strictly a2-3-linkage specific and produce 2,7-anhydro-Neu5Ac [20,44,45]. However, the substrate and linkage specificity of sialidases is often unknown due to reliance on artificial substrates such as 4-methylumbelliferyl-Neu5Ac (4MU-Neu5Ac) or 2-O-(p-Nitrophenyl)-a-D-Neu5Ac (PNP-Neu5Ac; Table 1).

The GH33 catalytic domains adopt a six-bladed p-propeller fold (Figures 3A and 3B). GH33 catalytic domains are often associated with additional domains [46] including membrane-binding domains [47] and carbohydrate-binding modules (CBMs) such as sialic acid-specific CBM40 [19,20] and broadly specific CBM32 [48] as classified in CAZy (www.cazy.org). CBMs are believed to mediate adherence of the enzyme to cognate carbohydrate substrates and enhance the hydrolase activity of the catalytic domains by increasing enzyme substrate proximity [49]. Both exo-and trans-sialidases share a set of active site residues and cleave the terminal a-linked sialic acid residue by the same catalytic mechanism. This conserved active site includes a glutamic acid-tyrosine charge relay with the tyrosine acting as the catalytic nucleophile [40] and an aspartate residue as the general acid/base (Figure 2). The incoming sialic acid residue is orientated in the active site via a trio of arginines,

which co-ordinates the sialic acid carboxylate moiety and a hydrophobic pocket which accommodates the ligand N-acetyl group (Figure 3C). Aspartic acid-boxes are motifs commonly found at the termini of sialidase p-propeller blades, they may stabilize the protein fold by providing inter-blade contacts [50,51].

Hydrolytic exo-sialidases

Among GI commensals, Bacteroidetes species are found at high abundance and many of them express sialidases in culture [17]. B. thetaiotaomicron spp. encode the sialidases required to cleave and release terminal sialic acid from the mucosal glycoconjugates, but do not encode the Nan cluster required to consume the liberated monosaccharide [21]. The purified sialidase from B. thetaiotaomicron VPI-5482 has been shown to hydrolyse sialylglycoconjugates including fetuin and transferrin [52] (Table 1). Presumably, the release of sialic acids allows B. thetaiotaomicron to access highly coveted underlying carbohydrates in the mucus. Recently, a sialidase from Bacteroides vulgatus BVU 4143 has been shown to be active against 4MU-Neu5Ac and PNP-Neu5Ac and inhibited by N-acetyl-2,3-didehydro-2-deoxyneuraminic acid (Neu5Ac2en) inhibitor [31] (Table 1). B. fragilis strains are among those bacteria that have been shown to possess the complete pathway of sialic acid catabolism including the sialidase. Several sialidases from

Table 1 Characterized gut commensal and pathogenic sialidases

Abbreviations: AGP- human alphal-acid glycoprotein; BSM, bovine submaxillary mucin; GM1, monosialotetahexosylganglioside; KDN, acid; PGM, pig gastric mucin.

2-keto-3-deoxy-D-glcero-D-galactonic acid; Neu5Prop, W-propionylneuraminic

Bacterial species and strain Protein name Uniprot/Genbank PDB Domains P/E* Substrates tested + Я References

Akkermansia muciniphila ATCC Amuc_0625/Am0707* B2UPI5 GH33 P 4MU-Neu5Ac, a2,3-, AGP, Fetuin + [20] [58]

BAA-835/DSM 22959 a2,6- linkages, +

asialofetuin -

Neu5Ac-,Neu5Gc-, Neu5Prop-, KDN- +

Amuc_1835/Am2085t B2UN42 GH33 P 4MU-Neu5Ac, a2,3-, AGP, Fetuin + [20] [58]

a2,6- linkages, +

asialofetuin -

Neu5Ac-, Neu5Gc-, Neu5Prop- +

KDN- -

Amuc_0623/Am0705§ B2UP13 GH33 P Neu5Ac-, Neu5Gc- + + [58]

Neu5Prop-, KDN- +

Am_1547/Am1757§ B2ULI1 GH33 P Neu5Ac-, Neu5Gc-, Neu6Prop-, KDN- + [58]

B. fragilis YCH46/ sialidase (BF1729) P31206 GH33 P 4MU-Neu5Ac + [50]

TAL2480

B. fragilis SBT3182* P colominic acid (a2-8) + + [51,52]

a2-3 and a2-6 Neu5Ac-Lac +

B. fragilis 4852* P a2,3-, linear + + [54]

2,6- and 2,8- linkages, branched +

sialylconjugates

GM1 and mixed ganglosides -

Mucin, fetuin, AGP and other sialylated +

glycoproteins

^-linked sialylconjugates -

B. fragilis YM4000* E 4MU-Neu5Ac + [53]

B. thetaiotaomicron VPI-5482 sialidase (BtsA;BTSA;BT0455) Q8AAK9 4BBW GH33 P a2,3-, 2,6- and 2,8- linked sialylconjugates + [49]

fetuin, AGP, transferrin +

B. vulgatus ATCC 8482/DSM BVU_4143 A6L7T1 GH33 4MU-Neu5Ac, PNP-Neu5Ac + [31]

1447/NCTC 11154*

B. bifidum JCM 1254 exo-a-sialidase (SiaBb2;BBP_0054) BAK26854.1 GH33 P 4MU-Neu5Ac + [56]

a2,3-, 2,6- and 2,8- linked sialylconjugates +

(2,3- linkages preferred),

gangliosides, fetuin, PGM, hen egg yolk +

N-glycans

also transfers Neu5Ac to 1-alkanols +

o o TD <T> Table 1 Continued

Bacterial species and strain Protein name Uniprot/Genbank PDB Domains P/E* Substrates tested + /-( References

s Cl. perfringens A99 sialidase 1 'small' P10481 GH33 P 4MU-Neu5Ac + [68]

l C. perfringens ATCC 10543 sialidase 2 (NanH) Q59311 GH33 P 4MU-Neu5Ac + [69]

C. perfringens ATCC 13124» sialidase (CPF_0721) Q0TT67 4L2E CBM40, GH33 P 4MU-Neu5Ac + [70]

ee C. perfringens str 13» exo-a-sialidase (NanI;CPSA;CPE0725) Q8XMG4 2BF6 CBM40, GH33 P Fetuin, BSM, colominic acid, + [68] [66]

d b • 2VK5 bovine brain gangliosides

P o 2VK6 Can also hydrate 2-deoxy- +

d 2VK7 2,3-dehydro-Neu5Ac acid to

P s Neu5Ac

3 e C. perfringens str 13/ ATCC exo-a-sialidase (NanJ;CPE0553 Q8XMY5 2V73[A,B] CBM32, CBM40, GH33 P Only the CBMs are [48]

d 13124» characterized

d Clostridium tertium ATCC sialidase (NanH;SiaH) P77848 CBM40, GH33 P 4MU-Neu5Ac + [78]

i 14573

ed R. gnavus ATCC 29149 RgNanH* A7B557 CBM40, GH33 P 4MU-Neu5Ac, a2,3-, AGP, + [20]

d Fetuin

e a2,6-linkages, asialofetuin -

Releases 2,7 +

3 anhydro-Neu5Ac

o 3 5. typhimurium TA262/LT2 sialidase (NanH;STSA) P29768 1DIL GH33 P 4MU-Neu5Ac + [60] [59][63]

3 o 1DIM a2-3 Neu5AcLac + +

3 2SIL a2-6 Neu5AcLac, +

u 2SIM gangliosides, mucin, fetuin, +

0' 3SIL colominic acid

o 4MU-Neu5Ac>MU-Neu5Gc

Can produce Neu5Ac2en

*P/E refers to whether the characterization is carried out with purified (P) enzymes (including recombinant enzyme) or with bacterial extract (E).tThis column indicates whether the enzyme is active (+) or not (-) against the substrates tested, + + is used to denote more activity than +, where relative activity is indicated.^These strains are 'flesh-eating' strains isolated from gangrene rather than gut bacteria but are included because more biochemical data are available.*Details of enzymes are not currently in CAZy 'characterized' page.

different B. fragilis strains have been characterized, showing a broad specificity with some preference for the a2-8 linkage (Table 1) [53-57]. Sialidases have also been identified in the genomes of infant-derived Bifidobacteria, including two intracellular sialidases from B. longum subsp. infantis ATCC 15697 [18], two predicted extracellular exo-a-sialidases of B. bifidum PRL 2010 [58] and a putative sialidase from B. breve UCC2003 [34]. However, the only sialidase from this group of infant-associated bacteria to be functionally characterized is SiaBb2 from B. bifidum JCM 1254, a strain for which the genome sequence is not yet publicly available. SiaBb2 has a strong preference for a2-6 linkages and was shown to be sufficient to confer B. longum 105-A with the ability to degrade human milk oligosaccharides (HMOs) [59]. This sialidase can also transfer Neu5Ac to 1-alkanols at high acceptor concentrations [59] (Table 1). All four putative sialidases annotated in the genome of the mucin-degrading bacteria A. muciniphila ATCC BAA-835 [60] have recently been characterized [20,61]. The enzymes are active against a range of sialylated substrates with either a2-3or a2-6 linkages (Table 1).

Among gut pathogens, NanH/STSA from S. typhimurium TA262/LT2 strain has been biochemically [62,63] (Table 1) and structurally [64,65] characterized (Figure 3A), revealing conservation of key catalytic residues with the GH34 viral sialidases, including the nucleophilic charge relay, the aspartic acid acid/base and the arginine triad. This enzyme shows kinetic preference for sialyl a2-3 linkages over sialyl a2-6 linkages [62] and preferentially cleaves Neu5Ac residues rather than N-glycolylneuraminic acid (Neu5Gc) residues [66] (Table 1). Some strains of C.perfringens encode multiple sialidases (Table 1) [67-74]. The evolutionary rationale for this is unclear but may be because the enzymes differ in their cellular location, properties and sensitivities to inhibitors [74]. NanI from C. perfringens is unusual in that it is a hydrolytic enzyme which can also hydrate the inhibitor Neu5Ac2en to Neu5Ac in vitro [69]. These enzymes differ from V. cholerae sialidases which can hydrolyse both a-2,3-and a-2,6-linked sialic acid substrates [75] and produce the Neu5Ac2en inhibitor [19] (Table 1). The active site has many features in common with other viral and bacterial sialidases but, uniquely, has an essential Ca2 + ion which plays a crucial structural role [19,76].

Trans-sialidases and IT-sialidases

Most trans-sialidases have been characterized from trypano-some species [77]. Trans-sialidases have not been reported in the gut microbiota. However a few examples of exo-sialidases from gut bacteria have been reported to perform trans-glycosylation reactions under certain experimental conditions. These include the aforementioned SiaBb2 from B. bifidum and NanI from C. perfringens [69] (Table 1).

IT-sialidases are unique in that they catalyse an intramolecular reaction in which the O7-hydroxy group of the bound sialic acid glycerol group attacks the positively charged C2 atom of the oxocarbenium intermediate [44,78].

The altered reaction pathway leads to release of 2,7-anhydro-Neu5Ac instead of Neu5Ac (Figure 2). The first example of this enzyme class was described in NanL [79,80], which is purported to be from the leech Macrobdella decora, but may be from a bacterial source in the leech GI tract, as previously suggested [81]. Three IT-sialidases have been biochemically and structurally described: NanL [44,78], NanB from Streptococcus pneumonia [45] and RgNanH from R. gnavus [20] (Figure 3B). The active site of IT-sialidases is characterized by a conserved threonine residue which sterically hinders the substrate glycerol group, forcing it into an axial position whence it can attack at the anomeric C2 carbon and form the intramolecular linkage [44,78] (Figure 3C). An additional characteristic feature of these enzymes is a hydrophobic rim close to the arginine triad, formed by tryptophan-tyrosine stack [20,44,45]. This feature provides strict specificity for a2-3-linked substrates and may also provide an important contribution to the reaction mechanism by providing a desolvated, hydrophobic environment. This allows the intramolecular reaction to proceed, as the O7 hydroxy of the glycerol group must outrun any incoming water molecules that would otherwise attack the C2 carbon and produce Neu5Ac (Figure 3C).

RgNanH from R. gnavus ATCC 29149 is the first example of an IT-sialidase functionally characterized in gut bacteria [20,28]. The enzyme produces 2,7-anhydro-Neu5Ac with strict specificity towards a2-3 glycosidic substrate linkages. RgNanH is a three-domain modular protein with an N-terminal lectin-like domain (L-domain) classified as a CBM40, a GH33 catalytic domain (N-domain) and a domain inserted into the catalytic domain (I-domain). The crystal structure of the RgNanH catalytic domain has been solved and demonstrates the six-bladed f-propeller fold characteristic of sialidases [20]. A domain of unknown function protrudes from between two blades of the f -propeller (I-domain). Crystal structures in complex with 2,7-anhydro-Neu5Ac and known inhibitors of hydrolytic sialidases, allowed interrogation of the active site. Of particular importance is the conservation of the active site threonine (Thr557), which is proposed to sterically force the substrate glycerol group into a position from where it can attack the C2 atom [20] (Figure 3C). This residue also impacts on the response to sialidase inhibitors, as shown by poor inhibition by Neu5Ac2en and micromolar inhibition by siastatin B [20].

Bioinformatics analyses revealed that the presence of IT-sialidases is shared by other members of the gut microbiota, in particular Blautia hansenii, Ruminococcus torques, all 10 strains of Clostridium perfringens with available genome data, C. sp. 7 2 43 FAA, C. celatum, C. nexile, C. spiroforme, three unclassified Lachnospiraceae, more than 100 strains of Streptococcus agalactiae and three of the genome-sequenced publicly available Lactobacillus salivarius strains. The detection of IT-sialidase homologues in at least 11 % of gut metagenomes of a population of diseased and healthy humans confirmed that this enzyme is widespread across gut bacteria, especially in Firmicutes. This analysis

also revealed a greater abundance of IT-sialidase encoding species in patients with inflammatory bowel diseases (IBD) as compared with healthy individuals [20]. The specific niche colonization of these bacteria may reflect their adaptation to particular mucin glycosylation profiles associated with intestinal inflammation and/or infection [82,83].

Conclusions and perspectives

Bacterial sialidases and their sialoglycan targets contribute to host-microbe interactions at the mucosal surface. An imbalance in the proportion of gut commensals able to modulate mucosal sialic acid levels or a change in host mucin sialylation is often associated with enteric infection or intestinal inflammation. Maintaining a balance in the ability of gut commensals to produce and/or consume sialic acid in the mu-cosal compartment is therefore essential to gut homoeostasis.

Further investigations of bacterial sialidases should clarify the type of sialylated structures that are accessible to the gut bacteria and the specificity of sialidases towards sialic acids with different modifications and in different linkages. These include gaining structural insights into the diversity of sialic acid derivatives that can be produced and/or taken up by commensal and pathogenic bacteria. Thus, for therapeutic purposes, modulation of sialidase expression might be effectively achieved by appropriate use of specific inhibitors or pro/prebiotic approaches targeting specific bacterial strains.

Funding

The work was supported by the Biotechnology and Biological Sciences Research Council [grant number BB/J004529/1].

References

1 Johansson, M.E., Larsson, J.M. 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 CrossRef PubMed

2 McGuckin, M.A., Linden, S.K., Sutton, P. and Florin, T.H. (2011) Mucin dynamics and enteric pathogens. Nat. Rev. Microbiol. 9, 265-278 CrossRef PubMed

3 Corfield, A.P. (2015) Mucins: a biologically relevant glycan barrier in mucosal protection. Biochim. Biophys. Acta 1850, 236-252 CrossRef PubMed

4 Jonckheere, N., Skrypek, N., Frenois, F. and Van Seuningen, I. (2013) Membrane-bound mucin modular domains: from structure to function. Biochimie 95, 1077-1086 CrossRef PubMed

5 Brockhausen, I., Schachter, H. and Stanley, P. (2009) O-GalNAc glycans. In Essentials of Glycobiology, 2nd edn (Varki, A., Cummings, R.D., Esko, J.D., Freeze, H.H., Stanley, P., Bertozzi, C.R., Hart, G.W. and Etzler, M.E., eds), Cold Spring Harbor, New York

6 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 CrossRef PubMed

7 Holmen Larsson, J.M., Thomsson, K.A., Rodriguez-Pineiro, A.M., Karlsson, H. and Hansson, G.C. (2013) Studies of mucus in mouse stomach, small intestine, and colon. III. Gastrointestinal Muc5acand Muc2 mucin O-glycan patterns reveal a regiospecific distribution. Am. J. Physiol. Gastrointest. Liver Physiol. 305, G357-G363 CrossRef PubMed

8 Larsson, J.M.H., Karlsson, H., Sjovall, H. and Hansson, G.C. (2009) A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn. Glycobiology 19, 756-766 CrossRef PubMed

9 Sekirov, I., Russell, S.L., Antunes, L.C. and Finlay, B.B. (2010) Gut microbiota in health and disease. Physiol. Rev. 90, 859-904 CrossRef PubMed

10 Jandhyala, S.M., Talukdar, R., Subramanyam, C., Vuyyuru, H., Sasikala, M. and Nageshwar, R.D. (2015) Role of the normal gut microbiota. World J. Gastroenterol. 21, 8787-8803 CrossRef PubMed

11 Marcobal, A., Southwick, A.M., Earle, K.A. and Sonnenburg, J.L. (2013) A refined palate: bacterial consumption of host glycans in the gut. Glycobiology 23, 1038-1046 CrossRef PubMed

12 Tailford, L.E., Crost, E.H., Kavanaugh, D. andJuge, N. (2015a) Mucin glycan foraging in the human gut microbiome. Front. Genet. 6, 81 CrossRef

13 Plumbridge, J. and Vimr, E. (1999) Convergent pathways for utilization of the amino sugars N-acetylglucosamine, N-acetylmannosamine, and N-acetylneuraminic acid by Escherichia coli. J. Bacteriol. 181, 47-54 PubMed

14 Brigham, C., Caughlan, R., Gallegos, R., Dallas, M.B., Godoy, V.G. and Malamy, M.H. (2009) Sialic acid (N-acetyl neuraminic acid) utilization by Bacteroides fragilis requires a novel N-acetyl mannosamine epimerase. J. Bacteriol. 191, 3629-3638 CrossRef PubMed

15 Almagro-Moreno, S. and Boyd, E.F. (2009) Insights into the evolution of sialic acid catabolism among bacteria. BMC Evol. Biol. 9, 118 CrossRef PubMed

16 Fraser (1978) Neuraminidase production by clostridia. J. Med. Microbiol. 11, 269-280 CrossRef PubMed

17 Moncla, B.J., Braham, P. and Hillier, S.L. (1990) Sialidase (neuraminidase) activity among gram-negative anaerobic and capnophilic bacteria. J. Clin. Microbiol. 28, 422-425 PubMed

18 Sela, D.A., Chapman, J., Adeuya, A., Kim, J.H., Chen, F., Whitehead, T.R., Lapidus, A., Rokhsar, D.S., Lebrilla, C.B., German, J.B. et al. (2008) The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome. Proc. Natl. Acad. Sci. U.S.A. 105, 18964-18969 CrossRef PubMed

19 Moustafa, I., Connaris, H., Taylor, M., Zaitsev, V., Wilson, J.C., Kiefel, M.J., von Itzstein, M. and Taylor, G. (2004) Sialic acid recognition by Vibrio cholerae neuraminidase. J. Biol. Chem. 279, 40819-40826

CrossRef PubMed

20 Tailford, L.E., Owen, C.D., Walshaw, J., Crost, E.H., Hardy-Goddard, J., Le Gall, G., de Vos, W.M., Taylor, G.L. and Juge, N. (2015b) Discovery of intramolecular trans-sialidases in human gut microbiota suggests novel mechanisms of mucosal adaptation. Nat. Commun. 6, 7624 CrossRef

21 Marcobal, A., Barboza, M., Sonnenburg, E.D., Pudlo, N., Martens, E.C., Desai, P., Lebrilla, C.B., Weimer, B.C., Mills, D.A., German, J.B. and Sonnenburg, J.L. (2011) Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways. Cell Host Microbe 10, 507-514 CrossRef PubMed

22 Sebaihia, M., Wren, B.W., Mullany, P., Fairweather, N.F., Minton, N., Stabler, R., Thomson, N.R., Roberts, A.P., Cerdeno-Tarraga, A.M., Wang, H. et al. (2006) The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat. Genet. 38, 779-786 CrossRef PubMed

23 Vimr, E.R., Kalivoda, K.A., Deszo, E.L. and Steenbergen, S.M. (2004) Diversity of microbial sialic acid metabolism. Microbiol. Mol. Biol. Rev. 68, 132-153 CrossRef PubMed

24 Ng, K.M., Ferreyra, J.A., Higginbottom, S.K., Lynch, J.B., Kashyap, P.C., Gopinath, S., Naidu, N., Choudhury, B., Weimer, B.C., Monack, D.M. and Sonnenburg, J.L. (2013) Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens. Nature 502, 96-99 CrossRef PubMed

25 Nakayama-Imaohji, H., Ichimura, M., Iwasa, T., Okada, N., Ohnishi, Y. and Kuwahara, T. (2012) Characterization of a gene cluster for sialoglycoconjugate utilization in Bacteroides fragilis. J. Med. Invest. 59, 79-94 CrossRef PubMed

26 Lewis, A.L. and Lewis, W.G. (2012) Host sialoglycans and bacterial sialidases: a mucosal perspective. Cell Microbiol 14, 1174-1182 CrossRef PubMed

27 Jermyn, W.S. and Boyd, E.F. (2002) Characterization of a novel Vibrio pathogenicity island (VPI-2) encoding neuraminidase (nanH) among toxigenic Vibrio cholerae isolates. Microbiology 148, 3681-3693 CrossRef PubMed

28 Crost, E.H., Tailford, L.E., Le Gall, G., Fons, M., Henrissat, B. and Juge, N.

(2013) Utilisation of mucin glycans by the human gut symbiont Ruminococcus gnavus is strain-dependent. PLoS One 8, e76341 CrossRef PubMed

29 Iguchi, A., Thomson, N.R., Ogura, Y., Saunders, D., Ooka, T., Henderson, I.R., Harris, D., Asadulghani, M., Kurokawa, K., Dean, P. et al. (2009) Complete genome sequence and comparative genome analysis of enteropathogenic Escherichia coli O127:H6 strain E2348/69. J. Bacteriol. 191, 347-354 CrossRef PubMed

30 Reister, M., Hoffmeier, K., Krezdorn, N., Rotter, B., Liang, C., Rund, S., Dandekar, T., Sonnenborn, U. and Oelschlaeger, T.A. (2014) Complete genome sequence of the gram-negative probiotic Escherichia coli strain Nissle 1917. J. Biotechnol. 187, 106-107 CrossRef PubMed

31 Huang, Y.L., Chassard, C., Hausmann, M., von Itzstein, M. and Hennet, T. (2015) Sialic acid catabolism drives intestinal inflammation and microbial dysbiosis in mice. Nat. Commun. 6, 8141 CrossRef PubMed

32 Hoyer, L.L., Hamilton, A.C., Steenbergen, S.M. and Vimr, E.R. (1992) Cloning, sequencing and distribution of the Salmonella typhimurium LT2 sialidase gene, nanH, provides evidence for interspecies gene transfer. Mol. Microbiol. 6, 873-884 CrossRef PubMed

33 Roggentin, P., Schauer, R., Hoyer, L.L. and Vimr, E.R. (1993) The sialidase superfamily and its spread by horizontal gene transfer. Mol. Microbiol. 9, 915-921 CrossRef PubMed

34 Egan, M., O'connell Motherway, M., Ventura, M. and Van Sinderen, D.

(2014) Metabolism of sialic acid by Bifidobacterium breve UCC2003. Appl. Environ. Microbiol. 80, 4414-4426 CrossRef PubMed

35 Severi, E., Hood, D.W. and Thomas, G.H. (2007) Sialic acid utilization by bacterial pathogens. Microbiology 153, 2817-2822 CrossRef PubMed

36 Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P.M. and Henrissat, B. (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42, D490-495 CrossRef PubMed

37 von Itzstein, M. (2007) The war against influenza: discovery and development of sialidase inhibitors. Nat. Rev. Drug Discov. 6, 967-974 CrossRef PubMed

38 Xu, G., Kiefel, M.J., Wilson, J.C., Andrew, P.W., Oggioni, M.R. and Taylor, G.L. (2011) Three Streptococcus pneumoniae sialidases: three different products. J. Am. Chem. Soc. 133, 1718-1721 CrossRef PubMed

39 Watts, A.G, Damager, I., Amaya, M.L., Buschiazzo, A., Alzari, P., Frasch, A.C. and Withers, S.G. (2003) Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: tyrosine is the catalytic nucleophile. J. Am. Chem. Soc. 125, 7532-7533 CrossRef PubMed

40 Amaya, M.F., Watts, A.G., Damager, I., Wehenkel, A., Nguyen, T., Buschiazzo, A., Paris, G., Frasch, A.C., Withers, S.G. and Alzari, P.M. (2004) Structural insights into the catalytic mechanism of Trypanosoma cruzi trans-sialidase. Structure 12, 775-784 CrossRef PubMed

41 Schenkman, S., Jiang, M.-S., Hart, G.W. and Nussenzweig, V. (1991) A novel cell surface trans-sialidase of trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells. Cell 65, 1117-1125 CrossRef PubMed

42 Cheng, J., Yu, H., Lau, K., Huang, S., Chokhawala, H.A., Li, Y., Tiwari, V.K. and Chen, X. (2008) Multifunctionality of Campylobacter jejuni sialyltransferase CstII: characterization of GD3/GT3 oligosaccharide synthase, GD3 oligosaccharide sialidase, and trans-sialidase activities. Glycobiology 18, 686-697 CrossRef PubMed

43 Cheng, J., Huang, S., Yu, H., Li, Y., Lau, K. and Chen, X. (2010) Trans-sialidase activity of Photobacterium damsela alpha2,6-sialyltransferase and its application in the synthesis of sialosides. Glycobiology 20, 260-268 CrossRef PubMed

44 Luo, Y., Li, S.C., Chou, M.Y., Li, Y.T. and Luo, M. (1998) The crystal structure of an intramolecular trans-sialidase with a NeuAc alpha2->3Gal specificity. Structure 6, 521-530 CrossRef PubMed

45 Gut, H., King, S.J. and Walsh, M.A. (2008) Structural and functional studies of Streptococcus pneumoniae neuraminidase B: An intramolecular trans-sialidase. FEBS Lett. 582, 3348-3352 CrossRef PubMed

46 Thobhani, S., Ember, B., Siriwardena, A. and Boons, G.J. (2003) Multivalency and the mode of action of bacterial sialidases. J. Am. Chem. Soc. 125, 7154-7155 CrossRef PubMed

47 Camara, M., Boulnois, G.J., Andrew, P.W. and Mitchell, T.J. (1994) A neuraminidase from Streptococcus pneumoniae has the features of a surface protein. Infect. Immun. 62, 3688-3695 PubMed

48 Boraston, A.B., Ficko-Blean, E. and Healey, M. (2007) Carbohydrate recognition by a large sialidase toxin from Clostridium perfringens. Biochemistry 46, 11352-11360 CrossRef PubMed

49 Ficko-Blean, E. and Boraston, A.B. (2012) Insights into the recognition of the human glycome by microbial carbohydrate-binding modules. Curr. Opin. Struct. Biol. 22, 570-577 CrossRef PubMed

50 Copley, R.R., Russell, R.B. and Ponting, C.P. (2001) Sialidase-like Asp-boxes: sequence-similar structures within different protein folds. Protein Sci. Publ. Protein Soc. 10, 285-292 CrossRef

51 Quistgaard, E.M. and Thirup, S.S. (2009) Sequence and structural analysis of the asp-box motif and asp-box beta-propellers; a widespread propeller-type characteristic of the vps10 domain family and several glycoside hydrolase families. BMC Struct. Biol. 9, 46 CrossRef PubMed

52 Park, K.H., Kim, M.G., Ahn, H.J., Lee, D.H., Kim, J.H., Kim, Y.W. and Woo, E.J. (2013) Structural and biochemical characterization of the broad substrate specificity of Bacteroides thetaiotaomicron commensal sialidase. Biochim. Biophys. Acta 1834, 1510-1519 CrossRef PubMed

53 Russo, T.A, Thompson, J.S., Godoy, V.G. and Malamy, M.H. (1990) Cloning and expression of the Bacteroides fragilis TAL2480 neuraminidase gene, nanH, in Escherichia coli. J. Bacteriol. 172, 2594-2600 PubMed

54 Tanaka, H, Ito, F and Iwasaki, T (1992) Purification and characterization of a sialidase from Bacteroides fragilis SBT3182. Biochem. Biophys. Res. Commun. 189, 524-529 CrossRef PubMed

55 Tanaka, H., Ito, F. and Iwasaki, T. (1994) Two sialidases which preferentially hydrolyze sialyl alpha 2-8 linkage from Bacteroides fragilis SBT3182. J. Biochem. 115,318-321 PubMed

56 Godoy, V.G., Dallas, M.M., Russo, T.A. and Malamy, M.H. (1993) A role for Bacteroides fragilis neuraminidase in bacterial growth in two model systems. Infect. Immun. 61, 4415-4426 PubMed

57 von Nicolai, H., Hammann, R., Werner, H. and Zilliken, F. (1983) Isolation and characterization of sialidase from Bacteroides fragilis. FEMS Microb. Lett. 17, 217-220

58 Turroni, F., Bottacini, F., Foroni, E., Mulder, I., Kim, J.H., Zomer, A., Sanchez, B., Bidossi, A., Ferrarini, A., Giubellini, V. 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 CrossRef PubMed

59 Kiyohara, M., Tanigawa, K., Chaiwangsri, T., Katayama, T., Ashida, H. and Yamamoto, K. (2011) An exo-alpha-sialidase from bifidobacteria involved in the degradation of sialyloligosaccharides in human milk and intestinal glycoconjugates. Glycobiology 21, 437-447

CrossRef PubMed

60 Van Passel, M.W., Kant, R., Zoetendal, E.G., Plugge, C.M., Derrien, M., Malfatti, S.A., Chain, P.S., Woyke, T., Palva, A., De Vos, W.M. and Smidt, H. (2011) The genome of Akkermansia muciniphila, a dedicated intestinal mucin degrader, and its use in exploring intestinal metagenomes. PLoS One 6, e16876 CrossRef PubMed

61 Huang, K., Wang, M.M., Kulinich, A., Yao, H.L., Ma, H.Y., Martinez, J.E., Duan, X.C., Chen, H., Cai, Z.P., Flitsch, S.L. et al. (2015) Biochemical characterisation of the neuraminidase pool of the human gut symbiont Akkermansia muciniphila. Carbohydr. Res. 415, 60-65

CrossRef PubMed

62 Hoyer, L.L., Roggentin, P., Schauer, R. and Vimr, E.R. (1991) Purification and properties of cloned Salmonella typhimurium LT2 sialidase with virus-typical kinetic preference for sialyl alpha 2 -- 3 linkages. J. Biochem. 110, 462-467 PubMed

63 Taylor, G., Vimr, E., Garman, E. and Laver, G. (1992) Purification, crystallization and preliminary crystallographic study of neuraminidase from Vibrio cholerae and Salmonella typhimurium LT2. J. Mol. Biol. 226, 1287-1290 CrossRef PubMed

64 Crennell, S.J., Garman, E.F., Laver, W.G., Vimr, E.R. and Taylor, G.L. (1993) Crystal structure of a bacterial sialidase (from Salmonella typhimurium LT2) shows the same fold as an influenza virus neuraminidase. Proc. Natl. Acad. Sci. U.S.A. 90, 9852-9856 CrossRef PubMed

65 Crennell, S.J., Garman, E.F., Philippon, C., Vasella, A., Laver, W.G., Vimr, E.R. and Taylor, G.L. (1996) The structures of Salmonella typhimurium LT2 neuraminidase and its complexes with three inhibitors at high resolution. J. Mol. Biol. 259, 264-280 CrossRef PubMed

66 Minami, A., Ishibashi, S., Ikeda, K., Ishitsubo, E., Hori, T., Tokiwa, H., Taguchi, R., leno, D., Otsubo, T., Matsuda, Y. et al. (2013) Catalytic preference of Salmonella typhimurium LT2 sialidase for N-acetylneuraminic acid residues over N-glycolylneuraminic acid residues. FEBS Open Bio. 3, 231-236 CrossRef PubMed

67 Shimizu, T., Ohshima, S., Ohtani, K., Shimizu, T. and Hayashi, H. (2001) Genomic map of Clostridium perfringens strain 13. Microbiol. Immunol. 45, 179-189 CrossRef PubMed

68 Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S. and Hayashi, H. (2002) Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc. Natl. Acad. Sci. U.S.A. 99, 996-1001

CrossRef PubMed

69 Newstead, S.L., Potter, J.A., Wilson, J.C., Xu, G., Chien, C.H., Watts, A.G., Withers, S.G. and Taylor, G.L. (2008) The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates. J. Biol. Chem. 283, 9080-9088 CrossRef PubMed

70 Li, J., Sayeed, S., Robertson, S., Chen, J. and McClane, B.A. (2011) Sialidases affect the host cell adherence and epsilon toxin-induced cytotoxicity of Clostridium perfringens type D strain CN3718. PLoS Pathog. 7, e1002429 CrossRef PubMed

71 Roggentin, P., Kleineidam, R.G. and Schauer, R. (1995) Diversity in the properties of two sialidase isoenzymes produced by Clostridium perfringens spp. Biol. Chem. Hoppe Seyler. 376, 569-575

CrossRef PubMed

72 Chien, C.H., Shann, Y.J. and Sheu, S.Y. (1996) Site-directed mutations of the catalytic and conserved amino acids of the neuraminidase gene, nanH, of Clostridium perfringens ATCC 10543. Enzyme Microb. Technol. 19, 267-276 CrossRef PubMed

73 Lee, Y., Ryu, Y.B., Youn, H.S., Cho, J.K., Kim, Y.M., Park, J.Y., Lee, W.S., Park, K.H. and Eom, S.H. (2014) Structural basis of sialidase in complex with geranylated flavonoids as potent natural inhibitors. Acta Crystallogr. D Biol. Crystallogr. 70, 1357-1365 CrossRef PubMed

74 Li, J. and McClane, B.A. (2014) The Sialidases of Clostridium perfringens type D strain CN3718 differ in their properties and sensitivities to inhibitors. Appl. Environ. Microbiol. 80, 1701-1709

CrossRef PubMed

75 Corfield, A.P., Higa, H., Paulson, J.C. and Schauer, R. (1983) The specificity of viral and bacterial sialidases for alpha(2-3)- and alpha(2-6)-linked sialic acids in glycoproteins. Biochim. Biophys. Acta 744, 121-126 CrossRef PubMed

76 Crennell, S., Garman, E., Laver, G., Vimr, E. and Taylor, G. (1994) Crystal structure of Vibrio cholerae neuraminidase reveals dual lectin-like domains in addition to the catalytic domain. Structure 2, 535-544 CrossRef PubMed

77 Schauer, R. and Kamerling, J.P. (2011) The chemistry and biology of trypanosomal trans-sialidases: virulence factors in Chagas disease and sleeping sickness. Chembiochem. 12, 2246-2264 CrossRef PubMed

78 Luo, Y., Li, S.-C., Li, Y.-T. and Luo, M. (1999) The 1.8 Â structures of leech intramolecular trans-sialidase complexes: evidence of its enzymatic mechanism. J. Mol. Biol. 285, 323-332 CrossRef PubMed

79 Li, Y.T., Nakagawa, H., Ross, S.A., Hansson, G.C. and Li, S.C. (1990) A novel sialidase which releases 2,7-anhydro-alpha-N-acetylneuraminic acid from sialoglycoconjugates. J. Biol. Chem. 265, 21629-21633 PubMed

80 Chou, M.Y., Li, S.C. and Li, Y.T. (1996) Cloning and expression of sialidase L, a NeuAcalpha2-&gt;3Gal-specific sialidase from the leech, Macrobdella decora. J. Biol. Chem. 271, 19219-19224 CrossRef PubMed

81 Grobe, K., Sartori, B., Traving, C., Schauer, R. and Roggentin, P. (1998) Enzymatic and molecular properties of the Clostridium tertium sialidase. J. Biochem. 124, 1101-1110 CrossRef PubMed

82 Kashyap, P.C., Marcobal, A., Ursell, L.K., Smits, S.A., Sonnenburg, E.D., Costello, E.K., Higginbottom, S.K., Domino, S.E., Holmes, S.P., Relman, D.A. et al. (2013) Genetically dictated change in host mucus carbohydrate landscape exerts a diet-dependent effect on the gut microbiota. Proc. Natl. Acad. Sci. U.S.A. 110, 17059-17064 CrossRef PubMed

83 Theodoratou, E., Campbell, H., Ventham, N.T., Kolarich, D., Puc ic -Bakovic, M., Zoldos, V., Fernandes, D., Pemberton, I.K., Rudan, I., Kennedy, N.A. et al. (2014) The role of glycosylation in IBD. Nat. Rev. Gastroenterol. Hepatol. 11, 588-600 PubMed

Received 13 November 201S doi:10.1042/BST20150226