The Interaction of a Carbohydrate-binding Module from a Clostridium perfringens N-Acetyl-β-hexosaminidase with Its Carbohydrate Receptor

Clostridium perfringens is a notable colonizer of the human gastrointestinal tract. This bacterium is quite remarkable for a human pathogen by the number of glycoside hydrolases found in its genome. The modularity of these enzymes is striking as is the frequent occurrence of modules having amino acid sequence identity with family 32 carbohydrate-binding modules (CBMs), often referred to as F5/8 domains. Here we report the properties of family 32 CBMs from a C. perfringens N-acetyl-β-hexosaminidase. Macroarray, UV difference, and isothermal titration calorimetry binding studies indicate a preference for the disaccharide LacNAc (β-d-galactosyl-1,4-β-d-N-acetylglucosamine). The molecular details of the interaction of this CBM with galactose, LacNAc, and the type II blood group H-trisaccharide are revealed by x-ray crystallographic studies at resolutions of 1.49, 2.4, and 2.3 Å, respectively. Clostridium perfringens is a notable colonizer of the human gastrointestinal tract. This bacterium is quite remarkable for a human pathogen by the number of glycoside hydrolases found in its genome. The modularity of these enzymes is striking as is the frequent occurrence of modules having amino acid sequence identity with family 32 carbohydrate-binding modules (CBMs), often referred to as F5/8 domains. Here we report the properties of family 32 CBMs from a C. perfringens N-acetyl-β-hexosaminidase. Macroarray, UV difference, and isothermal titration calorimetry binding studies indicate a preference for the disaccharide LacNAc (β-d-galactosyl-1,4-β-d-N-acetylglucosamine). The molecular details of the interaction of this CBM with galactose, LacNAc, and the type II blood group H-trisaccharide are revealed by x-ray crystallographic studies at resolutions of 1.49, 2.4, and 2.3 Å, respectively. Clostridium perfringens is a Gram-positive, spore-forming, non-motile, rod-shaped anaerobe. As a pathogen of humans, C. perfringens is often associated with gas gangrene, necrotic enteritis, and, most commonly, food poisoning (1Hatheway C.L. Clin. Microbiol. Rev. 1990; 3: 66-98Crossref PubMed Google Scholar, 3Rood J.I. Cole S.T. Microbiol. Rev. 1991; 55: 621-648Crossref PubMed Google Scholar). Determination of the genome sequence of C. perfringens (strain 13) (4Shimizu T. Ohshima S. Ohtani K. Hayashi H. Microbiol. Immunol. 2001; 45: 179-189Crossref PubMed Scopus (22) Google Scholar) has revealed at least 54 open reading frames encoding putative glycoside hydrolases falling into 24 known glycoside hydrolase families (see afmb.cnrs-mrs.fr/CAZY/index.html) (5Coutinho P.M. Henrissat B. Gilbert H.J. Davies G.J. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. The Royal Society of Chemistry, Cambridge1999: 3-12Google Scholar). Many encode intracellular proteins likely involved in the latter stages of sugar metabolism or proteins involved in peptidoglycan remodeling; however, roughly one-half are predicted to be secreted and are likely involved in the early stages of sugar metabolism. Although C. perfringens is most frequently thought of as a “flesh-eater,” its most common niche in humans is the gastrointestinal tract. However, very few, if any, of the secreted glycoside hydrolases have predicted substrate specificities consistent with metabolism of dietary polysaccharides in the human gut making gastric mucins, highly hydrated glycoproteins comprising up to 80% carbohydrate, the most likely target of the secreted C. perfringens enzymes. Indeed, the majority of these enzymes are predicted to have specificities appropriate for the degradation of complex glycans, suggesting that this bacterium is well equipped to attack the diverse sugar structures of the mucins in this environment. Consistent with this is the mucosal necrosis associated with severe enteritis caused by C. perfringens (6Gerding D.N. Johnson S. Rood J.I. McClane B.A. Songer J.G. Titball R.W. The Clostridia Molecular Biology and Pathogenesis. Harcourt Brace & Company, London1997: 117-140Google Scholar), which may be in part due to the arsenal of C. perfringens glycoside hydrolases. In turn, breaking down the mucosal barrier could improve access of other toxins, such as the pore-forming cpe (C. perfringens enterotoxin), to the epithelial layer. Thirteen of the predicted C. perfringens (strain 13) glycoside hydrolases (and notably 13 glycoside hydrolases for each of the sequenced Bacteroides sp. genomes (thetaiotaomicron, fragilis YCH46, and fragilis 25285)) are highly modular and have, in addition to catalytic domains, modules with amino acid sequence identity to family 32 carbohydrate-binding modules (CBMs) 3The abbreviations used are: CBM, carbohydrate-binding module; ITC, isothermal titration calorimetry; r.m.s.d., root mean square deviation. (7Boraston A.B. Notenboom V. Warren R.A. Kilburn D.G. Rose D.R. Davies G. J. Mol. Biol. 2003; 327: 659-669Crossref PubMed Scopus (64) Google Scholar). CBMs are generally considered to be modules with carbohydrate-binding function, but no catalytic activity, that are found within the modular architectures of glycoside hydrolases (8Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1537) Google Scholar). They are currently classified into 45 families based on amino acid sequence identity (see afmb.cnrs-mrs.fr/~cazy/CAZY/index.html.) and loosely grouped into three types, A (crystalline polysaccharide binding), B (polysaccharide chain binding), and C (small sugar binding or “lectin-like”), established by functional properties (8Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1537) Google Scholar). Based on limited biochemical evidence, the family 32 CBMs appear to be type C CBMs. The x-ray crystal structures of the Cladobotryum dendroides galactose oxidase and the Micromonospora viridifaciens sialidase (MvGH33) revealed the β-sandwich lectin-like folds of their cognate CBM32 modules (9Ito N. Phillips S.E. Stevens C. Ogel Z.B. McPherson M.J. Keen J.N. Yadav K.D. Knowles P.F. Nature. 1991; 350: 87-90Crossref PubMed Scopus (697) Google Scholar, 10Gaskell A. Crennell S. Taylor G. Structure. 1995; 3: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Co-crystallizations of MvGH33 with galactose showed the potential of its CBM32 module, here called MvCBM32, to bind galactose (10Gaskell A. Crennell S. Taylor G. Structure. 1995; 3: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 11Newstead S.L. Watson J.N. Bennet A.J. Taylor G. Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 1483-1491Crossref PubMed Scopus (43) Google Scholar), which was subsequently verified by functional studies (7Boraston A.B. Notenboom V. Warren R.A. Kilburn D.G. Rose D.R. Davies G. J. Mol. Biol. 2003; 327: 659-669Crossref PubMed Scopus (64) Google Scholar). Thus, the family 32 CBMs, which are often referred to as F5/8 domains, have been generally considered as galactose binding domains. The family 32 CBMs stand out among the CBM families, because they are frequently found appended to enzymes with “exotic” specificities (e.g. sialidases, β-hexosaminidases, mannosidases, and fucosidases) and are found in bacteria capable of causing disease in humans. In contrast, the vast majority of CBMs in other families are found appended to enzymes that are active on plant cell wall polysaccharides. In the context of the plant cell wall hydrolases, the function of CBMs has been repeatedly shown to be to localize the enzyme to an appropriate substrate (8Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1537) Google Scholar). By analogy to the plant cell wall hydrolases, the role of family 32 CBMs is likely to target their parent enzymes to carbohydrate substrates; however, with these CBMs the substrates are likely more complex glycans, such as gastric mucins in the case of C. perfringens and Bacteroides sp. Previous studies of family 32 CBMs have not addressed the possibility that the specificity of these CBMs extend beyond a preference simply for galactose and may actually include specificity for complex glycan chains. Thus, studies of family 32 CBMs from bacterial pathogens enter a new area of carbohydrate-binding module-mediated host-pathogen interactions and will extend our knowledge of this potentially complex family of carbohydrate-binding proteins. To better understand CBM32 structure and function we initiated studies of CpGH84C from C. perfringens (strain ATCC 13124). This enzyme, which comprises four modules defined on the basis of primary structure comparisons (see Fig. 1), was chosen as a model system, because, relative to other family 32 CBM-containing enzymes, it is reasonably small and has a simple modular architecture that is amenable to accurate definition of the modular boundaries. To facilitate structure-function studies, we dissected this protein at the genetic level to recombinantly produce isolated CpCBM32. The experimental results reveal the ability of the CBM to bind to terminal glycotopes commonly found in elaborated O- and complex N-glycans (12Gupta D. Kaltner H. Dong X. Gabius H.J. Brewer C.F. Glycobiology. 1996; 6: 843-849Crossref PubMed Scopus (101) Google Scholar, 14Robbe C. Capon C. Coddeville B. Michalski J.C. Biochem. J. 2004; 384: 307-316Crossref PubMed Scopus (257) Google Scholar), whereas the x-ray crystal structures of CpCBM32 in complex with sugar help uncover the molecular details that confer this binding ability. Structural comparisons with known CBM32s and other C. perfringens CBM32s suggest variations in glycan specificities, but all are based on a key terminal galactose residue. This work provides the first detailed structure-function analysis of a family 32 CBM and will provide a foundation for further studies of CBMs within this family. Materials—Unless otherwise stated, chemicals, carbohydrates, glycoproteins, and polysaccharides were purchased from Sigma. Cloning—The DNA fragment encoding the family 32 CBM (Fig. 1) of CpGH84C was amplified by PCR from C. perfringens genomic DNA (Sigma, ATCC 13124) using previously described methods (15Ficko-Blean E. Boraston A.B. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 834-836Crossref PubMed Scopus (20) Google Scholar). Nucleotides 1873-2301 of the cpgh84c gene, corresponding to the CBM (amino acid residues 625-767), were amplified with the oligonucleotide primers 5′-CACCAATCCAAGAACAGTAAAG-3′ (CBMF) and 5′-CTTTTATCCATGAACATTAACCTC-3′ (CBMR). The amplified gene fragment was ligated directly into the pET-150 TOPO Directional Cloning kit (Invitrogen) to generate pCBM32. The polypeptide (called CpCBM32) encoded by pCBM32 comprises a H6 tag fused to the CpCBM32 module by an enterokinase protease cleavage site. Protein Production and Purification—pCBM was transformed into the BL21star (DE3) Escherichia coli expression strain (Invitrogen). A 1.5-liter culture was grown in Luria-Bertani (LB) media, supplemented with ampicillin (100 μg/ml), to an optical density of ∼1 and induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside then grown overnight at 37 °C. The cells were harvested at 4,000 × g and resuspended in 20 ml of binding buffer containing 20 mm Tris, pH 8, and 0.5 m NaCl. Cells were lysed using a French pressure cell. Cell debris was removed by centrifugation for 1 h at 27,000 × g. The was to by with binding buffer containing and were on a and containing the polypeptide of were were and buffer was in a using a molecular by was Determination of Protein of proteins were by UV using H. Biochem. PubMed Scopus Google Scholar). was with was resuspended in to a of and and Clostridium and CpCBM32 were into m pH by using a of was with 1 of and the was to for 1 h at in the was into pH by using a to 1 of or of plant and glycoproteins were a were to then for h with ml of 20 in pH protein was to overnight at 13 in ml of 20 in pH were with ml of 20 in pH for of was at using the from was based on of Fig. UV UV of CpCBM32 were as described previously A.B. Warren R.A. Kilburn D.G. 2001; PubMed Scopus Google Scholar). were for and and at the appropriate were for further The for the were for each sugar The at three were by of the from the and the were carbohydrate for the three were with using a binding model for were at 20 in mm Tris, pH The are the and of three titration calorimetry was as described previously A.B. Warren R.A. Kilburn D.G. 2001; PubMed Scopus Google Scholar, A. J. Boraston A.B. 2004; PubMed Scopus Google Scholar) using a Protein were buffer mm Tris, pH were by in buffer from the protein protein and sugar were and to Protein were by UV as described of mm LacNAc were into CpCBM32 which C T. S. Biochem. PubMed Scopus Google Scholar). of mm type II H-trisaccharide were into CpCBM32 In this the C was due to the binding Based on the binding in the crystal the was at 1 in the analysis of this were with a binding and were using the to the H6 tag was removed from CpCBM32 by with enterokinase a The was a to the tag and protein from the were and as into 20 mm Tris, pH of CpCBM32 with galactose were with m and m Tris, pH were with in m was used to CpCBM32 LacNAc and the type II blood group H-trisaccharide of this the to be m a and the protein to be The used was m with were with a area to an x-ray with and an were with Acta Crystallogr. D Biol. Crystallogr. 55: PubMed Scopus Google Scholar). are in and of of sugar in a new was by molecular using the family 32 module from the viridifaciens sialidase (10Gaskell A. Crennell S. Taylor G. Structure. 1995; 3: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) as a The A. A. J. Crystallogr. Scopus Google Scholar) was to corresponding to the in the This model was and was by of using K. Acta Crystallogr. D Biol. Crystallogr. 2004; PubMed Scopus Google Scholar). was using Acta Crystallogr. D Biol. Crystallogr. PubMed Scopus Google Scholar). were using the of and to The model was used as a model to the structures of the other CpCBM32 sugar were or more were and were as to the in the CpGH84C model are in Carbohydrate of CpCBM32 from on its amino acid sequence identity with the family 32 module from the viridifaciens sialidase we that the CpCBM32 module in CpGH84C is a carbohydrate-binding This was by binding using glycoproteins and which revealed binding to type gastric and with relative binding of gastric Fig. knowledge of the glycan structures commonly found on these glycoproteins C. Capon C. Coddeville B. Michalski J.C. Biochem. J. 2004; 384: 307-316Crossref PubMed Scopus (257) Google Scholar) as a we the binding of and to CpCBM32 by and UV The addition of and to CpCBM32 in of the UV of the of in sugar binding A.B. Warren R.A. Kilburn D.G. 2001; PubMed Scopus Google Scholar) (Fig. and were but not the UV of CpCBM32 and are primary of CpCBM32. studies by UV showed an of roughly 1 × for and a preference for (Fig. and The of the group of not appear to confer to CpCBM32 and LacNAc galactose by of and respectively. The of LacNAc the of the group of the in the of the binding to we could due to of sugar but not by UV at 20 in mm pH × in a new The binding to LacNAc and the type II H-trisaccharide was further by (Fig. C and and The for LacNAc revealed the binding common to interactions Brewer C.F. Rev. PubMed Scopus Google Scholar, K. A. 2001; PubMed Scopus Google Scholar). The of binding was the analysis of which the of the in be this small is consistent with the majority of The of CpCBM32 for the type II H-trisaccharide was to the of on the basis of the LacNAc binding and x-ray (see this was at 1 for the analysis J. 2003; PubMed Scopus Google Scholar). with the of the type II H-trisaccharide is to binding relative to LacNAc or it not The roughly in due to the on the H-trisaccharide to by of a which is by a to of up to a in the of due to in the sugar or protein the of the but not their and, of the are by in mm pH × were and are from the are the from three were and are from the are the from three were and are from the are the from three was as a for the were and are from the are the from three This was as a for the in a new The of was in the of galactose, and its x-ray crystal structure was by molecular at The is that of a β-sandwich comprising a of three a of (Fig. The are the family 32 CBMs from the Cladobotryum dendroides galactose oxidase (9Ito N. Phillips S.E. Stevens C. Ogel Z.B. McPherson M.J. Keen J.N. Yadav K.D. Knowles P.F. Nature. 1991; 350: 87-90Crossref PubMed Scopus (697) Google the CBM is referred to as and the bacterial viridifaciens sialidase (10Gaskell A. Crennell S. Taylor G. Structure. 1995; 3: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (194) Google the CBM is referred to as for a more detailed are the family and CBMs as well as the sp. CpCBM32 the of and and the of and (Fig. This was most likely to be on the basis of three the because it could be in that the was not or commonly found associated with carbohydrate-binding by is most consistent with or as the B of this was consistent with the B of in the chains. This not appear to a role in binding carbohydrate, because it is quite from the carbohydrate binding site. on other CBMs have been removed have a role for such because are to bind carbohydrate A. J. Mol. Biol. 2004; PubMed Scopus Google Scholar, E. K. PubMed Scopus Google Scholar). is notable that the of this is in CBMs from family (7Boraston A.B. Notenboom V. Warren R.A. Kilburn D.G. Rose D.R. Davies G. J. Mol. Biol. 2003; 327: 659-669Crossref PubMed Scopus (64) Google Scholar, C. Gilbert H.J. Boraston A.B. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Boraston A.B. J. Mol. Biol. 2004; PubMed Scopus (20) Google Scholar), S. Boraston A.B. N. Davies G.J. Structure. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), and the sp. Struct. Biol. Google Scholar). CpCBM32 in with density of the galactose complex revealed a of and a which the the by on the of with the from the (Fig. for the of (and is by three potential to the of the sugar from the terminal of a terminal of and the from the of (Fig. are the of and and the of galactose a with the of The of the galactose binding to the blood group the interactions of CpCBM32 with galactose LacNAc and the type II blood group H-trisaccharide A of was used as the for of are shown as of the complex revealed that the and of the galactose were at this protein the sugar of LacNAc and the type II the of by CpCBM32 with these of CpCBM32 (Fig. The binding of LacNAc to relative to the galactose In the galactose structure the comprising residues was very and, in residues and could not be In the LacNAc this and the of and in on the sugar to with the galactose and residues and terminal of is to with the of The other terminal of with the of an for a to the of the The terminal of is to with the of galactose and the of The group of the not appear to be to However, this group a by the of the and and a number of A the group of and the of interactions are the likely of the of CpCBM32 for LacNAc The crystal structure of CpCBM32 in complex with the type II blood group H-trisaccharide revealed the interactions the LacNAc of this sugar and the protein to be to the interactions (Fig. The of the type II H-trisaccharide a and interactions with the protein were limited to the first by the of and the of (Fig. The is involved in a to the galactose and The is for potential with the of by the of Molecular of CpCBM32 CBM32 module from CpGH84C to have that the of The primary galactose but is to and it to provide the of the binding and provide the for the on the of this galactose in binding In the case of LacNAc, the is to the galactose, the by an of relative to galactose and is by the of the group of of a of the type blood group H-trisaccharide into the CpCBM32 binding using LacNAc as a that CpCBM32 may this sugar with the in the as for the of LacNAc In the case of the group of the in this disaccharide be to the group of Although is to the binding of it is if the interaction of this sugar be to of the LacNAc an of the 1 be in a to The structure of CpCBM32 with the type II H-trisaccharide revealed an that the residue. However, this to a The an but from the of this This is consistent with the of from the protein Indeed, the binding in the LacNAc, and type II H-trisaccharide the latter sugar results in the of at least that otherwise be on the protein in the of the residue. the of the blood group is by the CBM, it is not a CpCBM32 may have a that to the of the The of galactose is which may the of sugar to that acid is found to LacNAc, at the of a and to often as an of C. Capon C. Coddeville B. Michalski J.C. Biochem. J. 2004; 384: 307-316Crossref PubMed Scopus (257) Google Scholar). is that CpCBM32 may a acid within this putative binding with 32 of CpCBM32 are and with amino acid sequence of and and for of and (Fig. A and the of and very with the of a in CpCBM32 (Fig. A and the crystal structure of in complex with galactose was S.L. Watson J.N. Bennet A.J. Taylor G. Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 1483-1491Crossref PubMed Scopus (43) Google Scholar). Although a complex for is not a of the and structures and the residues involved in galactose binding of functional residues (Fig. However, in the of further is A of and CpCBM32 that the architecture of the carbohydrate-binding and the of interactions are very The majority of the galactose binding comprising and of CpCBM32 is well the proteins (Fig. A and in binding galactose CpCBM32 in is not to with the The comprising residues in CpCBM32 by the in Fig. and the in Fig. B and the residues and which appear key in binding the of (and this that is not the primary for these CBMs. This galactose is well among other CBM32s having amino acid sequence identity as as (Fig. enzymes specificities for galactose, and acid The of sequence in the of the that to the binding (Fig. This among these reasonably galactose is the primary but that on the galactose may be by the CBMs. This could the specificities of the enzymes to which these CBM32 modules are However, CBM32 preference the enzyme specificity is the specificity of the CpCBM32 from CpGH84C for terminal LacNAc not the specificity of which is an (15Ficko-Blean E. Boraston A.B. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 834-836Crossref PubMed Scopus (20) Google Scholar, Taylor Davies G.J. Struct. Mol. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). amino acid sequence identity a the of family 32 CBMs in C. perfringens and other However, at these of sequence identity the of functional involved in galactose is and amino to structure This that the specificity of family 32 CBMs is not on of the bacterial enzymes containing family 32 CBMs to a of glycoside hydrolases by the bacterial gut from of Clostridium and The role of these enzymes is likely in from dietary or By analogy to plant cell wall hydrolases, the role of the CBM32s in these enzymes is to the enzyme to a which in the case of CpGH84C is likely the terminal LacNAc common to the of This an of the enzyme to a the CBM the catalytic module to on that the of enzymes that family 32 carbohydrate binding with plant cell wall hydrolases and their CBMs, that the of catalytic specificities is by the of CBM32 Thus, CBM family 32 may be a to be for carbohydrate-binding proteins with a of specificity

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