João P Ribeiro

William Pau

Carlo Pifferi

Olivier Renaudet

Annabelle Varrot

Lara K Mahal
email: lkmahal@nyu.edu

CERMAV Anne Imberty
email: imberty@cermav.cnrs.fr

Characterization of a high-affinity sialic acid specific CBM40 from Clostridium perfringens and engineering of divalent form

Keywords: Carbohydrate binding domain, Clostridium perfringens, sialic acid, protein-glycan interactions, xray structure

Carbohydrate-binding modules (CBMs) are a class of polypeptides usually associated with carbohydrate-active enzymatic sites. We have characterized a new member of the family CBM40, coded from a section of the gene NanI from Clostridium perfringens. Glycan arrays revealed its preference towards α(2,3)linked sialosides, that was confirmed and quantified by calorimetric studies. The CBM40 binds to α(2,3)sialyllactose with a Kd ~ 30 μM, a leading affinity value for this class of proteins. Inspired by lectins structure and their arrangement as multimeric proteins, we have engineered a dimeric form of the CBM, and using SPR we have observed 6-11 fold binding increases due to the avidity affect. The structures of the CBM, resolved by X-ray crystallography, in complex with α(2,3)-or α(2,6)-sialyllactose explain its binding specificity and unusually strong binding.

INTRODUCTION

Sialic acids are the most abundant terminal carbohydrate residues present on glycoconjugates at the surface of eukaryotic cells [START_REF] Varki | Biological roles of oligosaccharides: all of the theories are correct[END_REF][START_REF] Traving | Structure, function and metabolism of sialic acids[END_REF]. The Nacetylneuraminic acid (Neu5Ac) monosaccharide is the most common form of sialic acid on mammalian cells. Sialylation is required for the structural stabilization of cells [START_REF] Varki | Essentials of Glycobiology[END_REF] and is especially important in early mammalian development [START_REF] Schnaar | Sialic Acids in the Brain: Gangliosides and Polysialic Acid in Nervous System Development, Stability, Disease, and Regeneration[END_REF][START_REF] Wang | The role and potential of sialic acid in human nutrition[END_REF]. In cancer, altered surface sialylation is a common hallmark of the malignant phenotype of tumor cells [START_REF] Wang | Altered Glycosylation in Cancer: Sialic Acids and Sialyltransferases[END_REF]. The analysis of total serum and lipid bound Neu5Ac is valuable in cancer staging, prognosis and progression, and elevated levels have been associated with cancer development [START_REF] Pearce | Sialic acids in cancer biology and immunity[END_REF][START_REF] Dall'olio | β-galactoside α2,6 sialyltransferase in human colon cancer: contribution of multiple transcripts to regulation of enzyme activity and reactivity with sambucus nigra agglutinin[END_REF]. Increase of the negative charges associated with Neu5Ac also contributes to the cell mobility by helping dispersion from the main tumor, influencing the metastatic potential of those cells [START_REF] Schultz | Regulation of the metastatic cell phenotype by sialylated glycans[END_REF].

Due to the importance of Neu5Ac, specific receptors for this epitope are of interest as biomarkers. Currently, only a handful of Neu5Ac binders are commercially available [START_REF] Ito | Enrichment strategies for glycopeptides[END_REF]. This includes proteins purified from plants, as the lectins from wheat germ (WGA), Maackia amurensis (MAA) and Sambucus nigra (SNA-I) [11,[START_REF] Nicholls | Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses[END_REF], from the mushroom Polyporus squamosus (PSL-I) [13,[START_REF] Kadirvelraj | Structure and binding analysis of Polyporus squamosus lectin in complex with the Neu5Ac(alpha)2-6Gal(beta)1-4GlcNAc human-type influenza receptor[END_REF] or from invertebrates such as Limax flavus (LFA) [START_REF] Knibbs | Binding determinants of the sialic acid-specific lectin from the slug Limax flavus[END_REF]. While SNA-I and PSL-I are specific for α2,6linked Neu5Ac (namely terminal 6'sialyllactosamine, 6'-SLN), the other lectins have a broader range of interactions, for example displaying cross reactivities with N-acetylglucosamine -GlcNAc -(WGA, LFA) or with 3'sulfolactose (MAA). As a consequence, the analysis of complex samples requires the use of several of these lectins. Additionally, these commercial lectins come with restrictions that arise from seasonal depletion of the natural sources, undesired protein glycosylation or batch inconsistency. Recombinant proteins have proven to be a valid and effective alternative, amending for the aforementioned complications [START_REF] Hsu | A simple strategy for the creation of a recombinant lectin microarray[END_REF].

Sialidases, a group of enzymes that cleave sialosides, are secreted by microbial organisms and mammalian cells. These include gut bacteria, either commensals or pathogens [START_REF] Lewis | Host sialoglycans and bacterial sialidases: a mucosal perspective[END_REF][START_REF] Juge | Sialidases from gut bacteria: a mini-review[END_REF], viruses such as influenza [START_REF] Matrosovich | Sialic Acid Receptors of Viruses[END_REF], and human cells [20]. The catalytic domain of these enzymes is often linked to one or more carbohydrate binding modules (CBMs) that reversibly binds the glycan substrate to increase its local concentration, consequently improving the catalytic efficiency of the enzyme [START_REF] Bourne | Glycoside hydrolases and glycosyltransferases: families and functional modules[END_REF]. CBMs are small sized, monovalent domains that can generally be easily expressed in recombinant systems [22] and may provide an interesting alternative to lectins. Developed by pathogens for binding and invading human tissues, these receptors could provide specific Neu5Ac binders. In addition, CBMs can be easily produced, and their low affinity binding can be compensated through avidity by fusing multiple CBMs together [START_REF] Connaris | Enhancing the receptor affinity of the sialic acidbinding domain of Vibrio cholerae sialidase through multivalency[END_REF].

CBMs are organized into 73 families based upon their amino acid sequence similarity (in http://www.cazy.org) [24]. Most interact with plant cell wall glycans, but five CBM families have been categorized as mammalian glycan binding domains: CBM32, CBM40, CBM47, CBM51 and CBM57. Within these, the members of CBM40 are known to bind to Neu5Ac, what was first verified with the CBM located at the N-terminus of GH33 sialidase from Vibrio cholerae [START_REF] Moustafa | Sialic acid recognition by Vibrio cholerae neuraminidase[END_REF]. Presently, there are only six structurally characterized CBM40s which are associated with the sialidases from Clostridium perfringens (Cp) NanJ [START_REF] Boraston | Carbohydrate recognition by a large sialidase toxin from Clostridium perfringens[END_REF], V. cholerae (Vc) NanH [START_REF] Connaris | Enhancing the receptor affinity of the sialic acidbinding domain of Vibrio cholerae sialidase through multivalency[END_REF], Macrobdella decora (Md) NanL [START_REF] Luo | The crystal structure of an intramolecular trans-sialidase with a NeuAc alpha2-->3Gal specificity[END_REF] and Streptococcus pneumoniae (Sp) NanA [START_REF] Yang | Structural characterization of the carbohydrate-binding module of NanA sialidase, a pneumococcal virulence factor[END_REF], NanB [29] and NanC [START_REF] Owen | Streptococcus pneumoniae NanC: STRUCTURAL INSIGHTS INTO THE SPECIFICITY AND MECHANISM OF A SIALIDASE THAT PRODUCES A SIALIDASE INHIBITOR[END_REF]. They all share a conserved β-sandwich fold consisting of two antiparallel β-sheets, with the exception of Vc NanH´s that shows a different architecture, which led researchers to suggest its integration into a new CBM40 subfamily [START_REF] Boraston | Carbohydrate recognition by a large sialidase toxin from Clostridium perfringens[END_REF].

We herein report the successful characterization of the CBM40 from C. perfringens (ATCC 13124) which is associated with NanI [START_REF] Myers | Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens[END_REF], for which only the catalytic domain has been structurally characterized [START_REF] Newstead | Crystallization and atomic resolution X-ray diffraction of the catalytic domain of the large sialidase, nanI, from Clostridium perfringens[END_REF]. The recombinant CBM40_NanI demonstrates unusual micromolar affinity towards sialylated oligosaccharides. The structural analysis of the complexes with 3'sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) provides insights into the molecular details responsible for this affinity. A pseudo-dimeric version of the CBM40 was also engineered as to enhance the affinity from multiple interactions between the CBM and glycans. This work presents a new probe towards sialoconjugates, expanding the repertoire of sialoside binders.

EXPERIMENTAL

Materials

Genomic DNA of C. perfringens ATCC® 13124 TM was bought from ATCC (#13124D-5 TM ). 3'-Sialyllactose sodium salt (CAS #128596-80-5) and 6'-sialyllactose (#35890-38-1) were purchased from Carbosynth. 3'-sialyl-N-acetyllactosamine (#81693-22-3) and 6'-sialyl-N-acetyllactosamine (#174757-71-2) were bought from Dextra UK and sialic acid (#131-48-6) from AppliChem. Biotinylated 3'sialyllactose (#0060-BM) and biotinylated 6'sialyllactose (#0063-BM) were obtained from Lectinity.

Propargyl sialic acid synthesis

All chemical reagents were purchased from Aldrich (Saint Quentin Fallavier, France) or Acros (Noisy-Le-Grand, France).

Moisture-sensitive reactions were performed under argon atmosphere by using oven-dried glassware and reactions were monitored by TLC using silica gel 60 F254 pre-coated plates (Merck). Spots were inspected by UV light and visualised by charring with 10% H2SO4 in EtOH. Silica gel 60 (0.063-0.2 mm or 70-230 mesh, Merck) was used for column chromatography. 1 H and 13 C NMR spectra were recorded on Bruker Avance 400 MHz or Bruker Avance III 500 MHz spectrometers and chemical shifts (δ) were reported in parts per million (ppm). Spectra were referenced to the residual proton solvent peaks relative to the signal of CDCl3 (δ 7.27 and 77.0 ppm for 1 H and 13 C) and D2O (4.79 ppm for 1 H), assignments were done by gCOSY and gHMQC experiments.

A route for the synthesis of the propargyl sialic acid (2-propynyl 5-acetamido-3,5-dideoxy-Dα-D-galacto-2-nonulopyranosidonic acid) starting from N-acetylneuraminic acid is shown in scheme 1. All the reaction conditions, mass and NMR spectra and their assignments are in supplementary information.

Scheme 1

Gene cloning

The following oligonucleotide primers were used: 5'-GAC GAC GGA TCC CAT GTT AAG TTC ACT AGG AGA ATA TAA GGA TAT-3' (45-mer) and 5'-TCC CAT CTC GAG TTA TTT TGT CTC TCC AGT CTT ACT AAG TAA ATA ATC ATC-3' (51-mer) for the construction of pNanI. They were designed as to include BamHI and XhoI restriction sites (underlined sequences) in the CBM40_NanI gene sequence. PCRs were done using the Q5 Hot Start High Fidelity polymerase (New England Biolabs) and genomic DNA from Clostridium perfringens strain ATCC® 13124 TM as a template. The same cloning protocol was followed for pNanJ, using the following oligonucleotide primers 5'-AC TAC GGA TCC AGG GTA AAT ATA ACA GGT GAT T -3' (33-mer) and 5'-G CAT CTC GAG TTT AGT TTC TCC TGT TTT TCT TA-3' (36-mer) which contain SacI and XhoI restrictions sites respectively. To engineer a CBM40_NanI with a multivalent presentation, we

Protein Expression and Purification

E. coli BL21(DE3) cells transformed with plasmids pNanI, pNanJ or p2NanI were cultured in TB medium at 37 °C. When reaching an OD600 of 0.5 -0.7, the cultures were moved to a 16 °C incubator and IPTG was added to a final concentration of 1mM. After overnight incubation, cells were pelleted down, washed and resuspended in cold PBS pH 7.4. The cells were lysed by sonication on ice three times at 20% intensity with 0.5 cycle for 2 minutes or with a Cell Disruption System (Constant Systems Ltd) at 1900 bars. After 30 min centrifugation at 4 °C and 50000 g, the supernatants were submitted to nickel affinity chromatography. After being allowed to bind the matrix (Ni-NTA agarose, Qiagen), the proteins were washed (50 mM NaH2PO4.H2O pH 8.0, 300 mM NaCl and 10 mM imidazole) and eluted (using the same buffer with an increased 250 mM imidazole concentration). The eluted fractions were filtered, checked for absorbance at 280 nm and concentrated by centrifugation using Vivaspin (Startorius) with a 10 kDa cut-off filter, before being further purified by size-exclusion chromatography. A Superdex 75 prep grade column was equilibrated with PBS pH 7.4 and used for the estimation of aggregate states and molecular weights of the purified proteins. A calibration curve for molecular size estimation -done with cytochrome c, myoglobin, ovalbumin and BSA, previously to the experiments -confirmed the expected molecular weight values. Purified proteins were run in denaturing protein gels in a 12% (w/v) acrylamide matrix in alkaline buffer conditions (Tris.HCl, pH 8.8).

Protein estimation

Protein concentrations were determined with a Nanodrop spectrometer ND2000 (Ozyme) with absorbance readings at 280 nm wavelengths, using molar extinction coefficient values of 11.92, 21.89, and 32.78 (x10 3 M -1 cm -1 ) for Cp CBM40_NanJ, Cp CBM40_NanI and Cp di-CBM40_NanI, respectively, as well as their corresponding molecular weights of 23.69, 24.62 and 46.55 kDa (values for calculations that include the N-terminal tagged amino acids).

Thermostability

To examine their stability, protein solutions of Cp CBM40_NanI, Cp CBM40_NanJ and Cp di-CBM40_NanI were assayed using a Thermal Shift Assay (TSA), adapting a protocol previously described [START_REF] Dupeux | A thermal stability assay can help to estimate the crystallization likelihood of biological samples[END_REF]. Samples were diluted to a final protein concentration of 0.1 mg mL -1 in the chosen buffer and 5× SYPRO Orange (Molecular Probes, CA) was added to a final volume of 25 μl. The samples were subjected to thermal denaturation using a Real Time PCR machine (Mini Opticon, Bio-Rad Laboratories) with a temperature gradient starting at 25 °C and ascending to 100 °C at a heating rate of 1 °C/min. Protein unfolding was followed by the increase of the fluorescence values given by the SYPRO Orange probe. Different buffer effects were analyzed in a pH range of 3-10.

Crystallization and data collection

Solutions of CBM40_NanI (5.5 mg mL -1 ) in PBS where sent to the High Throughput Crystallization laboratory (HTXlab) in Grenoble, France, to screen for crystallization conditions. The protein/ligand mixture was tested against the JCSG (Qiagen), Wizard I and II (Rigaku reagents), PACT (Qiagen), plate 1 (Qiagen), plates 4 and 5 (Hampton) screens. Hits were obtained with the use of the Clear Strategy TM Screens, (Molecular Dimensions Ltd) at pH 4.5. Crystals of CBM40_NanI with 3'-SL appeared in the form of plates after several days in CSSI-12 containing 0.1M sodium acetate pH 4.5, 0.2M calcium acetate and 8% of both polyethylene glycol (PEG) 550 monomethyl ether (MME) and PEG 20000. In the same time period, parallelepiped crystals of CBM40_NanI in complex with 6'-SL grew in 0.1M sodium acetate pH 4.5, 0.3M sodium acetate and 25% PEG 2000 MME. Afterwards, larger crystals were then grown with homemade solutions. All the diffraction experiment data sets were collected at the European Synchrotron Radiation Facility (ESRF). Final data for CBM/3'-SL was collected to 1.9 Å on BM30A beamline using an ADSC Q315r detector whilst the CBM/6'-SL complex was obtained at 2.0 Å on the ID29 beamline using a Pilatus detector. Diffraction images were integrated using program XDS [START_REF] Leslie | Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography[END_REF][START_REF] Kabsch | Xds[END_REF] and all further processing was done using the CCP4 program suite [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF].

Structure solution and refinement

Coordinates of molecule A of the CBM40 from C. perfringens ATCC® 13124 TM NanJ (PDB ID 2V73) were used as model for molecular replacement with PHASER [START_REF] Mccoy | Phaser crystallographic software[END_REF] in a search for six monomers per asymmetric units for the 6'-SL complex. The coordinates of molecule A of the CBM40/3'-SL were used to search for the three copies of the CBM40/6'-SL complex. Five percent of the observations were set aside for cross-validation analysis, and hydrogen atoms were added in their riding positions and used for geometry and structure-factor calculations. The initial model was optimized using ARP/wARP [START_REF] Langer | Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7[END_REF] prior restrained maximum likelihood refinement with REFMAC 5.8 [START_REF] Murshudov | REFMAC5 for the refinement of macromolecular crystal structures[END_REF] iterated with manual rebuilding in Coot [START_REF] Emsley | Features and development of Coot[END_REF]. Incorporation of the ligand was performed after inspection of the 2Fo-DFc weighted maps. Water molecules, introduced automatically using Coot, were inspected manually. The quality of the models was assessed using the PDB validation server (http://wwpdbvalidation.wwpdb.org/validservice/) and coordinates were deposited in the Protein Data Bank under codes 5FRE and 5FRA.

Hemagglutination and minimal concentration inhibition assays

The hemagglutination activity for the Cp CBM40_NanI and Cp di-CBM40_NanI was determined using 2-fold serial dilutions of the protein with rabbit erythrocytes (Biomérieux). In those tests, 25 µL of the serial dilutions of the protein were incubated with 25 µL of 2% rabbit erythrocytes suspensions in 100 mM NaCl for 30 min at 37 °C plus 30 min at room temperature. After identifying the highest dilution of the protein that exhibited hemagglutination, a concentration 4 times higher was used in the subsequent minimal inhibition concentration (MIC) experiments. Serially dilutions of 12.5 µL of different saccharide solutions were added to the 12.5 µL solutions of protein of identical concentrations. These 25 µL mixtures were incubated with 25 µL of 2% rabbit erythrocytes suspensions, following the same development steps as above.

Glycan array

Cp CBM40_NanI and Cp di-CBM40_NanI were labeled with Alexa Fluor 488 (Invitrogen) using manufacturer's protocols and sent to the Consortium of Functional Glycomics (CFG). Binding specificity was determined using the printed mammalian glycan array v5.2, which contains 609 glycans [START_REF] Wang | Cross-platform comparison of glycan microarray formats[END_REF]. The scanner response is linear to a maximum of ~50,000 RFU. Both proteins were incubated with the arrays at 200 µg mL -1 . The extracted data was then transformed to give Z scores for each glycan on the array (Z= x-mean/st dev). A threshold of Z=1.645 (p≤0.05) was used to establish significance.

Isothermal Titration Calorimetry

ITC experiments were performed using a Microcal ITC200 microcalorimeter (Malvern Instruments Ltd). All titrations were carried out in PBS pH 7.4, at 25 °C. Aliquots of 10 µL of each carbohydrate, dissolved in the same buffer were added to the protein solution present in the calorimeter cell, in 2 min intervals. For the different experiments, protein concentrations were prepared in solution with concentrations that varied from 100 to 415 µM, and the carbohydrates samples ranged from 2.1 to 6.0 mM. Data was fitted with MicroCal Origin 7 software, following standard procedures. This yielded the stoichiometry (N), the association constant (K a) and enthalpy of binding (ΔH). Other thermodynamic parameters (changes in free energy, ΔG, and entropy, ΔS, were calculated from the equation ΔG = ΔH-TΔS = -RTln Ka, being R = 8.314 J/mol/K.

Surface Plasmon Resonance

SPR experiments were performed using a Biacore X100 biosensor instrument (GE Healthcare) at 25°C. Biotinylated 3'-SL and 6'-SL were immobilized on CM5 chips (GE Healthcare) that were previously coated with streptavidin, following the same protocol as previously described [START_REF] Lameignere | Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia[END_REF]. Each monovalent biotinylated sugar, 3'-SL or 6'-SL, was diluted to 1 µg mL -1 in HEPES Buffer with 0.05% Tween 20 (HBS-T) before being injected in one of the flow cells of the chip. Low immobilization levels of 94 and 64 response units were obtained for 3'-SL and for 6'-SL, respectively. A reference surface was always present in flow cell 1, thus allowing for the subtraction of bulk effects and non-specific interactions with streptavidin. The running buffer consisted of the same HBS-T pH 7.4. The purified proteins were injected over the flow cell surface at 30 µL min -1 in series of 2-fold dilutions. The dissociation of this analyte was done by passing running buffer during 4 -6 min. Surfaces were regenerated with one or two consecutive 30 second injections of 100 mM Neu5Ac, also at 30 µL min -1 . The information on the affinity was determined by assuming Langmuir 1:1 binding, using the BIAevaluation software.

RESULTS AND DISCUSSION

Sequences identified as belonging to the family 40 CBM were selected from the CAZY (Carbohydrate-Active enZYmes) database [24] and used to build a phylogenetic tree (http://phylogeny.lirmm.fr). The topology of the tree (see supplementary information, SI 2), shows the CBM40s cluster into one main group with few outliers. The CBM from Cp NanI clusters with the other CBM from the same organism, Cp CBM40_NanJ, with a phylogenetic similarity of 0.942. As previously noted by other authors, the CBM of Vc NanH is more distantly related to the other CBM40s, among those with validated Neu5Ac specificity and available structural information [START_REF] Boraston | Carbohydrate recognition by a large sialidase toxin from Clostridium perfringens[END_REF]. The BLAST protein alignment between the Cp CBM40_NanI sequence and the other structurally characterized CBM40s -Cp NanJ (PDB ID: 2V73), Sp NanA (4C1W), NanB (2VW0) and NanC (4YZ5), and Md nanL (1SLI) -shows that CBM40_NanI shares 56% identity with the CBM40_NanJ from the same organism (C. perfringens), 23%, 30% and 27% homology values with the S. pneumoniae proteins, and 33% with the CBM of Macrobdella decora leech. Cp CBM40 NanI appears therefore sufficiently different from previously characterized CBMs of family 40 to substantiate a full characterization.

After cloning of genomic DNA of C. perfringens ATCC® 13124 TM , recombinant Cp CBM40_NanI was purified with a yield of approximately 10 mg L -1 . The Cp CBM40_NanJ was produced in the same conditions for comparison. The expressed proteins appear as one strong band on SDS-PAGE gels, with apparent molecular weights close to 25 kDa marker, in agreement with the theoretical values of 23.7 and 24.6 kDa for Cp CBM40_NanJ and Cp CBM40_NanI, respectively. The proteins were separated from aggregates by gel exclusion chromatography and collected as a single peak with a retention time representative of the monomers (see SI 3). The thermal stability of the Cp CBM40_NanI and Cp CBM40_NanJ was assessed by Thermal Shift assays (TSA). The highest stability was obtained in PBS buffer at pH 7, with melting temperatures of 54° C for Cp CBM40_NanJ and 65° C for Cp CBM40_NanI. One issue with the use of CBMs as glycan probes is their low affinities. One method to overcome this is to engineer constructs containing multiple CBM domains [START_REF] Connaris | Enhancing the receptor affinity of the sialic acidbinding domain of Vibrio cholerae sialidase through multivalency[END_REF]. Thus, a divalent version of the protein was engineered. The pseudo-dimeric Cp CBM40_NanI has a 12 amino acid-long linker (ALNGSELGSGSG) inserted between the two copies to provide flexibility between the binding modules. Under identical expression protocol, a yield of ~ 5 mg L -1 purified di-CBM40_NanI was obtained. The expected band for a 46 kDa weight protein was seen in the SDS-PAGE gel (SI 3). The engineered Cp di-CBM40_NanI was stable in PBS buffer pH 7.0, with an observed Tm of 66 °C (SI 4).

Characterization of binding specificity demonstrates strong preference for 3-linked sialic acids

An initial evaluation of glycan binding was performed by inhibition of hemagglutination using rabbit erythrocytes. As expected, hemagglutinating activity was not observed with monomeric Cp CBM40_NanI as agglutination requires a multivalent binding to crosslink the cells. In contrast, Cp di-CBM40_NanI showed positive hemagglutination down to a 50 µg mL -1 concentration. Among the glycans tested as HA inhibitors, 3'-SL was the strongest inhibitor, with minimal inhibitory concentration (MIC) of 0.78 mM. 6'-SL was also able to inhibit hemagglutination with a MIC value of 1.56 mM. Lactose, galactose, glucose, mannose and fucose (tested up to 50 mM) had no inhibitory effect (see SI 5).

To gain further insight into the binding motifs of Cp CBM40_NanI, both monomeric and dimeric CBMs were tested on the Consortium for Functional Glycomics' glycan microarray v5.2. This array contains a panel of 609 mammalian, including α2,3-, α2,6-and α2,8-sialosides [START_REF] Blixt | Printed covalent glycan array for ligand profiling of diverse glycan binding proteins[END_REF]. Monomeric Cp CBM40_NanI gave only a very weak signal with clear binding to only a few epitopes bearing α2,3sialic acid on a type II N-acetyllactosaminyl core (Neu5Acα2,3Galβ1,4GlcNAc) (SI 6). Previous assays involving monomeric Cp CBM40_NanJ were also unsuccessful due to low affinity [START_REF] Boraston | Carbohydrate recognition by a large sialidase toxin from Clostridium perfringens[END_REF]. The signal-tonoise ratio was considerably improved when assaying the engineered dimeric protein. The Cp di-CBM40_NanI screen confirms the specificity of this binding domain towards α2,3-sialosides (Figure 1). Of the 27 glycans that had significant binding (p ≤ 0.05 as determined by Z-score analysis) on the array, 24 contained an α2,3-sialoside. The remaining 3 were an anomalous chitin epitope, sialic acid bound directly to a linker and a single biantennary α2,6-epitope detected. Both Nglycolyl-and 9-O acetyl-containing α2,3-sialosides acids were tolerated. Binding was enhanced by an additional nearby negative charge (sulfation or sialylation).

Cp CBM40_NanI displays unusually strong affinity for sialylated oligosaccharides

The glycan-binding activities of Cp CBM40_NanI and di-CBM40_NanI were confirmed by isothermal titration calorimetry at 25°C. All interactions showed exothermic behaviors with a steep decrease in the exothermic heat of binding until saturation is achieved (see Figure 2). The stoichiometry values (N) for sialylated compounds titrated in Cp CBM40_NanI and di-CBM40_NanI are close to 1 and 2 respectively, in agreement with the expected presence of one or two binding sites (Table 1). Both proteins have similar behavior with strong affinity for 3'-SL (Kd value ~30 μM) and weaker affinity for 6'-SL (Kd ~ 60 μM). Values in the same data range were obtained for 3'-and 6'sialyllactosamine (SI 7). Free Neu5Ac monosaccharide could not be assayed directly due to pH variations during the titration. To assay the monosaccharide, a derivative of Neu5Ac with a propargyl aglycon (SiaOPr) was synthesized and assayed for binding. The CBMs showed weaker binding (Kd values of 170 and 189 μM for monomeric and dimeric CBM, respectively) to the monosaccharide conjugate than to the sialylated trisaccharides. To directly compare binding affinities, ITC experiments were also performed for Cp CBM40_NanJ. As expected, lower binding affinities were obtained with Kd values of 1.48 and 2.67 mM for 3'-SL and 6'-SL, respectively (SI 7), confirming the previously published estimations of low affinity [START_REF] Boraston | Carbohydrate recognition by a large sialidase toxin from Clostridium perfringens[END_REF].

Protein

Ligand (µM)

Kd (µM) Cp CBM40_NanI 3'-SL 14.4 Cp CBM40_NanI 6'-SL 36.0 Cp di-CBM40_NanI 3'-SL 1.3 Cp di-CBM40_NanI 6'-SL 6.1
The binding interactions were also analyzed by Surface Plasmon Resonance spectroscopy. Commercial available monomeric biotinylated 3'-SL and 6'-SL were immobilized on a streptavidin coated chip to obtain high-density coverage. The sensorgrams show a typical shape for fast association and dissociation events after the injection of increasing concentrations of Cp CBM40_NanI or Cp di-CBM40_NanI. The binding kinetics of the experiments were too fast to be reliably measured by SPR and steady-state analysis was performed to evaluate the dissociation constants (see SI 8). The data confirmed the stronger binding of Cp CBM40_NanI and Cp di-CBM40_NanI with the 3'-SL-versus the 6'-SLfunctionalized surface. The monomeric CBM showed a Kd of 14.4 µM for 3'-SL, whereas 6'-SL appears to bind at a 2.5 times lower affinity, with a dissociation value of 36 µM, in agreement with ITC data (Table 2). For the Cp di-CBM40, an increase of more than one order of magnitude in affinity (11fold) is observed for the interaction with 3'-SL and a 6-fold for 6'-SL, resulting in affinities of 1.3 and 6.1 µM, respectively. Experiments conducted with Cp CBM40_NanJ did not yield sensorgrams of sufficient quality to evaluate, most likely due to the low affinity of Cp CBM40_NanJ-sialoside interactions.

Structural basis for specificity and affinity

To gain insight into the structural basis for sialoside binding of Cp CBM40_NanI, crystal structures were obtained. An original screening of 600 conditions, using a high throughput crystallization robot, led to isolation of thin crystals that diffracted at low resolution. Upon optimization with the Crystal Clear Strategy Screen (Molecular Dimension Ltd) bigger and thicker crystals of Cp CBM40_NanI complexed with 3'-SL and 6'-SL were obtained. The protein crystalized in P21 space group for both complexes, with 3 and 6 independent monomers in the asymmetric unit for crystals of the CBM40_NanI complexed with 3'-SL or 6'-SL, respectively (see table in SI 9). The structure of the CBM/6'-SL was solved using PDB ID 2V73 (Cp CBM40_NanJ) as a model, and subsequently used to solve the structure of the Cp CBM40_NanI/3'-SL. The monomers superimposed with root-meansquare deviation (r.m.s.d.) values between 0.12 and 0.16 Å for Cp CBM40/3'SL and between 0.15 and 0.20 Å for Cp CBM40_NanI/6'-SL.

The Cp CBM40_NanI adopts the characteristic β-sandwich fold of CBM40 members, formed by two β-sheets of five and six antiparallel β-strands and two α-helices, one within the sheets and another at the C-terminus, packed against βstrand 7. The carbohydrate recognition site is located on the concave face of the 5-stranded βsheet, in a shallow depression, in which the ring of the sialic acid lays parallel to the surface of the protein (Figure 3A). Examination of the electron density clearly allowed the identification of the sialic acid and galactose residues in all binding sites in the CBM/3'-SL complex and in half of the molecules in the 6'-SL, whilst only the Neu5Ac moiety could be defined with confidence for the others sites. The glucose moieties could not be modeled as they were too disordered.

The majority of the interactions between the protein and the ligand involve the sialic acid moiety. In both complexes (see Figures 3B and3C), Ile46, Tyr65 and His93 side chains form a hydrophobic pocket that accommodates the methyl of the Nacetyl group. Arg76 and Arg153 make electrostatic interactions with the carboxylate group. The Asn158 residue establishes hydrogen bonds with the glycerol moiety. The hydroxyl group of Tyr160 makes hydrogen bonds to the carboxylate group and the nitrogen of the N-acetyl group. Glu74 is involved in two hydrogen bonds with the nitrogen of the N-acetyl group and the O4 hydroxyl and the NH1 and NH2 atoms of Arg153 make H-bonds with the carboxylate group. The carboxylate makes water mediated interactions with the main chain nitrogen of Asn158 and the NH2 atom of Arg76. Comparison between the binding sites of Cp CBM40_NanI and NanJ show that the amino acids making direct contacts with the ligand are conserved in both structures (see SI 10). perfringens NanI with structurally related structures. Cp NanI (in red, boxed), Cp NanJ (in cyan), Sp NanA (in yellow), Sp NanB (in green), Sp NanC (in brown) and Md NanL (in purple). The loop 5-6 in CBM40_NanI is identified with an arrow; E) Multiple amino acid sequence alignment between CBM40_NanI (shown as NanI) and the CBM40s from C. perfringens ATCC® 13124 TM 's NanJ, S. pneumoniae's NanA, NanB and NanC, and Macrobdella decora NanL. The secondary structure regions of the CBM40_NanI are identified on top. Shaded residues show the conservation between the proteins. The CBM40_NanI amino acids making key contacts with the sialic acid are identified with blue triangles. The region between β-strands 5 and 6 is boxed within red limits and residue Gln85 is marked with a red star due to their particular importance as part of the binding domain. The sequence of V. cholerae NanH is not shown due to its poor alignment (see SI 8). The figure was created with Espript 3.0 [START_REF] Robert | Deciphering key features in protein structures with the new ENDscript server[END_REF].

In the complex with 3'SL, the galactose does not interact directly with the protein but is involved in a dense network of water-bridged hydrogen bonds. Six water molecules are conserved in the three independent monomers where three of them are directly involved in stabilizing the galactose residue (SI 11). These three water molecules are responsible for locking the conformation of the galactosyl moiety and creating a further network with other waters, bridging both inter-amino acid and intra-ligand interactions. Gln85 is a key amino acid in this network, interacting with two water molecules which bridge with the O4 hydroxyl of the galactosyl moiety. Lys58 NH makes another water mediated H-bond with the OH6 group of the galactose. Also the Ser157 interacts with a chain of water molecules connecting to the carboxylate of the NeuAc and the O4 hydroxyl of the galactose.

In the CBM/6'-SL complex, two of the three resolved 6'-SL molecules have the same conformation whereas the third shows different dihedrals angles (SI 12). This is an effect of the highest solvent exposure and absence of contacts between the galactose ring of the ligand and the protein. It can be seen in figures 3B-C that the galactose ring is oriented outside the recognition domain of the protein.

Comparison with the other CBM40 proteins

The overall macromolecular architecture of the Cp CBM40_NanI is well conserved when comparing to other members of CBM40 family apart NanH which belongs to another branch of the phylogenetic tree as discussed above. Sequence alignment show that the recognition domains of the CBM40s share common key residues that provide the protein its binding specificity (figure 3E and SI 13). Within these binding domains, residues Glu74 and Arg153 (Cp CBM40_NanI numeration) are particularly important, being spatially conserved in all CBMs (with the exception of Md NanL, which has an aspartic instead of glutamic acid), making a total of four H-bonds with the Neu5Ac (two with the N-acetyl and O4 hydroxyl, and two with the carboxylate group, respectively). A highly variable region between all of the CBM40s is the loop between β-strands 5 and 6 (Figure 3D and E, see starred box). In Cp CBM40_NanI, this loop is longer by 6-8 amino acids and has a unique spatial arrangement compared to the other CBM40 members. In fact, this is a major point of difference between C. perfringens CBM40_NanI and CBM40_NanJ, which share considerable sequence identity and bind sialic acid in virtually the same position on their protein surfaces. Despite this, Cp CBM40_NanI displays a much higher affinity to sialylated oligosaccharides (~ 100-fold) than its counterpart. This difference in affinity appears to be due to an increased water mediated H-bond setup inside the binding site of Cp CBM40_NanI.

As seen in the crystal structures, the loop between β-strands 5 and 6, exclusive to Cp CBM40_NanI, introduces an additional surface in the recognition domain. In this loop, residue Gln85 creates water mediated interactions with both the sialyl carboxylate and galactosyl hydroxyl groups. In contrast to NanI, the crystal structure of Cp CBM40_NanJ does not show such water-mediated H-bond network (SI 10).

Sialylated oligosaccharides are flexible epitopes, due to the possibility of therefore conformations at the α2-3 and α2-6 linkages [START_REF] Imberty | Structure, Conformation, and Dynamics of Bioactive Oligosaccharides: Theoretical Approaches and Experimental Validations[END_REF]. It is therefore of interest to compare if different CBMs recognized the same epitope. Sp CBM40_NanA and NanC were also successfully cocrystalized with the 3'-SL and 6'-SL ligands (PDB ID: 4C1W and 4C1X, and 4YZ5 and 4YW2, respectively), among others [START_REF] Yang | Structural characterization of the carbohydrate-binding module of NanA sialidase, a pneumococcal virulence factor[END_REF][START_REF] Owen | Streptococcus pneumoniae NanC: STRUCTURAL INSIGHTS INTO THE SPECIFICITY AND MECHANISM OF A SIALIDASE THAT PRODUCES A SIALIDASE INHIBITOR[END_REF]. Probably due to the exposure of the ligands and the few contacts made with the protein, different conformations are seen for the 3'-SL and 6'-SL when bound Cp CBM40_NanI, Sp CBM40_NanA and Sp CBM40_NanC. 3'-SL shares similar conformations in NanI and NanA (in NanC the galactose ring is in a full inverted position relative to the others, whereas 6'-SL structure is alike in NanI and NanC (see SI 14).

CONCLUSION

Although the use of CBMs in biotechnology is fairly recent, they have been described in the paper, textile and food industries [START_REF] Oliveira | Recombinant CBM-fusion technology -Applications overview[END_REF]. Their potential application in health sciences has also been proved in different scenarios as molecular probes [START_REF] Von Schantz | Structural basis for carbohydrate-binding specificity--a comparative assessment of two engineered carbohydrate-binding modules[END_REF], in microarrays [START_REF] Ofir | Versatile protein microarray based on carbohydrate-binding modules[END_REF], as tools for the immobilization of proteins and cells [START_REF] Shoseyov | Carbohydrate binding modules: biochemical properties and novel applications[END_REF] or assisting in vitro glyco-catalysed reactions leading to enhanced yields [START_REF] Oliveira | Recombinant CBM-fusion technology -Applications overview[END_REF][START_REF] Volkov | Prospects for practical application of substrate-binding modules of glycosyl hydrolases (A review)][END_REF][START_REF] Codera | Carbohydrate-binding module assisting glycosynthase-catalysed polymerizations[END_REF]. Recent studies have shown the therapeutic potential of multivalent CBMs in the prevention of infection by the influenza virus, through the masking of sialic acid receptors [51,[START_REF] Connaris | Prevention of influenza by targeting host receptors using engineered proteins[END_REF].

This study presents a new family 40 CBM that binds specifically to NeuAc with a preference towards α2,3-sialosides. It was easily engineered and expressed in multimeric fashion by fusing consecutives copies of its gene, leading to a substantial increase of its affinity (11-fold) over the monomeric version. Although the previously described C. perfringens NanJ is highly similar to NanI, the binding affinity of NanI to sialosides acid was much stronger, making it a better candidate to develop as a glycan binding reagent. Our studies suggest that the variable loop between β-strands 5 and 6 may be responsible for this difference in affinity and may contribute to the unusual ligand specificity observed. The binding Cp CBM40_NanI towards sialosides is considerably more restricted than any other published CBM40, with a distinct preference for α2,3-sialosides, and its interactions at micromolar values are comparable only with the CBM of Vibrio cholerae's NanH, which shows a broad specificity to all sialosides [START_REF] Connaris | Enhancing the receptor affinity of the sialic acidbinding domain of Vibrio cholerae sialidase through multivalency[END_REF]. The binding of α2,3-sialosides -but not 3'-O-sulfated onesmakes this reagent more specific for sialic acid residues than the commonly used plant lectin from Maackia amuerensis MAL-1 [START_REF] Wang | Cross-platform comparison of glycan microarray formats[END_REF].

The present study unravels the molecular basis for the difference in affinity and specificity for two closely related CBM40s from the same organisms. In addition, the high affinity of Cp CBM40_NanI for 3'SL and its availability as monomer or as engineered dimer makes it an excellent tool for detection and characterization of sialylation for biotechnology use.

Figure 1 -

 1 Figure 1 -Glycan array profile of Cp di-CBM40_NanI. The Alexa Fluor 488-fused protein was tested against a panel of 609 glycans. Some examples of α(2,3)-linked sialosides are shown (more details at CFG request nº16976, supplementary information file CFG_diCBM40_NanI.xls).

Figure 2 -

 2 Figure 2 -ITC isotherms showing the binding of A) CBM40_NanI to 3'-SL and 6'-SL; B) di-CBM40_NanI to 3'-SL and 6'-SL; and C) CBM40_NanI to SiaOPr.

Figure 3 -

 3 Figure 3 -A) Structure of the CBM40 from C. perfringens ATCC® 13124 TM NanI in complex with 3'-SL and 6'-SL (at the left and right, respectively). Ligands are shown with a 2Fo -Fc density map contoured at 1.0 r.m.s.; B-C) Zoomed-in view on the binding domain occupied with 3'-SL and 6'-SL; D) Comparison of the CBM40 of C.perfringens NanI with structurally related structures. Cp NanI (in red, boxed), Cp NanJ (in cyan), Sp NanA (in yellow), Sp NanB (in green), Sp NanC (in brown) and Md NanL (in purple). The loop 5-6 in CBM40_NanI is identified with an arrow; E) Multiple amino acid sequence alignment between CBM40_NanI (shown as NanI) and the CBM40s from C. perfringens ATCC® 13124 TM 's NanJ, S. pneumoniae's NanA, NanB and NanC, and Macrobdella decora NanL. The secondary structure regions of the CBM40_NanI are identified on top. Shaded residues show the conservation between the proteins. The CBM40_NanI amino acids making key contacts with the sialic acid are identified with blue triangles. The region between β-strands 5 and 6 is boxed within red limits and residue Gln85 is marked with a red star due to their particular importance as part of the binding domain. The sequence of V. cholerae NanH is not shown due to its poor alignment (seeSI 8). The figure was created with Espript 3.0[START_REF] Robert | Deciphering key features in protein structures with the new ENDscript server[END_REF].

Table 1 -

 1 Thermodynamics of binding of Cp CBM40_NanI, Cp di-CBM40_NanI Cp CBM40_NanJ to different sialylated ligands, as determined by ITC at 25 °C.

	Protein	[P] Ligand	[L]	N	Ka	Kd	ΔH	TΔS	ΔG
		(µM)		(mM)		(x10 4 M -1 )	(µM)	(kcal/mol) (kcal/mol) (kcal/mol)
		400	3'-SL	5.0	0.86 ± 0.01	3.16 ± 0.09	32	-10.49 ± 0.14	-4.35	-6.14
		400	6'-SL	3.6	0.95 ± 0.01	1.48 ± 0.05	68	-8.69 ± 0.05	-3.01	-5.68
	Cp CBM40_NanI	50	3'-SLN	0.9	0.98 ± 0.05	2.68 ± 0.20	37	-9.38 ± 0.06	-3.34	-6.04
		50	6'-SLN	0.8	0.92 ± 0.08	2.23 ± 0.25	49	-8.81 ± 0.10	-2.88	-5.93
		100 SiaOPr	5.0	1.00*	0.59± 0.05	170	-19.95± 0.05	-14.81	-5.14
		185	3'-SL	5.0	1.94 ± 0.01	2.84 ± 0.08	35	-11.20 ± 0.01	-5.16	-6.07
	Cp di-CBM40_NanI	185	6'-SL	3.6	1.88 ± 0.01	1.71 ± 0.06	59	-9.74 ± 0.01	-3.96	-5.78
		50 SiaOPr	5.0	2.00*	0.51 ± 0.06	196	n/d	n/d	n/d
						(x10 3 M -1 )	(mM)
	Cp CBM40_NanJ	100	3'-SL 10.0	1.00*	0.67 ± 0.04	1.48	-1.51 ± 0.05	2.35	-3.86
		100	6'-SL 10.0	1.00*	0.44 ± 0.06	2.23	-2.72 ± 0.27	0.89	-3.61

Table 2 -

 2 Binding affinities of Cp CBM40_NanI and Cp di-CBM40_NanI to immobilized 3'-SL and 6'-SL receptors, asdetermined by SPR at 25 °C.

Acknowledgements

The research leading to these results has received funding from the European Community's 7th Framework Programme (FP7/2007-2013) under the Marie Curie International Outgoing Fellowship for Career Development (PIOF-GA-2011-298910), BioStruct-X (grant agreement N°283570), and ERASynbio program SynGlycTis (ANR-14-SYNB-0002-02) grants. The authors are also grateful to the Labex Arcane for the financial support (ANR-11-LABX-0003-01).

We also thank the participation of the Protein-glycan Interaction Resource of the CFG and the supporting grant R24 GM098791.

Crystal data collection was performed at the European Synchrotron Radiation Facility, Grenoble, France and we are thankful for the access to beamlines BM30A-FIP and ID29 as well as for the technical support of David Cobessi on BM30A.

Conflict of Interest:

The authors declare that they have no conflicts of interest with the contents of the publication.

Author Contributions:

LKM conceived the project. JPR, LKM and AI coordinated the paper. JPR and WP performed cloning, protein expressions and purifications. JPR ran all the biophysical experiments and provided data analysis. JPR and AV resolved the crystal structures. CP and OR synthesized and characterized the SiaOPr. JPR analyzed, generated the figures and tables and wrote the body of the paper. AV, LKM and AI revised the paper leading to the final version. All authors reviewed the results and approved the manuscript for submission.