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

Article abstract of DOI:10.1042/BCJ20160340

CBMs(carbohydrate-binding modules) are a class of polypeptides usually associated with carbohydrate-active enzymatic sites. We have characterized a new member of the CBM40 family, coded from a section of the gene NanI from Clostridium perfringens. Glycan arrays revealed its preference towards α(2,3)-linked sialosides, which was confirmed and quantified by calorimetric studies. The CBM40 binds to α(2,3)-sialyl-lactose with a Kd of -30 μM, the highest 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 (surface plasmon resonance) 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)-sialyl-lactose explain its binding specificity and unusually strong binding.

Full text of DOI:10.1042/BCJ20160340

Biochem. J. (2016) 473, 2109–2118 doi:10.1042/BCJ20160340
2109
Characterization of a high-affinity sialic acid-specific CBM40 from
Clostridium perfringens and engineering of a divalent form
1
1
Jo a˜ o P. Ribeiro*†, William Pau*, Carlo Pifferi‡, Olivier Renaudet‡, Annabelle Varrot†, Lara K. Mahal* and Anne Imberty†
*Biomedical Chemistry Institute, New York University Department of Chemistry, 100 Washington Square East, Room 1001, New York, NY 10003, U.S.A.
†
CERMAV, UPR5301, CNRS and Universit e´ Grenoble Alpes, 601 rue de la Chimie, BP 53, 38041, Grenoble, France
Universit e´ Grenoble Alpes, DCM, BP 53, 38041 Grenoble cedex 9, France
‡
CBMs (carbohydrate-binding modules) are a class of polypeptides
usually associated with carbohydrate-active enzymatic sites. We
have characterized a new member of the CBM40 family, coded
from a section of the gene NanI from Clostridium perfringens.
Glycan arrays revealed its preference towards α(2,3)-linked
sialosides, which was confirmed and quantified by calorimetric
using SPR (surface plasmon resonance) 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)-sialyl-lactose explain its binding specificity and
unusually strong binding.
studies. The CBM40 binds to α(2,3)-sialyl-lactose with a K
d
of
∼
30 μM, the highest 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
Key words: carbohydrate-binding domain, Clostridium perfrin-
gens, protein–glycan interactions, sialic acid, X-ray structure.
INTRODUCTION
glycosylation or batch inconsistency. Recombinant proteins have
proven to be a valid and effective alternative, compensating for
the aforementioned complications [16].
Sialic acids are the most abundant terminal carbohydrate residues
present on glycoconjugates at the surface of eukaryotic cells
Sialidases, a group of enzymes that cleave sialosides, are
secreted by microbial organisms and mammalian cells. These
include gut bacteria, either commensals or pathogens [17,18],
viruses such as influenza [19] and human cells [20]. The catalytic
domain of these enzymes is often linked to one or more
CBMs (carbohydrate-binding modules) that reversibly binds the
glycan substrate to increase its local concentration, consequently
improving the catalytic efficiency of the enzyme [21]. CBMs are
small 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 for through avidity
by fusing multiple CBMs together [23].
CBMs are organized into 73 families on the basis of their
amino acid sequence similarity (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, which was
first verified with the CBM located at the N-terminus of GH33
(glycoside hydrolase 33) sialidase from Vibrio cholerae [25]. At
present, there are only six structurally characterized CBM40s
which are associated with the sialidases from Clostridium
perfringens NanJ [26], V. cholerae NanH [23], Macrobdella
decora NanL [27] and Streptococcus pneumoniae NanA [28],
NanB [29] and NanC [30]. They all share a conserved β-sandwich
fold consisting of two antiparallel β-sheets, with the exception of
[
1,2]. The N-acetylneuraminic acid (Neu5Ac) monosaccharide
is the most common form of sialic acid on mammalian cells.
Sialylation is required for the structural stabilization of cells
[
[
3] and is especially important in early mammalian development
4,5]. In cancer, altered surface sialylation is a common hallmark
of the malignant phenotype of tumour cells [6]. 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 [7,8]. An increase in the
negative charges associated with Neu5Ac also contributes to
the cell mobility by helping dispersion from the main tumour,
influencing the metastatic potential of those cells [9].
Owing 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 [10]. This
includes lectins purified from plants, such as WGA (wheatgerm
agglutinin), MAA (Maackia amurensis agglutinin) and SNA-I
(
Sambucus nigra agglutinin I) [11,12], mushrooms, such as PSL-
I (Polyporus squamosus lectin I) [13,14], or invertebrates such
as LFA (Limax flavus agglutinin) [15]. Whereas SNA-I and PSL-
ꢀ
I are specific for α2,6-linked Neu5Ac [namely terminal 6 -SLN
ꢀ
(
6 -sialyl-N-acetyl-lactosamine)], the other lectins have a broader
range of interactions, for example displaying cross reactivities
with N-acetylglucosamine (GlcNAc) (WGA and 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
ꢀ
Abbreviations: CBM, carbohydrate-binding module; CFG, Consortium for Functional Glycomics; GlcNAc, N-acetylglucosamine; HBS-T, Hepes-buffered
saline with Tween 20; ITC, isothermal titration calorimetry; LFA, Limax flavus agglutinin; MAA, Maackia amurensis agglutinin; MIC, minimal inhibition
concentration; MME, monomethyl ether; Neu5Ac, N-acetylneuraminic acid; PSL-I, Polyporus squamosus lectin I; SiaOPr, Neu5Ac derivative with a
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
propargyl aglycon; 3 -SL, 3 -sialyl-lactose; 6 -SL, 6 -sialyl-lactose; 3 -SLN, 3 -sialyl-lactosamine; 6 -SLN, 6 -sialyl-lactosamine; SNA-I, Sambucus nigra
agglutinin I; SPR, surface plasmon resonance; TSA, thermal shift assay; WGA, wheatgerm agglutinin.
1
Correspondence may be addressed to either of these authors (email lkmahal@nyu.edu or imberty@cermav.cnrs.fr).
ꢁ
c 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

2110
J.P. Ribeiro and others
+
Scheme 1 Synthesis of 2-propynyl 5-acetamido-3,5-dideoxy-D-α-D-galacto-2-nonulopyranosidonic acid 4: a) MeOH, Amberlite 120 H ; b) AcCl, HCl(g); c)
Propargyl alcohol, AgOTf, 4 A˚ MS; d) NaOMe, MeOH; e) LiOH, H
O.
2
V. cholerae NanH which shows a different architecture, which led
researchers to suggest its integration into a new CBM40 subfamily
Gene cloning
ꢀ
The following oligonucleotide primers were used: 5 -GAC GAC
GGA TCC CAT GTT AAG TTC ACT AGG AGA ATA TAA
[
26].
In the present paper, we report the successful characterization
ꢀ
ꢀ
GGA TAT-3 (45-mer) and 5 -TCC CAT CTC GAG TTA TTT
of the CBM40 from C. perfringens (ATCC 13124) which
is associated with NanI [31], for which only the catalytic
domain has been structurally characterized [32]. The recombinant
CBM40_NanI demonstrates unusual micromolar affinity towards
sialylated oligosaccharides. The structural analysis of the
ꢀ
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®
ꢀ
ꢀ
ꢀ
ꢀ
complexes with 3 -SL (3 -sialyl-lactose) and 6 -SL (6 -sialyl-
lactose) provides insights into the molecular details responsible
for this affinity. A pseudo-dimeric version of the CBM40 was also
engineered so as to enhance the affinity from multiple interactions
between the CBM and glycans. The present study describes a
new probe towards sialoconjugates, expanding the repertoire of
sialoside binders.
13124TM 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 changed the 5 - and 3 -termini of the
gene sequences to integrate 36 additional codons (representing a
12 amino acid long linker that will be present between two CBM
ꢀ
ꢀ
EXPERIMENTAL
Materials
copies). The PCRs were done using the oligonucleotides 1-For:
ꢀ
5
-GAC GAC GGA TCC CAT GTT AAG TCC ACT AGG ATA
®
TM
ꢀ
ꢀ
Genomic DNA of C. perfringens ATCC 13124 was bought
TAA GGA TAT-3 (45-mer, BamHI); 1-Rev: 5 -G CAC GAG CTC
GCT GCC GTT CAG CGC TTT TGT CTC TCC AGT-3 (40-
mer, SacI); 2-For: 5 -C GTC GAG CTC GGC AGC GGC AGC
GGC TTA AGT TCA CTA GGA-3 (40-mer, SacI), 5 -TCC CAT
CTC GAG TTA TTT TGT CTC TCC AGT CTT ACT AAG TAA
TM
ꢀ
ꢀ
from the A.T.C.C. (#13124D-5 ). 3 -SL sodium salt (CAS
128596-80-5) and 6 -SL (#35890-38-1) were purchased from
Carbosynth. 3 -SLN (3 -sialyl-N-acetyl-lactosamine) (#81693-
2-3) and 6 -SLN (#174757-71-2) were from Dextra UK and
sialic acid (#131-48-6) was from AppliChem. Biotinylated 3 -SL
#0060-BM) and biotinylated 6 -SL (#0063-BM) were obtained
ꢀ
ꢀ
#
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2
ꢀ
ꢀ
ATA ATC ATC-3 (51-mer, XhoI). After the two sequences were
ꢀ
(
made, the digestion by the same endonuclease allowed the ligation
of the two CBM40_NanI copies in tandem. All the generated
inserts were subjected to endonuclease treatment and introduced
in the multiple cloning region of the pET45b( + ) vector, yielding
to pNanI, pNanJ and p2NanI plasmids that encode for a copy
of Cp CBM40_NanI, Cp CBM40_NanJ or Cp di-CBM40_NanI,
respectively, fused to a polyhistidine tag at the N-terminus. The
constructs were verified by DNA sequencing (Genewiz, US or
Eurofins, EU). The DNA of the positive clones was amplified in
Escherichia coli XL1 blue cells and then used to transform E. coli
BL21(DE3) cells for protein production.
from Lectinity.
Propargyl sialic acid synthesis
All chemical reagents were purchased from Aldrich or Acros.
Moisture-sensitive reactions were performed under an 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 visualized by
charring with 10% H SO in ethanol. Silica gel 60 (0.063–0.2 mm
or 70–230 mesh; Merck) was used for column chromatography.
2
4
1
13
H and C NMR spectra were recorded on Bruker Avance 400
Protein expression and purification
MHz or Bruker Avance III 500 MHz spectrometers and chemical
shifts (δ) were reported in p.p.m. Spectra were referenced to the
E. coli BL21(DE3) cells transformed with plasmids pNanI, pNanJ
◦
residual proton solvent peaks relative to the signal of CDCl
3
1
(δ
or p2NanI were cultured in terrific broth medium at 37 C. When
1
13
2
◦
7
.27 and 77.0 p.p.m. for H and C) and H
2
O (4.79 p.p.m. for H),
reaching a D600 of 0.5–0.7, the cultures were moved to a 16 C
assignments were made by gCOSY (gradient-selected COSY) and
gHMQC (gradient-selected HMQC) experiments.
incubator and IPTG was added to a final concentration of 1 mM.
After overnight incubation, cells were pelleted down, washed
and resuspended in ice-cold PBS (pH 7.4). The cells were lysed
by sonication on ice three times at 20% intensity with 0.5
cycle for 2 min or with a Cell Disruption System (Constant
Systems) at 1900 bars (1 bar = 100 kPa). After centrifugation
A route for the synthesis of the propargyl sialic acid (2-propynyl
-acetamido-3,5-dideoxy-D-α-D-galacto-2-nonulopyranosidonic
5
acid) starting from N-acetylneuraminic acid is shown in
Scheme 1. All reaction conditions, mass and NMR spectra and
their assignments are in the Supplementary Online Data (SI 1).
◦
at 50000 g for 30 min at 4 C, the supernatants were submitted
ꢁ
c 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

High-affinity sialic acid-specific CBM40 from Clostridium perfringens
2111
to nickel-affinity chromatography. After being allowed to bind
solutions. All diffraction experiment datasets were collected at
the European Synchrotron Radiation Facility (ESRF), Grenoble,
2
+
the matrix (Ni -nitrilotriacetate–agarose; Qiagen), the proteins
were washed with 50 mM NaH PO O, pH 8.0, 300 mM NaCl
ꢀ
2
4
·H
2
France. Final data for CBM/3 -SL were collected at 1.9 Å (1 Å =
and 10 mM imidazole, and eluted using the same buffer but
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
0.1 nm) on the BM30A beamline using an ADSC Q315r detector
ꢀ
whereas 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 [34,35] and all further processing
was carried out using the CCP4 program suite [36].
1
0 kDa molecular-mass cut-off filter, before being purified further
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 masses of the purified
proteins.
Structure solution and refinement
A calibration curve for molecular size estimation created
with cytochrome c, myoglobin, ovalbumin and BSA, before the
experiments, confirmed the expected molecular mass values.
Purified proteins were run in denaturing protein gels in a
Co-ordinates of molecule A of the CBM40 from C. perfringens
®
TM
ATCC 13124 NanJ (PDB code 2V73) were used as a model
for molecular replacement with PHASER [37] in a search
ꢀ
for six monomers per asymmetric units for the CBM40–6 -SL
1
2% (w/v) acrylamide matrix under alkaline buffer conditions
ꢀ
complex. The coordinates of molecule A of the CBM40–3 -
(
Tris/HCl, pH 8.8).
SL were used to search for the three copies of the CBM40–
ꢀ
6
-SL complex. A total of 5% 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 [38] before restrained maximum likelihood
refinement with REFMAC 5.8 [39] iterated with manual
rebuilding in Coot [40]. Incorporation of the ligand was performed
Protein estimation
Protein concentrations were determined with a Nanodrop
spectrometer ND2000 (Ozyme) with absorbance readings at
2
80 nm wavelengths, using molar absorption coefficient values
−
1
− 1
of 11920, 21890 and 32780 M ·cm
for C. perfringens
CBM40_NanJ, C. perfringens CBM40_NanI and C. perfringens
di-CBM40_NanI respectively, as well as their corresponding
molecular masses of 23.69, 24.62 and 46.55 kDa (values for
calculations that include the N-terminally tagged amino acids).
after inspection of the 2F
o
− F
c
weighted maps. Water molecules,
introduced automatically using Coot, were inspected manually.
The quality of the models was assessed using the PDB validation
server (http://wwpdb-validation.wwpdb.org/validservice/) and
co-ordinates were deposited in the PDB under codes 5FRE and
5FRA.
Thermostability
To examine their stability, protein solutions of C. perfringens
CBM40_NanI, C. perfringens CBM40_NanJ and C. perfringens
di-CBM40_NanI were assayed using a TSA (thermal shift
assay), adapting a protocol described previously [33]. Samples
Haemagglutination and minimal concentration inhibition assays
Haemagglutination activity for C. perfringens CBM40_NanI
and C. perfringens di-CBM40_NanI was determined using 2-
fold serial dilutions of the protein with rabbit erythrocytes
(Biom e´ rieux). In those tests, 25 μl of the serial dilutions of the
protein were incubated with 25 μl of 2% rabbit erythrocyte
−
1
were diluted to a final protein concentration of 0.1 mg·ml
in the chosen buffer and 5× SYPRO Orange (Molecular
Probes/Invitrogen) 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
◦
suspensions in 100 mM NaCl for 30 min at 37 C and 30 min
◦
◦
a temperature gradient starting at 25 C and rising to 100 C at a
at room temperature. After identifying the highest dilution of
the protein that exhibited haemagglutination, a concentration 4-
fold higher was used in the subsequent MIC (minimal inhibition
concentration) experiments. Serial 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 erythrocyte suspensions, following the
same development steps as above.
◦
heating rate of 1 C/min. Protein unfolding was followed by the
increase in the fluorescence values given by the SYPRO Orange
probe. Different buffer effects were analysed in the pH range of
3
–10.
Crystallization and data collection
−
1
Solutions of CBM40_NanI (5.5 mg·ml ) 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
Glycan array
C. perfringens CBM40_NanI and C. perfringens di-
CBM40_NanI were labelled with Alexa Fluor 488 (Molecular
®
(
Qiagen), plates 4 and 5 (Hampton) screens. Hits were obtained
TM
with the use of the Clear Strategy
Dimensions) at pH 4.5. Crystals of CBM40_NanI with 3 -SL
appeared in the form of plates after several days in CSSI-
Screens, (Molecular
Probes/Invitrogen) using the manufacturer’s protocols and sent
to the Consortium for Functional Glycomics (CFG). Binding
specificity was determined using the printed mammalian glycan
array version 5.2, which contains 609 glycans [41]. The scanner
response is linear to a maximum of ∼50000 RFU (relative
fluorescent units). Both proteins were incubated with the arrays at
ꢀ
1
2 containing 0.1 M sodium acetate, pH 4.5, 0.2 M calcium
acetate and 8% of both PEG550 MME (monomethyl ether)
and PEG20000. In the same time period, parallelepiped crystals
of CBM40_NanI in complex with 6 -SL grew in 0.1 M sodium
ꢀ
− 1
200 μg·ml . The extracted data was then transformed to give Z
acetate, pH 4.5, 0.3 M sodium acetate and 25% PEG2000 MME.
Afterwards, larger crystals were then grown with home-made
scores for each glycan on the array (Z = x-mean/S.D.). A threshold
of Z = 1.645 (P ꢀ 0.05) was used to establish significance.
ꢁ
c 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

2112
J.P. Ribeiro and others
Isothermal titration calorimetry (ITC)
different from previously characterized CBMs of family 40 to
substantiate a full characterization.
ITC experiments were performed using a MicroCal ITC200
microcalorimeter (Malvern Instruments). All titrations were
carried out in PBS, pH 7.4, at 25 C. Aliquots of 10 μl of
®
After cloning of genomic DNA of C. perfringens ATCC
TM
1
3124 , recombinant C. perfringens CBM40_NanI was purified
◦
−
1
with a yield of approximately 10 mg·l . The C. perfringens
CBM40_NanJ was produced under the same conditions for
comparison. The expressed proteins appear as one strong band
on SDS/PAGE gels, with apparent molecular masses close to
the 25 kDa marker, in agreement with the theoretical values
of 23.7 and 24.6 kDa for C. perfringens CBM40_NanJ and
C. perfringens 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 (Supplementary Online Data SI 3). The thermal
stability of the C. perfringens CBM40_NanI and C. perfringens
CBM40_NanJ was assessed by TSAs. The highest stability was
each carbohydrate dissolved in the same buffer were added to
the protein solution present in the calorimeter cell at 2 min
intervals. For the different experiments, protein concentrations
were prepared in solution with concentrations that varied from
1
00 to 415 μM, and the carbohydrate samples ranged from 2.1
to 6.0 mM. Data were fitted using MicroCal Origin 7 software,
following standard procedures. This yielded the stoichiometry
(
N), the association constant (K ) and enthalpy of binding (ꢀH).
a
Other thermodynamic parameters (changes in free energy, ꢀG,
and entropy, ꢀS), were calculated from the equation ꢀG =
−
1
−1
ꢀ
H − TꢀS = − RTlnK , with R = 8.314 J·mol ·K .
a
◦
obtained in PBS buffer at pH 7, with melting temperatures of 54 C
◦
for C. perfringens CBM40_NanJ and 65 C for C. perfringens
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 [23].
Thus a divalent version of the protein was engineered. The
pseudo-dimeric C. perfringens CBM40_NanI has a 12-amino-
acid-long linker (ALNGSELGSGSG) inserted between the two
copies to provide flexibility between the binding modules. Using
Surface plasmon resonance (SPR)
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
coated previously with streptavidin, following the same protocol
as described previously [42]. Each monovalent biotinylated sugar,
ꢀ
ꢀ
− 1
3
-SL or 6 -SL, was diluted to 1 μg·ml in HBS-T (Hepes-
− 1
an identical expression protocol, a yield of ∼5 mg·l purified
buffered saline, pH 7.4, with 0.05% Tween 20) before being
injected into one of the flow cells of the chip. Low immobilization
di-CBM40_NanI was obtained. The expected band for a 46 kDa
protein was seen in the SDS/PAGE gel (Supplementary Online
ꢀ
levels of 94 and 64 response units were obtained for 3 -SL and
Data SI 3). The engineered C. perfringens di-CBM40_NanI
ꢀ
6
-SL respectively. A reference surface was always present in
◦
was stable in PBS (pH 7.0), with an observed T
Supplementary Online Data SI 4).
m
of 66 C
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. The purified proteins were injected
(
−
1
over the flow cell surface at 30 μl·min in a series of 2-fold
dilutions. The dissociation of this analyte was achieved by passing
running buffer for 4–6 min. Surfaces were regenerated with one
or two consecutive 30-s injections of 100 mM Neu5Ac, also at
Characterization of binding specificity demonstrates strong
preference for 3-linked sialic acids
An initial evaluation of glycan binding was performed by
inhibition of haemagglutination using rabbit erythrocytes. As
expected, haemagglutinating activity was not observed with
monomeric C. perfringens CBM40_NanI as agglutination
requires a multivalent binding to cross-link the cells. In
contrast, C. perfringens di-CBM40_NanI showed positive
−
1
3
0 μl·min . The information on the affinity was determined
by assuming Langmuir 1:1 binding, using the BIAevaluation
software.
−
1
haemagglutination down to a concentration of 50 μg·ml
.
RESULTS AND DISCUSSION
ꢀ
Among the glycans tested as haemagglutination inhibitors, 3 -SL
was the strongest inhibitor, with an MIC of 0.78 mM. 6 -SL was
ꢀ
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
also able to inhibit haemagglutination with an MIC of 1.56 mM.
Lactose, galactose, glucose, mannose and fucose (tested up to
50 mM) had no inhibitory effect (Supplementary Online Data
SI 5).
(
Supplementary Online Data SI 2), shows the CBM40s cluster into
one main group with few outliers. The CBM from C. perfringens
NanI clusters with the other CBM from the same organism,
C. perfringens CBM40_NanJ, with a phylogenetic similarity of
To gain further insight into the binding motifs of C.
perfringens CBM40_NanI, both monomeric and dimeric CBMs
were tested on the CFG glycan microarray version 5.2.
This array contains a panel of 609 mammalian glycans,
including α2,3-, α2,6- and α2,8-sialosides [43]. Monomeric
0
.942. As previously noted by other authors, the CBM of V.
cholerae NanH is more distantly related to the other CBM40s,
among those with validated Neu5Ac specificity and available
structural information [26]. The BLAST protein alignment
between the C. perfringens CBM40_NanI sequence and the other
structurally characterized CBM40s, i.e. C. perfringens NanJ (PDB
code 2V73), S. pneumoniae NanA (PDB code 4C1W), NanB
C. perfringens CBM40_NanI gave only
a very weak
signal with clear binding to only a few epitopes bearing
α2,3-sialic acid on a type II N-acetyl-lactosaminyl core
(Neu5Acα2,3Galβ1,4GlcNAc) (Supplementary Online Data
SI 6). Previous assays involving monomeric C. perfringens
CBM40_NanJ were also unsuccessful due to low affinity [26].
The signal-to-noise ratio was considerably improved when
assaying the engineered dimeric protein. The C. perfringens 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
(
PDB code 2VW0) and NanC (PDB code 4YZ5) and M. decora
NanL (PDB code 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 M. decora
leech. C. perfringens CBM40 NanI therefore appears sufficiently
ꢁ
c 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

High-affinity sialic acid-specific CBM40 from Clostridium perfringens
2113
Figure 1 Glycan array profile of C. perfringens 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 number 16976, and in
Supplementary Online Data file CFG_diCBM40_NanI.xls).
Z-score analysis) on the array, 24 contained an α2,3-sialoside.
The remaining three were an anomalous chitin epitope, sialic
acid bound directly to a linker and a single biantennary α2,6-
epitope detected. Both N-glycolyl- and 9-O-acetyl-containing
α2,3-sialosides acids were tolerated. Binding was enhanced by
an additional nearby negative charge (sulfation or sialylation).
and 189 μM for monomeric and dimeric CBM respectively) to the
monosaccharide conjugate than to the sialylated trisaccharides.
To compare binding affinities directly, ITC experiments were
also performed for C. perfringens CBM40_NanJ. As expected,
lower binding affinities were obtained with K values of 1.48 and
d
ꢀ
ꢀ
2.67 mM for 3 -SL and 6 -SL respectively (Supplementary Online
Data SI 7), confirming the previously published estimations of low
affinity [26].
The binding interactions were also analysed by SPR
spectroscopy. Commercially available monomeric biotinylated
C. perfringens CBM40_NanI displays unusually strong affinity for
sialylated oligosaccharides
ꢀ
ꢀ
3
-SL and 6 -SL were immobilized on a streptavidin-coated
sensorchip to obtain high-density coverage. The sensorgrams
show a typical shape for fast association and dissociation events
after the injection of increasing concentrations of C. perfringens
CBM40_NanI or C. perfringens 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
dissociationconstants (SupplementaryOnline Data SI8). Thedata
confirmed the stronger binding of C. perfringens CBM40_NanI
The glycan-binding activities of C. perfringens CBM40_NanI
and di-CBM40_NanI were confirmed by ITC at 25 C. All
interactions showed exothermic behaviours with a steep decrease
in the exothermic heat of binding until saturation was achieved
◦
(
Figure 2). The stoichiometry values (N) for sialylated compounds
titrated in C. perfringens 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
ꢀ
ꢀ
and C. perfringens di-CBM40_NanI with the 3 -SL- compared
similar behaviour with strong affinity for 3 -SL (K
d
∼30 μM)
ꢀ
ꢀ
with the 6 -SL-functionalized surface. The monomeric CBM
and weaker affinity for 6 -SL (K
d
∼60 μM). Values in the same
ꢀ
ꢀ
ꢀ
ꢀ
showed a K of 14.4 μM for 3 -SL, whereas 6 -SL appears to bind
data range were obtained for 3 - and 6 -SLN (Supplementary
Online Data 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
d
with 2.5-fold lower affinity, with a K of 36 μM, in agreement with
d
ITC data. For the C. perfringens di-CBM40, an increase of more
than one order of magnitude in affinity (11-fold) is observed for the
ꢀ
ꢀ
interaction with 3 -SL and a 6-fold increase for 6 -SL, resulting in
affinities of 1.3 and 6.1 μM respectively. Experiments conducted
binding. The CBMs showed weaker binding (K values of 170
d
ꢁ
c 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

2114
J.P. Ribeiro and others
ꢀ
ꢀ
ꢀ
ꢀ
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
ꢁ
c 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

High-affinity sialic acid-specific CBM40 from Clostridium perfringens
2115
Table 1 Thermodynamics of binding of C. perfringens CBM40_NanI, C. perfringens di-CBM40_NanI C. perfringens CBM40_NanJ to different sialylated
◦
ligands, as determined by ITC at 25 C
ND, not determined. 1 kcal = 4.184 kJ.
(
a)
4
− 1
Protein
[Protein] (μM)
Ligand
[Ligand] (mM)
N
K
a
(×10 M
)
K
d
(μM)
ꢀH (kcal/mol)
TꢀS (kcal/mol)
ꢀG (kcal/mol)
ꢀ
C. perfringens CBM40_NanI
400
3 -SL
5.0
3.6
0.9
0.8
5.0
5.0
3.6
5.0
0.86 + 0.01
3.16 + 0.09
32
68
37
49
170
35
− 10.49 + 0.14
− 4.35
− 3.01
− 3.34
− 2.88
− 14.81
− 5.16
− 3.96
ND
− 6.14
− 5.68
− 6.04
− 5.93
− 5.14
− 6.07
− 5.78
ND
−
0.95 + 0.01
−
−
−
ꢀ
4
5
5
1
00
0
0
6 -SL
1.48 + 0.05
− 8.69 + 0.05
−
−
ꢀ
3 -SLN
0.98 + 0.05
−
2.68 + 0.20
− 9.38 + 0.06
−
−
ꢀ
6 -SLN
0.92 + 0.08
2.23 + 0.25
− 8.81 + 0.10
−
−
0.59 + 0.05
−
−
00
SiaOPr
1.00*
− 19.95 + 0.05
−
− 11.20 + 0.01
−
ꢀ
C. perfringens di-CBM40_NanI
185
3 -SL
1.94 + 0.01
2.84 + 0.08
−
−
1.88 + 0.01
−
ꢀ
1
5
85
0
6 -SL
1.71 + 0.06
59
196
− 9.74 + 0.01
−
−
SiaOPr
2.00*
0.51 + 0.06
ND
−
(
b)
3
− 1
Protein
[Protein] (μM)
Ligand
[Ligand] (mM)
N
K
a
(×10 M
)
K
d
(mM)
ꢀH (kcal/mol)
TꢀS (kcal/mol)
ꢀG (kcal/mol)
ꢀ
C. perfringens CBM40_NanJ
100
3 -SL
10.0
10.0
1.00*
1.00*
0.67 + 0.04
1.48
2.23
− 1.51 + 0.05
2.35
0.89
− 3.86
− 3.61
−
0.44 + 0.06
−
−
− 2.72 + 0.27
−
ꢀ
1
00
6 -SL
7
6
153
with C. perfringens CBM40_NanJ did not yield sensorgrams of
sufficient quality to evaluate, probably due to the low affinity of
C. perfringens CBM40_NanJ–sialoside interactions.
acetyl group. Arg and Arg make electrostatic interactions
158
with the carboxylate group. Asn establishes hydrogen bonds
with the glycerol moiety. The hydroxy group of Tyr makes
hydrogen bonds with the carboxylate group and the nitrogen
of the N-acetyl group. Glu is involved in two hydrogen
bonds with the nitrogen of the N-acetyl group and the O4
hydroxy group, and the NH1 and NH2 atoms of Arg make
hydrogen bonds with the carboxylate group. The carboxylate
makes water-mediated interactions with the main-chain nitrogen
of Asn and the NH2 atom of Arg . Comparison between
the binding sites of C. perfringens CBM40_NanI and NanJ
show that the amino acids making direct contacts with the
ligand are conserved in both structures (Supplementary Online
Data SI 10).
160
74
Structural basis for specificity and affinity
153
To gain insight into the structural basis for sialoside binding of
C. perfringens 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 Dimensions), bigger and thicker
158
76
ꢀ
crystals of C. perfringens CBM40_NanI complexed with 3 -
ꢀ
SL and 6 -SL were obtained. The protein crystalized in P2
1
ꢀ
In the complex with 3 SL, the galactose does not interact
space group for both complexes, with three and six independent
monomers in the asymmetric unit for crystals of the CBM40_NanI
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
Supplementary Online Data 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. Gln is a key
amino acid in this network, interacting with two water molecules
which bridge with the O4 hydroxy group of the galactosyl moiety.
Lys NH makes another water-mediated hydrogen bond with the
OH6 group of the galactose. Also, Ser interacts with a chain
ꢀ
ꢀ
complexed with 3 -SL or 6 -SL respectively (Supplementary
ꢀ
Online Data SI 9). The structure of the CBM–6 -SL was solved
using PDB code 2V73 (C. perfringens CBM40_NanJ) as a model,
and subsequently used to solve the structure of the C. perfringens
(
ꢀ
CBM40_NanI–3 -SL. The monomers superimposed with RMSD
ꢀ
values between 0.12 and 0.16 Å for C. perfringens CBM40–3 SL
85
and between 0.15 and 0.20 Å for C. perfringens CBM40_NanI–
ꢀ
6
-SL.
The C. perfringens CBM40_NanI adopts the characteristic β-
58
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 β-strand
157
of water molecules connecting to the carboxylate of the Neu5Ac
and the O4 hydroxy group of the galactose.
7. The carbohydrate-recognition site is located on the concave
ꢀ
ꢀ
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 (Supplementary Online Data 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 and 3C that the galactose ring is
oriented outside the recognition domain of the protein.
face of the five-stranded β-sheet, in a shallow depression, in
which the ring of the sialic acid lies 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, whereas only the Neu5Ac
moiety could be defined with confidence for the other sites.
The glucose moieties could not be modelled as they were too
disordered.
Comparison with the other CBM40 proteins
The majority of the interactions between the protein and
the ligand involve the sialic acid moiety. In both complexes
The overall macromolecular architecture of the C. perfringens
CBM40_NanI is well conserved when comparing with other
members of CBM40 family, apart NanH which belongs to another
46
65
93
(
Figures 3B and 3C), Ile , Tyr and His side chains form a
hydrophobic pocket that accommodates the methyl of the N-
ꢁ
c 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

2116
J.P. Ribeiro and others
ꢀ
ꢀ
Figure 3 3D structure of CBM40 from Clostridium perfringens NanI in complex with 3 -SL and 6 -SL and sequence comparison with characterized CBMs from
the same family
(
A) Structure of the CBM40 from C. perfringens ATCC® 13124TM NanI in complex with 3 -SL and 6 -SL (at the left and right respectively). Ligands are shown with a 2F
ꢀ
ꢀ
o
− F
c
density map contoured
ꢀ
ꢀ
at 1.0 σ. (B and C) Close-up view of the binding domain occupied by 3 -SL and 6 -SL. (D) Comparison of the CBM40 of C. perfringens NanI with structurally related structures. C. perfringens
NanI (in red, boxed), C. perfringens NanJ (in cyan), S. pneumoniae NanA (in yellow), S. pneumoniae NanB (in green), S. pneumoniae NanC (in brown) and M. decora NanL (in purple). The loop
–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 NanJ, S.
TM
5
pneumoniae NanA, NanB and NanC, and M. decora NanL. The secondary-structure regions of the CBM40_NanI are identified on top. Shaded residues show the conservation between the proteins.
8
5
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 Gln is marked with a
red star due to its particular importance as part of the binding domain. The sequence of V. cholerae NanH is not shown due to its poor alignment (Supplementary Online Data SI 8). The Figure was
created with ESPript 3.0 [53].
branch of the phylogenetic tree as discussed above. Sequence
alignments show that the recognition domains of the CBM40s
share common key residues that provide the protein its binding
specificity (Figure 3E and Supplementary Online Data SI 13).
CBM40_NanI numeration) are particularly important, being
spatially conserved in all CBMs (with the exception of M.
decora NanL, which has an aspartic acid instead of glutamic
acid), making a total of four hydrogen bonds with the Neu5Ac
(two with the N-acetyl and O4 hydroxy group, and two with
74
153
Within these binding domains, Glu and Arg (C. perfringens
ꢁ
c 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

High-affinity sialic acid-specific CBM40 from Clostridium perfringens
2117
the carboxylate group respectively). A highly variable region
between all of the CBM40s is the loop between β-strands 5
and 6 (Figures 3D and 3E, see starred box). In C. perfringens
CBM40_NanI, this loop is longer by six to eight amino acids and
has a unique spatial arrangement compared with 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, C.
perfringens CBM40_NanI displays a much higher affinity for
sialylated oligosaccharides (∼100-fold) than its counterpart. This
difference in affinity appears to be due to an increased water-
mediated hydrogen-bond set-up inside the binding site of C.
perfringens CBM40_NanI.
with the CBM of Vibrio cholerae NanH, which shows a broad
specificity for all sialosides [23]. The binding of α2,3-sialosides,
but not 3 -O-sulfated ones, makes this reagent more specific for
sialic acid residues than the commonly used MAL-1 (Maackia
amurensis lectin I) [41].
ꢀ
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 C. perfringens 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.
As seen in the crystal structures, the loop between β-strands
and 6, exclusive to C. perfringens CBM40_NanI, introduces
5
an additional surface in the recognition domain. In this loop,
AUTHOR CONTRIBUTION
85
Gln creates water-mediated interactions with both the sialyl
carboxylate and galactosyl hydroxy groups. In contrast with
NanI, the crystal structure of C. perfringens CBM40_NanJ
does not show such a water-mediated hydrogen-bond network
Lara Mahal conceived the project. Jo a˜ o Ribeiro, Lara Mahal and Anne Imberty co-
ordinated the paper. Jo a˜ o Ribeiro and William Pau performed cloning, protein expression
and purification. Jo a˜ o Ribeiro ran all of the biophysical experiments and provided data
analysis. Jo a˜ o Ribeiro and Annabelle Varrot resolved the crystal structures. Carlo Pifferi
and Olivier Renaudet synthesized and characterized the SiaOPr. Jo a˜ o Ribeiro analysed
data, generated the Figures and Tables and wrote the body of the paper. Annabelle Varrot,
Lara Mahal and Anne Imberty revised the paper leading to the final version. All authors
reviewed the results and approved the paper for submission.
(
Supplementary Online Data SI 10).
Sialylated oligosaccharides are flexible epitopes, due to the
possibility of their conformation at the α2-3 and α2-6 linkages
44]. It is therefore of interest to compare whether different CBMs
recognized the same epitope. S. pneumoniae CBM40_NanA and
[
ꢀ
ꢀ
NanC were also successfully co-crystalized with the 3 -SL and 6 -
SL ligands (PDB codes 4C1W and 4C1X, and 4YZ5 and 4YW2
respectively), among others [28,30]. Probably due to the exposure
of the ligands and the few contacts made with the protein, different
ACKNOWLEDGEMENTS
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.
ꢀ
ꢀ
conformations are seen for 3 -SL and 6 -SL when bound to C.
perfringens CBM40_NanI, S. pneumoniae CBM40_NanA and S.
ꢀ
pneumoniae CBM40_NanC. 3 -SL shares similar conformations
FUNDING
in NanI and NanA [in NanC, the galactose ring is in a full inverted
ꢀ
The research leading to these results has received funding from the European Community’s
position relative to the others, whereas the 6 -SL structure is alike
7
th Framework Programme (FP7/2007–2013) under the Marie Curie International
in NanI and NanC (Supplementary Online Data SI 14)].
Outgoing Fellowship for Career Development [grant number PIOF-GA-2011-298910],
BioStruct-X [grant number 283570] and ERASynbio program SynGlycTis [grant number
ANR-14-SYNB-0002-02]. WearealsogratefultotheLabexArcaneforthefinancialsupport
[
grant number ANR-11-LABX-0003-01]. We are also grateful for the participation of the
Protein–Glycan Interaction Resource of the Consortium for Functional Genomics (CFG)
supporting grant number R24 GM098791].
Conclusion
[
Although the use of CBMs in biotechnology is fairly recent, they
have been described in the paper, textile and food industries [45].
Their potential application in health sciences has also been proved
in different scenarios as molecular probes [46], in microarrays
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Received 14 April 2016/13 May 2016; accepted 16 May 2016
Accepted Manuscript online 17 May 2016, doi:10.1042/BCJ20160340
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c 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.