Báo cáo khoa học: NMR studies on the interaction of sugars with the C-terminal domain of an R-type lectin from the earthworm Lumbricus terrestris pot

NMR studies on the interaction of sugars with the
C-terminal domain of an R-type lectin from the earthworm
Lumbricus terrestris
Hikaru Hemmi
1
, Atsushi Kuno
2
, Shigeyasu Ito
2,3
, Ryuichiro Suzuki
2,3
, Tsunemi Hasegawa
3
and Jun Hirabayashi
2
1 National Food Research Institute, National Agriculture and Food Research Organization (NARO), Tsukuba, Japan
2 Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan
3 Department of Material and Biological Chemistry, Yamagata University, Yamagata, Japan
Sugar-binding proteins, known as lectins, exist ubiqui-
tously in both animals and plants, but lectins from the
annelid phylum have rarely been reported. A 29 kDa
lectin (EW29) was isolated from the earthworm
Lumbricus terrestris using affinity chromatography on
asialofetuin–agarose in the screening of galectin-like
proteins. The protein consists of two homologous
domains (14 500 Da), i.e. N- and C-terminal domains,
which show 27% identity with each other [1]. Both
domains of EW29 form a tandem-repeat type structure
and contain triple-repeated QXW motifs [2,3]. This
short motif has been found in many carbohydrate-
recognition proteins including the plant lectin ricin

B-chain [4]. The 3D structures of R-type lectins have
been determined [2,5–19], and these proteins possess
common b-trefoil fold structures, although their
sugar-binding affinities differ depending on their ligand
specificities.
Recent biochemical data on EW29 and its truncated
mutants showed it to be a single-domain type of
Keywords
earthworm Lumbricus terrestris;
hemagglutinating activity; NMR titration;
R-type lectin; sugar
Correspondence
H. Hemmi, National Food Research
Institute, National Agriculture and Food
Research Organization (NARO), 2-1-12
Kannondai, Tsukuba, 305-8642 Ibaraki,
Japan
Fax: +81 298 387996
Tel: +81 298 388033
E-mail:
(Received 1 December 2008, revised 30
January 2009, accepted 2 February 2009)
doi:10.1111/j.1742-4658.2009.06944.x
The R-type lectin EW29, isolated from the earthworm Lumbricus terrestris,
consists of two homologous domains (14 500 Da) showing 27% identity
with each other. The C-terminal domain (Ch; C-half) of EW29 (EW29Ch)
has two sugar-binding sites in subdomains a and c, and the protein uses
these sugar-binding sites for its function as a single-domain-type hemagglu-
tinin. In order to determine the sugar-binding ability and specificity for
each of the two sugar-binding sites in EW29Ch, ligand-induced chemical-

shift changes in EW29Ch were monitored using
1
H–
15
N HSQC spectra as
a function of increasing concentrations of lactose, melibiose, d-galactose,
methyl a-d-galactopyranoside and methyl b-d-galactopyranoside. Shift
perturbation patterns for well-resolved resonances confirmed that all of
these sugars associated independently with the two sugar-binding sites of
EW29Ch. NMR titration experiments showed that the sugar-binding site in
subdomain a had a slow or intermediate exchange regime on the chemical-
shift timescale (K
d
=10
)2
to 10
)1
mm), whereas that in subdomain c had
a fast exchange regime for these sugars (K
d
= 2–6 mm). Thus, our results
suggest that the two sugar-binding sites of EW29Ch in the same molecule
retain its hemagglutinating activity, but this activity is 10-fold lower than
that of the whole protein because EW29Ch has two sugar-binding sites in
the same molecule, one of which has a weak binding mode.
Abbreviations
Ch (C-half), the C-terminal domain; EW29, earthworm 29 kDa lectin; a-Me-Gal, methyl a-
D-galactopyranoside; b-Me-Gal, methyl
b-
D-galactopyranoside; STD, saturation transfer difference.

FEBS Journal 276 (2009) 2095–2105 ª 2009 The Authors Journal compilation ª 2009 FEBS 2095
hemagglutinin, differing from other tandem repeat-type
proteins in the R-type lectin family, such as ricin [2],
abrin [20] and Sambucus sieboldina agglutinin [21], the
exception being the ricin B1 domain which has two
sugar-binding sites in the same molecule [22,23]. Based
on these structural features, R-type lectins generally
contain one sugar-binding site per domain, suggesting
that the truncated mutant, which comprises a single
domain, may have no hemagglutinating activity
[2,5,20]. However, the C-terminal domain (Ch; C-half)
of EW29 (EW29Ch) bound to asialofetuin–agarose as
strongly as the whole protein and retained its hemag-
glutinating activity, although at a level 10-fold lower
than that of the whole protein [1]. The crystal struc-
tures of the complex between EW29Ch and lactose or
N-acetylgalactosamine (PDB: 2ZQN or 2ZQO)
reported recently indicate that the overall structure of
EW29Ch resembles the characteristic pseudo-threefold
symmetry of the three subdomains designated a, b and
c, and that the protein has two sugar-binding sites in
subdomains a and c [24,25]. Therefore, determination
of the sugar-binding ability and specificity for each of
the two sugar-binding sites in EW29Ch is expected to
elucidate the molecular basis of the carbohydrate
cross-linking properties of the lectin.
In this study, the sugar-binding ability and specific-
ity of each of the two sugar-binding sites in EW29Ch
for certain sugars were determined using NMR titra-
tion experiments in combination with recent crystallo-

graphic studies [25]. The NMR titration experiments
showed that the a sugar-binding site has a much tigh-
ter sugar-binding mode than the c sugar-binding site.
Furthermore, saturation transfer difference (STD)–
NMR experiments for a mixture of the protein with
sugar revealed the epitope of the sugar for the sugar-
binding protein. Thus, our results suggest that the two
sugar-binding sites of EW29Ch in the same molecule
retained its hemagglutinating activity, but this activity
was 10-fold lower than that of the whole protein
because EW29Ch has two sugar-binding sites in the
same molecule, one of which has a weak binding
mode.
Results
Resonance assignments
Complete resonance assignments for EW29Ch have
been reported elsewhere [26]. In this study, we
observed chemical shifts for some residues in sub-
domain a as a pair of resonance signals in the
unbound state and the bound state. Furthermore, the
resonance signal of residues Gly21 and Asn23 in sub-
domain a, which was assigned because of lactose con-
tamination in the previous study [26], disappeared in
the completely sugar-free state (Fig. S1). The reso-
nance signals of EW29Ch in the completely sugar-free
state were therefore reassigned using multidimensional
and multinuclear NMR spectroscopy, as described
elsewhere [26]. Figure 1 shows the
1
H–

15
N HSQC spec-
trum for EW29Ch in the completely sugar-free state.
The assignment data previously deposited in BMRB
under accession number 6226 [26] were corrected and
re-deposited under the same accession number.
Fig. 1.
1
H–
15
N HSQC spectrum of the C-ter-
minal domain of EW29 in the sugar-free
state. A 600 MHz 2D
1
H–
15
N HSQC spec-
trum of the 0.9 m
M C-terminal domain of
EW29 at pH 6.1 and 298K in the sugar-free
state. Cross-peaks are labeled based on an
analysis of through-bond connectivities. The
side chains of NH
2
resonances of aspara-
gines and glutamines are connected by
horizontal lines. The side chains of NH
resonances of tryptophan are marked by
‘sc’.
Interaction of EW29Ch with sugars H. Hemmi et al.

2096 FEBS Journal 276 (2009) 2095–2105 ª 2009 The Authors Journal compilation ª 2009 FEBS
Identification of sugar-binding sites
The interaction of
15
N-labeled EW29Ch with lactose,
melibiose, galactose, methyl a-d-galactopyranoside
(a-Me-Gal) and methyl b-d-galactopyranoside (b-Me-
Gal) was monitored using
1
H–
15
N HSQC spectros-
copy. An overlay of 10
1
H–
15
N HSQC spectra showed
progressive chemical-shift changes for some amide
resonances of EW29Ch upon the addition of lactose.
Overlaid spectra showed two types of chemical
exchange (slow and fast) on the chemical-shift time-
scale (Fig. 2A). Figure 2B shows the overall effect of
lactose binding by mapping the observed main- and
side-chain
15
N chemical-shift changes on the crystal
structure of EW29Ch. Residues showing a slow
exchange regime in EW29Ch were located in subdo-
main a, whereas those showing a fast exchange regime
were located in subdomain c (Fig. 2B). Larger chemi-

cal-shift changes in the fast exchange regime were
observed for backbone amides, as well as side-chain
amide and indole groups, of residues within or adja-
cent to the sugar-binding site of subdomain c, identi-
fied from the crystal structure of lactose-liganded
EW29Ch. In the case of other sugars used in this
study, chemical exchanges in subdomain a showed a
slow exchange regime for b-Me-Gal and an intermediate
exchange regime for melibiose, galactose and a-Me-Gal,
whereas those in subdomain c showed a fast exchange
A
B
Unbound
Bound
Slow exchange
Fast exchange
Fig. 2. Chemical-shift changes in NMR titration of EW29Ch with lactose. (A) Ten
1
H–
15
N HSQC spectra of
15
N-labeled EW29Ch in the pres-
ence of protein ⁄ lactose molar ratios of 0 (red), 0.5 (green), 1.0 (blue), 2.0 (magenta), 4.0 (gold), 6.0 (orange), 10.0 (pink), 20.0 (purple), 40.0
(coral) and 80.0 (cyan) are overlaid. Overlay spectra show that free and bound forms of some residues in the protein are in a slow exchange
on the NMR time scale and those of other residues are in a fast exchange. Representative residues for slow and fast exchanges are labeled.
The arrow indicates the direction in which amide
1
H–
15

N peaks shift with the adding of sugar. (B) Mapping of the
1
H
N
and
15
N chemical-shift
changes upon the addition of excess lactose on a ribbon diagram of the crystal structure of EW29Ch (PDB: 2ZQN) generated by
MOLMOL
[53]. Spheres represent
15
N atoms of the main chain and side chains of each residue in the protein. Residues showing a slow exchange
regime are in red and those showing a fast exchange regime and D
av
⁄ D
max
> 0.2, where D
av
is the normalized weighted average of the
1
H
and
15
N chemical-shift changes and D
max
is the maximum observed weighted shift difference (0.549 p.p.m. for side chain amide cross peak
of N124), are in green. Residues showing a fast exchange regime and 0.1 £ D
av
⁄ D
max

£ 0.2 are in light green, and others are in gray. Key
residues showing chemical-shift changes in slow and in fast exchange regimes are labeled.
H. Hemmi et al. Interaction of EW29Ch with sugars
FEBS Journal 276 (2009) 2095–2105 ª 2009 The Authors Journal compilation ª 2009 FEBS 2097
regime for all sugars used. No chemical-shift changes
were observed for any sugars in subdomain b. These
results showed that each of the two sugar-binding sites
(a and c) of EW29Ch had a distinct chemical exchange
on the chemical-shift timescale.
Site-specific dissociation constants determined
by NMR
The site-specific binding constants and chemical
exchange regimes of EW29Ch with sugars used in this
study are given Table 1. Upon the addition of sugars,
the chemical-shift changes in subdomain a were in a
slow exchange regime or an intermediate exchange
regime, as described above. For lactose and b-Me-Gal,
the second signal corresponding to the bound state
clearly appeared at [sugar] ⁄ [EW29Ch]  0.3; the first
signal corresponding to the unbound state disappeared
completely at [sugar] ⁄ [EW29Ch]  1.3 and only the
second signal was observed (Fig. 3A). This phenome-
non indicated a stoichiometric interaction between
EW29Ch and lactose at a  1 : 1 ratio. The dissocia-
tion constants (K
d
) of the a sugar-binding site for
lactose and b-Me-Gal were calculated in a similar way
using NMR titration experiments. From the theoretical
calculations of K

d
for the ratios of sugar-free to sugar-
bound peak intensities as a function of sugar concentra-
tion, the protein concentration was lowered to 0.05 mm
to obtain the K
d
more precisely for the sugar-binding
site in the slow exchange regime. However, the K
d
of
the a sugar-binding site could not be calculated using
nonlinear regression fitting to the binding isotherm
because the protein concentration was too low to detect
the peak intensities accurately (Fig. S2). Therefore, the
K
d
values of the a sugar-binding site (residues Asp18,
Ser28, Trp33 and Gln44) were approximated to 0.01–
0.07 mm for lactose and 0.02–0.08 mm for b-Me-Gal.
This was similar to the previously reported K
d
value of
0.016 mm for lactose using total binding constants of
the two sugar-binding sites in EW29Ch, measured by
frontal affinity chromatography analysis [24].
For other sugars, the first signal corresponding to
the unbound state began to broaden at [sugar] ⁄
[EW29Ch]  0.5, the center of the broadened signals
shifted to the position of the second signal correspond-
ing to the bound state during titration ([sugar] ⁄

[EW29Ch] = 0.5–3) and the resonance signal of the
side-chain NH of Trp33 disappeared. The broadened
signals sharpened at the position of the second signal
at [sugar] ⁄ [EW29Ch] = 3–4 and the resonance signal
of the side-chain NH of Trp33 appeared at the posi-
tion of the second signal corresponding to the bound
state (Fig. 3B). Because the signals were in an interme-
diate exchange, of which the K
d
should have a medium
value between that in the slow exchange regime and
that in the fast exchange regime, titration data indi-
cated that the interaction with sugars had a K
d
of
 10
)1
mm. Furthermore, at the a sugar-binding site,
the binding specificity for an anomer was observed; the
chemical exchange for lactose and b-Me-Gal was in
the slow exchange regime, whereas that for melibiose
and a-Me-Gal, as anomers of lactose and b-Me-Gal,
was in the intermediate exchange regime. Thus, the
configuration at the hemiacetal carbon of galactose
may affect the dissociation constants.
By contrast, because chemical-shift changes upon
the addition of sugars at subdomain c were in the fast
exchange regime, K
d
values describing the interaction

of lactose, melibiose, galactose, a-Me-Gal and b-Me-
Gal with EW29Ch were calculated using nonlinear
least-square fitting of the chemical shift titration data
to the binding isotherm [27]. A plot of the weighted
average chemical-shift changes of
1
H and
15
N reso-
nances for the cross-peaks of Gly122, as a function of
the molar ratio of each sugar to EW29Ch, is shown in
Fig. 4. The K
d
values for each sugar were calculated
from the titration curves measured for the main chain
and amide proton groups of Ile102, Ile104, Cys115,
Trp117, Lys118 and Gly122, and the side chain nitro-
gen and amide proton group of Asn124 in the c sugar-
binding site. These residues, which exhibited the most
significant perturbations in the
15
N and amide proton
chemical shifts upon sugar binding, all lie within or
adjacent to the sugar-binding sites of EW29Ch identi-
fied by the crystal structure of the complex. Average
c-site dissociation constants calculated for each sugar
Table 1. Average site-specific dissociation constants calculated for
EW29Ch with sugar ligands. Data obtained at 25 °C and pH 6.1 in
50 m
M of potassium phosphate and a 10% D

2
O ⁄ 90% H
2
O mix-
ture. The chemical exchange regime in parentheses is based on
the observed alterations in NMR signal positions and intensities.
Sugar
K
d
(mM)
a Site
a
c Site
b
Lactose 0.01–0.07 (slow) 2.66 ± 0.30 (fast)
Melibiose  10
)1
(intermediate) 5.34 ± 0.81 (fast)
Galactose  10
)1
(intermediate) 3.89 ± 0.37 (fast)
a-Me-Gal  10
)1
(intermediate) 4.48 ± 0.38 (fast)
b-Me-Gal 0.02–0.08 (slow) 2.88 ± 0.21 (fast)
a
Dissociation constants could not be calculated accurately due to
the slow or intermediate exchange regime, so the K
d
is shown as

an approximation.
b
The reported K
d
values are the average of the
those determined from the
15
N and H
N
chemical shift perturbations
of Ile102, Ile104, Cys115, Trp117, Lys118, Gly122 and Asn124. The
error range is the standard deviation.
Interaction of EW29Ch with sugars H. Hemmi et al.
2098 FEBS Journal 276 (2009) 2095–2105 ª 2009 The Authors Journal compilation ª 2009 FEBS
were analyzed in accordance with a simple model of
each of the two sugar-binding sites in EW29Ch inter-
acting with one sugar molecule in an independent or
non-cooperative manner, because this assumption was
supported by evidence (monophasic changes in chemi-
cal shifts upon the addition of each sugar and crystal-
lographic studies of the protein-sugar complex) similar
to that reported by Scha
¨
rpf et al. [28]. The K
d
values
for the c sugar-binding site were 2–6 mm for all sugars
in this study (Table 1), so these results indicated that
the a sugar-binding site of EW29Ch is a high-affinity
site and the c sugar-binding site is a low-affinity site.

Interactions of sugars with EW29Ch by
STD–NMR experiments
STD–NMR experiments were conducted to determine
the binding epitope of lactose and b-Me-Gal to
A
B
Fig. 3. Close-up of the
1
H–
15
N HSQC regions showing the chemical exchange for Asp18 or Trp33sc with increasing amounts of some sug-
ars. Peak movements of main chain amide and amide proton of Asp18, and side chain amide and amide proton (marked by ‘sc’) of Trp33 in
EW29Ch during the titration of lactose (A, top), b-Me-Gal (A, bottom), melibiose (B, top) and a-Me-Gal (B, bottom). Shown are regions of the
1
H–
15
N HSQC spectra corresponding to Asp18 and Trp33sc in EW29Ch at [sugar] ⁄ [EW29Ch] molar ratios indicated at the top. The behavior
of Asp18 and Trp33sc during the titration of lactose and b-Me-Gal corresponds to a slow exchange regime (A) and those during titration of
melibiose and a-Me-Gal correspond to an intermediate exchange regime (B).
H. Hemmi et al. Interaction of EW29Ch with sugars
FEBS Journal 276 (2009) 2095–2105 ª 2009 The Authors Journal compilation ª 2009 FEBS 2099
EW29Ch because NMR titration experiments of
EW29Ch with sugars showed that the chemical
exchange of the a sugar-binding site upon the addi-
tion of lactose or b-Me-Gal was in the slow exchange
regime. The STD effect for sugar arose from the con-
tribution of both the a and c sugar-binding sites
because of the mixture of lactose and EW29Ch at a
ratio of 100 : 1. At first, 2D STD–TOCSY and 2D
STD–[

1
H,
13
C] HSQC spectra were obtained for
lactose with EW29Ch to assign the STD–NMR signals
completely, because the proton chemical shifts of the
galactose and glucose residues in lactose partly over-
lap. In the STD–TOCSY and STD–[
1
H,
13
C] HSQC
spectra, the H1-Gal, H2-Gal, H3-Gal, H4-Gal,
H5-Gal and H6-Gal resonances were assigned unambi-
guously (Fig. 5). In both 2D spectra, resonance signals
from the glucose residue in lactose were not observed.
However, the crystal structure of EW29Ch with
lactose showed that the glucose residue of the lactose
molecule interacted with subdomain c of EW29Ch
[25]. This interaction may be an artifact caused by the
crystallization of lactose-liganded EW29Ch because:
(a) in the other EW29Ch molecule of the crystal
structure (each crystal contained two molecules A and
B) the interaction between the glucose residue and
subdomain c of EW29Ch was not observed; (b) the
B-factor of the side chain of Lys105 was high, indicat-
ing that the side chain of Lys105 is flexible; (c) in this
NMR study, the K
d
of the c sugar-binding site for

b-Me-Gal was the same as that for lactose; and (d)
the STD–NMR data in this study showed that the epi-
tope of lactose for EW29Ch is the galactose residue.
Thus, these results showed that both sugar-binding
sites of EW29Ch only interact with the galactose
residue.
1D
1
H STD–NMR was conducted for the
EW29Ch–b-Me-Gal complex to quantitatively analyze
the epitopes of sugars interacting with the protein,
because the resonance signals of the glucose residue
overlapping with those of the galactose residue within
the lactose affected the subtraction of the free induction
0
0.1
0.2
0.3
0.4
0
.5
0 50 100 150
Δav (p.p.m.)
[su
g
ar] / [EW29Ch]
Fig. 4. Dissociation constants (K
d
) of EW29Ch for some sugars. K
d

values of EW29Ch for some sugars were determined by nonlinear
regression fitting of the chemical-shift change versus the sugar
concentration to the binding isotherm describing the binding of one
ligand molecule to a single protein site using the Solver function of
EXCEL 2002. The weighted average of the
1
H and
15
N chemical-shift
changes of Gly122 given by D
av
={(D
NH
2
+ D
N
2
⁄ 25) ⁄ 2}
1 ⁄ 2
[50] is
plotted as a function of sugar ⁄ protein molar ratios of added lactose
()), melibiose (h), galactose (4), a-Me-Gal (·) and b-Me-Gal (s).
Fig. 5. 2D STD–TOCSY and STD–[
1
H,
13
C] HSQC spectra for the
mixture of lactose (5 m
M) and EW29Ch (50 lM). (A) Reference
TOCSY spectrum of the mixture of lactose and EW29Ch at a ratio

of 100 : 1. (B) The STD–TOCSY spectrum of the same sample was
collected in an alternative fashion. (C) STD–[
1
H,
13
C] HSQC spec-
trum at a 10-fold higher concentration of the same sample.
Interaction of EW29Ch with sugars H. Hemmi et al.
2100 FEBS Journal 276 (2009) 2095–2105 ª 2009 The Authors Journal compilation ª 2009 FEBS
decay values with on- and off-resonance protein satu-
ration. Figure 6 shows the 1D
1
H NMR spectrum of
b-Me-Gal incubated with EW29Ch at a ratio of
100 : 1, and the corresponding 1D STD spectrum.
The integral value of the H4 proton, the largest signal
of b-Me-Gal, was set to 100%. Figure 6C shows the
relative degree of saturation of individual protons
normalized to that of H4. The H3, H5, H6a and H6b
protons had similar STD intensities of between 41%
and 54%. The H2 proton had a smaller STD intensity
of 30%. The lowest intensities corresponded to the
H1 proton and protons of the O-methyl group, which
reached only 18% and 11%, respectively. These
results indicated that EW29Ch recognizes the region
from H2 to H6a ⁄ H6b, particularly H4, and barely
interacts with the region of H1 and the O-methyl
group. The crystal structure of EW29Ch with lactose
showed that the O2, O3 and O4 atoms of the galac-
tose residue of lactose formed hydrogen bonds with

EW29Ch, and the C3, C4, C5, C6, O3 and O6 atoms
of the galactose residue formed stacking interactions
with both the a and c sugar-binding sites of EW29Ch
[26]. Therefore, our results confirmed that GalO2–
GalO6 of the galactose residue are epitopes for bind-
ing to EW29Ch.
Discussion
Binding of an individual lectin site (monovalent bind-
ing) to a monosaccharide is extremely weak, with K
d
values typically in the range of 0.1–10 mm [29–31]. In
the R-type lectin family, the dissociation constants of
ricin and RCA120 have been determined mainly by
equilibrium dialysis studies and fluorescence polariza-
tion studies [32–42]. Ricin has at least two binding
sites in its molecule. The K
d
values of ricin for lactose
at 4 °C are  0.03 mm for the high-affinity site and
 0.3 mm for the low-affinity site. Recently, a third
binding site has been found in ricin; thus, the ricin B1
domain of the ricin B chain has two sugar-binding
sites in the same molecule [22,23]. Because sugars
bound at the third sugar-binding site of the ricin B
chain were not observed, it is speculated that K
d
for
the third sugar-binding site of the ricin B1 domain is
more than one order of magnitude larger than that for
the low-affinity site of ricin, like the K

d
value for the
c sugar-binding site of EW29Ch. In this study, K
d
of
the a sugar-binding site of EW29Ch for lactose at
25 °C was 0.01–0.07 mm and that of the c sugar-bind-
ing site was 2.66 mm (Table 1). K
d
for the a sugar-
binding site of EW29Ch was almost the same as that
for the high-affinity sites of ricin and RCA120,
whereas K
d
for the c sugar-binding site of EW29Ch is
very similar to that for the third binding site of the
ricin B chain and has the lowest binding ability.
Although previous structural studies using X-ray
crystallography have clearly shown the mechanism of
galactoside-binding to the two binding sites, it is still
unclear why one site binds lactose more strongly [2].
In this study, we observed slight differences between
the binding modes of the a and c sugar-binding sites
in EW29Ch from the sugar complex structure of
EW29Ch [25]. The residue at the a sugar-binding site,
Gln22, interacts with GalO2 of lactose, but the corre-
sponding residue in the c sugar-binding site was not
observed. Lys36 in subdomain a is replaced by His120
in subdomain c, and one hydrogen bond toward the
O2 atom was deduced. These results suggested that the

a sugar-binding site has a tighter interaction with lac-
tose than the c sugar-binding site because of the num-
ber of intermolecular hydrogen bonds and of residues
interacting with lactose. Our results agreed well with
those from the crystal structure of the complex
between EW29Ch and lactose [25]. However, it
remains unclear why the a sugar-binding site binds to
lactose much more strongly. Future studies will aim to
determine both the refined sugar-free structure and
the refined complex structure of EW29Ch with lactose
in a solution state by using residual dipolar coupling
Fig. 6. 1D STD–NMR spectrum for the mixture of b-Me-Gal (5 mM)
and EW29Ch (50 l
M). (A) Reference NMR spectrum of the mixture
of b-Me-Gal and EW29Ch at a ratio of 100 : 1. (B) STD–NMR spec-
trum of the same sample. Prior to acquisition, a 30 ms spin–lock
pulse was applied to remove residual protein resonance. (C) Struc-
ture of b-Me-Gal and the relative degree of saturation of individual
protons normalized to that of the H4 proton as determined from
the 1D STD–NMR spectrum (B).
H. Hemmi et al. Interaction of EW29Ch with sugars
FEBS Journal 276 (2009) 2095–2105 ª 2009 The Authors Journal compilation ª 2009 FEBS 2101
constants by NMR to analyze the interaction between
the protein and lactose in a solution state.
As mentioned above, one of two sugar-binding sites
of EW29Ch, the a sugar-binding site, has a tight bind-
ing mode, but the c sugar-binding site has a weak
binding mode. This manner of binding reflected the
dissociation constants of EW29Ch in previous frontal
affinity chromatography analysis and corresponds to

that of the higher binding site by NMR, even if the
sugar-binding ability of one of the two binding sites is
much higher than that of the other. However, the
hemagglutinating activity of lectin is related to its mul-
tivalency for the cross-linking of cells. This means that
the hemagglutinating activity depends on the weaker
of the two sugar-binding sites because both must bind
to sugars on the surface of cells to cross-link and
agglutinate cells. Consequently, EW29Ch retains its
hemagglutinating activity but at level 10-fold lower than
that of the whole protein, whereas EW29Ch binds to
asialofetuin–agarose as strongly as the whole protein.
A common feature of lectins is their multivalent
binding properties. As a consequence, lectin binding to
cells leads to cross-linking and aggregates of glycopro-
tein and glycolipid receptors. Thus, the carbohydrate
cross-linking properties of lectins are a key feature of
their biological activities [30,43,44]. R-type lectins are
reported to have physiological functions such as
enzyme targeting and glycoprotein hormone turnover
[45]. The physiological function of EW29, however,
remains unknown. Clarification of the sugar-binding
ability and specificity of the two sugar-binding sites,
which was determined by NMR titration studies and
STD–NMR experiments, is expected to provide clues to
understand the precise physiological function of EW29.
Experimental procedures
Sample preparation
15
N-labeled or

13
C,
15
N-labeled EW29Ch was expressed and
purified using
15
N-labeled or doubly labeled CHL medium
(Chlorella Industry Co., Tokyo, Japan) as described else-
where [26]. In this study, purified EW29Ch was dialyzed in
distilled water many more times than had been done
previously, because a pair of resonance signals in the sugar-
free state and the sugar-bound state were observed for
some residues in the a subdomain owing to lactose
contamination from affinity chromatography using lactose–
agarose. The final product contains the full-length 131
amino acid EW29Ch sequence of Lumbricus terrestris (resi-
dues Lys130)Glu260 in EW29) [1], with an N-terminal
methionine residue (total length of 132 amino acids).
Residues are numbered from the N-terminal methionine
residue (Met1–Lys2–Pro3 ). Residue Lys2 of EW29Ch in
this study corresponds to residue Lys130 of EW29 or
residue Lys130 of the crystal structure of EW29Ch (PDB:
2ZQN or 2ZQO) [25].
NMR spectroscopy
Purified EW29Ch was dissolved in 50 mm of potassium
phosphate buffer (pH 6.1) and a protease inhibitor cocktail
(Sigma Chemical Co, St Louis, MO, USA) in either a 90%
H
2
O ⁄ 10%

2
H
2
O mixture or 99.96%
2
H
2
O. The final con-
centration of the protein was 0.9 mm. NMR spectra were
acquired at 25 °C on Bruker DRX600 and Avance 800
NMR spectrometers. All
1
H dimensions were referenced to
internal 4,4-dimethyl-4-silapentane-1-sulfate, and
13
C and
15
N were indirectly referenced to 4,4-dimethyl-4-silapen-
tane-1-sulfate [46]. All multidimensional NMR spectra were
acquired in the phase-sensitive mode using the States–time-
proportional phase increment method [47] or the echo-anti-
echo mode [48]. Shifted sine-bell window functions were
applied to NMR data prior to zero-filling and Fourier
transformation. NMR data were processed using felix2000
software (Accelrys, San Diego, CA, USA) or the nmrpipe
package [49], and analyzed using sparky software (God-
dard and Kneller, sparky 3, University of California, San
Francisco, CA, USA).
1
H,

13
C and
15
N assignments were
obtained from standard multidimensional NMR methods
as described elsewhere [26].
Titration of EW29Ch with sugars monitored
by NMR
The binding of each of sugar; lactose, melibiose, galactose
(all from Wako Chemicals, Tokyo, Japan), a-Me-Gal and
b-Me-Gal (both from Seikagaku Co., Tokyo, Japan), to
EW29Ch at 25 °C (pH 6.1) was measured quantitatively
using
1
H–
15
N HSQC NMR spectroscopy. Each sugar stock
solution used in this study was prepared by weight in a
sample buffer of 50 mm of potassium phosphate (pH 6.1).
Aliquots of these solutions (starting protein concentration
of 300–350 lm) were added directly to uniformly
15
N-
labeled EW29Ch contained in an NMR tube. For each
titration, 20
1
H–
15
N HSQC spectra were recorded consecu-
tively with increasing concentrations of each sugar. For the

progressive chemical-shift changes of EW29Ch under condi-
tions of fast exchange on the chemical-shift timescale,
15
N
and
1
H
N
chemical-shift changes in EW29Ch were calculated
using the equation D
av
={(D
NH
2
+ D
N
2
⁄ 25) ⁄ 2}
1 ⁄ 2
, where
D
NH
is the chemical-shift change of the amide proton and
D
N
that of the nitrogen [50]. Sugar-binding constants (K
d
)
were calculating using the Solver function of excel 2002
(Microsoft, Redmond, WA, USA) for the c sugar-binding

site by nonlinear regression fitting of the chemical-shift
change versus sugar concentration to the binding isotherm
Interaction of EW29Ch with sugars H. Hemmi et al.
2102 FEBS Journal 276 (2009) 2095–2105 ª 2009 The Authors Journal compilation ª 2009 FEBS
describing the binding of one ligand molecule to a single
protein site [27]. Similarly, assuming that sugar binding
to EW29Ch is a reversible single-step transition under
conditions of slow exchange on the chemical-shift timescale,
the dissociation constant, K
d
, is given by
K
d
¼½P½L=½PL
Here, [P], [L] and [PL] are the respective concentrations
of free EW29Ch, free sugar and the EW29Ch–sugar com-
plex. [P] ⁄ [PL] ratios were determined as a function of [L]
from free and bound peak intensities [51], because the two
signals in sugar-free and sugar-bound forms were observed
separately under the slow exchange regime. K
d
values were
also calculated using the Solver function of excel 2002 for
the a sugar-binding site by nonlinear regression fitting of
the ratio of free and bound peak intensities versus sugar
concentration to the binding isotherm. Throughout the
titration for calculating K
d
values under the slow exchange
regime, the concentration of EW29Ch was maintained at

0.05 mm and lactose or b-Me-Gal was added incrementally
from 0 to 0.15 mm.
STD–NMR experiments
Non-labeled EW29Ch was expressed using Luria–Bertani
medium and purified using the same method as the prepa-
ration of labeled EW29Ch [26]. To a sample of EW29Ch in
phosphate-buffered solution (50 mm of potassium phos-
phate buffer pH 6.1 and a protease inhibitor cocktail in
99.96%
2
H
2
O) was added lactose or b-Me-Gal. The final
sugar concentration was 5 mm at a sugar-to-protein ratio
of 100 : 1. 1D and 2D STD–NMR spectra were obtained
as described previously [52]. The time dependence of the
saturation transfer was determined by recording 1D STD
spectra with 1000 scans and saturation times from 0.25 to
6.0 s. The irradiation power in all STD–NMR experiments
was set to  0.15 W. Relative STD values were calculated
by dividing STD signal intensities by the intensities of the
corresponding signals in a reference spectrum of the same
sample recorded with 64 scans. All STD–NMR spectra for
epitope mapping were acquired using a series of equally
spaced 50 ms Gaussian-shaped pulses for saturation with
1 ms intervals and a total saturation time of  2 s. On-res-
onance irradiation of the protein was conducted at a chemi-
cal shift of –0.4 p.p.m. and off-resonance at a chemical
shift of 30 p.p.m. where no protein signal was present. Free
induction decay values with on- and off-resonance protein

saturation were recorded in an alternative fashion. Subtrac-
tion of the 1D STD spectra was achieved via phase cycling.
Protein resonance was suppressed by the application of a
30 ms spin–lock pulse before acquisition. 2D STD–TOCSY
and STD–[
13
C,
1
H] HSQC spectra at natural abundance
with on- and off-resonance protein saturation were
recorded with 128 scans or 512 scans per t
1
increment in an
alternative fashion. The 2D spectra were acquired with
spectra widths of 10 p.p.m. in
1
H and 80 p.p.m. in
13
C, and
128 (t
1
) and 2048 (t
2
) complex points or 64 (t
1
) and 1024
(t
2
) complex points for STD-TOCSY or STD-[
13

C,
1
H]
HSQC spectra. An MLEV mixing time of 100 ms was
applied in STD–TOCSY spectra.
Acknowledgements
We thank Drs Rintaro Suzuki and Toshimasa Yama-
zaki (National Institute of Agrobiological Sciences)
and Dr Chojiro Kojima (Nara Institute of Science and
Technology) for help in calculating sugar-binding
constants from NMR titration data. We also thank
Ms Sachiko Unno (AIST) for the preparation of the
proteins. This work was supported in part by Grant-
in-Aid for Scientific Research (C) (18580342 and
20580373) from the Japan Society for the Promotion
of Science (to HH and AK).
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Supporting information
The following supplementary material is available:
Fig. S1. Close-up of the
1
H–
15
N HSQC regions show-
ing the chemical exchange for Gly21 with increasing
amounts of some sugars.
Fig. S2. Dissociation constants (K
d
) of the a sugar-
binding site in EW29Ch for lactose (A) and b-Me-Gal
(B).
This supplementary material can be found in the

online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
H. Hemmi et al. Interaction of EW29Ch with sugars
FEBS Journal 276 (2009) 2095–2105 ª 2009 The Authors Journal compilation ª 2009 FEBS 2105