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

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.

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–

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-

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

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] ⁄

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

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.

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

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

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

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

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%

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

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

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

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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