Biochimica et Biophysica Acta 1780 (2008) 1131–1142

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Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

Caenorhabditis elegans galectins LEC-1–LEC-11: Structural features andsugar-binding properties
Yoko Nemoto-Sasaki a,⁎, Ko Hayama b, Hiroyuki Ohya a, Yoichiro Arata a,1, Mika Kato Kaneko c,
Naruya Saitou d, Jun Hirabayashi b, Ken-ichi Kasai aaDepartment of Biological Chemistry, Teikyo University School of Pharmaceutical Sciences, 1091-1 Suarashi, Sagamiko, Sagamihara, Kanagawa 229-0195, Japan
Lectin Application and Analysis Team, Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba 305-8568, Japanc
Glycogene Function Team, Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8568, Japand
Division of Population Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japanba r t i c l e

i n f oArticle history:
Received 5 March 2008
Received in revised form 28 June 2008
Accepted 7 July 2008
Available online 14 July 2008
Keywords:
Caenorhabditis elegans
Galectin
LectinFrontal afﬁnity chromatography
Carbohydrate-binding propertya b s t r a c t
Galectins form a large family of β-galactoside-binding proteins in metazoa and fungi. This report presents acomparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes (lec-2–5 and 8–11) from a
λZAP cDNA library. Among them, lec-2–5 were found to encode 31–35-kDa polypeptides containing two
carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8–11 were
found to encode 16–27-kDa polypeptides containing a single carbohydrate-recognition domain and a
C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1–4, -6, and 8–10 wereexpressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using afﬁnity
adsorbents with various β-galactosides, i.e., N-acetyllactosamine (Galβ1-4GlcNAc), lacto-N-neotetraose(Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and asialofetuin, demonstrated that LEC-1–4, -6, and -10 have a signiﬁcant
afﬁnity for β-galactosides, while the others have a relatively lower afﬁnity. These results indicate that the
integrity of key amino acid residues responsible for recognition of lactose (Galβ1-4Glc) or N-acetyllactosamine
in vertebrate galectins is also required in C. elegans galectins. However, analysis of their ﬁne oligosaccharidebinding properties by frontal afﬁnity chromatography suggests their divergence towards more specialized
functions.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
Galectins are a group of lectins that show β-galactoside-binding
activity and have at least one evolutionarily conserved carbohydraterecognition domain (CRD) [1]. They were ﬁrst found in the electricorgan of the electric eel in 1975 [2] and then identiﬁed in a wide range of
species, including invertebrates and fungi [3–6]. The ﬁrst invertebrate
galectin was identiﬁed in the primitive model animal Caenorhabditis
elegans, a nematode, as a 32-kDa protein (N32, LEC-1) consisting of two

Abbreviations: CRD, carbohydrate-recognition domain; FAC, frontal afﬁnity chromatography; Lac, lactose; LacNAc, N-acetyllactosamine; LN2, 3, and 5, N-acetyllactosamine oligomers (Galβ1-4GlcNAc)2, (Galβ1-4GlcNAc)3, and (Galβ1-4GlcNAc)5,
respectively; LNT, lacto-N-tetraose; LNnT, lacto-N-neotetraose; LNFP-I, II, and III, lactoN-fucopentaose I, II, and III, respectively; LNDFH, lacto-N-difucohexaose; NA2, 3, and4, biantennary, triantennary, and tetraantennary asialo type N-glycans, respectively;
ORF, open reading frame; PA, pyridylaminated⁎ Corresponding author. Tel.: +81 42 685 3741; fax: +81 42 685 3742.
E-mail address: ynsasaki@pharm.teikyo-u.ac.jp (Y. Nemoto-Sasaki).
1
Present address: Laboratory of Molecular Immunology, Faculty of Pharmaceutical
Sciences, Josai University, Sakado 350-0295, Japan.
0304-4165/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2008.07.003

CRDs. The CRDs showed 25–35% amino acid-sequence similarities tovertebrate galectin CRDs. This was also the ﬁrst study on a “tandemrepeat type” galectin [7]. Later, another nematode galectin (N16, LEC-6)
consisting of two identical 16-kDa subunits was isolated from theworm, classiﬁed as a “proto-type,” and characterized [8].
Thus far, 14 members of the galectin family have been identiﬁed in
mammals (e.g., human, mouse, rat, bovine, etc.) and named galectins-1–
10 and 12–15 in the order of identiﬁcation. The name galectin-11 is not
used at present [9]. Galectins are categorized into three structural types
on the basis of the global architecture (not sequence similarity), i.e.,“proto” (galectins-1, -2, -5, -7, -10, -13, -14, and -15), “chimera” (galectin3), and “tandem-repeat” types (galectins-4, -6, -8, -9, and -12) [9,10].
Galectins that have been characterized thus far have the following
properties in common: (1) they bind β-galactosides, e.g., N-acetyllactosamine (LacNAc; Galβ1-4GlcNAc); (2) they are soluble proteins with noapparent membrane-anchoring region; (3) they do not require metal
ions for sugar binding; and (4) they have characteristics distinctive oftypical cytoplasmic proteins, i.e., N-terminal acetylation, absence of asignal sequence for secretion, no glycosylation, preservation of freecysteine residue(s) rather than formation of disulﬁde bond(s) [4].

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With regard to structure and function, extensive studies onvertebrate galectins have established the presence of eight conserved
amino acids (His44, Asn46, Arg48, Val59, Asn61, Trp68, Glu71, andArg73; the numbers correspond to those of human galectin-1) thatplay a crucial role in the binding of sugars [11–18]. Therefore, it is ofevolutionary interest to know whether this holds true in the case of
invertebrate galectins. It is also important from a comparative
viewpoint to know the divergence of the galectin proteins ininvertebrates.
Based on the above concept, we initiated “comparative galectinomics” focusing on the nematode C. elegans (some data has beenpresented previously, [19,20]). The C. elegans genome [21] contains asigniﬁcant number of potential galectin genes, as previously suggested[22,23]. Thus far, however, it has not been investigated from acomparative standpoint to determine whether all of the “potential”
galectin genes are actually expressed in vivo and function as βgalactoside-binding lectins. In this study, we targeted 11 potential C.
elegans galectin genes (designated lec-1 to lec-11), including thosepreviously characterized (lec-1 and lec-6), for further functional
analysis. The analysis included the isolation of full-length cDNA
clones to compare their amino acid sequences and production ofrecombinant proteins to assess their sugar-binding properties. We
isolated full-length cDNA clones, except for lec-7. Although we failed
to produce a recombinant protein of LEC-5, we successfully produced
recombinant proteins of LEC-2–4 and LEC-8–11. By using afﬁnity
chromatography, we showed that LEC-2–4 and -10 are able to bind βgalactosides. Thus, it became evident that most potential C. elegans
galectin genes (lec-1–4, -6, and -10) are functional. Moreover, LEC-8, -9,
and -11 showed extremely weak afﬁnities for the β-galactosides
examined thus far, though they might have afﬁnities for othercarbohydrates besides those used in this study. The results clearly
indicated that β-galactoside-binding properties have been strongly
conserved across species.
2. Materials and methods
2.1. Database search for the C. elegans galectin-like genes
The TBLASTN search was performed for potential galectin genes in
the C. elegans genome database with amino acid sequences of LEC-1(GenBank accession number: M94671) and LEC-6 (D63575) as queries.
Among the genes showing signiﬁcant scores (E values b 0.1), those
encoding polypeptides containing at least four of the eight criticalamino acids (His44, Asn46, Arg48, Val59, Asn61, Trp68, Glu71, andArg73; residue numbers correspond to those of human galectin-1)
were considered for further analysis.
2.2. Reverse transcriptase-polymerase chain reaction
Total RNA was prepared from 0.2 g (wet weight) of wild-type
Bristol N2 strain (mixed stages) by using the SV Total RNA isolation
system (Promega), and reverse transcription (RT) was performed witha 1st-strand™ cDNA synthesis kit (Clontech). Appropriate sets ofinternal primers were designed to amplify the DNA fragments ofapproximately 0.2 kbp (for details, see Supplementary Table 1).
Polymerase chain reaction (PCR) was performed by using LA-Taq DNA
polymerase (Takara Bio), and the cycling conditions were as follows:
92°C, 30 s; 62°C, 30 s; and 72°C, 1 min. Thereafter, a ﬁnal extension
was conducted at 72°C for 10 min.
2.3. Screening of cDNAs of lec-2–5 and 7–11
The ampliﬁed 0.2-kbp cDNA fragments were used as probes toscreen a previously described λZAP cDNA library [24] after labelingwith [α-32P] dCTP using the Megaprime™ DNA labeling system(Amersham). Plaque hybridization was performed either indepen-dently (lec-2, -10, and -11) or in groups (lec-3–5 and 7–9) by mixing
relevant probes. In vivo excision for conversion to the plasmid
pBluescript was performed as previously described [25]. Colony PCR
was performed to screen positive clones. These positive clones were
subjected to sequence analysis by a conventional dideoxy method as
described previously [8]. To determine the trans-spliced leader
sequence in the 5′-untranslated region, nested PCR was performed
as described previously [26].
2.4. Prediction of hydrophobic regions and signal peptides
For prediction of hydrophobic regions and signal peptides, the
SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/), the SignalP ver. 3.0(http://www.cbs.dtu-dk/services/SignalP/), and the PSORT II programs(http://psort.ims.u-tokyo.ac.jp/) were used.
2.5. Phylogenetic analysis of LEC-1–11 and DC2.3a
The Clustal W program was used for multiple alignments [27] of the
LEC-1–11 and DC2.3a amino acid sequences, and the neighbor-joining
method [28] was used to construct a phylogenetic tree as described
previously [29]. To validate the conﬁdence levels in the constructed
tree, bootstrap analysis was performed. By bootstrap analysis, a total of1000 trees were generated from the initial data set, and the percentageof trees containing a particular clade was measured.
2.6. Production of recombinant proteins LEC-2–4 and 8–11
Full-length coding regions of the lec-2, -3b, -4, -8, -9, -10, and -11
genes were ampliﬁed by a standard PCR procedure. A plasmid
containing a full-length cDNA of each galectin-like gene was used asa template; the primers used are described in Supplementary Table 2.
In the case of lec-8, a truncated mutant LEC-8ΔC, which lacks a Cterminal tail (residues 142–180), was also constructed by using lec-8F
and lec-8ΔCR primers (Supplementary Table 2). Expression ofrecombinant proteins was performed as described previously[30,31]. Brieﬂy, ampliﬁed PCR fragments were ligated into a pET21aor pET21d vector (Novagen) that was digested with relevant
restriction enzymes (i.e., NdeI/XhoI or NdeI/BamHI). The resultant
expression constructs were used to transform Escherichia coli BL21
(DE3) cells. Production of recombinant proteins was induced by theaddition of isopropyl-β-D-thiogalactoside.
2.7. Preparative afﬁnity chromatography on asialofetuin-agarose
Preparative asialofetuin-agarose chromatography was performed
as described previously [30,31]. Brieﬂy, after induction of recombinant proteins (37°C for 4 h or 25°C for 16 h), E. coli cells were
harvested by centrifugation and disrupted by sonication (30 min, onice) with ﬁve volumes of MEPBS (4 mM β-mercaptoethanol, 2 mM
EDTA, 20 mM sodium phosphate, pH 7.2, 150 mM NaCl). Aftercentrifugation (15,000 rpm, 4°C, 25 min), the derived supernatantsolution was applied to an asialofetuin-agarose column (10 ml bed
volume, 9 mg fetuin immobilized per ml of agarose gel). Afterextensive washing of the column, the adsorbed protein was eluted
with MEPBS containing 20 mM lactose. The protein concentration
was determined and each fraction was subjected to analysis bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) using a 14% gel. In the case of experiments for LEC-8–11, Trisbuffered saline (TBS; 50 mM Tris–HCl, pH 7.5, 150 mM NaCl) wasused instead of MEPBS. For recombinant proteins, which have arelatively weak afﬁnity for asialofetuin, i.e., LEC-4, -8, -9, and -11,
retarded fractions containing these proteins were pooled, concentrated by ultraﬁltration using the Millipore Centricon Plus 20 (PL-10,
10,000 Da), and subjected to rechromatography on the same
column. In the cases of LEC-8, -10, and -11, the use of metal-chelate

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afﬁnity chromatography proved to be useful (Hayama, K.; unpublished data).
2.8. Frontal afﬁnity chromatography
The principle and basic procedures of frontal afﬁnity chromatography (FAC) have been described previously [31–36];
Kd ¼ Bt =Vf −V0 −½A0ð1Þwhere Kd is the dissociation constant (M) between the analyte (A inthis study; either recombinant galectins or pyridylaminated-oligosaccharides (PA-oligosaccharides)) and immobilized ligand (B in thisstudy; either glycans or recombinant galectins). Bt is the total amountof effectively immobilized ligand (mol), whereas [A]0 is the initialconcentration of the analyte A (M). Vf is the elution volume of A (ml),which can be determined as described previously [34] by monitoring
the ﬂuorescence signal based on either tryptophan (excitation andemission wavelengths, 285 and 350 nm, respectively) if A is arecombinant galectin or PA-oligosaccharides (excitation and emission
wavelengths, 310 nm and 380 nm, respectively) if A is a PAoligosaccharide. Under the assumption that [A]0 is small relative to
Kd, Eq. (1) is simpliﬁed into Eq. (2):
Kd ¼ Bt =ðVf −V0 Þð2Þ

For analytical afﬁnity chromatography, three types of column(4 × 10 mm; column volume, 0.126 ml) packed with either asialofetuinagarose (2 mg/ml gel), Synsorb 34 (N-acetyllactosamine, LacNAc;
Galβ1-4GlcNAc), or Synsorb 101 (lacto-N-neotetraose, LNnT; Galβ14GlcNAcβ1-3Galβ1-4Glc) were purchased from Wako Pure Chemicals,Tokyo, Japan. Binding of the puriﬁed recombinant proteins LEC-1, -2, 3b, -4, -6, -8ΔC, -9, and -10 to these columns was analyzed by FAC. FAC
was carried out as previously described [33,34]. Brieﬂy, 2 ml of
puriﬁed recombinant galectin solution was dissolved in EDTA-PBS at aconcentration of 1 mg/ml and applied to the columns. The elution of
the protein was monitored as described above. The ﬂow rate and thecolumn temperature were kept at 0.25 ml/min and 20°C, respectively.
To analyze the ﬁne oligosaccharide-binding properties, puriﬁed
recombinant proteins of LEC-2, -3b, -4, -8ΔC, -9, and -10 were
immobilized on HiTrap N-hydroxysuccinimide-activated (NHS-activated) Sepharose cartridge columns (1 ml; GE Healthcare Biosciences)according to the manufacturer's instructions at appropriate concentrations (1–7 mg/ml gel), as previously described [31]. PA-oligosaccharides were obtained as described previously [35]. The association
constants between the lectin-immobilized agarose beads and PAoligosaccharides were determined by the above FAC equation, as
previously described [31].

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encodes N16. Since the overall sequence similarity between lec-1 andlec-6 is relatively low (~ 25% identity), the observed overlap ismeaningful. These galectin-like genes are classiﬁed under the protein
family (Pfam, http://pfam.sanger.ac.uk/) with the identiﬁer PF00337.
Among the extracted candidate genes, we carefully examined thepreservation of key amino acids, i.e., His44, Asn46, Arg48, Val59,
Asn61, Trp68, Glu71, and Arg73 (numbers correspond to those ofhuman galectin-1), that likely form the sugar-binding site of the C.
elegans galectins. On the basis of this assumption, we chose thefollowing nine genes for further analysis: F52H3.7, ZK892.1, C44F1.3,
ZK1248.16, R07B1.2, R07B1.10, C16H3.2, W01A11.4, and F38A5.3.
According to GeneFinder prediction, F52H3.7, ZK892.1, C44F1.3, andZK1248.16 were strongly suggested to encode tandem-repeat-type
galectins like LEC-1, and were thus designated as lec-2–5, respectively.
On the other hand, R07B1.2, R07B1.10, C16H3.2, W01A11.4, andF38A5.3 were predicted to encode a single galectin CRD and were
therefore designated as lec-7–11 (Supplementary Table 3). We
excluded DC2.3 as a candidate from the cDNA cloning study becauseof errors in the predicted sequence. However, an updated prediction
revealed that this gene met the criteria, and the critical amino acids
were found to be conserved. Therefore, we used this sequence data forcomparison with the cloned cDNAs.
3.2. mRNA expression and cDNA cloning of lec-2–5 and 7–11
mRNA expression of the candidate galectin genes (lec-2–5 and 7–11) was examined by RT-PCR. Transcripts of lec-2, -4, -5, -8, -9, -10,and -11 were detectable after 30 cycles. However, transcripts of lec-3
and lec-7 were not detectable even after 35 cycles (Fig. 1).
After conﬁrming the expression of the candidate genes, we
attempted to isolate cDNA clones by screening a λ phage librarypreviously used [7,8]. The 0.2-kbp PCR fragments obtained wereradiolabeled and used as probes. Full-length cDNAs were successfully
obtained for lec-2, -4, -5, -8, -9, -10, and -11 using an initial phagesuspension containing 6 × 105 phages. However, no positive clone was
detected despite repeated trials for lec-3 and lec-7. In case of lec-3,
however, the entire coding region predicted for this gene could be
ampliﬁed by direct RT-PCR using lec-3F and lec-3R primers (Supplementary Table 2; data not shown). Further, no ampliﬁcation productwas obtained for lec-7 even after 35 cycles of ampliﬁcation.
Many mRNAs of C. elegans are known to attach to the trans-spliced
leader sequence at the 5′-end [37–39]. In our previous study, the lec-1
transcript has been shown to possess the 22-nucleotide consensus
trans-spliced leader sequence “SL1” at positions −52 to −31 (where“A” of the initiation codon ATG indicates position 1) [26]. Nucleotide
sequencing of lec-2, -4, and -5 cDNAs showed that all of them

3. Results
3.1. Novel galectin-like genes in C. elegans
The presence of many galectin-like genes other than lec-1(originally named N32) [7] and lec-6 (similarly named N16) [8] in C.
elegans was ﬁrst implicated by Cooper, D.N.W. (University ofCalifornia, San Francisco; personal communication) [22,23]. Our
TBLASTN analysis using the lec-1 sequence as the query resulted in
the identiﬁcation of a number of genes with signiﬁcant E values(b0.1): W09H1.6 (= lec-1), F52H3.7, ZK892.1, C44F1.3, ZK1248.16,
C53D6.7, DC2.3, R07B1.2, R07B1.10, C16H3.2, W01A11.4, T02G6.7,
F41D3.6, F38A5.3, F46A8.4, F49F1.11, F49F1.10, F49F1.9, and F46A8.3,
where W09H1.6 encodes N32. When the lec-6 sequence was used as
the query, the identiﬁed genes largely overlapped: i.e., Y55B1AR.1(=lec-6), ZK892.1, F52H3.7, W01A11.4, W09H1.6, R07B1.10, C16H3.2,
C44F1.3, ZK1248.16, DC2.3, F41D3.6, F46A8.3, F49F1.11, F46A8.4,
F49F1.10, T02G6.7, R07B1.2, F49F1.9, and F38A5.3, where Y55B1AR.1

Fig. 1. RT-PCR analysis of lec-1–11. To conﬁrm the mRNA expression of the C. elegans
potential galectin genes lec-1–11, forward and reverse primers were designed for eachgene, as described in Materials and methods. Approximately 0.2 kbp products wereanalyzed after 35 PCR cycles. The arrow indicates the position of the 0.2-kbp ampliﬁed
products.

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contained an SL1 sequence in their 5′-non-coding regions (Supplementary Fig. 1). RT-PCR with two nested reverse (antisense) primers
speciﬁc for either lec-3, -6, -8, -9, -10, or -11 and either SL1 or SL2
primers revealed that lec-3, -6, -9, -10, and -11 cDNAs contain the SL1
sequence (Supplementary Fig. 1). On the other hand, we failed todetect the trans-spliced leader sequence of lec-8. Since the obtained
lec-8 cDNA clone had only a 17-bp 5′-non-coding region (Supplementary Fig. 1), it is possible that lec-8 is also subjected to trans-splicing.
On the basis of the established cDNA sequences for lec-1–6 and 8–
11, genome organizations of these genes have been provided in
Supplementary Fig. 2. Exon/intron boundaries were in accordancewith the consensus “GT/AG” rule (data not shown).
Except for lec-3 (described below), all the cloned galectin-like genetranscripts were unique, and thus, each encoded only one distinct
amino acid sequence. Nucleotide sequences of lec-2–5 and 8–11
cDNAs, including the SL1 sequence, as well as the deduced amino acidsequences were registered in GenBank DNA databases. Accession
numbers are shown in Supplementary Table 3.
3.3. Splicing variants of lec-3
As described above, lec-3 cDNA was directly ampliﬁed by RT-PCR
by using forward (lec-3F) and reverse primers (lec-3R) designed on

the basis of the GeneFinder prediction. However, 5′- rapid ampliﬁcation of cDNA ends (RACE) analysis revealed that the lec-3 gene has anadditional splicing variant, designated lec-3b (the former transcript isnamed lec-3a hereafter). Except for the difference in the 5′-region,
both transcripts had the same sequence (Supplementary Fig. 1). These
splicing variants encoded polypeptides that differed only in their Nterminal amino acid sequences, i.e., MAEPKSF in LEC-3a andMPSVWHCAKYI in LEC-3b (Supplementary Fig. 1).
3.4. Deduced amino acid sequences of LEC-1–11 and DC2.3a
Amino acid sequences deduced from cloned cDNA structures areshown in Fig. 2. The cDNAs of lec-2, -3a, -3b, -4, and -5 encode
tandem-repeat-type polypeptides, consisting of 278, 297, 301, 283,
and 314 amino acids, respectively, similar to lec-1. The cDNA of lec-9
encodes a mono-CRD type polypeptide, similar to that encoded by lec6, with 140 amino acids. On the other hand, the cDNAs of lec-8, -10,and -11 encode 180, 192, and 232 amino acid proteins, respectively.
They have an N-terminal CRD and an unknown functional region at
the C-terminal, and thus, they are considered to be novel chimeratype galectin-like proteins. Although LEC-9 lacks a C-terminal tail, we
consider LEC-9 to be a member of the chimera-type galectin-likeproteins based on the amino acid similarity (See Discussion). The

Fig. 2. Amino acid-sequence alignments of LEC-1–11 and DC2.3a. Amino acid sequences deduced from the cDNA structures of lec-1–11 were aligned to produce maximal identity by
the Clustal W program. Tandem-repeat domains of LEC-1–5 are separately aligned. Sequences of LEC-7 and DC2.3a reported by the C. elegans sequencing consortium have also been
included for comparison. Highly conserved and similar amino acids are shown as white characters on a black and shaded background, respectively, using BOXSHADE program. The
eight highly conserved residues (H, N, R, V, N, W, E, and R), which have been shown to play an important role in the binding of sugars, are denoted by asterisks. The position of aconsensus tripeptide for adhesion in LEC-4N, LEC-5N, and LEC-11 is denoted as “RGD.” The potential signal peptide of LEC-5 is underlined. The positions of the β-strands forming two
β-sheets (S1–S6 and F1–F5) of the N-terminal CRD of LEC-1 based on the X-ray crystal structure [44] are shown at the top of the sequences. Hydrophobic segments observed in Cterminal tail regions of LEC-10 and -11 are underlined with wavy lines.

Y. Nemoto-Sasaki et al. / Biochimica et Biophysica Acta 1780 (2008) 1131–1142

deduced amino acid sequences of LEC-7 and DC2.3a registered by theC. elegans sequencing consortium are also included for comparison in
Fig. 2.

Table 1Conservation of critical amino acids and binding to asialofetuin
Critical amino acids

3.5. Predicted signal peptide of LEC-5
LEC-1

Galectins identiﬁed to date do not have any signal peptides, except
for a few exceptions such as LEC-5 [23]. Moreover, Kaji et al. [40] andFan et al. [41] recently identiﬁed LEC-5 as an N-linked glycoprotein.
Therefore, we searched for signal peptides and transmembraneregions within the deduced amino acid sequences of the cloned
galectin-like genes using the programs SOSUI, PSORT, and SignalP. The
SOSUI program predicted that LEC-1–11 are soluble proteins. The
PSORT program predicted that LEC-5 had a signal peptide, and thepredicted cleavage site was between Cys17 and Gln18 at the Nterminal. No other genes examined were predicted to have signal
peptides. The same results were obtained by SignalP-NN analysis. The
SignalP-HMM program also predicted that LEC-5 had a signal peptide,
but the predicted cleavage site was between Gly19 and Asp20.

LEC-2
LEC-3
LEC-4
LEC-5
LEC-6
LEC-7b
LEC-8
LEC-9
LEC-10
LEC-11DC2.3aba3.6. Phylogenetic relationship of LEC-1–11 and DC2.3abc

The deduced amino acid sequences of LEC-1–11 and DC2.3a werealigned by Clustal W and a phylogenetic tree was constructed (Fig. 3).
The N-terminal and C-terminal CRDs of LEC-1–5 were separately
analyzed. The polypeptides were classiﬁed into three groups: (i)tandem-repeat-type galectin-like proteins LEC-1, -2, -3, -4, and -5; (ii)
LEC-6, -7, -8, -9, -10, and DC2.3a; and (iii) LEC-11. We constructed thephylogenetic tree using the lec-11 gene as an outgroup. In thesubgroup (i), the N-terminal and C-terminal CRDs were separately
clustered. This result indicates that these CRDs were generated
tandemly in the ancestral gene of this subgroup. After divergence ofthe lec-1, -2, -3, -4, and -5 genes, lec-1, -2, and -3 showed relatively
slower evolutionary rates than those of the lec-4 and -5 genes. The
topologies of the N-terminal and C-terminal CRDs of LEC-1, -2, and -3
are not the same in the tree. However, the bootstrap value at the nodeconnecting the N-terminal CRD of LEC-1, -2, and -3 is very low (35%)

1135

N-CRD
C-CRD
N-CRD
C-CRD
N-CRD
C-CRD
N-CRD
C-CRD
N-CRD
C-CRD

H.

N.

R...

V.

N...

W..

E.

R

H.
H.
H.
H.
H.
H.
H.
H.
H.
Q.
H.
H.
H.
H.
H.
H.
H.

S.
N.
S.
N.
S.
N.
K.
N.
S.
S.
N.
K.
N.
N.
N.
S.
N.

R...
R...
R...
R...
R...
R...
N...
R...
R...
R...
R...
E...
R...
R...
R...
R...
R...

V.
I.
V.
V.
V.
V.
I.
V.
I.
V.
V.
V.
A.
A.
V.
V.
V.

N...
N...
N...
N...
N...
N...
N...
N...
N...
N...
N...
N...
N...
N...
N...
N...
N...

W..
W..
W..
W..
W..
W..
W..
W..
W..
W..
W..
W..
W..
W..
W..
W..
W..

E.
E.
E.
E.
E.
E.
E.
E.
D.
E.
E.
Q.
E.
E.
E.
E.
E.

R
R
R
R
R
R
R
R
Q
R
R
R
R
S
R
H
R

Number of
substitution

Relative afﬁnity
for asialofetuina

1
1
1
0
1
0
3
0
4
2
0
3
1
2
0
2
0

100
112
122
67
NTc
198
NTc
21
9
137
NTc
NTc

Data from the results of Supplementary Fig. 4A are shown.
LEC-7 and DC2.3a are included for comparison. See Supplementary Table 3.
NT, not tested.

and that at the C-terminal CRD is also relatively low (52%). These three
genes appeared to have originated at the same time.
3.7. Production and puriﬁcation of recombinant galectin-like proteins
To investigate the sugar-binding properties of the C. elegans
galectin-like proteins, recombinant proteins were expressed in E. coli,
and their soluble extracts were applied to a column of asialofetuin
agarose, as in the previous cases of LEC-1 [7] and LEC-6 [8]. We
successfully produced recombinant proteins of LEC-2, -3b, -4, -8, -9, -10,and -11, but failed to produce a recombinant LEC-5 protein. Aliquots of
ﬂow-through, retarded, and adsorbed fractions were subjected to SDSPAGE to verify the binding ability of individual galectin-like proteins toasialofetuin. LEC-2, -3b, and -10 were found to be strongly adsorbed on
the column and were eluted speciﬁcally with lactose (Supplementary
Fig. 3). A considerable portion of proteins leaked from the column
before lactose elution in the cases of LEC-4, -8, and -11 (Supplementary
Fig. 3). LEC-9 was shown to have the lowest afﬁnity for asialofetuin
because it was scarcely adsorbed on the column. Nevertheless, its
elution was signiﬁcantly retarded when compared with the bulk of theE. coli proteins.
In this study, a deletion mutant LEC-8ΔC lacking the C-terminal tailregion was also used because its intact form (intact LEC-8) was very
sensitive to proteolysis (data not shown). However, the results for intact
LEC-8 and LEC-8ΔC were almost the same (data of LEC-8 not shown). The
above galectin-like proteins (LEC-1, -2, -3b, -4, -6, -8ΔC, -9, -10, and -11)
were puriﬁed to an apparent homogeneity by single or repeated steps ofasialofetuin-agarose chromatography.
3.8. Analytical afﬁnity chromatography

Fig. 3. A phylogenetic tree of the C. elegans galectin family. The phylogenetic tree wasconstructed as described under Materials and methods. For the analysis of tandemrepeat-type galectin-like proteins LEC-1–5, independent analysis was performed for Nterminal CRDs and C-terminal CRDs. The numbers on the individual branches of the tree
indicate bootstrap values. The scale bar corresponds to 0.1 substitutions per site.

In order to examine the sugar-binding ability of LEC-1–11
quantitatively, FAC [32] was performed with a recently reinforced
system using a miniature column packed with asialofetuin-agarose,and the elution fronts were analyzed as described previously [33,34].
The obtained results (Supplementary Fig. 4A) were consistent with the
behavior of these proteins in preparative experiments using asialofetuin-agarose (Supplementary Fig. 3), and were found to be associatedwith the integrity of the critical amino acids demonstrated in the caseof vertebrate galectins (His44, Asn46, Arg48, Val59, Asn61, Trp68,
Glu71, and Arg73; numbers correspond to those of human galectin-1),
as shown in Table 1. LEC-6 and -10, in which these amino acids are1136

Y. Nemoto-Sasaki et al. / Biochimica et Biophysica Acta 1780 (2008) 1131–1142

completely conserved, showed the highest afﬁnity for asialofetuin.
LEC-9, which has two substitutions, showed signiﬁcant but poor
afﬁnity. LEC-4 has three substitutions in the N-terminal CRD, whereas
its C-terminal CRD is perfectly conserved. It showed intermediate
afﬁnity. Therefore, galectins with the eight critical amino acids were
found to retain their sugar-binding afﬁnity toward asialofetuin.
However, since fetuin carries distinct sugar chains, i.e., three Nlinked [42] and three O-linked oligosaccharides [43], it is not clear
whether the above discussion is applicable to only complex-type Nglycans or can be extended to more extensive β-galactosides. To clarify

this point, we also analyzed the binding afﬁnity of individual galectins
for immobilized N-acetyllactosamine (LacNAc, Galβ1-4GlcNAc) andlacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc). The
results were largely consistent with each other and also with that of
the asialofetuin-agarose experiment (Supplementary Fig. 4). However,there were some differences: (1) LEC-2 showed a relatively lower
afﬁnity for both LacNAc and LNnT relative to LEC-1. (2) LEC-6 showed ahigher afﬁnity for LacNAc than LEC-1, while the former had a relatively
poor afﬁnity toward LNnT. These differences may reﬂect specializedfeatures of individual galectins, as described before [35].

Fig. 4. Schematic representation of PA-oligosaccharides used in FAC analysis. Note that the reducing terminals are pyridylaminated for FAC analysis. Symbols used to representmonosaccharides are shown in the inset.

Y. Nemoto-Sasaki et al. / Biochimica et Biophysica Acta 1780 (2008) 1131–1142

3.9. Fine oligosaccharide-binding properties of LEC-1, -2, -3b, -4, -6,
and -10
We have reported the detailed oligosaccharide-binding properties ofextensive galectins, including LEC-1 [31], LEC-6, and galectins from otherspecies [35] by FAC using a panel of 41 PA-oligosaccharides (for structures,
see Fig. 4). In this study, we extended the FAC analysis to other membersof the C. elegans galectin family, i.e., LEC-2, -3b, -4, -8, -9, and -10, whichwere obtained in sufﬁcient amounts as soluble forms. Among the tested
proteins, LEC-2, -3b, -4, and -10 showed signiﬁcant afﬁnity for some of thePA-oligosaccharides used in this study. For convenience, the sugarbinding afﬁnities of these C. elegans galectins, including the previously
characterized LEC-1 and LEC-6, were compared (Table 2, Fig. 5).
3.9.1. Conserved features of all LECs
All LECs and CRDs shown in Table 2 and Fig. 5 recognized basic
oligosaccharides such as lacto-N-neotetraose (LNnT, 26), lacto-N-tetraose(LNT, 27), and a complex-type N-glycan with biantennary Galβ1-4GlcNAc(NA2, 01).
To examine the effect of the number of branching, afﬁnities for tri(NA3, 02) and tetraantennary glycans (NA4, 04) were compared withthose for a biantennary (NA2, 01) glycan. Relative afﬁnities withrespect to LNnT (26) are shown in Table 3. All LECs and CRDs showed

1137

higher afﬁnities for the tri- (02) and tetraantennary glycans (04) thanfor the biantennary (01) glycan. LEC-3b and -6 showed much higher
afﬁnities for the tetraantennary (04) glycan than for the triantennaryglycan (02) (Table 3).
Next, the effect of the number of repeats of N-acetyllactosamine wasanalyzed. Relative afﬁnities of (LacNAc)2 (39), (LacNAc)3 (40), and(LacNAc)5 (41) to LNnT (26) are shown in Table 3. All the LECs and CRDs
examined, except for LEC-10, exhibited enhanced afﬁnities as the numberof repeats increased. Afﬁnities of LEC-10 for these oligolactosamines were
low, and no signiﬁcant difference was observed.
Substitutions at the 6-OH groups of nonreducing terminal galactoses
with sialic acids (01 vs. 10–12) eliminated their binding afﬁnities (Table 4).
On the other hand, α1–2 fucosylation of nonreducing terminal galactose of
LNT increased (LEC-1N, -3b, and -10) or did not change (LEC-1, -1C, -2, -4,
and -6) the afﬁnity (27 vs. 28) (Table 4).
Substitutions at 3-OH groups of the GlcNAc residues of Galβ14GlcNAc (type 2 chain) by fucose in complex-type N-glycans reduced
their binding afﬁnities (02 vs. 05, 04 vs. 06) (Table 2, Fig. 5). The
decrease in afﬁnity caused by α1–3 fucosylation was also seen at the
GlcNAc residue of the type 2 chain in lacto-N-neotetraose (LNnT) (26vs. 30) (Table 2, Fig. 5). In the case of α1–4 fucosylation at the GlcNAc
residue of Galβ1-3GlcNAc (type 1 chain) in lacto-N-tetraose (LNT), theafﬁnity decreased (LEC-1, -1N, -1C, -4, -6, and -10) or remained

Table 2
Dissociation constants (Kd's) determined by frontal afﬁnity chromatography for PA-oligosaccharides and galectinsa
PA-sugar no.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
a

LEC-1b

Trivial name

NA2NA3NA3 (type 1)
NA4NA3 (LeX)
NA4 (LeX)
NA2 (1-6Fuc)
NA3 (1-6Fuc)
NA4 (1-6Fuc)
NA2 (monosialo)
NA2 (monosialo)
NA2 (disialo)
GA3 (lactose)
GA2
GA1
GM3
6′-Sia-Lac
GM2
GM1
GD1a
GD1b
Gb3
Gb4
Forsmann penta
Galili penta
LNnT
LNT
LNFP-ILNFP-IILNFP-III
LNDFH
A-hexaA-hepta2′-Fuc-Lac
α-Gal-Lac
B-tetraB-pentaA-tetra
LN2
LN3
LN5

Whole

LEC-1N

LEC-1C

220
100
80
110
230
230
230
120
110
960
–
–
–
–
680
–
–
–
880
–
–
–
530
–
54
210
91
60
300
780
240
58
250
–
290
190
–
–
120
68
31

680
370
260
380
920
780
580
370
370

150
84
52
77
210
150
200
82
78
950
1300
–
–
–
670
–
–
–
1100
–
–
–
500
–
100
200
77
65
240
890
190
810
170
–
97
270
–
–
97
51
18

–
–
–
–
–
–
–
–
–
–
–
–
–
2900
–
72
1000
440
130
–
–
–
48
1800
–
420
350
–
–
940
590
350

Kd's are given in μM.
Data from our previous report are included for comparison (Ref. [31,35]).
c
NT, not tested.
b

LEC-2

LEC-3

LEC-4

LEC-6b

LEC-10

61
26
20
23
70
58
51
25
21
110
–
–
–
–
740
–
NTc
–
–
–
–
–
–
300
17
140
35
36
38
910
12
24
12
–
680
340
–
–
NTc
19
10

300
110
100
41
100
240
300
140
100
1000
1400
–
–
–
1900
–
–
–
3800
–
–
3800
2100
700
70
630
250
90
230
–
570
37
650
3800
440
77
–
330
430
230
170

74
26
22
27
80
55
71
30
25
280
350
4100
600
890
56
1800
–
1300
54
540
86
–
130
44
170
25
83
65
570
630
780
72
830
500
92
33
600
360
210
110
40

93
40
36
27
100
54
82
46
38
590
710

130
70
81
63
140
110
130
78
60
470
550

–
2000
–
580
3400
–
–
630
–
1600
–
1300
3400
160
320
190
170
–
–
–
1600
–
3800
850
1500
–
–
370
230
140

–
5500
–
–
–
–
–
2700
–
–
–
–
470
130
290
320
120
–
–
2700
75
2700
1800
4300
860
–
240
1100
1200
1400

1138

Y. Nemoto-Sasaki et al. / Biochimica et Biophysica Acta 1780 (2008) 1131–1142

Fig. 5. Comparison of ﬁne oligosaccharide-binding properties of the C. elegans galectins, LEC-1, -1N, -1C, -2, -3b, -4, -6, and -10. Puriﬁed recombinant galectins were immobilized on
NHS-activated Sepharose 4FF, and FAC analysis was performed with 41 PA-oligosaccharides (Fig. 4), as described in Materials and methods. Association constants were calculated
from the results shown in Table 2. Note that the ordinate scales are variable, reﬂecting the overall binding strengths of galectins. ⁎Not tested.

unchanged (LEC-2 and -3b) (27 vs. 29) (Table 4). Fucosylation of the
reducing terminal GlcNAc of complex-type N-glycans had no effect(01 vs. 07, 02 vs. 08) (Table 2, Fig. 5).

A-hexasaccharide (32) and B-tetrasaccharide (36) were observed ascompared to those of the other LECs and CRDs (Table 2, Fig. 5).

3.9.2. Specialized features of individual LECs

LEC-4 exhibited a strong afﬁnity for the oligosaccharides GA1 (15),GM1 (19), and GD1b (21) containing Galβ1-3GalNAc at the nonreducing terminal, while the other LECs and CRDs used in this study
showed a weak or no afﬁnity (Table 2, Fig. 5). LEC-4 also strongly
recognized the Forssman pentasaccharide (24), which was weakly
recognized or not recognized by other LECs or CRDs. In contrast, LEC-4
showed a relatively weak afﬁnity for the Galili pentasaccharide (25)among the C. elegans galectins (Table 2, Fig. 5). The addition ofgalactose to the 3-OH group of the terminal galactose of lacto-Nneotetraose (LNnT) (26) decreased the afﬁnity of LEC-4, while it
increased the afﬁnities of the other LECs (Galili (25)/LNnT (26)) (Table4). Moreover, only LEC-4 exhibited a distinct preference for Nacetyllactosamine (type 2 chain, 26) over lacto-N-biose (type 1
chain, 27) (Table 5). Although LEC-10 showed similar afﬁnity for thetype 1 and type 2 chains, other LECs and CRDs preferred the type 1(1) LEC-2
LEC-2 exhibited some distinct features as compared to the other
LECs and CRDs. As shown in Table 4, the substitution at the 4-OH
group of GlcNAc in the type 1 chain by fucose (LNDFH, Leb (31)/LNFP-I(28) or A-heptasaccharide (33)/A-hexasaccharide (32)) increased the
afﬁnity of LEC-2. The substitution at the nonreducing terminal Gal of
lacto-N-fucopentaose II by fucose (LNDFH, Leb (31)/LNFP-II, Lea (29))
resulted in 3.2-fold increase in the afﬁnity of LEC-2 (Table 2, Fig. 5).
(2) LEC-3b
LEC-3b showed striking enhancement of afﬁnity for NA4 (04) ascompared to that for NA3 (02) (Table 3). Relatively strong afﬁnities for(3) LEC-4

Y. Nemoto-Sasaki et al. / Biochimica et Biophysica Acta 1780 (2008) 1131–1142

1139

Table 3
Effects of branching and repeating of lactosamine units on galectin afﬁnitya
Number of lactosamine unit

Branch2
3
4
Repeat
2
3
5
a
b

Ratio

LEC-1

LEC-2

LEC-3

LEC-4

LEC-6

LEC-10

1.3
2.4
2.6

2.3
5.4
6.1

2.1
5.7
15

0.34
0.96
0.93

3.4
8.0
12

2.2
4.1
4.6

2.1
3.9
11

NTb
7.4
14

1.5
2.7
3.7

0.12
0.23
0.63

0.86
1.4
2.3

0.26
0.24
0.21

Whole

LEC-1N

LEC-1CNA2 (01)/LNnT (26)
NA3 (02)/LNnT (26)
NA4 (04)/LNnT (26)

0.95
2.1
1.9

1.5
2.7
2.6

LN2 (39)/LNnT (26)
LN3 (40)/LNnT (26)
LN5 (41)/LNnT (26)

1.8
3.1
6.8

1.1
1.7
2.9

Relative afﬁnities with respect to LNnT (26) were calculated based on Kd's presented in Table 2.
NT, not tested.

Although the preference of A-hexasaccharide (32) for LNFP-I (28)
was remarkable (Table 4), the overall LEC-10 afﬁnity for oligosaccharides was very similar to that of LEC-6 (Table 2, Fig. 5). LEC-10 was
unique in that it preferred A-tetrasaccharide (38) to B-tetrasaccharide(36) (Table 5).

There was no discrepancy with regard to the nucleotide sequencesof the galectin-like cDNAs obtained in this study and those publishedin the genome database. Four galectin-like genes, lec-1, -2, -3, and -5
were mapped to chromosome II, and lec-4 and -6 were mapped tochromosome III. lec-8–9, lec-10, and lec-11 were mapped to chromosome X, V, and IV, respectively (Supplementary Table 3). Apparently,
lec-7 and -8 are gene duplication products because both mapped to
the X chromosome within 2 kbp, and they were identiﬁed in the same
cosmid with identiﬁers R07B1.2 (lec-7) and R07B1.10 (lec-8), respectively. On the WormBase website (http://www.wormbase.org/), a C.
elegans database, there exists 1–5 gene models of transcripts for each
galectin-like gene, and some of these models have been conﬁrmed by
the C. elegans sequencing consortium. cDNAs isolated by this study(except for lec-3b as described below) correspond to some of the genemodels shown in WormBase (Supplementary Table 3). There are ﬁvesplicing variants of lec-3 registered by the C. elegans sequencing
consortium in WormBase (ZK892.1a-e; GenBank cDNA accession no.
NM_063758, NM_001027360-3). lec-3a corresponds to ZK892.1a(NM_063758) (Supplementary Table 3), whereas lec-3b has nocorresponding cDNA in WormBase.

4. Discussion

4.2. Predicted features from deduced amino acid sequences

4.1. Novel galectin-like genes in C. elegans

From the deduced amino acid sequences, we can describe somefeatures of the individual galectin-like proteins. From a structural
viewpoint, the C-terminal CRD of LEC-3 and DC2.3a are assumed tohave an extra loop consisting of 12 and 10 amino acids, respectively,
between two β-strands, S4 and S5 (Fig. 2) on the basis of X-ray
crystallography of LEC-1 [44]. This loop region probably affects the
carbohydrate-binding property because this region is adjacent to thecarbohydrate-binding site. Insertion of such an extra-loop region has
also been found in a tandem-repeat-type galectin (Hco-GAL-4) of theparasitic nematode Haemonchus contortus [45].
A notable feature shared by LEC-4, -5, and -11 is the presence of an“RGD” sequence at equivalent positions (Fig. 2). Interestingly, ovine
galectin-15/OVGAL11 also has an RGD motif at an equivalent position

chain to the type 2 chain (Table 5). Taken together, LEC-4 exhibited themost distinctive carbohydrate-binding property among the C. elegans
galectins analyzed in the present study.
(4) LEC-6
The main feature of LEC-6 was the relative preference for complextype N-glycans (Table 2, Fig. 5). Its low afﬁnity for A-hexasaccharide(32) was another notable feature. Substitutions at the 3-OH group of
the terminal galactose of lacto-N-fucopentaose I (LNFP-I, 28) by Nacetylgalactosamine decreased the afﬁnities of LEC-1C and LEC-6,
while they increased or did not change the afﬁnities of the other LECs
(A-hexasaccharide (32)/LNFP-I (28)) (Table 4).
(5) LEC-10

According to a genome database search by Cooper, D.N.W. [23], anumber of potential galectin genes have been nominated in somemodel organisms, including C. elegans. However, it is unknown
whether such candidate genes are actually expressed in vivo andwhether they have substantial sugar-binding activity as that of the
galectins, i.e., β-galactoside-binding proteins.
We isolated the full-length cDNAs of potential galectin genes (lec2–5 and 8–11) in the present study. We also added the sequence dataof lec-7 and DC2.3a registered by the C. elegans sequencing consortiumfor comparison with the cloned galectin-like genes (Table 1, Figs. 2, 3,
Supplementary Table 3, Fig. 2).

Table 4
Effects of substitutions in lactosamine units on galectin afﬁnitya
Substitution

Siaα2-6Gal

Fucα1-2Gal
GalNAcα1-3Gal
Fucα1-4GlcNAc

Galβ1-3Gal
a

Ratio

NA2 (monosialo) (10)/NA2 (1)NA2 (monosialo) (11)/NA2 (1)NA2 (disialo) (12)/NA2 (1)
LNFP-I (28)/LNT (27)A-hexa (32)/LNFP-I (28)
LNFP-II (29)/LNT (27)
LNDFH (31)/LNFP-I (28)A-hepta (33)/A-hexa (32)
Galili (25)/LNnT (26)

LEC-1
Whole

LEC-1N

LEC-1C

0.23
0
0
1.5
1.0
0.30
0.25
0.23
3.9

0
0
0
3.4
2.7
0
0
0.03
14

0.16
0.12
0
1.2
0.08
0.32
0.34
4.8
2.0

Relative afﬁnities for relevant pairs were calculated based on Kd's presented in Table 2.

LEC-2

LEC-3

LEC-4

LEC-6

LEC-10

0.55
0
0
0.97
1.5
0.92
3.0
2.0
8.2

0.30
0.21
0
2.8
2.4
1.1
0.16
0.06
9.0

0.26
0.21
0.02
1.3
0.90
0.15
0.08
0.09
0.15

0.16
0.13
0
1.1
0.11
0
0
0
2.0

0.28
0.24
0
2.7
1.6
0
0.04
0.03
2.2

1140

Y. Nemoto-Sasaki et al. / Biochimica et Biophysica Acta 1780 (2008) 1131–1142

Table 5
Binding preference of galectinsa
Ratio

LNT (27)/LNnT (26) (type 1/type 2)
A-tetra (38)/B-tetra (36)
a

LEC-1
Whole

LEC-1N

LEC-1C

2.3
0

2.3
0

2.6
0

LEC-2

LEC-3

LEC-4

LEC-6

LEC-10

4.0
0

2.5
0.23

0.30
0.09

1.7
0

0.91
3.6

Relative afﬁnities were calculated based on Kd's presented in Table 2.

[10]. This position apparently corresponds to the boundary between
S2 and F1 β-strands of N-terminal CRDs based on the X-ray crystal
structure [44] (Fig. 2). Since the region is expected to be exposed on
the surface of the protein, it is possible that the RGD motif actuallyplays a role in cell/substratum adhesion like the other RGD-containing
proteins such as ﬁbronectin, vitronectin, laminin, and von Willebrandfactor [46]. In that case, their potential receptors, i.e., integrins, may
function together. Actually, ovine galectin-15/OVGAL11 is reported tomediate cell adhesion in an RGD sequence-dependent manner [47].
PSORT and SignalP analysis revealed that only LEC-5 had a signal
peptide among the C. elegans galectin-like proteins examined. Therefore,
LEC-5 can be regarded as an unusual galectin along with the sponge
galectin GCLT1, which also has the putative signal peptide [48]. Recently,
Kaji et al. [40] and Fan et al. [41] identiﬁed LEC-5 as an N-linked
glycoprotein. The glycosylation site identiﬁed was Asn110 [41]. These
results suggest that LEC-5 is a unique galectin family member because
most of the galectins isolated to date exhibit no glycosylation. Although
localization of the LEC-5 protein in C. elegans cells has not been elucidated,
LEC-5 is probably secreted extracellularly through a classical endoplasmic
reticulum (ER)/Golgi pathway that is different from those used by the mostother galectins. Trp111 in the N-terminal CRD of LEC-5 is one of theindispensable amino acids for carbohydrate binding among galectins fromvarious species. Therefore, the glycosylation at Asn110 may inhibit
carbohydrate binding of the N-terminal CRD of LEC-5 because of sterichindrance. In the present study, we failed to produce a recombinant LEC-5
protein and analyze its galactose-binding ability. At present, we cannot
estimate the proportion of endogenous N-glycosylated LEC-5. However, it
seems important to assess whether the endogenous N-glycosylated LEC-5
protein has a carbohydrate-binding ability or some other biological
function independent of carbohydrate binding.
A notable feature in LEC-7, -8, -10, and -11 is the presence of anadditional C-terminal region of varied length, i.e., 40, 41, 53, and 90 aminoacids, respectively. The similarity in this region between LEC-8 and LEC-10
is apparently high (48%), whereas that between the others are much lower(5–22%).
As another distinct feature is that signiﬁcantly high contents ofhistidine were observed in LEC-8 (14%), LEC-10 (14%), and LEC-11 (11%).
In addition, LEC-11 showed notable distinctions in its tail region. The tailregion of LEC-11 is considerably hydrophobic and it lacks the charged
residues spanning from Ile153 to Pro216 (Fig. 2). Nevertheless, theregion does not seem to form the usual membrane-integrated region
since it has 17 proline residues that are α-helix breakers. Analysis with
the SOSUI program also conﬁrmed that the predicted LEC-11 is a soluble
protein. The hydrophobic tail is followed by a distinct sequence “His–
His–His–Arg–His–His–His” (residues 225–231).
Although the size of LEC-9 is very similar to that of LEC-6, similarity
between LEC-6 and LEC-9 is considerably low (28% identity). On the other
hand, LEC-9 shows much higher similarities to LEC-7, -8, and -10 (36, 52,
and 46% identities, respectively) despite the lack of a C-terminal tail. At
present, it is not known whether LEC-9 forms a noncovalent dimer likeLEC-6. Therefore, we consider LEC-9 as a member of chimera-type
galectin-like proteins such as LEC-7, -8, -10, and -11.
4.3. Oligosaccharide-binding property
The present study demonstrated that most of the C. elegans
galectin-like genes investigated in this work (lec-1, -2, -3, -4, -6, and -10) proved to be functional in terms of their β-galactoside-binding
ability. However, it is notable that some expressed proteins have only apoor sugar-binding activity (e.g., LEC-8, -9, and -11). The former typeof galectin-like proteins have a perfectly or almost perfectly conserved
set of eight critical amino acid residues that are indispensable for thesugar-binding function of vertebrate galectins (i.e., His44, Ans46,
Arg48, Val59, Asn61, Trp68, Glu71, and Arg73; numbers correspond to
those of human galectin-1). Therefore, the presence of these conserved essential amino acids could be a key in identifying functional
galectins when screening other databases. DC2.3a, which was notcloned in the present investigation, seems to be another galectin
candidate that has a β-galactoside-binding ability because all thecritical amino acids are conserved in this protein. It was also shownthat individual members of the C. elegans galectin family are divergent
in terms of both molecular structure and sugar-binding properties.
FAC in the present study indicated that the C. elegans galectins
share basic carbohydrate-binding properties with mammalian
galectins [35], which are summarized as follows. (1) All LECs and
CRDs in this study recognize basic oligosaccharides such as lactoN-neotetraose, lacto-N-tetraose, and a complex-type N-glycan
with biantennary Galβ1-4GlcNAc to some extent (Kd values; 25–1000 μM). (2) All LECs and CRDs show higher afﬁnities for tri- andtetraantennary glycans than for biantennary glycan. Similarly,
afﬁnity is enhanced as the number of repeats of N-acetyllactosamine increases. (3) Substitutions at 6-OH groups of nonreducing
terminal galactoses and at 3-OH groups of the GlcNAc residues ofGalβ1-4GlcNAc (type 2 chain) results in a decrease in the afﬁnitiesof all LECs and CRDs. In contrast, substitutions at 4-OH groups of
the GlcNAc residue of Galβ1-3GlcNAc (type 1 chain) results indecreased afﬁnities in most cases, while there are some exceptions
like LEC-1C, LEC-2, and LEC-3.
LEC-2 shows relatively high afﬁnities for lacto-N-difucohexaose(LNDFH, Leb) and A-heptasaccharide. Not only C. elegans galectins
but also those from other species showed no or relatively weak
afﬁnity for these saccharides in our previous study [35]. Here, LEC2 is considered a novel-type galectin. Thus far, the carbohydratebinding properties of invertebrate galectins have not been
extensively investigated in detail. The result suggests the possible
binding of invertebrate galectins to glycans that cannot berecognized by mammalian galectins.
Among the C. elegans galectins examined in the present study, LEC4 exhibits many distinct features. For instance, LEC-4 has a relatively
high afﬁnity for variable glycolipid-type oligosaccharides. Moreover,
only LEC-4 prefers the type-2 chain compared to the type-1 chain
among the C. elegans galectins. However, these features have also
been observed in some galectins from other species [35].
In the above FAC study, LEC-8 and -9 were not observed to haveany signiﬁcant sugar-binding activity. However, since almost all thePA-oligosaccharides used are of mammalian origin, it is probable that
LEC-8 and -9 have a remarkable afﬁnity for some endogenous glycans
existing in C. elegans, which have structures different from those in
mammals. Alternatively, chimera-type galectin-like proteins of C.
elegans may have diverse functions that are unknown because they
have a C-terminal tail whose function is unknown. It was reported
that the expression of lec-8 mRNA was elevated after a Serratia
marcescens infection [49]. This result implies that LEC-8 may play arole in defense against bacterial infections.

Y. Nemoto-Sasaki et al. / Biochimica et Biophysica Acta 1780 (2008) 1131–1142

4.4. Expression of galectin-like genes in C. elegans
With regard to the expression of the C. elegans galectin-like genes
in vivo, we detected mRNAs of lec-1, -2, -4, -5, -6, -8, -9, -10, and -11
by RT-PCR. According to the Nematode Expression Pattern DataBase(NEXTDB) (http://nematode.lab.nig.ac.jp/), mRNA expressions of lec1–6 and 8–11 are spatiotemporally diverse. The 32-kDa galectin
protein was shown to be abundantly localized in the cuticle and in theterminal valve of the pharynx of adult worms [50]. However, thus far,the analysis of the spatiotemporal localization of each galectin protein
has not been performed from a comparative viewpoint.
4.5. Concluding remarks
We previously identiﬁed several glycoprotein ligands for LEC-6
from an extract of C. elegans [51,52]. In the present study, other C.
elegans galectins were found to possess the conserved fundamental
properties, i.e., ability to recognize β-galactosides such as Nacetyllactosamine. It is intriguing that a relatively simple multicellularorganism like C. elegans has numerous galectins. In the present study,
we revealed that they have diverse carbohydrate-binding properties.
These results suggest that individual galectins have distinct roles invivo. To further conﬁrm the concept, we wish to examine protein
localizations and glycoconjugate ligands for each C. elegans galectin invivo as a future study.
Acknowledgements
The authors thank Drs. Tadasu Urashima (Obihiro University ofAgriculture and Veterinary Medicine) and Kei-ichi Yoshida (Seikagaku Kogyo, Co. Ltd.) for the gift of oligosaccharides used in the
FAC analysis. The authors also thank Dr. Tomoharu Takeuchi(Teikyo University School of Pharmaceutical Sciences) for helpful
discussions. This work was supported by a grant (No. 10178102; toJ.H.) from the program Grants-in-Aid for Scientiﬁc Research on
Priority Areas of the Ministry of Education, Science, Sports, and Culture of Japan; Kato Memorial Bioscience Foundation; and Mizutani
Foundation.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbagen.2008.07.003.
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