(Received for publication, September 7, 1995; and in revised form, November 10, 1995)
From the
In our previous study (Hirabayashi, J., Satoh, M., Ohyama, Y.,
and Kasai, K.(1992) J. Biochem. (Tokyo) 111,
553-555), two -galactoside-binding lectins (apparent subunit
molecular masses, 16 and 32 kDa, respectively) were identified in the
nematode Caenorhabditis elegans. The subsequent study revealed
that the 32-kDa lectin is a member of the galectin family. Since the
32-kDa galectin was found to consist of two homologous domains (
16
kDa), 16-kDa lectin was thought to be a degradation product of the
32-kDa galectin. To clarify this, the 16-kDa lectin was purified by an
improved procedure employing extraction with a calcium-supplemented
buffer. The purified 16-kDa lectin was found to exist as a dimer
(
30 kDa) and showed hemagglutinating activity toward trypsinized
rabbit erythrocytes, which was inhibited by lactose. Almost the whole
sequence of the 16-kDa polypeptide (approximately 95%, 135 amino acids)
was determined after digestion with various proteases. Based on the
obtained information, a full-length cDNA was cloned with the aid of
RNA-polymerase chain reaction. The clone encoded 146 amino acids
including initiator methionine (calculated molecular mass, 15,928 Da).
Based on these results, it was concluded that the 16-kDa lectin is a
novel member of the galectin family, but not a degradation product of
the 32-kDa galectin as had previously thought. However, the 16-kDa
galectin showed relatively low sequence similarities to both the
N-terminal and the C-terminal domains of the 32-kDa galectin (28% and
27% identities, respectively) and to various vertebrate galectins
(14-27%). Nonetheless, all of the critical amino acids involved
in carbohydrate binding were conserved. These observations suggest
that, in spite of phylogenic distance between nematodes and
vertebrates, both the 16-kDa and 32-kDa nematode isolectins have
conserved essentially the same function(s) as those of vertebrate
galectins, probably through recognition of a key disaccharide moiety,
``N-acetyllactosamine.''
Galectins are a group of soluble animal lectins, which exhibit
specificity for -galactosides and have significant sequence
homology (Barondes et al., 1994a). They conserve a
characteristic carbohydrate-recognition domain (CRD) (
)(Drickamer and Taylor, 1993; Hirabayashi, 1993;
Hirabayashi and Kasai, 1993; Hirabayashi, 1994; Barondes et
al., 1994b). Although galectins had been initially investigated as
developmentally regulated lectins mainly in vertebrates; e.g. electric eel (Teichberg et al., 1975), chick (Kobiler and
Barondes, 1978; Oda and Kasai, 1983), bovine (De Waard et al.,
1976), rat (Clerch et al., 1988), mouse (Raz et al.,
1988; Willson et al., 1989), and human (Hirabayashi and Kasai,
1984), some galectins have also been found recently in invertebrates; a
homologous galactose-binding protein (32 kDa) was identified in the
nematode Caenorhabditis elegans as the first invertebrate
galectin (Hirabayashi et al., 1992a; Hirabayashi et
al., 1992b). Furthermore, two galectin cDNAs were cloned in a
mesozoan, the marine sponge Geodia sydnium (Pfeifer et
al., 1993). These findings support the idea that galectins form an
extensive protein family in the animal kingdom. Notably, these
non-mammalian galectins also show saccharide-binding specificity for
-galactosides. Such conservative properties are in contrast to
another large protein family of animal lectins, i.e. C-type
lectins (for comparative reviews, see Hirabayashi, 1993; Hiralsayashi,
1994)).
C. elegans, belonging taxonomically to the pseudocoelomates of Protostomia, has a simple body structure (2 mm in length) consisting of only about 1,000 cells. Nevertheless, it develops a primitive nerve-muscular system. The worm is experimentally useful, because it is transparent and hermaphroditic, and has a short generation time (3 days). Such features are apparently advantageous for studies on anatomy, development, fertilization, and, in particular, genetics (Coulson et al., 1988), and the nematode has become an important model animal since the initial proposal by Brenner in 1965 (reviewed by Wood(1988)).
From the viewpoint of protein
architecture, galectins can be classified into three structural types
(Hirabayashi et al., 1992b; Hirabayashi and Kasai, 1993); that
is (i) proto type (subunit molecular mass, 14,000-16,000
Da), (ii) chimera type (29,000-35,000 Da), and (iii) tandem-repeat type (32,000-36,000). The proto type,
having a single CRD, includes a number of small galectins of both
mammalian and non-mammalian species. Galectin-3, which has been found
only in mammals, is the sole member of the chimera type that consists
of an N-terminal proline-, glycine-, and tyrosine-rich repetitive
domain and a C-terminal CRD that shows homology to other members of the
galectins (reviewed by Anderson and Wang(1992) and Hughes(1994)).
Tandem-repeat type was firstly identified in C. elegans (32-kDa galectin; Hirabayashi et al., 1992b), but more
recently similar galectins were also found in mammals. Rat (Oda et
al., 1993; Tardy et al., 1995a) and porcine (Chiu et
al., 1994) galectin-4, rat galectin-6 (Gitt et al.,
1995a), and rat galectin-8 (Hadari et al., 1995) are
categorized into this type. However, the latter mammalian galectins are
not complete homologues of the nematode 32-kDa galectin, since sequence
homology between them is relatively low, and the mammalian
tandem-repeat type galectins (both galectin-4 and -8) have an extra
linker domain between the two repeated CRDs. The sequence of a cDNA for
a probable homologue in Onchocerca volvulus to the C.
elegans 32-kDa galectin has recently been deposited in the
GenBank(TM) data base (accession number U04046). Among these
galectin CRDs (14 kDa), several amino acid residues are strictly
conserved. All of them are located in a central region, which has been
shown to be encoded by a single exon (Ohyama and Kasai, 1988; Gitt and
Barondes, 1991; Gitt et al., 1992; Gritzmacher et
al., 1992). Many of these conserved residues, i.e. His-44, Asn-46, Arg-48, Asn-61, Trp-68, Glu-71, and Arg-73
(residue numbers are those of human galectin-1) were shown to be
critical for the saccharide binding by our systematic mutagenesis
studies (Hirabayashi and Kasai, 1991, 1994). In recent x-ray
crystallographic studies, all of them were proved to be involved in the
interaction between lactose and human galectin-2 (Lobsanov et
al., 1993), and in that between N-acetyllactosamine and
human (Liao et al., 1994) or bovine galectin-1 (Bourne et
al., 1994).
In our previous study (Hirabayashi et al.,
1992a), we found that the nematode contained two soluble,
metal-independent -galactoside-specific lectins (apparent subunit
molecular masses estimated by SDS-PAGE, 16,000 Da and 32,000 Da,
respectively (designated hereafter as nematode 16-kDa and 32-kDa
lectins, respectively). They were purified by a procedure essentially
similar to that applied to galectin-1 purification (Hirabayashi and
Kasai, 1984; Hirabayashi et al., 1987); briefly, the nematode
lectins were extracted with lactose-containing MEPBS (20 mM lactose, 4 mM
-mercaptoethanol, 2 mM EDTA,
20 mM sodium phosphate, pH 7.2, 150 mM NaCl) and were
adsorbed on asialofetuin-agarose. Although cDNA cloning revealed that
the 32-kDa protein is the first invertebrate member of the galectin
family, it remains to be determined whether the 16-kDa lectin also
belongs to the same protein family or not. Since preliminary amino acid
analysis showed that the 16-kDa lectin is very similar to 32-kDa
galectin, it seemed likely that the 16-kDa protein was derived from the
32-kDa species through limited proteolysis during preparation, as in
the case of galectin-4 (Leffler et al., 1989; Oda et
al., 1993; Tardy et al., 1995a). On the other hand,
antiserum raised against nematode 32-kDa galectin reacted only poorly
with 16-kDa lectin. A major difficulty in answering the above question,
however, was that the 16-kDa lectin was a minor component; the yields
of 16-kDa and 32-kDa lectins in the previous purification procedure
were approximately 20-30 and 200 µg, respectively, from 10 g
of the worm. After trial-and-error studies, we found that the use of
calcium-supplemented buffer (Ca-TBS) for extraction instead of the
previously used MEPBS greatly improved the yield of 16-kDa lectin. In
this paper, we describe an improved purification of the 16-kDa lectin,
its protein-chemical characterization, and cDNA cloning, as well as
preliminary identification of endogenous glycoprotein receptors for the
lectin. It was concluded that the 16-kDa lectin is a novel member of
the galectin family in the nematode C. elegans, belonging to
the proto type category.
Two -galactoside-binding lectins (16 and
32 kDa) were purified essentially as described previously (Hirabayashi et al., 1992a). However, to improve the purification yield of
the 16-kDa lectin, Ca-TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, supplemented with 5 mM CaCl2) was used instead of
MEPBS. After the worms were disrupted by sonication in 5 volumes of
Ca-TBS, most of the soluble proteins were removed by centrifugation
(15,000 rpm, 4 °C, 25 min), and lectins were extracted with
lactose/Ca-TBS (Ca-TBS containing 20 mM lactose) by shaking
the suspension for 30 min at 4 °C. The derived supernatant
containing lectins (sup-2) was dialyzed extensively against Ca-TBS to
remove lactose, and then applied to an asialofetuin-agarose column (10
ml volume, approximately 9 mg of fetuin was immobilized/ml of gel;
prepared according to De Waard et al.(1976)). After extensive
washing of the column with Ca-TBS, the bound lectins were eluted with
lactose/Ca-TBS. Protein was determined by the method of Bradford(1976).
To prepare a membrane fraction, which was assumed to contain lectin ligand glycoconjugates, frozen nematodes (10 g wet weight) were thawed, and soluble proteins including 16-kDa lectin were removed, as described under ``Purification of 16-kDa Lectin.'' To the resultant pellet, Ca-TBS containing 1% Triton X-100 (Triton/Ca-TBS) was added in a ratio of 2.5 volumes to the original weight of worms. Membrane fraction, designated sup-3, was obtained after shaking (30 min at 4 °C) and centrifugation (16,000 rpm at 4 °C for 25 min).
The prepared membrane fraction was
applied to a column (volume, 2 ml) packed with the recN16-agarose.
After extensive washing of the column with Ca-TBS, the bound protein
was eluted first with the same buffer containing 0.1 M sucrose, then with that containing 0.1 M lactose. The
eluted fractions were subjected to protein determination (Bradford,
1976), SDS-PAGE, and lectin-probed Western blotting analysis described
below.
We also found that the
purification yield of 16-kDa nematode lectin was greatly improved by
replacing the extraction buffer lactose/MEPBS with lactose/Ca-TBS. When
the nematode was homogenized with lactose/Ca-TBS, the yield of 16-kDa
lectin was approximately 200 µg from 10 g of worms, while it was
20-30 µg when lactose/MEPBS was used. On the other hand, the
yield of the 32-kDa lectin was greatly reduced (20-30 µg from
10 g of worms) compared with that by lactose/MEPBS extraction (200
µg from 10 g of worms) (Fig. 1B). This may be
largely attributed to calcium-activated proteolysis of 32-kDa lectin,
because time-dependent decrease in this lectin was observed in Western
blotting analysis of the extract prepared in the presence of calcium
(data not shown). However, it was not associated with the increase in
16-kDa lectin band detected with anti-16-kDa lectin antiserum. This
result suggests that the 16-kDa lectin is not a degradation product of
the 32-kDa galectin, though it seemed likely that a region linking the
two homologous CRDs (16 kDa) of 32-kDa galectin is susceptible to
proteolysis, as has been demonstrated for galectin-4 (Oda et
al., 1993; Tardy et al., 1995a). It is not clear why the
yield of 16-kDa lectin was greatly increased when Ca-supplemented
buffer was used. Although calcium was effective for the purification,
this does not mean that the lectin requires calcium for activity,
because the bound 16-kDa lectin was not eluted from the
asialofetuin-agarose column with EDTA (Fig. 1A).
Figure 1:
A, purification of C. elegans lectins (16 and 32 kDa) on asialofetuin-agarose by an improved
procedure. In the present experiment, lectins (16 and 32 kDa) were
extracted with 0.1 M lactose dissolved in Ca-TBS instead of in
MEPBS. Subsequent steps were as described previously (Hirabayashi et al., 1992a, 1992b). The figure shows a representative
chromatogram. Arrows indicate the starting positions of EDTA
and lactose elution in that order. The two lectins were eluted only by
the latter eluent. B, comparison of the results obtained by
the previous (lane 1) and present (lane 2) procedures
on SDS-PAGE. Purified lectin fractions were subjected to conventional
SDS-PAGE under denaturing conditions in the presence of
-mercaptoethanol using 14% gel. Protein was stained with silver.
Positions of 16-kDa and 32-kDa lectins are indicated by arrowheads.
Figure 2: Effects of saccharides (lactose, galactose, and melibiose) of various concentrations on extraction of 16-kDa lectin from C. elegans. The 16-kDa lectin was extracted with either lactose (lanes 1-3), galactose (lanes 4-6), or melibiose (lanes 7-9) and was subjected to a conventional Western blotting analysis to see how much of the lectin had been extracted. Polyacrylamide gel (14%) and specific antiserum against the nematode 16-kDa lectin were used. Saccharide concentrations: lanes 1, 4, and 7 (1 mM); lanes 2, 5, and 8 (10 mM); lanes 3, 6, and 9 (100 mM). As a control, an extract in the absence of sugar (sup-1) was included for lectin extraction (lane 10). To each lane, 20 µg of protein was applied.
To confirm the above result and to analyze undetermined regions, the N-terminal (Lys-5) and C-terminal (Lys-4) fragments were subjected to second digestions with trypsin, AspN, or Staphylococcus V8 protease. The derived peptides were purified on a TSK-ODS-80TM column, and sequenced (see results in Table 1). Finally, 130 amino acids were determined with relevant overlapping sequences, and the result showed unambiguously that the nematode 16-kDa lectin is a novel member of the galectin family (described in more detail under ``Discussion''). The total residue number comprised 95% of that finally obtained by cDNA cloning (described below), because we failed to obtain a small N-terminal peptide.
Figure 3:
Nucleotide sequence of the clone 7B1
obtained by screening a ZAP cDNA library and deduced amino acid
sequence. The clone was isolated by plaque hybridization (see
``Experimental Procedures'') with a 0.4-kbp probe obtained by
RNA-PCR (F1 and R1 primers were used). Other PCR primers used for
analyses are also indicated. Codons corresponding to amino acid
residues determined directly by peptide analyses (see also Table 1) are underlined. A putative initiation site ATG
is also underlined. A termination codon TAA is indicated with asterisks, and a possible poly(A) additional signal AATAA is double-underlined.
After three rounds of
screening, a single clone, designated 7B1, was obtained. The clone had
an insert of 0.55 kbp and was positive in Southern hybridization using
the 0.4-kbp probe and in a PCR check using F1 and R1 primers (which
amplified a 0.4-kbp fragment). The nucleotide sequence of the clone 7B1
was determined by the dideoxy termination method with a DNA sequencer
(Applied Biosystems 373S) after automatic subcloning into
pBluescript(SK).
The deduced amino acid sequence
agreed completely with that directly determined by peptide analysis
except for the N-terminal region, which was determined only by cDNA
analysis (Fig. 3). The clone had a putative initiation codon ATG
for initiator methionine (iMet). This methionine was followed by a
decapeptide,
Ile-Gly-Gly-Gly-Ile-Gly-Ile-Ser-Phe
-Cys, and
further by the previously determined tryptic peptide sequence
(Lys-5-T-8),
Asn
-Glu-Phe-Phe-Asn-Pro-Gln-Thr-Pro-Val
-Asn-Ile-Pro-Val-Gln-Gly-Phe-Ser-Asn-Gly
-Ala-Arg.
This result was unexpected in two respects. (i) The N-terminal
decapeptide sequence is unusual in that it is relatively rich in
glycine and isoleucine, and has no charged amino acid; (ii) this
decapeptide sequence ended with cysteine, but not with arginine or
lysine. We could not obtain any corresponding fragment by
reversed-phase chromatography after digestion of Lys-5 with various
proteases. The unusual amino acid composition may partly explain the
unexpected proteolysis and failure to recover the N-terminal peptide,
because such a glycine-rich structure does not seem to form stable
secondary structures and may be extremely susceptible to proteolysis.
Including this missing N-terminal decapeptide, the calculated molecular
weight and total residue number of the nematode 16-kDa galectin are
15,928 Da and 146 amino acid residues, respectively (initiator
methionine included). The deduced amino acid sequence had no consensus
sequence (Asn-X-Ser/Thr) for attachment of asparagine-linked
oligosaccharide.
Figure 4:
Northern hybridization analysis. A 0.4-kbp
PCR fragment produced by using F1 and R1 primers was labeled with
[P]dCTP and used as a probe for a transcript of
the nematode 16-kDa galectin. For this purpose, 10 µg of total RNA
was electrophoresed and hybridized as described under
``Experimental Procedures.'' A 0.7-kb species gave a stable
hybridization signal. Positions of six reference RNAs are shown with arrowheads.
Genomic Southern hybridization was performed after digestion of the nematode genomic DNA with BamHI, EcoRI, HindIII, or PstI. After hybridization with the 0.4-kbp probe used above, each digest gave a single hybridization signal in autoradiography (Fig. 5). The result, though not definitive, suggests that the nematode 16-kDa galectin gene is unique.
Figure 5: Genomic Southern hybridization analysis. Ten-microgram aliquots of genomic DNA of the nematode were digested with restriction endonucleases, BamHI (B), EcoRI (E), or HindIII (H). The digests were electrophoresed and hybridized with the same 0.4-kbp probe as that used in Fig. 7.
Figure 7: Comparison of amino acid sequences of representative animal galectins. Mammalian galectins are numbered according to the previous proposal (Barondes et al., 1994a). Highly conserved amino acids are shaded. Among them, critical amino acid residues, which have been shown to be involved in interaction with lactose (Lobsanov et al., 1993) and N-acetyllactosamine (Liao et al., 1994; Bourne et al., 1994), are also emphasized with ``#'' (hydrogen bonding) or ``@'' (hydrophobic interaction). Amino acid identities to C. elegans 16-kDa lectin are also shown.
Purified recN16 showed significant hemagglutination
activity toward trypsinized rabbit erythrocytes prepared according to
Kobiler and Barondes(1978). The activity was completely inhibited by
the addition of 100 mM lactose. The minimum concentration of
recN16
required for hemagglutination was about 10 µg of the
recombinant fusion protein/ml (equivalent to 7.6 µg of intact
protein/ml); in terms of specific activity, 140
titer
/mg/ml. This value is somewhat lower than those
obtained for vertebrate galectins (i.e. approximately 1,000
titer
/mg/ml). Lactose showed the highest inhibitory
effect on hemagglutination (concentration required for 50% inhibition,
6 mM), and melibiose and galactose were much less inhibitory
(both 50 mM).
As the next step, C. elegans membrane fraction was prepared by using 1% (w/v) Triton X-100. Since all of the soluble proteins including galectins had previously been extracted with lactose, the fraction did not contain nematode 16-kDa galectin. The membrane fraction was applied to a column (2 ml) equilibrated with Ca-TBS containing 0.1% Triton X-100. After extensive washing of the column (10 volumes), the bound materials were eluted first with 0.1 M sucrose, and then with 0.1 M lactose dissolved in the same buffer (Fig. 6A). Analysis of the eluted proteins by SDS-PAGE followed by silver staining revealed the presence of multiple protein bands only in the lactose eluate. The bands were mainly in the molecular mass range larger than 30 kDa (Fig. 6B, lane 1). A few of them (approximately 300 and 150 kDa, shown with closed triangles in lane 2) were dominant in the fraction. Since these proteins were not eluted with 0.1 M sucrose prior to the lactose elution, the binding is considered to be specific. Yields of these glycoprotein receptor candidates were 160 µg from 2 ml of the detergent-solubilized membrane fraction (equivalent to 0.8 g of the nematode).
Figure 6:
Isolation of recN16-binding
glycoproteins on an immobilized-lectin column. A, a typical
chromatogram obtained with 2 ml of membrane fraction and 2 ml of
immobilized lectin gel prepared as described under ``Experimental
Procedures.'' Fraction volume was 2 ml. Triton X-100 (1%) extract
(2 ml of the membrane fraction) was applied to the column (packed with
2 ml of recN16
-immobilized gel) equilibrated with Ca-TBS
containing 0.1% Triton X-100. After extensive washing of the column
with the equilibration buffer, the bound glycoproteins were eluted with
0.1 M sucrose and then 0.1 M lactose. B,
analysis of the eluted glycoproteins by SDS-PAGE followed by either
silver staining (lane 1) or lectin-probed Western blotting (lanes 2 and 3; for details, see ``Experimental
Procedures''). In the latter analysis, recN16
binding was
performed both in the absence (lane 2) and in the presence (lane 3) of 0.1 M lactose. Various lectin-probed
bands are observed (shown with triangles), among which most
significant ones are represented with closed
triangles.
To confirm the above result, lectin-probed Western
blotting analysis was performed by using free recN16 as described
under ``Experimental Procedures.'' When the lactose eluate
was used, a few protein bands, mostly corresponding to the above
observed ones (i.e. 300, 150, 100, and 70 kDa), were
visualized (Fig. 6B, lane 2). In the analysis,
lectin binding was greatly reduced in the presence of 0.1 M lactose (Fig. 6B, lane 3).
In our previous work, in which lactose/MEPBS was used for lectin extraction, 16-kDa lectin was purified only as a minor component relative to 32-kDa lectin. Since the latter consists of two homologous domains (i.e. CRDs) as a tandem-repeat type galectin, it seemed likely that the 16-kDa lectin was derived from the 32-kDa galectin by limited proteolysis. In fact, the 16-kDa species became a major component when MEPBS was substituted with Ca-TBS. This observation suggested the presence of a Ca-activated protease(s) responsible for the formation of 16-kDa lectin from the 32-kDa galectin. However, this was not the case, and the 16-kDa lectin was confirmed here to be a novel member of the galectin family. The 16-kDa galectin reacted only poorly with anti-32-kDa galectin antibody, and it showed significant but relatively low amino acid sequence similarity to the 32-kDa galectin. Since the C. elegans 32-kDa galectin lacks an apparent linker domain, a linking region between the two CRDs which exists in mammalian tandem-repeat type galectins, i.e. galectin-4 (Oda et al., 1993; Tardy et al., 1995a) and galectin-8 (Hadari et al., 1995), it may not be really cleaved. However, it remains unclear why calcium increases the yield of 16-kDa galectin but not that of 32-kDa galectin.
Although the sequence of nematode 16-kDa galectin was homologous to other galectin family members of both nematode and vertebrates, observed similarities were not very high: i.e. in terms of amino acid identities, the nematode 16-kDa galectin is homologous to the N-terminal (28%) and C-terminal domains (27%) of 32-kDa nematode galectin, human galectin-1 (19%), human galectin-2 (20%), human galectin-3 (23%), and the N-terminal (26%) and C-terminal domains (27%) of rat galectin-4 (see Fig. 7). Therefore, we cannot assign the nematode 16-kDa galectin as a possible ancestral molecule of small proto type galectins. Rather, as far as amino acid identities are concerned, nematode 16-kDa galectin is more similar to either the chimera (e.g. galectin-3) or the tandem-repeat type (e.g. galectin-4) than the proto type (i.e. galectins-1 and -2). In this context, Ahmed and Vasta(1994) have recently pointed out that galectin-1 is distinct from other galectin family members from the viewpoints of both saccharide-binding specificity and sequence similarity.
C. elegans 16-kDa galectin was shown to have a short redundant N-terminal region. The region is not as hydrophobic as usual signal sequences, nor does it resemble any known functional protein motif. Its function is not yet known, but may be unique to this lectin molecule. In this context, it is notable that many other galectins have an extra segment (non-CRD segment) in the N-terminal region, but not in the C-terminal region, as typically represented by the glycine-, proline-, and tyrosine-rich repetitive domain of galectin-3. A short, redundant N-terminal region is also observed in nematode 32-kDa galectin (tandem-repeat type), recently cloned galectin-8 (tandem-repeat type) (Hadari et al., 1995), and galectin-5 (monomeric proto type) (Gitt et al., 1995b). These facts imply that the N-terminal region can more readily be linked to other segments than the C-terminal region. Such N-terminal modifications may contribute to developing the ``identities'' of individual galectin molecules.
According to the previous
classification, which was made simply based on protein architecture
(Hirabayashi et al., 1992b), the 16-kDa nematode galectin
belongs to the proto type. Here, ``proto'' does not
correspond to ``primitive'' in terms of molecular evolution.
However, the fact that all animal species contain this type of
galectin(s) (summarized in Table 2) implies that the present
galectins have actually evolved from such a proto type ancestral
protein, although the present members should have diverged greatly, and
thus, have become much more specific in their functions. So far
identified prototype galectins have been shown to exist as non-covalent
dimers with a few exceptions of recently cloned rat galectin-5 (Gitt et al., 1995b) and human galectin-7 (Madson et al.,
1995; Magnald et al., 1995) that are monomeric. In the present
study, C. elegans 16-kDa galectin was also found to exist as a
dimer. Furthermore, recombinant 16-kDa galectin (recN16) caused
hemagglutination of trypsinized rabbit erythrocytes, although its
specific activity was somewhat lower than those of vertebrate
galectins.
Why are there two galectins, 16-kDa and 32-kDa, in C. elegans? It seems meaningful that all animal species in which galectins have been found have more than one galectin, although this tendency seems to be more prominent in mammals (see Table 2). Notably, all of them have conserved the fundamental ability to recognize a key disaccharide structure, N-acetyllactosamine. In this regard, Poirier and Robertson(1993) recently reported that deficiency of one of the mouse isolectins (galectins-1 and -3) was possibly compensated by the other. These authors generated null-mutant mice as regards galectin-1 gene by a procedure of homologous recombination, but these ``knocked-out'' mice were found to be completely vital and also proliferative. In this case, galectin-3 was suggested to substitute for galectin-1. Surprisingly, such unexpected results have been found increasingly for various developmental factors, such as tenascin (Saga et al., 1992), PrP (Bueler et al., 1992), and myoD (Rudnicki et al., 1992). These observations strongly suggest that many important proteins, including galectins, have evolutionally become more and more redundant (by making multiple gene copies), essentially because they are fundamental for the life of multicellular animals. In this context, the effect of destruction of either (and both) of the nematode galectin genes is of great interest.
Although the 32-kDa galectin has been
shown to be most abundantly expressed in the epidermal layer of adult
worms, ()the localization of the 16-kDa galectin is not
known at present. When the 16-kDa galectin was extracted from the
nematode, galactose and melibiose were also effective to some degree.
However, this was not the case for 32-kDa galectin, because even 100
mM of these saccharides failed to extract the 32-kDa galectin
(data not shown). Therefore, the 16-kDa galectin may have a looser
specificity than 32-kDa galectin. Alternatively, these isolectins may
have distinct kinetic properties in saccharide binding. In this regard,
we recently found that the 32-kDa galectin has considerably slower
on/off rates (i.e. in terms of k
and k
) in binding to immobilized asialofetuin than the
16-kDa nematode galectin, through analysis with a Biacore biosensor. (
)Thus, co-occurrence of proto type and tandem-repeat type
galectin in many animal species could be meaningful from various
viewpoints, i.e. (i) saccharide binding specificity, (ii)
kinetic properties, and (iii) cross linking features (homologous or
heterologous binding to branched saccharides), as has recently been
demonstrated (Gupta and Brewer, 1994; Bourne et al., 1994;
Dessen et al., 1995).
Although C. elegans was
chosen as a model animal to elucidate the basic features of complicated
multicellular systems, basic biochemical studies on the worm are not
well advanced. This is particularly true in the field of glycobiology.
In fact, there has been almost no information on glycoconjugates and
glycoenzymes of the nematode. In the present study, however, we
identified several candidate glycoprotein ligands for C. elegans 16-kDa galectin, and showed that the binding was metal-independent
and lactose-sensitive. Considering the conservative properties of
galectins, the results imply the presence of N-acetyllactosamine-type glycoconjugates even in the nematode.
In this context, comparative studies of the two C. elegans galectins from both biochemical ()and genetic
viewpoints (
)are in progress.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D63575[GenBank].