From the Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan
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ABSTRACT |
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Novel type lectins were found in the phylum Annelida, i.e. in the earthworm, tubifex, leech, and lugworm. The lectins (29-31 kDa) were extracted from the worms without the use of detergent and purified by affinity chromatography on asialofetuin-agarose. On the basis of the partial primary structures of the earthworm Lumbricus terrestris 29-kDa lectin (EW29), degenerate primers were synthesized for use in the reverse transcriptase-polymerase chain reaction. An amplified 155-base pair fragment was used to screen a cDNA library. Four types of full-length clones were obtained, all of which encoded 260 amino acids, but which were found to differ at 29 nucleotide positions. Since three of them resulted in non-silent substitutions, EW29 mRNA was considered to be a mixture of at least three distinct polynucleotides encoding the following proteins: Ala44-Gln197-Ile213 (clone 5), Gly44-Gln197-Val213 (clone 7), and Ala44-His197-Ile213 (clones 8 and 9; different at the nucleotide level, but encoding an identical polypeptide). Genomic polymerase chain reaction using DNA from a single worm revealed that the single worm already had four sets of cDNAs. The EW29 protein showed two features. First, the lectin was composed of two homologous domains (14,500 Da) showing 27% identity with each other. When each of the domains was separately expressed in Escherichia coli, the C-terminal domain was found to bind to asialofetuin-agarose as strongly as the whole protein, whereas the N-terminal domain did not bind and only retardation was observed. EW29 was found to exist as a monomer under non-denaturing conditions. It had significant hemagglutinating activity, which was inhibited by a wide range of galactose-containing saccharides. Second, EW29 contained multiple short conserved motifs, "Gly-X-X-X-Gln-X-Trp." Similar motifs have been found in many carbohydrate-recognizing proteins from an extensive variety of organisms, e.g. plant lectin ricin B-chain and Clostridium botulinum 33-kDa hemagglutinin. Therefore, these carbohydrate-recognition proteins appear to form a protein superfamily.
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INTRODUCTION |
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Galactose-binding proteins (lectins) represent a distinguished group among lectins because statistics have shown that more than 60% of the lectins reported thus far are galactose-specific (1). A possible explanation for such a preference is that galactose was selected as an important "recognition" saccharide especially in higher organisms. In fact, galactose is used more frequently in higher organisms like mammals, whereas glucose and mannose are recognized by microorganisms such as bacteria and fungi. From a glycochemical viewpoint, galactose has a nature inherently distinct from that of glucose, mannose, or fructose. The latter three monosaccharides are related by "Lobry de Bruyn-Alberta van Ekenstein transformation" (2). It is well established that in N-linked oligosacharide biosynthesis galactose is incorporated at later stages after removal of glucosyl and mannosyl residues from the common precursor Glc3Man9GlcNAc2 (3, 4). These observations led one of the authors to present a hypothesis on the origin of elementary hexoses that galactose is a "latecomer" saccharide relative to glucose and mannose (5). Galactose is exposed at outermost spaces of cells unless it is masked by sialic acids, so that it is easily recognized by various communication molecules by homophilic carbohydrate-carbohydrate (6) or heterophilic carbohydrate-protein interactions (7).
Among galactose-specific lectins, galectins are unique in that all of the members belonging to this family are galactose-specific (8, 9). In general, they are soluble and require no metal ion for their activity. Galectins bind most preferentially to lactosamine-containing saccharides of both glycoproteins and glycolipids. Although galectins had long been believed to occur in vertebrates only, they proved to be distributed in much lower organisms as well (10-15). Although the biological roles of galectins are not fully understood, they are supposed to play multiple roles basic to multicellular organisms, such as in development, differentiation, tumorigenesis, metastasis, apoptosis, etc. In this regard, if galectins are essential for these biological processes, they would be expected to exist in all animal species. However, there have been only few reports of galectins other than in two animal phyla representing deuterostomes and protostomes, i.e. Vertebrata and Nematoda, respectively. On the other hand, if this is not the case, a possibility emerges that some other galactose-binding lectins compensate for the absence of galectins in such animal phyla.
We have undertaken screening of galectin-like proteins from the phylum Annelida, by employing the same purification strategy as that for galectins, i.e. lactose-specific extraction in the absence of detergent and metal ion, and affinity chromatography on asialofetuin-agarose. As a result, 29-31-kDa lectins were purified from four annelids as follows: earthworm, tubifex (both belonging to the class Oligochaeta), lugworm (Polychaeta), and leech (Hirudinea). These proteins immunologically cross-reacted with one another and had similar biochemical properties as galectins with respect to specificity, solubility, and metal independence. Detailed structural analysis including cDNA cloning was carried out for 29-kDa lectin from the earthworm Lumbricus terrestris. As a result, the earthworm 29-kDa lectin (designated hereafter EW29)1 proved to be a "tandem repeat"-type lectin, which consists of two tandemly repeated homologous domains (14.5 kDa). Contrary to our expectation, it showed no sequence homology to the known galectins. However, it showed resemblance to some carbohydrate-relating proteins such as ricin B-chain, C. botulinum hemagglutinin, etc. It also contained conserved multiple repeats of short motifs.
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EXPERIMENTAL PROCEDURES |
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Worms-- Earthworms (Allolobophora japonica and L. terrestris), tubifex (Tubifex hattai), and lugworm (Neanthes japonica) were purchased from local fishing shops. Leeches (Glossiphonia complanata) were harvested from shallow irrigation water in Toyama Prefecture, Japan. The worms were rinsed with cold PBS, weighed, and processed immediately for lectin purification.
Purification of Annelid Lectins--
Lectins were extracted from
worms by essentially the same procedure previously described for
galectins (16, 17); briefly, worms were disrupted by homogenization in
a Polytron (Kinematica) with 5 volumes of cold EDTA-MEPBS (4 mM -mercaptoethanol, 2 mM EDTA, 20 mM sodium phosphate, pH 7.2, 150 mM NaCl).
After the bulk of soluble proteins had been removed by centrifugation
(15,000 rpm, 4 °C, 25 min), the lectins were specifically extracted
from the precipitate (ppt-1) with EDTA-MEPBS containing 20 mM lactose by shaking for 30 min at 4 °C. After
centrifugation as above, the obtained extract (sup-2) was extensively
dialyzed to remove lactose and then applied to a column of
asialofetuin-agarose (bed volume, 10 ml) prepared according to De Waard
et al. (18). After washing of the column with EDTA-MEPBS
(400 ml), bound protein was eluted with the same buffer containing 20 mM lactose.
Separation of EW29 by High Performance Gel-permeation Chromatography-- The main component (29 kDa) of the affinity purified lectin fraction from earthworms (EW29) was further purified by high performance gel-permeation chromatography on a TSK-G2000SWXL column (7.5 × 300 mm). The column was equilibrated and eluted with EDTA-PBS both in the presence and absence of 20 mM lactose at a flow rate of 0.5 ml/min. Protein elution was monitored by absorbance at 280 nm. For the subsequent hemagglutination assay, lactose was removed by extensive dialysis against EDTA-PBS.
Hemagglutination Assay-- Basically, the conventional assay system described by Nowak et al. (19) was used; briefly, 25 µl of serially diluted samples was mixed in each well of a 96-well microtiter V plate with 25 µl of EDTA-PBS, 25 µl of 1% (w/v) bovine serum albumin in saline, and 25 µl of trypsinized rabbit erythrocytes. After the plate had stood at room temperature for 1 h, "dot" (no agglutination) or "mat" (agglutination) formation was judged. For assessment of the inhibitory effect of various mono- and oligosaccharides, 25 µl of maximally diluted lectin solution that gave mat formation and 25 µl of serially diluted inhibitor saccharides were used in place of serially diluted samples and EDTA-PBS, respectively. Minimum concentrations that gave negative dot formation under the above conditions were defined as I50.
Effect of Saccharides on Extraction of Earthworm Lectin-- The effect of various saccharides on lectin extraction was investigated as described previously (12); earthworm lectin was extracted from small portions of ppt-1 (equivalent to 1-g wet weight of the worm) in the presence of a 0.1 M concentration of various sugars. After centrifugation, the supernatant solutions were subjected to SDS-PAGE, followed by Western blotting on a nitrocellulose membrane. The lectin was stained by a conventional double antibody method using anti-tubifex lectin antiserum (described below) and horseradish peroxidase-conjugated goat anti-rabbit IgG antiserum (Seikagaku Co., Tokyo). Both antisera were used at a 1,000-fold dilution. For peroxidase detection, POD Immunostain Set or High Sensitive Immunoblotting Kit (both from Wako Chemicals, Tokyo) was used.
Production of Antisera-- Antisera were raised in rabbits against either affinity purified tubifex lectin or affinity and gel filtration-purified EW29 by injecting the animals several times at 10-14-day intervals with 0.1-0.2 mg of lectin emulsified with Freund's complete adjuvant. Titer of the produced antisera was evaluated by both dot and Western blotting analyses. The antisera were stored at 4 °C in the presence of 0.02% NaN3.
Protein Structural Analyses-- Affinity purified lectins from earthworm, tubifex, and leech were further purified by reversed phase chromatography on a TSK TMS250 column (4.6 × 75 mm). Protein was eluted by a linear gradient of acetonitrile (20-60%, v/v) in 0.1% trifluoroacetic acid. The separated lectin fractions were lyophilized, dissolved in 10 mM Tris-HCl, pH 9.0, and digested with Achromobacter protease I (Wako Chemicals, Tokyo). Generated peptides were separated by reversed phase chromatography on a TSK-ODS-80TM column (7.5 × 250 mm) and were analyzed by a protein sequencer (Applied 477A) as described (17). The peptides were designated "Lys"-1, -2, -3 and so on, in the order of elution. In addition, in the case of earthworm 29-kDa lectin, reduction and S-carboxymethylation was also performed prior to the protease digestion. Thus derived peptides were prefixed "Cm-Lys."
Preparation of Genomic DNA from Earthworms-- Genomic DNA was prepared from two earthworm species, A. japonica and L. terrestris, by a conventional procedure (20). A few worms were used for the preparation from A. japonica, and a single worm was used for that from L. terrestris to assess the observed gene polymorphism (described below).
cDNA Cloning of EW29-- A probe DNA was prepared by means of the polymerase chain reaction (PCR). For amplification of a part of earthworm lectin gene, four sets of convergent oligonucleotide primers were synthesized based on the determined peptide sequences (Lys-4, Lys-7, Lys-8, and Lys-10), designated as Lys-4F/4R, Lys-7F/7R, Lys-8F/8R, and Lys-10F/10R (see Table IV for detail). PCR was performed by use of all combinations of the above forward (F) and reverse (R) primers and a Takara LA-PCR Kit (94 °C, 30 s; 52 °C, 1 min, 72 °C, 3 min for 35 cycles, then 72 °C, 10 min). Genomic DNA prepared from the earthworm A. japonica was used as a template. The amplified DNA (155 bp) obtained with primers Lys-4F and Lys-7R was cloned into pCRII (Invitrogen) on the basis of TA-cloning strategy according to the manufacturer's instruction, and the nucleotide sequence was confirmed with an Applied 373S DNA sequencer.
For cloning of full-length cDNAs, aProduction of Recombinant Earthworm Lectins, Wh, Nh, and
Ch--
Expression plasmids for recombinant EW29 protein (Wh),
N-terminal domain (Nh; meaning N-half), and C-terminal domain (Ch) were
constructed by a PCR procedure; for the production of Wh, a pair of
oligonucleotide primers were designed, i.e. G ATG GCT GGA
AGG CCT TTT CTG (Met1-Ala-Gly-Arg-Pro-Phe-Leu; designated
Wh-F; see Table IV) and CGAGTGGAGT TTA CTC GGA TTC G (antisense,
Glu258-Ser-Glu-Ter; Wh-R), and a full-length fragment
(794-bp) encoding 260 amino acids was amplified with a full-length
plasmid (clone 5) used as a template. The derived fragment was cloned
into pCRII in frame with -galactosidase
-peptide, and the derived
plasmid was used to transform Escherichia coli TOP10F'
(Invitrogen). Positive clones were selected by colony immunoblot
analysis using specific antiserum against EW29 and horseradish
peroxidase-conjugated goat anti-rabbit IgG as described previously
(21). The cloned E. coli was proliferated to full growth in
1 liter of LB medium in the presence of antibiotics at 37 °C. The
recombinant protein was induced by adding isopropyl-
-thiogalactoside
to give a final concentration of 0.2 mM and incubating the
culture for 2 h at the same temperature. The cells were collected
and lysed by sonication, and recombinant protein was adsorbed to
asialofetuin-agarose, as described previously (10).
Single Worm Genomic PCR-- In order to assess a cause for the polymorphism found in the earthworm lectin cDNAs (described under "Results"), genomic DNA was prepared from a single worm, and genomic PCR was performed with primers Wh-F and Wh-R to amplify a full-length region. After cloning of the derived fragments (0.8 kilobase pairs) into pCRII and transformation of E. coli TOP10F', 9 independent genomic clones were selected and their nucleotide sequences were compared with one another and to those derived from the cDNA cloning.
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RESULTS |
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Identification of Galactose-binding Lectins in Annelids-- Based on the concept that galectins are bound to insoluble glycoconjugates but are solubilized with a competing sugar, lactose, we applied the same procedure to purify annelid lectins as employed for galectins (10, 16, 17) as follows: (i) galactose-specific extraction in the absence of metal ions, and (ii) affinity chromatography on asialofetuin-agarose. A typical result of affinity chromatography on asialofetuin-agarose is shown for earthworm lectin in Fig. 1A, and the results of SDS-PAGE for all four annelid speceis, i.e. earthworm, tubifex, leech and lugworm, are shown in Fig. 1B. Major molecular species observed after lactose elution from the column were the following: 29 kDa (earthworm), 31 kDa (tubifex), 30 kDa (leech), and 30 kDa (lugworm). The observed sizes were also similar to those reported for either mammalian chimera-type (29-35 kDa) (22) or nematode tandem repeat-type galectins (32 kDa; see Refs. 10, 11, and 13). Since these lectins were obtained in the absence of metal ion, they were considered not to be C-type lectins. The fact that no detergent was necessary for the extraction also excluded the possibility that the annelid lectins are membrane-bound proteins. The yields of purified lectins from the four annelid species are summarized in Table I.
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Comparison of Structures of Annelid Lectins-- In order to elucidate the structural relationship between the annelid lectins, we first attempted Western blotting analysis by using anti-tubifex lectin antiserum. As shown in Fig. 2, all of the lectins purified from the other annelids were also positive, together with the smaller fragments described above. The degree of cross-reactivities with leech (Hirudinea) and lugworm (Polychaeta) proteins was relatively weak, probably reflecting phylogenic kinship between tubifex and earthworm (both belonging to the same class, Oligochaeta). When crude lectin extracts (sup-2) were used instead of purified fractions, only high molecular species (29-31 kDa) were observed. This is consistent with the above assumption that smaller fragments were digestion products.
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Effect of Saccharides on Lectin Extraction--
Since it became
evident that annelid lectins are structurally related, subsequent
studies were focused on the earthworm 29-kDa lectin (EW29), because its
purification yield was the best among the species examined (Table I).
First, various saccharides were tested for their ability to extract the
lectin from the earthworm precipitate (ppt-1) as described under
"Experimental Procedures." As a result, among various simple
saccharides, only galactose, lactose, and melibiose were found to be
effective. On the other hand, glucose, -methylmannoside,
L-fucose, maltose, and sucrose had no effect even at the
highest concentration used (100 mM; data not shown). As the
next step, concentrations of the effective saccharides were changed
(i.e. 1, 10, and 100 mM), and the amounts of
extracted lectin were compared (Fig. 3).
Lactose and melibiose showed almost the same ability to extract the
lectin at 10 and 100 mM, whereas galactose showed slightly
poorer ability. It is notable that EW29 was extractable with not only
lactose, but also galactose and melibiose (Gal
1,6Glc), a linkage
isomer of lactose (Gal
1,4Glc). Such a feature has never been
observed for galectins.
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Oligomeric Structure and Hemagglutinating Activity of EW29-- High performance gel filtration analysis was carried out to estimate the molecular organization of EW29 under non-denaturing conditions. The affinity purified earthworm lectin (a mixture of major 29-kDa and minor 15-17-kDa fragments) was applied to a TSK G2000SWXL column in the presence of 20 mM lactose (Fig. 4A), since in its absence the protein elution was considerably retarded. Fractions showing UV adsorption (280 nm) were subjected to SDS-PAGE analysis (Fig. 4B). By comparing eluting positions of marker proteins, the 29-kDa lectin was considered to exist as a monomer (calculated mass, 30,000 Da). SDS-PAGE analysis showed that the purified fraction was >95% pure and almost free of the 16-kDa species. We used this fraction as highly purified EW29 for the production of specific antiserum and for the following hemagglutination test.
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cDNA Cloning of EW29-- Based on the peptide sequences of EW29 (Lys4, Lys7, Lys8, and Lys9), four pairs of degenerate primers were synthesized (for their sequences, see Table IV). PCR was performed by using all combinations of these forward and reverse primers,, and genomic DNA was prepared from the earthworm as a template. A significant amplification product (155 bp) was obtained only when the combination of primers, Lys-4F and Lys-7R, was used (data not shown). The amplified fragment was cloned into pCRII, and the nucleotide sequence was determined. The deduced amino acid sequence was found to follow the sequence of Lys-4 (used as a forward primer) with no termination codon in frame and also included the determined sequence of Lys-3, "Asp-Val-Val-His-His-Arg-Asn-Asp-Lys" (data included in Fig. 5). This confirmed the derived fragment is relevant as a PCR product corresponding to EW29.
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Single Worm Genomic PCR-- To clarify whether the polymorphism described above occurs at the species or individual level, we performed genomic PCR using DNA prepared from a single worm of L. terrestris. The entire coding region was amplified by using newly synthesized primers, Wh-F and Wh-R (Table IV), and the derived fragments (0.8 kilobase pairs) were analyzed after cloning into pCRII. As a result, all of the 9 genomic clones analyzed proved to be correlated to one of the four types of cDNA clones as follows: 1, 2, 3, and 3 genomic clones were assigned to cDNA clone 5, 7, 8, and 9, respectively. This result clearly demonstrates that the observed polymorphism already existed at the individual level.
Bacterial Expression of Recombinant Earthworm Lectins, Wh, Nh, and
Ch--
To prove that the obtained cDNAs were derived from
mRNAs for functional 29-kDa lectin, we chose one of the cDNAs
(clone 5) for an expression experiment in E. coli. As
expected, a 35-kDa protein (a fusion product with -galactosidase
-peptide) was produced and purified by affinity chromatography on
asialofetuin-agarose (Fig. 6). However,
many smaller fragments (15-17 kDa) were again observed, as in the case
of natural proteins (Fig. 1). Since these small fragments were able to
bind to the affinity column, they were considered to be either
N-terminal or C-terminal domains that retained binding ability even
after degradation. To confirm this, we transferred the small fragments
that had been adsorbed to asialofetuin-agarose to a polyvinylidene
difluoride membrane (indicated by a, b,
c, and d in Fig. 6), and subjected them to direct
sequence analysis. Partial N-terminal sequences suggested that the
bands a, c, and d were C-terminal
fragments beginning from Lys105, His127, and
Lys132, respectively. Since all these positions were
located in the connecting region between N-terminal and C-terminal
domains, this result indicates that only the C-terminal domain retained
binding activity. On the other hand, the band "b" did
not give any significant amino acid peak even when a sufficient amount
of protein was applied. It might be attributable to artificial
N-terminal cyclization to form pyroglutamate, because many glutamine
residues, i.e. Gln110, Gln122, and
Gln123, are located in the connecting region.
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DISCUSSION |
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When we first chose tubifex (class Oligochaeta) for purification of galactose-binding proteins, multiple protein bands around 15-17 kDa together with a faint 31-kDa band were observed on SDS-PAGE after purification by asialofetuin-agarose chromatography. However, antiserum raised against this mixed fraction detected only the 31-kDa species in the lactose extract of tubifex (data not shown). This implied that the smaller fragments were degradation products generated by proteolysis during purification from the parental 31-kDa protein. This antiserum also detected approximately 30-kDa proteins in the lactose extracts prepared from other annelids, i.e. earthworm (Oligochaeta), lugworm (Polychaeta), and leech (Hirudinea).
During the course of this study, Cole and Zipser (24, 25) reported the
presence of similar galactose-specific lectins, LL16, LL35, and LL63,
from the leech Hemopis marmorata. LL16 and LL35 seem to have
biochemical properties similar to those of the earthworm lectin
investigated in the present study, i.e. in terms of
molecular weight, metal independence for sugar binding, and specificity
for simple saccharides. As they used detergent to extract leech
lectins, it should have solubilized endogenous ligand glycoconjugates
to which the lectins were bound. They also reported that LL16 and LL35
showed broader sugar binding specificity than LL63; binding of the
former lectins to asialofetuin-agarose was inhibited by galactosamine
and -methylgalactoside as in the case of our preparation, whereas
that of the latter was not inhibited by these saccharides. Since
the reported molecular size (35 kDa) of LL35 was close to those of our
annelid lectins (29-31 kDa), and specific antiserum raised against
highly purified EW29 reacted significantly with
LL35,3 we consider LL35 to be
a leech homologue of the earthworm lectin. Galactose-binding lectins
recently identified in N. japonica and Marphysa
sanguinea (both belonging to Polychaeta; see Ref. 26) as well as
Urechis unicinctus (belonging to Echiuroidae; see Ref. 27)
may also represent couterpart molecules in these annelids.
Although these annelid lectins were prepared essentially by the same
strategy as used for galectins, partial peptide analyses suggested that
they are not related to galectins but form another protein family
having the key consensus motif
Gly-X-X-X-Gln-X-Trp. In
fact, the earthworm lectin could be extracted with both galactose and
melibiose as well as lactose, whereas galectins in general have only
weak affinity to the former saccharides. Although we added
-mercaptoethanol to the buffer for lectin extraction, it proved to
be unimportant, because its removal did not significantly reduce the
recovery or activity of the earthworm lectin.
The result of cDNA cloning of EW29 was striking in several aspects.
First of all, the lectin proved not to be a member of the galectin
family, contrary to our expectation. Moreover, the annelid lectins were
suggested to be members of a larger family that includes many other
carbohydrate-recognition proteins. At least two groups of proteins have
been shown to contain the multiple short consensus motif
Gly-X-X-X-Gln-X-Trp (Fig.
8). The first group consists of
hemagglutinins (29-35 kDa) including EW29 investigated in this work
and HA33 (a 33-kDa major component of hemagglutinin from C. botulinum type C; see Ref. 28) as well as the B-chain of a series
of plant toxins. A number of plant toxins consisting of A-chain
(ribosome-inactivating protein) and B-chain (lectin) have been
reported, e.g. ricin (or RCA60; see Ref. 29), agglutinin (or
RCA120; see Ref. 30) from Ricinus communis and their related homologues in elderberry (Sambucus sieboldina), S. sieboldina agglutinin (31), and sieboldin-b (32). Modeccin (33),
abrin (34), and viscumin (35) are also this type of toxin found in
other plants. The second group consists of various hydrolytic enzymes
(see Fig. 8): (i) glycosidases Oerskovia xanthineolytica -1,3-glucanase (36) and Streptomyces lividans xylanase A
(37), and (ii) proteases Rarobacter faecitabitus protease I
(38) and
subunit of horseshoe crab coagulation factor G (
subunit is a serine protease; see Ref. 39). Notably, all members of the lectin group are known to be specific for
galactose/N-acetylgalactosamine except for S. sieboldina agglutinin. Since S. sieboldina agglutinin binds to sialic acid that is
2,6-linked to galactose (Sia
2,6Gal), it may be a variant having deviated from an ancestral
galactose-specific protein. All of these members including EW29 consist
of two homologous domains (14-16 kDa), both of which retain
carbohydrate binding activity as far as examined. This fact implies
that acquirement of multivalency is critical for preservation of
efficient lectin function(s). On the other hand, carbohydrate-binding
properties are not known for most of the enzyme group. As a sole
exception, the CRD of R. faecitabitus protease I has been
shown to have mannose binding activity (38). The possibility is
excluded that EW29 exists as a larger conjugate via disulfide bridge(s)
like ricin and coagulation factor, because Western blotting detected
only the 29-kDa species even when
-mercaptoethanol was omitted
throughout the experiment.
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X-ray crystallographic study has been carried out for ricin complexed with lactose (40). As described, the ricin B-chain has two galactose-binding sites corresponding to the two homologous domains. Amino acid residues forming the binding sites are as follows: Asp22, Gln35, Trp37, Lys40, and Asn46 for N-terminal CRD, and Asp234, Asn255, and Tyr248 (shown as inverted boldface letters in Fig. 8B). These residues are mapped to the first repeat segment of the N-terminal CRD and to the third one of the C-terminal CRD, respectively. Inclusion of tryptophan (or tyrosine) residues at the seventh position of the consensus motif is also intriguing, because such aromatic residues are frequently found in carbohydrate-recognition sites of various sugar-binding proteins, particularly in galactose-specific lectins (41, 42).
Although total amino acid identities between these carbohydrate-recognition proteins were low (<10%), it is noteworthy that these CRDs had striking similarity in their segment architecture; all of the CRDs consisted of three segments each consisting of 40-50 amino acids (5 kDa) and containing 2 consensus motifs in the latter positions. Since the segment size agrees with a moderate "exon" size (43, 44), it is possible to speculate that the original CRD (15 kDa) was the result of several gene duplication events of this ancestral peptide, or "module," which is presumed to have had primitive carbohydrate binding activity. According to this scenario, one group of the descendants might have evolved as "lectins" (30 kDa) by acquiring divalency by further duplication of the 15-kDa CRD, whereas the other group might have evolved to "enzymes" by ligation with some peptides having catalytic domain(s). Their CRDs could have been useful, e.g. for facilitated binding to target cells covered by carbohydrate chains, as has been suggested for R. faecitabitus protease I (38). X-ray crystallography studies should support this hypothesis. "Ricin superfamily" or "R-type" lectin (where R indicates for ricin and repeat) seem to be appropriate as the name for this novel superfamily.
The present results on cDNA (genome) cloning showed that EW29 is encoded by multiple genes, i.e. genome polymorphism. The analyzed 8 cDNA and 9 genomic clones completely overlapped except for cDNA clone 2 (Table V), which differed from the major cDNA type (clones 1, 3, 5, and 10) at only two positions. Therefore, the actual number of EW29 genes in a single worm would not seem to far exceed four (i.e. the four types represented by cDNA clones 5, 7, 8 and 9; or genomic clones 1, 5, 2, and 3, respectively; see Table V). Difference among the four types of genes (transcripts) spans over the whole coding region at 29 positions, although most of them (26 cases) were silent substitutions. Even for the non-silent substitutions, they were rather homologous ones, and therefore, the generated three polypeptides would not be expected to show very different properties, although their stability or fine sugar-binding specificity might be different. In this regard, three closely similar bands detected by anti-tubifex antibody in the extraction experiment (Fig. 3) might represent these three polypeptides. On the other hand, the fact that half of the obtained cDNA clones were identical to clone 5 suggests that the corresponding gene is more strongly expressed than other genes (corresponding to cDNA clones 7, 8, and 9). Apparently, further functional studies are necessary to evaluate these variant genes.
The physiological function(s) of the investigated annelid lectins is not known. However, based on the observation that LL35 is localized in leech photoreceptors in sensory afferents, Zipser and colleagues (45) speculated that the lectin has some function in the axon targeting in the central nervous system of this organism. At the moment, it is not known whether the earthworm and other annelids have galectins or not. However, if they do, such galectins would not seem to be dominant with respect to the investigated lectins. In this context, the hypothesis described under the Introduction has again emerged that absence of galectins in some invertebrate phyla can be compensated by similar but distinct types of galactose-binding lectins. Inversely, it is of interest to note that no homologues to the investigated annelid lectins have been reported so far in vertebrates and nematodes, in which galectins are dominant. Alternatively, galectins do exist in almost all animal species but cannot be extracted with simple saccharides such as lactose, as has been demonstrated in some tandem repeat-type galectins (46, 47). In any case, the present study has shown the occurrence of relatively abundant, novel galactose-binding lectins in the phylum Annelida. Our results add further evidence of the ubiquitous occurrence of galactose-binding proteins in multicellular organisms and thus evidence for fundamental importance of "galactose recognition."
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Giebing (Düsserdorf) for kindly providing us with a cDNA library for the earthworm L. terrestris; Yasuhiro Arai, Sachiko Yamamoto, and Ko Hayama for their technical assistance. Fusako Honda and Ai Hirabayashi are also acknowledged for their collaboration in the worm collection.
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FOOTNOTES |
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* This work was supported in part by Grant 05274101 from the program for Scientific Research on Priority Areas (05274101) from the Ministry of Education, Science, Culture and Sports, Japan, and by grants from the Naito Foundation, Mitsubishi Foundation, and Kato Memorial Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-426-85-3741;
Fax: 81-426-85-3742; E-mail: j-hira{at}pharm.teikyo-u.ac.jp.
§ Recipient of a Japan Society for the Promotion of Science fellowship. Present address: Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Rd., Calcutta, India 700 032.
1 The abbreviations used are: EW29, earthworm 29-kDa lectin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; CRD, carbohydrate-recognition domain; bp, base pair; PBS, phosphate-buffered saline.
2 H. Ahmed, et al., unpublished observations.
3 A. Chapman and B. Zipser, personal communication.
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