Purification, characterization, and cDNA cloning of {alpha}-N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera

Mari Kakiuchi2, Nozomu Okino2, Noriyuki Sueyoshi2, Sachiyo Ichinose3, Akira Omori3, Shun-ichiro Kawabata4, Kuniko Yamaguchi2 and Makoto Ito1,2

2Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; 3Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida 194–8511, Tokyo, Japan; and 4Department of Biological Science, Graduate School of Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received on June 14, 2001; revised on August 27, 2001; accepted on August 29, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 References
 
We report here the purification, characterization, and cDNA cloning of a novel N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera. The purified lectin showed 19-kDa, 41-kDa, and 60-kDa protein bands on SDS–PAGE, possibly corresponding to a monomer, homodimer, and homotrimer. Interestingly, on 4–20% native PAGE the lectin showed at least nine protein bands, among which oligomers containing six to nine subunits had potent hemagglutination activity for sheep erythrocytes. The hemagglutination activity of the lectin was specifically inhibited by N-acetylgalactosamine, Tn antigen, and blood group A trisaccharide, but not by N-acetylglucosamine, galactose, galactosamine, or blood group B trisaccharide. The specificity of the lectin was further examined using various glycosphingolipids and biotin-labeled lectin. The lectin was found to bind to Gb5Cer, but not Gb4Cer, Gb3Cer, GM1a, GM2, or asialo-GM2, indicating that the lectin specifically binds to the terminal {alpha}-GalNAc at the nonreducing end. The hemagglutination activity of the lectin was completely abolished by chelation with EDTA or EGTA and completely restored by the addition of CaCl2. cDNA cloning of the lectin showed that the protein is composed of 168 amino acids, including a signal sequence of 18 residues, and possesses the typical C-type lectin motif. These findings indicate that the protein is a C-type lectin. The recombinant lectin, produced in a soluble form by Escherichia coli, showed binding activity for asialomucin in the presence of Ca2+ but no hemagglutination.

Key words: C-type lectin/{alpha}-GalNAc-specific lectin/oligomerization/starfish/Tn antigen


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 References
 
Lectins are a group of proteins (or glycoproteins) that bind specifically and reversibly to carbohydrates, particularly the sugar moiety of glycoconjugates, resulting in cell agglutination and precipitation of glycoconjugates (Goldstein et al., 1980Go). They are ubiquitously distributed in nature, being found in viruses, bacteria, fungi, plants, insects, crustaceans, and vertebrates (Sharon and Lis, 1989Go). Vertebrate Ca2+-dependent lectins, named C-type lectins, share structural features such as homology in amino acid sequence, the positions of disulfide bonds, and calcium binding sites, although they show different carbohydrate-binding specificities. Carbohydrate recognition domains of C-type lectins are conserved over species, although the degree of similarity in overall sequences is generally low (Drickamer, 1988Go; Harrison, 1991Go; Weis et al., 1991Go). C-type lectins have also been isolated from invertebrates (Giga et al., 1987Go; Muramoto and Kamiya, 1990Go; Suzuki et al., 1990Go; Hatakeyama et al., 1994Go). Although the functions of invertebrate lectins are ambiguous at present, several lines of evidence suggested that lectins have significant roles in the differentiation and development of invertebrates via mediation of cell–cell and/or cell–substrate interactions (Komano et al., 1980Go) and in neutralization and exclusion of foreign substances through binding to their carbohydrate components (Wu et al., 1997Go)

Besides having biological roles in cell recognition and host defense, lectins have long been used in research as proteins that recognize specific sugar chains of glycoconjugates, including bacterial lipopolysaccharides and cell surface glycoproteins as well as glycolipids (Gabius et al., 1998Go). Some of them would be useful for detecting tumor-specific or associate antigens, and developmentally regulated sugar residues (Konska et al., 1998Go).

We report here a novel {alpha}-N-acetylgalactosamine (GalNAc)-specific lectin from starfish, Asterina pectinifera. The lectin tends to make oligomers having molecular masses of 40–250 kDa composed of 19-kDa monomers. The activity to bind GalNAc was found in the monomer as well as oligomers, whereas hemagglutination activity was only found in the oligomers composed of six to nine subunits. The starfish lectin, consisting of 168 amino acid residues with a molecular mass of 18,935 Da, required Ca2+ for both binding and hemagglutination activity and contained the typical C-type lectin motif, indicating that the protein is a C-type lectin.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 References
 
Purification of the starfish lectin
A crude extract of starfish A. pectinifera was applied to a column of GalNAc-Sepharose CL-4B previously equilibrated with Tris-buffered saline (TBS) containing 5 mM CaCl2 (Figure 1). One protein peak was eluted with ethylenediamine tetra acetic acid disodium salt (EDTA) and showed hemagglutination activity with sheep erythrocytes. As shown in Table I, the lectin was purified 130-fold with 219% recovery. An increase in the recovery may indicate that inhibitor(s) were removed from the lectin fraction by the affinity chromatography. The purified lectin showed three protein bands on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) corresponding to molecular masses of 19 kDa, 41 kDa, and 60 kDa after staining with both Coomassie brilliant blue (Figure 2A) and silver staining solution (data not shown) under reducing conditions. The 19-kDa band is the major band followed by the 41-kDa band, whereas the 60-kDa band is very faint. It is likely that the three bands are derived from the lectin per se and represent a monomer, homodimer, and homotrimer, respectively, because all three bands were specifically visualized by western blotting with the anti-starfish lectin antibody (Figure 2C). These results also suggested the presence of SDS-resistant disulfide bonds under reducing conditions in the lectin molecule. On the other hand, under nonreducing conditions, two protein bands having molecular masses of 31 kDa and 57 kDa were visualized after staining with Coomassie brilliant blue (Figure 2B).



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Fig. 1. Affinity chromatography of starfish lectin on a GalNAc-Sepharose CL-4B column. Crude extract of starfish was applied to a column equilibrated with TBS containing 5 mM CaCl2. The adsorbed proteins were eluted with TBS containing 20 mM EDTA. Hemagglutination activity was measured with sheep erythrocytes. The fractions indicated by a horizontal bar were pooled as the active fraction. Squares, agglutination units (AUs), defined as the reciprocal value of the end point dilution causing hemagglutination; circles, protein content determined by the bicinchoninic acid method.

 

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Table I. Purification of starfish lectin
 


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Fig. 2. SDS–PAGE and immunoblot analysis of starfish lectin. Coomassie-stained SDS–PAGE of reduced (A) and nonreduced (B) starfish lectin eluted from the GalNAc-Sepharose column with 20 mM EDTA. Lane 1, molecular weight markers; lanes 2, 3, lectin eluted with EDTA. (C) Western immunoblot analysis using anti-starfish lectin rabbit polyclonal antibody.

 
Oligomerization of the starfish lectin
Interestingly, the purified lectin showed at least nine protein bands corresponding to molecular masses of 20–252 kDa when analyzed by 4–20% native PAGE using Tris-glycine buffer (Figure 3A). The lectin was eluted from the gel after electrophoresis, and its hemagglutination activity was determined using sheep erythrocytes. The highest activity was found in the fraction corresponding to the 135-kDa band, whereas bands corresponding to the molecular masses below 57 kDa showed little or no hemagglutination activity. Although the lectin apparently formed multiple complexes with different numbers of subunits, the complexes were converted to two major bands having 19 kDa and 41 kDa on SDS–PAGE under reducing conditions (Figure 3B). These results indicate that the formation of multiple complexes is necessary for the lectin to express hemagglutination activity, with the complexes composed of six to nine subunits showing especially strong activity (Figure 3A).



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Fig. 3. Oligomerization of starfish lectin. (A) Native PAGE (4–20% gradient) of the starfish lectin eluted from the GalNAc-Sepharose column with 20 mM EDTA. Proteins were stained with Coomassie brilliant blue. Unstained gel was cut into 2-mm slices, and each fraction was assayed for hemagglutination activity using sheep erythrocytes. Standard proteins used for molecular markers (molecular mass in parentheses) were thyroglobulin (669 kDa), ferritin (443 kDa), lactate dehydrogenase (140 kDa), bovine serum albumin (66 kDa), and trypsin inhibitor (20 kDa). (B) SDS–PAGE of several fractions of native PAGE. +, positive for hemagglutination activity, –, negative for hemagglutination activity.

 
Specificity of the starfish lectin
To characterize the carbohydrate-binding specificity of the purified lectin, three different approaches were used; the first was evaluation of its activity by hemagglutination assay using sheep erythrocytes, and the second was by thin-layer chromatography (TLC) overlay assay using various glycosphingolipids and biotin-labeled lectin. The third method was to use enzyme-linked immunosorbent assay (ELISA) with asialomucin-coated plates. The third approach was also used to evaluate the activity of the recombinant monomeric lectin for binding asialomucin.

Hemagglutination assay.
Table II shows the inhibition of the hemagglutination activity of the lectin by various monosaccharides, oligosaccharides, and glycopeptides. For monosaccharides, the most potent inhibitor was GalNAc, which blocked hemagglutination at 0.195 mM, whereas GlcNAc did not show any inhibition at 100 mM. Gal was found to inhibit the hemagglutination, but its inhibition was 500 times less than that of GalNAc. GalN, the de-acetylated form of GalNAc, showed no inhibition. The dimer, trimer, and tetramer of GalNAc by {alpha}-linkages were found to be slightly more effective than the monomer. Neither Glc, GlcN, L-Fuc, NeuAc, nor Man showed inhibition. Interestingly, D-Fuc, a methyl-pentose not found in nature, showed weak inhibition. Among the oligosaccharides and glycoproteins tested, blood group A trisaccharide and Tn antigen showed potent inhibitory effects, whereas blood group B trisaccharide (12.5 mM), fetuin (1 mg/ml), and asialofetuin (1 mg/ml) demonstrated no inhibition.


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Table II. Inhibition of hemagglutination activity of starfish lectin
 

TLC overlay assay.
The specificity of the lectin was further examined by TLC overlay assay using various glycosphingolipids (Figure 4). The lectin was found to bind Gb5Cer (Forssman antigen; GalNAc{alpha}1,3GalNAcß1,3Gal{alpha}1,4Gal-ß1,4Glcß1,1'Cer), but not Gb4Cer (globoside; GalNAc-ß1,3Gal{alpha}1,4Galß1,4Glcß1,1'Cer), Gb3Cer (Gal{alpha}1,4Gal-ß1,4Glcß1,1'Cer), GM1a (Galß1,3GalNAcß1,4[NeuAc-{alpha}2,3]Galß1,4Glcß1,1'Cer), GM2 (GalNAc-ß1,4[NeuAc-{alpha}2,3]Galß1,4Glcß1,1'Cer), or asialo-GM2 (GalNAc-ß1,4Galß1,4Glcß1,1'Cer). These results clearly indicate that the lectin specifically binds the terminal {alpha}-GalNAc, but not ß-GalNAc or {alpha}/ß-Gal, at the nonreducing end of glycosphingolipids. Interestingly, one of the receptors for the lectin on sheep erythrocytes seemed to be Gb5Cer, because the glycolipid having the same Rf as Gb5Cer was visualized by the lectin binding assay (Figure 4B) and Gb5Cer was actually isolated from sheep erythrocytes (Fraser and Mallette, 1974Go).



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Fig. 4. TLC overlay binding assay. Various glycosphingolipids (each 2.5 nmol) were applied to a TLC plate, which was developed with chloroform/methanol/0.02% CaCl2 (5:4:1, v/v/v). The glycosphingolipids were visualized by orcinol-H2SO4 (A) and biotin-labeled lectin (B). Details are described in Materials and methods. Lane 1, lipid extracts from sheep erythrocyte membranes; lane 2, Gb5Cer (Forssman antigen, GalNAc{alpha}1,3GalNAcß1,3Gal{alpha}1,4Galß1,4Glcß1,1'Cer); lane 3, Gb4Cer (globoside, GalNAcß1,3Gal{alpha}1,4Galß1,4Glcß1,1'Cer); lane 4, Gb3Cer (Gal{alpha}1,4Galß1,4Glcß1,1'Cer); lane 5, GM1a (Galß1,3GalNAcß1,4[NeuAc{alpha}2,3]Galß1,4Glcß1,1'Cer); lane 6, GM2 (GalNAcß1,4[NeuAc{alpha}2,3]Galß1,4Glcß1,1'Cer); lane 7, asialo-GM2 (GalNAcß1,4Galß1,4Glcß1,1'Cer).

 

ELISA.
To examine the specificity of the lectin by ELISA, we used asialomucin-coated plates, because asialomucin is the most potent inhibitor of hemagglutination of sheep erythrocytes by the lectin (Table II). The binding of the biotin-labeled lectin to asialomucin was strongly inhibited by GalNAc in a concentration-dependent manner, but not by Gal, GlcNAc, Man, NeuAc, or D-Fuc up to 200 µM (Figure 5A). When various glycopeptides were examined for haptens, asialomucin, mucin, and Tn antigen were found to be potent; among them asialomucin had the greatest inhibitory effect. Fetuin and T antigen showed no inhibitory effects up to 50 µg/ml (Figure 5B). These findings support those obtained from the hemagglutination assay (Table II). Tn antigens have the terminal GalNAc residues that are directly conjugated with the serine or threonine of backbone peptides by {alpha}-linkage, and the GalNAc residues were masked with Gal in the T antigen. The structure of the sugar moiety of asialomucin used in this study is not clear, but {alpha}-GalNAc residues are likely to be exposed at the nonreducing end (D'arcy et al., 1989Go; Savage et al., 1990Go).



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Fig. 5. Inhibition of the binding of starfish lectin to the asialomucin-coated plate by several carbohydrates and glycopeptides. Biotin-labeled starfish lectin (0.2 µg) was incubated at 4°C with various concentrations of the indicated carbohydrates in 100 µl of TBS containing 5 mM CaCl2. (A) closed circles, GalNAc; open circles, Gal; open circles, NeuAc; closed squares, Man; open triangles, GlcNAc; closed triangles, Fuc. (B) closed circles, mucin; open circles, asialomucin; closed squares, T antigen; open squares, Tn antigen; closed triangles, fetuin. Details are described in Materials and methods.

 

Ca2+ dependence of the starfish lectin
No hemagglutination activity was found when either EDTA or ethylene glycol bis(2-aminoethyl ether)-tetra acetic acid (EGTA) was added to the reaction mixture at 2.5 mM as the final concentration. The activity of the lectin was restored completely when chelating reagents were removed by dialysis and 5 mM CaCl2 was added to the reaction mixture. These results clearly indicate that Ca2+ is indispensable for the expression of the hemagglutination activity of the lectin and thus the lectin should be classified as type C.

Cloning of the starfish lectin cDNA
Using the N-terminal and internal amino acid sequences, sense and antisense primers were designed and polymerase chain reaction (PCR) was performed. As a result, the 114-bp PCR product was specifically amplified. To obtain the 5'- and 3'-terminal segments of the cDNA, 5'- and 3'-RACE were performed. A 231-bp PCR product was amplified from the 5'-RACE, and a 875-bp product from the 3'- rapid amplification of cDNA ends (RACE). The sequences of the overlapping cDNA fragments contained an initiation codon in agreement with the Kozak rule (Kozak, 1997Go) and a termination codon. However, several clones were found to exhibit minor variations. Thus, cDNA cloning of the lectin was performed using a starfish ovary cDNA library by plaque hybridization to confirm the sequence obtained by RACE-PCR. After the screening of 740,000 colonies, five positive plaques were obtained and converted to plasmids using the VCSM13 helper phage. One plasmid (designated pApL) contained a full-length cDNA encoding the lectin.

DNA and deduced amino acid sequences of the starfish lectin
Figure 6A shows the nucleotide and deduced amino acid sequences of pApL. The open reading frame of 504 nucleotides encoded a polypeptide of 168 amino acids. The presence of the putative signal sequence (18 amino acid residues) was found just before the N-terminus of the lectin. The presence of the hydrophobic motif near the N-terminus was also clearly indicated by hydrophobicity plot analysis (Figure 6B). No potential N-glycosylation sites were found in the sequence. A polyadenylation signal was found in the 3'-untranslated region (Figure 6A). From the deduced amino acid sequence, the molecular mass and pI of the lectin were calculated to be 18,935 and 4.50, respectively.



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Fig. 6. Nucleotide and deduced amino acid sequences of starfish lectin. (A) The nucleotide and deduced amino acid sequences. Nucleotide (upper) and deduced amino acid (lower) residues are numbered on the right. Amino acid residues are numbered begining with the first methionine. Asterisks and +1 represent the translation termination codon and the N-terminal amino acid, respectively. Single and double underlines represent amino acid sequences determined by microsequencing after digestion with lysylendopeptidase AP-1 and poly(A) additional signal, respectively. (B) Hydrophobicity plots for starfish lectin determined by the method of Kyte and Doolittle (1982)Go.

 
Alignment of the deduced amino acid sequence of the starfish lectin with those of other C-type lectins
The similarity in sequence to other C-type lectins was analyzed using CLUSTAL W software (Thompson et al., 1994Go). Figure 7 shows the alignment of the deduced amino acid sequence of the starfish lectin with those of C-type lectins; sea urchin lectin (echinoidin), acorn barnacle lectin (BRA-3), and human proteoglycan (brevican). It was revealed that starfish lectin exhibited identities of 39% for echinoidin, 35% for BRA-3, and 34% for brevican. Typical conserved sequence in C-type lectins was also found in the sequence of the starfish lectin (asterisks in Figure 7). These results again indicate that the starfish protein is a C-type lectin.



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Fig. 7. Comparison of the amino acid sequence of starfish lectin with those of C-type lectins. Alignment of the sequences was done with the aid of the computer program CLUSTAL W. Echinoidin, the sea urchin (Anthocidaris crassispina) lectin; brevican-human, proteoglycan from human; BRA-3, the acorn barnacle (Megabalanus rosa) lectin. Identical amino acids are indicated by white type on black background and chemically similar amino acids by small letters. Gaps inserted into the sequences are indicated by dots. An asterisk represents the typical C-type lectin motif.

 
Variants of the starfish lectin
RACE-PCR using starfish ovary mRNA suggested the presence of variants of the lectin. Thus, reverse transcriptase (RT)-PCR was performed with two primers designed based on sequences outside of the open reading frame as described in Materials and methods. Thirteen clones sequenced were classified into five groups. Minor variations were a change of T to G at position 10, T to C at 44, C to A at 48, T to A at 200, A to G at 250, and C to T at 458 of the prototype, respectively, with the result that Phe4, Phe15, Leu67, Thr84, and Ala153 could be replaced with Val, Ser, Gln, Ala, and Val, respectively.

Expression of the recombinant starfish lectin
The recombinant plasmid containing the starfish lectin cDNA, pTApL, was transfected into Escherichia coli JM109 cells which were grown at 37°C in the presence of isopropyl 1-thio-ß-D-galactoside (IPTG). The expression of proteins in cell lysates was analyzed by SDS–PAGE under reducing conditions (Figure 8A) and the activity was assayed by ELISA using asialomucin-coated plates (Figure 8B). The expression of the 19-kDa protein (monomeric form of the lectin) gradually increased with time after addition of IPTG, although dimeric and trimeric forms of the recombinant lectin were not detected by western blot (Figure 8A). Parallel with the expression of the 19-kDa protein, the activity to bind asialomucin was found to increase with time in the presence of 5 mM CaCl2, whereas no activity was found in the presence of EDTA (Figure 8B). These results indicate that the recombinant lectin binds to asialomucin in the presence of Ca2+. However, the recombinant lectin did not show any hemagglutination activity (data not shown), suggesting that it does not present as an oligomeric form.



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Fig. 8. Production of recombinant starfish lectin in E. coli JM109 carrying pTApL (A) and its binding activity against asialomucin (B). (A) Time course for production of the recombinant lectin after induction by IPTG. After addition of 0.1 mM IPTG, the culture was continued at 37°C for 16 h. At the indicated times, cell lysates were subjected to immunoblot analyses using anti-starfish lectin antibody. (B) Time course for the expression of the binding activity for the recombinant lectin after induction by IPTG. After addition of 0.1 mM IPTG, the culture was continued at 37°C for 2 h. At the indicated times, cell lysates were subjected to ELISA to measure the binding activity for asialomucin as described in Materials and methods. Closed circles, binding activity for asialomucin; squares, binding activity for asialomucin in the presence of 20 mM EDTA; open circles, binding activity for asialomucin of mock transfectant.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 References
 
In this study, we purified and cloned a GalNAc-specific lectin from starfish. The lectin seems to be a C-type lectin, because (1) Ca2+ is indispensable for the lectin to express the activity for agglutinating sheep erythrocytes as well as binding asialomucin, and (2) the primary structure of the lectin deduced from the cDNA represents a typical C-type lectin motif. Among the C-type lectins reported to date, the starfish lectin was found to be unique in forming oligomers. It showed two major protein bands corresponding to 19 kDa and 41 kDa and one minor 60-kDa band on SDS–PAGE under reducing conditions in the presence of 2-mercaptoethanol (2ME) after visualization with Coomassie brilliant blue and western blotting using anti-starfish lectin antibody (Figure 2). Based on cDNA cloning of the lectin, the molecular mass was estimated to be 18,935 and the presence of nine Cys residues was deduced. These results may indicate that the 19-kDa, 41-kDa, and 60-kDa proteins are a monomer, homodimer, and homotrimer, respectively, and SDS/2ME-resistant intermolecular disulfide bonds are present that could form oligomers. No possible N-glycosylation site was found in the sequence, and thus interaction between the carbohydrate-recognition domain and N-glycans of the lectin molecules is unlikely. Interestingly, the lectin showed at least nine protein bands corresponding to molecular masses of 20–252 kDa on 4–20% native PAGE (Figure 3A), among which oligomeric lectins composed of six to nine subunits were found to exhibit potent hemagglutination activity, suggesting that the lectin is present as an oligomeric structure in nature. A lectin with multimeric structure has also been reported in rainbow trout serum, although the specificity of the trout lectin is completely different from that of starfish lectin. The trout lectin is specific to Glc, GlcNAc, and Man (Jensen et al., 1997Go).

The specificity of the lectin is summarized as follows:

1. Substitution of C2 with an N-acetyl group is essential, because GalN, Gal, and blood group B trisaccharide (Gal{alpha}1,3[Fuc{alpha}1,2]Gal) do not potently inhibit the hemagglutination or lectin-mediated binding and the lectin does not bind to Gb3Cer (Gal{alpha}1,4Galß1,4Glcß1,1'Cer) or GM1a (Galß1,3GalNAcß1,4[NeuAc{alpha}2,3]Galß1,4Glcß1,1'Cer);

2. The OH group at C4 should be axial, because the activity of the lectin is specific to GalNAc but not to its C4 epimer GlcNAc;

3. The lectin specifically binds to terminal {alpha}-GalNAc, but not ß-GalNAc, at the nonreducing end, because the lectin binds to Gb5Cer (GalNAc{alpha}1,3GalNAcß1,3Gal{alpha}1,4Galß1,4Glcß1,1'Cer) and blood group A trisaccharide (GalNAc{alpha}1,3[Fuc{alpha}1,2]Gal), but not to Gb4Cer (GalNAcß1,3Gal{alpha}1,4Galß1,4Glcß1,1'Cer) or asialo-GM2 (GalNAcß1,4Galß1,4Glcß1,1'Cer);

4. Oligomerization of GalNAc by {alpha}1,4-linkages slightly increased the affinity of the lectin; and

5. GalNAc{alpha}-Ser/Thr at the nonreducing end in glycopeptides showed strong affinity to the lectin, but the masking of the terminal GalNAc residue with Gal or NeuAc greatly reduce the affinity.

In summary, we conclude that the activity of the starfish lectin is specific to the terminal {alpha}-GalNAc at the nonreducing end of glycoconjugates.

Interestingly, the starfish lectin specifically binds to the tumor-associated Tn antigen but not T antigen (Thomsen-Friedenreich antigen), whose determinants are GalNAc{alpha}-Ser/Thr and Galß1,3GalNAc{alpha}-Ser/Thr, respectively. Tn antigen is normally a cryptic structure in the peptide core of O-glycoproteins. It was first discovered on the red blood cells of a patient with hemolytic anemia and found to be responsible for the agglutination of erythrocytes caused by anti-Tn antibodies, which are universally present in human sera (Tn syndrome) (Berger, 1999Go). It is now widely recognized that Tn antigen, expressed in more than 70% of human adenocarcinomas (Springer, 1984Go), is one of the most specific human carcinoma-associated antigens. Thus, the starfish lectin would be useful to detect Tn antigens that might be abnormally expressed in human carcinoma (Konska et al., 1998Go).

It was clearly shown that the starfish lectin specifically binds the {alpha}-GalNAc residue, but not ß-GalNAc or {alpha}/ß-Gal, of glycoconjugates. Thus, the specificity of the lectin is completely different from that of Wistaria floribunda agglutinin (WFA), which specifically binds the ß-GalNAc (Kurokawa et al., 1976Go). We also found that the glycolipid-derived receptor for the starfish lectin on sheep erythrocytes is likely to be Gb5Cer. Because WFA could not bind the terminal {alpha}-GalNAc of Gb5Cer, which is a major glycolipid of sheep erythrocytes, this finding may explain why WFA does not cause aggregation of sheep erythrocytes.

Monovalent lectins are useful for elucidating the functions of cell-surface glyco-receptors and sometimes mimic the ligand for receptors because they specifically bind to receptors without aggregation of target cells (Kaku and Shibuya, 1992Go). Because the recombinant starfish lectin produced in E. coli had a monomeric structure on SDS–PAGE (Figure 8) and no hemagglutination activity, it could be used as a specific ligand to reveal the biological functions of cell-surface glyco-receptors having GalNAc at nonreducing terminal ends. However, the binding activity of the recombinant lectin for asialomucin was found to be ~15–20 times lower than that of the native one, suggesting that the recombinant lectin does not form the appropriate disulfide bonds necessary for sugar binding and hemagglutination.

The biological role of the starfish lectin is not clear at present. It is interesting to note that a GalNAc-specific lectin of Codium fragile subspecies tomentosides completely precipitated the Streptococcus type C polysaccharide (Wu et al., 1997Go). Thus, it is possible that the starfish lectin functions in defense against Gram-positive bacteria.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 References
 
Samples and reagents
Starfishes (A. pectinifera) were collected in April 1999 on the north coast of Kyushu in Japan. Sheep and guinea pig blood samples were obtained from Japan Biotest Laboratories (Tokyo). Various glycopeptides and monosaccharides, including bovine submaxillary-gland mucin, fetal calf serum fetuin and asialofetuin, D-glucosamine (GlcNAc), D-galactosamine (GalNAc), D-fucose (Fuc), L-fucose, sialic acid (NeuAc), and lactose were purchased from Sigma (St. Louis, MO). Sepharose CL-4B was a product of Amersham Pharmacia Biotech (Buckinghamshire, UK). Methyl-2-acetamido-2-deoxy-{alpha}-galactopyranoside and methyl-2-acetamido-2-deoxy-ß-galactopyranoside were from Calbiochem Novabiochem (Darmstadt, Germany). N-acetyl-{alpha}1,4-galactosamino-dimer, -trimer, and -tetramer were obtained from Funakoshi (Tokyo). E. coli strain JM109 and plasmid pTV118N were obtained from Takara Shuzo (Shiga, Japan). T antigen was obtained from Seikagakukogyo (Tokyo). Tn anigen was prepared by the method described by Asakawa et al. (1989)Go and also purchased from Calbiochem. Gb5Cer and asialo-GM2 were kindly donated from Drs. H. Higashi (RIKEN/Mitsubishi Kasei Institute of Life Sciences, Japan) and Y. Hirabayashi (RIKEN). Blood group A and B trisaccharides were from Calbiochem. Other reagents were of the highest analytical grade.

Preparation of GalNAc-Sepharose CL-4B
GalNAc-Sepharose CL-4B was prepared using divinylsulfone as described by Teichberg et al. (1988)Go.

Extraction and purification of starfish lectin
The lectin was extracted from internal organs except gonads and digestive organs with 20 mM Tris–HCl buffer, pH 7.5, with 0.15 M NaCl (TBS) containing 5 mM CaCl2, 3 µg/ml leupeptin, and pepstatin A. The extract was centrifuged at 8500 x g for 30 min at 4°C. The supernatant was filtered through absorbent cotton. The crude extract thus obtained was successively applied to a column of GalNAc-Sepharose CL-4B (10 ml) previously equilibrated with TBS containing 5 mM CaCl2. The column was washed with the same buffer and then the adsorbed proteins were eluted with TBS containing 20 mM EDTA.

Preparation of anti-starfish lectin
Purified lectin was dialyzed against distilled water before being used for immunization. Antiserum was obtained from a rabbit immunized with 1 mg of the purified lectin.

SDS–PAGE and immunoblotting
SDS–PAGE was performed according to the method of Laemmli (1970)Go. The proteins on SDS–PAGE were visualized with Coomassie brilliant blue or silver staining solution. For immunoblotting, gels were transferred to nitrocellulose membranes for 1 h at 15 V using an electroblot apparatus (Bio-Rad, CA). The membranes were treated with 1% skim milk in TBS containing 0.02% Tween 20 for 1 h at room temperature, treated with anti-starfish lectin antiserum (raised in rabbit) and incubated with anti-rabbit IgG–horseradish peroxidase (Santa Cruz, CA). Visualization was performed using a peroxidase staining kit, according to the protocol provided by the manufacturer (Nacalai Tesque, Kyoto, Japan).

Native PAGE
The purified starfish lectin was applied onto a 4–20% Tris-glycine polyacrylamide gel (Invitrogen, Groningen, The Netherlands). A duplicate gel was cut into 2 mm slices. Each slice was crushed in 150 µl of TBS containing 5 mM CaCl2. After centrifugation at 15,000 rpm for 5 min, an aliquot (60 µl) of the supernatant was subjected to hemagglutination assay using sheep erythrocytes.

Hemagglutination and inhibition assays
The hemagglutination assay was performed using sheep erythrocytes. Thirty-microliter aliquots of serial twofold dilutions of the lectins in TBS containing 5 mM CaCl2 were mixed with the same volume of a 10% (v/v) suspension of erythrocytes in the same buffer solution. After incubation at 37°C for 1 h, the extent of agglutination was examined visually. The hemagglutination activity was expressed as a titer, agglutination units (AUs), the reciprocal of the highest dilution giving detectable agglutination. The hemagglutination inhibition assay was performed by incubating 30 µl aliquots of the lectins (4 AU) in TBS containing 5 mM CaCl2 and various concentrations of mono- or oligosaccharides with the same volume of a 10% (v/v) suspension of sheep erythrocytes in the same buffer solution.

TLC overlay binding assay
Various glycosphingolipids (each 2.5 nmol) were applied to a TLC plate (PE SIL G, Whatman, Kent, England), which was developed with chloroform/methanol/0.02% CaCl2 (5:4:1, v/v/v). The TLC plate was blocked with 3% skim milk–TBS for 1 h at room temperature and then incubated with 5 µg of biotin-labeled lectin in TBS containing 0.02% Tween 20 (TBS-Tween) for 1 h at room temperature. After washing with TBS-Tween three times, streptavidin-labeled alkaline phosphatase (x1000 dilution, Sigma) was added and incubated for 1 h at room temperature. The lectins bound to glycosphingolipids were then visualized by addition of nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate as a substrate.

ELISA
Microtiter plates were coated with asialomucin (1 µg/100 µl in TBS containing 0.02% Tween 20), by incubating overnight at 4°C. After a wash with the same buffer, the plates were blocked with 1% bovine serum albumin in the same buffer, and biotin-labeled lectins were added, incubated at room temperature for 2 h, and then washed with the same buffer. Streptavidin-conjugate horseradish peroxidase (Invitrogen) was added and incubated at room temperature for 15 min. The enzyme activity of horseradish peroxidase was measured with 2,2'-azino bis (3-ethylbenzthiazoline-6-sulfonic acid) as a substrate at 415 nm, using a microplate reader, model 550 (Bio-Rad).

Ca2+ dependence of the starfish lectin
Starfish lectin was incubated with 5 mM EDTA or EGTA on ice for 1 h. Then the mixtures were incubated with 5% (v/v) sheep erythrocytes in 60 µl of TBS at 37°C for 1 h. To examine the reversibility, chelating reagents were removed by dialysis against TBS containing 5 mM CaCl2 for 16 h, and then the activity was assayed using sheep erythrocytes in the presence of 2.5 mM CaCl2 as described in Hemagglutination and inhibition assays.

Amino acid microsequencing
The lectin preparation from GalNAc-Sepharose CL-4B was further purified with 2D gel electrophoresis. The protein preparation (950 µl) was concentrated by acetone precipitation, dried, and dissolved in 400 µl of 7 M urea, 2 M thiourea, 2% Triton X-100, 1% Pharmalite (pH 3–10), 0.1% dithiothreitol, complete mini EDTA(-) (a protein inhibitor mixture; 1 tablet/10 ml, F. Hoffmann-La Roche, Basel, Switzerland). One hundred microliters of the solution was loaded on a sample cup for the first dimensional IPG gel strip (pH 3–10, 11 cm, Amersham Pharmacia Biotech). A vertical gel (16 x 16 cm) was used for the second dimension. After electrophoresis, the gel was blotted on a polyvinylidene difluoride membrane (ProBlott, Applied Biosystems, CA) and stained with Coomassie brilliant blue. The 19-kDa protein band was cut out and treated in situ with lysylendopeptidase AP-1 (Wako Pure Chemical Industries, Osaka, Japan). For the determination of peptide sequence T-11, trypsin was used instead of lysylendopeptidase. Peptides released from the membrane were fractionated with a reverse-phase high-performance liquid chromatography column of C8 (RP-300, 1.0 x 100 mm, Applied Biosystems) and sequenced with a pulse-liquid phase protein sequencer (Procise 492 cLC, Applied Biosystems).

First-strand cDNA synthesis
To obtain a partial cDNA sequence encoding the starfish lectin, we synthesized the first strand cDNA from the starfish. Total RNA and mRNA were obtained from 500 mg of gonad of the starfish using Sepasol RNA I (Nacalai Tesque) and a FastTrack 2.0 kit (Invitrogen), respectively. First strand cDNA was synthesized from 2 µg of mRNA using an AMV Reverse Transcriptase First-strand cDNA Synthesis kit (Life Sciences, FL).

Isolation of a partial cDNA encoding the starfish lectin
Based on the amino acid sequences of peptides derived from the purified starfish lectin, degenerate primers of both sense and antisense strands were designed. The sense oligonucleotide primer, Ap/C729TC (5'-TGGCARCCNGAYTGYTC-3'), was synthesized from the N-terminal peptide sequences. Antisense primers, Ap/C727in (5'-TCNGCYTCRTCRTANGT-3') and Ap/C727med (5'-GTRAAYTCYTGRCARTG-3'), were synthesized based on the internal amino acid sequence of C727. For the second round of nested PCR, Ap/C729TC (5'-TGGCARCCNGAYTGYTC-3') and Ap/C727in were used. The first round of PCR was performed using a set of the primers (Ap/C729TC and Ap/C727med) with first-strand cDNA as a template in a GeneAmp PCR System 9700 (Applied Biosystems) using AmpliTaq Gold (Applied Biosystems). The PCR products were cloned into pGEM T-easy vector (Promega, WI), and their DNA sequences were determined.

5'- and 3'-RACE
To obtain the 5'- and 3'-end, SMART RACE cDNA Amplification kit (Clontech, CA) was used. 5'-RACE first-strand cDNA was primed from 0.75 µg of mRNA with Superscript II reverse transcriptase (Invitrogen) using a SMART II oligonucleotide and a 5'-RACE cDNA synthesis primer. The 5'-end of the cDNA was amplified by PCR with gene-specific primer 1 (L85: 5'-GCCAGTGCCGAAGTAACGGTAGCA-3') and a universal primer mix and 5'-RACE first strand cDNA as a template, using the Advantage 2 PCR kit (Clontech). The 3'-RACE first-strand cDNA was primed using 3'-RACE cDNA synthesis primer. The 3'-end of the cDNA was obtained by PCR using a universal primer mix and gene-specific primer 2 (U100: 5'-CGGCACTGGCAAGACCTATGATGAA-3') and 3'-RACE first-strand cDNA as a template. The 5'- and 3'-RACE PCR products were cloned into pGEM T-easy vector, and their DNA sequences were determined.

cDNA library construction
The mRNA (5 µg) was subjected to synthesis of double-stranded cDNA using a cDNA Synthesis Kit (Strategene, CA), followed by ligation with EcoRI adaptor and with Uni ZAP XR Vector. After in vitro packaging with Gigapack III Gold Cloning Kit, the library was amplified once before use.

Probe preparation
Digoxygenin (DIG) labeling was performed using a PCR DIG Probe Synthesis Kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions.

Screening of the cDNA library
Plaques (3.7 x 105) of the starfish cDNA library were transferred onto Biodyne A membranes (Pall, NY). After baking at 120°C for 30 min, the membranes were prehybridized for 2 h at 65°C in a solution containing 5x SSC (1x SSC is 15 mM sodium citrate and 150 mM NaCl, pH 7.0), 0.1% sodium N-lauroylsarcosinate, 0.02% SDS, and 1% DIG blocking reagent, followed by hybridization with DIG-labeled DNA probes at 65°C for 16 h. After a wash with 0.1x SSC containing 0.1% SDS at 65°C for 30 min, colorimetric detection was performed using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate in the DIG DNA detection kit as described in the manufacturer’s instructions. Positive plaques were converted to plasmids using the VCSM13 helper phage (Stratagene).

Sequence variations in the starfish lectin mRNA
To evaluate the sequence variations in the starfish lectin mRNA, RT-PCR was done with primers outside of the open reading frame (U42: 5'-GGCTTGTGACACTCAACGGA-3' and L593: 5'-TCGTCCATTGCGGAGCCGAT-3'). PCR was performed in 50 µl of a reaction mixture containing each primer at 0.5 µM, 3 ng of template DNA, 2 mM dNTPs (dATP, dGTP, dCTP, and dTTP each at 0.5 µM), 2 mM MgCl2, and 2.5 U of AmpliTaq Gold (Applied Biosystems). PCR products were extracted from 1% agarose gel, and TA cloning was achieved using pGEM T-easy vector.

Construction of expression plasmid with starfish lectin cDNA
An insert that included the full-length of the open reading frame with NcoI and EcoRI sites (nucleotide position, 1–525) was amplified using a set of primers: Ap/UNcoI (5'-AAACCACCATGGCTTTCTTTCGGGCCTT-3') and Ap/LEcoRI (5'-CGAATTCGTCCATTGCGGAGCCGATTTA-3'). The amplified fragment was treated with NcoI and EcoRI, and subcloned into pTV118N (Takara). The recombinant plasmid was purified and designated pTApL.

Expression of starfish lectin cDNA
E. coli JM109 cells transformed with pTApL were grown at 37°C in Luria-Bertani medium containing 100 µg/ml ampicillin until the optical density at 600 nm reached about 0.5. Then, IPTG was added to the culture at a final concentration of 0.1 mM, and cultivation was continued for an additional 16 h at 37°C. Cells were harvested by centrifugation, suspended in TBS containing 5 mM CaCl2, sonicated, and used as the crude lectin solution.

Homology search
A computer-assisted homology search of starfish lectin was made using the DNA Data Bank of Japan homology search system and programs FASTA version 1.50 and BLAST version 2.00. The nucleotide sequences were aligned using CLUSTAL W (Thompson et al., 1994Go).

Other methods
Protein concentrations were determined with bicinchoninic acid using bovine serum albumin as a standard (Smith et al., 1985Go). Nucleotide sequences were determined on both strands by the dideoxynucleotide chain termination method with a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and a DNA Sequencer (model 377, Applied Biosystems). The nucleotide and amino acid sequences were evaluated using the DNASIS computer program developed by Hitachi Software Engineering (Tokyo).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 References
 
We thank for Drs. K. Kamiya (Kitazato University), T. Hatakeyama (Nagasaki University), H. Nakata (Kyoto Sangyo University), M. Nakao (Kyushu University), and K. Furukawa (Tokyo Metropolitan Institute of Gerontology) for their valuable discussions and suggestions. We thank the kind gifts of glycosphingolipids from Drs. Y. Hirabayashi (RIKEN) and H. Higashi (RIKEN/Mitubishi Kasei Institute of Life Sciencies). Thanks are also due to Ms. W. Kamada (Kyushu University) for technical assistance. The authors also acknowledge Dr. T. Nakamura (Kyushu University) for encouragement throughout the course of this study. This work was supported in part by Grant-in Aid for Scientific Research on (B) (13460044) from the Ministry of Education, Culture, Sport, Science and Technology, Japan. The nucleotide sequence reported in this article is under submission to the GenBankTM/EBI Data Bank.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 References
 
AU, agglutination unit; DIG, digoxygenin; EDTA, ethylenediamine tetra acetic acid disodium salt; EGTA, ethylene glycol bis(2-aminoethyl ether)-tetra acetic acid; ELISA, enzyme-linked immunosorbent assay; GalNAc, N-acetyl-D-galactosamine; GlcNAc, N-acetyl-D-glucosamine; IPTG, isopropyl 1-thio-ß-D-galactoside; 2ME, 2-mercaptoethanol; NeuAc, sialic acid; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse-transcriptase PCR; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; TLC, thin-layer chromatography; WFA, Wistaria floribunda agglutinin.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 References
 


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 References
 
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