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 1948511, 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: C-type lectin/-GalNAc-specific lectin/oligomerization/starfish/Tn antigen
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1998). Some of them would be useful for detecting tumor-specific or associate antigens, and developmentally regulated sugar residues (Konska et al., 1998
).
We report here a novel -N-acetylgalactosamine (GalNAc)-specific lectin from starfish, Asterina pectinifera. The lectin tends to make oligomers having molecular masses of 40250 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
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 -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.
|
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; GalNAc1,3GalNAcß1,3Gal
1,4Gal-ß1,4Glcß1,1'Cer), but not Gb4Cer (globoside; GalNAc-ß1,3Gal
1,4Galß1,4Glcß1,1'Cer), Gb3Cer (Gal
1,4Gal-ß1,4Glcß1,1'Cer), GM1a (Galß1,3GalNAcß1,4[NeuAc-
2,3]Galß1,4Glcß1,1'Cer), GM2 (GalNAc-ß1,4[NeuAc-
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
-GalNAc, but not ß-GalNAc or
/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, 1974
).
|
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 -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
-GalNAc residues are likely to be exposed at the nonreducing end (D'arcy et al., 1989
; Savage et al., 1990
).
|
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, 1997) 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.
|
|
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 SDSPAGE 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.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (Gal1,3[Fuc
1,2]Gal) do not potently inhibit the hemagglutination or lectin-mediated binding and the lectin does not bind to Gb3Cer (Gal
1,4Galß1,4Glcß1,1'Cer) or GM1a (Galß1,3GalNAcß1,4[NeuAc
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 -GalNAc, but not ß-GalNAc, at the nonreducing end, because the lectin binds to Gb5Cer (GalNAc
1,3GalNAcß1,3Gal
1,4Galß1,4Glcß1,1'Cer) and blood group A trisaccharide (GalNAc
1,3[Fuc
1,2]Gal), but not to Gb4Cer (GalNAcß1,3Gal
1,4Galß1,4Glcß1,1'Cer) or asialo-GM2 (GalNAcß1,4Galß1,4Glcß1,1'Cer);
4. Oligomerization of GalNAc by 1,4-linkages slightly increased the affinity of the lectin; and
5. GalNAc-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 -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-Ser/Thr and Galß1,3GalNAc
-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, 1999
). It is now widely recognized that Tn antigen, expressed in more than 70% of human adenocarcinomas (Springer, 1984
), 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., 1998
).
It was clearly shown that the starfish lectin specifically binds the -GalNAc residue, but not ß-GalNAc or
/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., 1976
). 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
-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, 1992). Because the recombinant starfish lectin produced in E. coli had a monomeric structure on SDSPAGE (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
1520 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., 1997). Thus, it is possible that the starfish lectin functions in defense against Gram-positive bacteria.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of GalNAc-Sepharose CL-4B
GalNAc-Sepharose CL-4B was prepared using divinylsulfone as described by Teichberg et al. (1988).
Extraction and purification of starfish lectin
The lectin was extracted from internal organs except gonads and digestive organs with 20 mM TrisHCl 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.
SDSPAGE and immunoblotting
SDSPAGE was performed according to the method of Laemmli (1970). The proteins on SDSPAGE 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 IgGhorseradish 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 420% 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 milkTBS 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 310), 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 310, 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 manufacturers 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 manufacturers 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, 1525) 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., 1994).
Other methods
Protein concentrations were determined with bicinchoninic acid using bovine serum albumin as a standard (Smith et al., 1985). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berger, E.G. (1999) Tn-syndrome. Biochim. Biophys. Acta, 1455, 255268.[ISI][Medline]
Darcy, S.M., Donoghue, C.M., Koeleman, C.A.M., van den Eijnden, D.H., and Savage, A.V. (1989) Determination of the structure of a novel acidic oligosaccharide with blood group activity isolated from bovine submaxillary-gland mucin. Biochem. J., 260, 389393.[ISI][Medline]
Drickamer, K. (1988) Two distinct classes of carbohydrate-recognition domains in animal lectins. J. Biol. Chem., 263, 95579560.
Fraser, B.A. and Mallette, M.F. (1974) Structure of Forssman hapten glycosphingolipid from sheep erythrocytes. Immunochemistry, 11, 581593.[CrossRef][ISI][Medline]
Gabius, H.J., Unverzagt, C., and Kayser, K. (1998) Beyond plant lectin histochemistry : preparation and application of markers to visualize the cellular capacity for protein-carbohydrate recognition. Biotech. Histochem., 73, 263277.[ISI][Medline]
Giga, Y., Ikai, A., and Takahashi, K. (1987) The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. J. Biol. Chem., 262, 61976203.
Goldstein, I.J., Hughes, R.C., Monsigny, M., Osawa, T., and Sharon, N. (1980) What should be called lectin? Nature, 285, 66.[ISI]
Harrison, F.L. (1991) Soluble vertebrate lectins: ubiquitous but inscrutable proteins. J. Cell Sci., 100, 914.[ISI][Medline]
Hatakeyama, T., Kohzaki, H., Nagatomo, H., and Yamazaki, N. (1994) Purification and characterization of four calcium-dependent lectins from the marine invertebrates, Cucumaria echinata. J. Biochem., 116, 209214.[Abstract]
Jensen, L.E., Thiel, S., Petersen, T.E., and Jensenius, J.C. (1997) A rainbow trout tectin with multimeric structure. Comp. Biochem. Physiol., 116B, 385390.
Kaku, H. and Shibuya, N. (1992) Preparation of a stable subunit of Japanese elderberry (Sambucus sieboldiana) bark lectin and its application for the study of cell surface carbohydrates by flow cytometry. FEBS Lett., 306, 176180.[CrossRef][ISI][Medline]
Komano, H., Mizuno, D., and Natori, S. (1980) Purification of lectin induced in the hemolymph of Sarcophaga peregrina larvae on injury. J. Biol. Chem., 255, 29192924.
Konska, G., Guillot, J., De Latour, M., and Fonck, Y. (1998) Expression of Tn antigen and N-acetyllactosamine residues in malignant and benign human breast tumors detected by lectins and monoclonal antibody 83D4. Int. J. Oncol., 12, 361367.[ISI][Medline]
Kozak, M. (1997) Recognition of AUG and alternative initiator codons is augmented by G in position D 4 but is not generally affected by the nucleotides in positions +5 and + 6. EMBO J., 16, 24822492.
Kurokawa, T., Tsuda, M., and Sugino, Y. (1976) Purification and characterization of a lectin from Wistaria floribunda seeds. J. Biol. Chem., 251, 56865693.[Abstract]
Kyte, J. and Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol., 157, 105132.[ISI][Medline]
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
Muramoto, K. and Kamiya, H. (1990) The amino-acid sequences of multiple lectins of acorn barnacle Megabalanus rosa and its homology with animal lectins. Biochim. Biophys. Acta, 1039, 4251.[ISI][Medline]
Savage, A.V., Donoghue, C.M., Darcy, S.M., Koeleman, C.A.M., and van den Eijnden, D.H. (1990) Structure determination of five sialylated trisaccharides with core type 1, 3, or 5 isolated from bovine submaxillary mucin. Eur. J. Biochem., 192, 427432.[ISI][Medline]
Sharon, N. and Lis, H. (1989) Lectins as cell recognition molecules. Science, 246, 227234.[ISI][Medline]
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Garter, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., and Klenke, D.C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem., 150, 7685.[ISI][Medline]
Springer, G.F. (1984) T and Tn, general carcinoma autoantigens. Science, 224, 11981206.[ISI][Medline]
Suzuki, T., Takagi, T., Furukohri, T., Kawamura, K., and Nakauchi, M. (1990) A calcium-dependent galactose-binding lectin from the tunicate Polyandrocarpa misakiensis. J. Biol. Chem., 265, 12741281.
Teichberg, V., Aberdam, D., Erez, U., and Pinelli, E. (1988) Affinity-repulsion chromatography. Principle and application to lectins. J. Biol. Chem., 263, 1408614092.
Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22, 46734680.[Abstract]
Weis, W.I., Kahn, R., Fourme, R., Drickamer, K., and Hendrickson, W.A. (1991) Structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by MAD phasing. Science, 254, 16081615.[ISI][Medline]
Wu, A.M., Song, S.C., Chang, S.C., Wu, J.H., Chang, K.S.S., and Kabat, E.A. (1997) Further characterization of the binding properities of a GalNAc specific lectin from Codium fragile subspecies tomentosoides. Glycobiology, 7, 10611066.[Abstract]