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Lectins from Tropical Sponges

2000; Elsevier BV; Volume: 275; Issue: 38 Linguagem: Inglês

10.1074/jbc.m001366200

1083-351X

Pedro Bonay Miarons, Manuel Fresno,

Glycosylation and Glycoproteins Research

Only a few animal phyla have been screened for the presence and distribution of lectins. Probably the most intensively studied group is the mollusk. In this investigation, 22 species from 12 families of tropical sponges collected in Los Roques National Park (Venezuela) were screened for the presence of lectins. Nine saline extracts exhibited strong hemagglutinating activity against pronase-treated hamster red blood cells; five of these reacted against rabbit red blood cells, four with trypsin-treated bovine red blood cells, and five with human red blood cells regardless of the blood group type. Extracts from the three species studied from genusAplysina (archeri, lawnosa, andcauliformis) were highly reactive and panagglutinating against the panel of red blood cells tested. The lectins from A. archeri and A. lawnosa were purified to homogeneity by ammonium sulfate fractionation, affinity chromatography onp-aminobenzyl-β-1-thiogalactopyranoside-agarose, and gel filtration chromatography. Both lectins exhibited a native molecular mass of 63 kDa and by SDS-polyacrylamide gel electrophoresis under reducing conditions have an apparent molecular mass of 16 kDa, thus suggesting they occur as homotetramers. The purified lectins contain 3–4 mol of divalent cation per molecule, which are essential for their biological activity. Hapten inhibition of hemagglutination was carried out to define the sugar binding specificity of the purifiedA. archeri lectin. The results indicate a preference of the lectin for nonreducing β-linked d-Gal residues being the best inhibitors of red blood cells binding methyl-β-d-Gal and thiodigalactoside (Galβ1–4-thiogalactopyranoside). The behavior of several glycans on immobilized lectin affinity chromatography confirmed and extended the specificity data obtained by hapten inhibition. Only a few animal phyla have been screened for the presence and distribution of lectins. Probably the most intensively studied group is the mollusk. In this investigation, 22 species from 12 families of tropical sponges collected in Los Roques National Park (Venezuela) were screened for the presence of lectins. Nine saline extracts exhibited strong hemagglutinating activity against pronase-treated hamster red blood cells; five of these reacted against rabbit red blood cells, four with trypsin-treated bovine red blood cells, and five with human red blood cells regardless of the blood group type. Extracts from the three species studied from genusAplysina (archeri, lawnosa, andcauliformis) were highly reactive and panagglutinating against the panel of red blood cells tested. The lectins from A. archeri and A. lawnosa were purified to homogeneity by ammonium sulfate fractionation, affinity chromatography onp-aminobenzyl-β-1-thiogalactopyranoside-agarose, and gel filtration chromatography. Both lectins exhibited a native molecular mass of 63 kDa and by SDS-polyacrylamide gel electrophoresis under reducing conditions have an apparent molecular mass of 16 kDa, thus suggesting they occur as homotetramers. The purified lectins contain 3–4 mol of divalent cation per molecule, which are essential for their biological activity. Hapten inhibition of hemagglutination was carried out to define the sugar binding specificity of the purifiedA. archeri lectin. The results indicate a preference of the lectin for nonreducing β-linked d-Gal residues being the best inhibitors of red blood cells binding methyl-β-d-Gal and thiodigalactoside (Galβ1–4-thiogalactopyranoside). The behavior of several glycans on immobilized lectin affinity chromatography confirmed and extended the specificity data obtained by hapten inhibition. Aplysina archeri lectin polyacrylamide gel electrophoresis In animals, only a few phyla have been screened for the presence and distribution of lectins. In particular, the number of lectins that have isolated from invertebrate organisms is quite small as compared with the great variety of lectins isolated from plant origin and have been limited to those partially characterized from mollusks and crustaceans. Since the discovery of hemagglutinins in sponges by Doddet al. (1Dodd R.Y. MacLennan A.P. Hawkins D.C. Vox Sang. 1968; 15: 386-391Crossref PubMed Scopus (24) Google Scholar), there have been some reports on the partial characterization (serological and immunoelectrophoretic properties) of lectins from the oldest multicellular animals (2Müller W.E. Müller I.M. Gamulin V. Braz. J. Med. Biol. Res. 1994; 27: 2083-2096PubMed Google Scholar, 3Müller W.E. Naturwissenschaften. 1998; 85: 11-25Crossref PubMed Scopus (112) Google Scholar) from the Mediterranean Sea or Japan (4Bretting H. Kabat E.A. Liao J. Pereira M.E. Biochemistry. 1976; 15: 5029-5038Crossref PubMed Scopus (62) Google Scholar, 5Bretting H. Kabat E.A. Biochemistry. 1976; 15: 3228-3236Crossref PubMed Scopus (76) Google Scholar, 6Diehl-Seifert B. Uhlenbruck G. Geisert M. Zahn R.K. Muller W.E. Eur. J. Biochem. 1985; 147: 517-523Crossref PubMed Scopus (38) Google Scholar, 7Kamiya H. Muramoto K. Hoshino T. Yamazaki M. Raj U. Bull. Jpn. Soc. Sci. Fish. 1986; 52: 2205-2206Crossref Scopus (4) Google Scholar, 8Kamiya H. Muramoto K. Goto R. Bull. Jpn. Soc. Sci. Fish. 1990; 56: 1159-1169Crossref Google Scholar). However, their properties and specificities have not been clearly defined. In this report, we present data on an investigation undertaken to search for novel lectins in marine organisms, which could show unique properties. In addition, we describe the purification and characterization of the lectins present in two species of tropical sponges, Aplysina lawnosaand Aplysina archeri. We present information on the nature and specificity of their combining site as examined by hapten inhibition experiments and affinity chromatography. The sponges were collected in the Los Roques National Park (Venezuela), in the Caribbean Sea. The sponge tissue was dried at room temperature (about 20 °C for 3 days). The dry material was reduced to powder in a mortar and stored at −70 °C until used. The dry tissue was extracted by stirring 50 g with 1 liter of 0.9% NaCl (containing 1 mm each CaCl2, MgCl2, and MnCl2 plus 0.02% NaN3) overnight at 4 °C. The extract was clarified by centrifugation at 15000 × g for 30 min at 4 °C. The supernatant, filtered through 0.2-μm filters, is referred to as crude extract. All glycoproteins were from Sigma. Monosaccharides and disaccharides were from Sigma and/or Dextra Laboratories. Oligosaccharides other than specified were from Dextra Laboratories. Terminal residues of neuraminic acid were removed by mild acid hydrolysis (10 mm HCl, 70 °C, 1 h) or digestion with Vibrio cholerae neuraminidase (Oxford Glycosystems, Oxford, UK). Oligosaccharides were reductively labeled with NaB3H4 or for oligosaccharides 18, 21, and 23 by galactose oxidase followed by NaB3H4reduction. Oligosaccharide 6 was obtained from oligosaccharide 4 by digestion with jack bean β-galactosidase (Oxford Glycosystems). Oligosaccharides 14 and 15 were obtained from mutant Ric21 of BHK (baby hamster kidney) cells as described previously (12Sato S. Anmasahum T. Hughes R. J. Biol. Chem. 1991; 266: 11485-11494Abstract Full Text PDF PubMed Google Scholar) and treated with α-fucosidase (bovine epididymis, Oxford Glycosystems, UK). Oligosaccharide 17 was obtained from Oxford Glycosystems. Oligosaccharide 18 was obtained from human transferrin by Pronase digestion as described (9Bayard G.S.B. Fournet B. Strecker G. Bouquelet S. Montreuil J. FEBS Lett. 1975; 50: 296-299Crossref PubMed Scopus (302) Google Scholar); in some instances, we obtained oligosaccharide 16 by hydrazinolisis of oligosaccharide 18. Oligosaccharides 19 and 20 were purified from bovine prothrombin (10Mizuochi T. Yamashita K. Fujikawa K. Kisiel W. Kobata A. J. Biol. Chem. 1979; 254: 6419-6425Abstract Full Text PDF PubMed Google Scholar), oligosaccharide 21 from BHK cells, and oligosaccharides 22 and 23 from bovine fetuin (11Takasaki S. Kobata A. Biochemistry. 1986; 25: 5706-5715Crossref Scopus (123) Google Scholar) as described (12Sato S. Anmasahum T. Hughes R. J. Biol. Chem. 1991; 266: 11485-11494Abstract Full Text PDF PubMed Google Scholar). Oligosaccharide 24 was prepared from the tetrasialylated fraction of human α1-acid glycoprotein after hydrazinolisis (13Yoshima H. Matsumoto A. Mizuochi T. Kawasaki T. Kobata A. J. Biol. Chem. 1981; 256: 8476-8484Abstract Full Text PDF PubMed Google Scholar). Red blood cells from the following species were used: bovine, rat, hamster, rabbit and human blood group A, B, and O. The blood was collected in 3.8% sodium citrate and washed three times with 0.9% NaCl by centrifugation at 3000 ×g for 5 min. the final pellet was resuspended with 0.9% NaCl to give a 4% cell suspension. The hemagglutination was performed at room temperature by serial dilution (1:2 with 0.9% NaCl) with a Takatsy microtitrator (Dynatech Laboratories Inc., Chantilly, VA) using 0.025-ml loops and 4% suspension of erythrocytes. A Coulter counter was used to count the remaining cells in suspension after a 1-h incubation; 1 hemagglutinating unit is defined as the amount of lectin able to agglutinate and hence precipitate 75% of the red blood cells in suspension after 60 min. For semiquantitative results (as during the purification), the hemagglutinating activity was defined as the reciprocal of the end point dilution giving a clear macroscopic agglutination at 60 min. Where indicated, the 4% red blood cell suspension was treated with Pronase (1 mg/ml; Sigma) or trypsin (250 μg/ml; Sigma) in 20 mm sodium acetate, pH 5.8, 150 mm NaCl containing 10 mm CaCl2 or in 100 mm Tris, pH 8.1, 100 mm NaCl, respectively, for 1 h at 37 °C. The red blood cells were washed three times before being resuspended at 4% in phosphate-buffered saline. The clear, dialyzed 15–30% ammonium sulfate fraction (250 ml) was passed through ap-aminobenzyl-β-thiogalactopyranoside-agarose (Sigma) column (2 × 8 cm) equilibrated in Buffer A (Hepes 50 mm, NaCl 100 mm, pH 7.6, containing CaCl2, MgCl2, and MnCl2, each at 1 mm) and washed with the same buffer until the optical density at 280 nm was below 0.020. Specific elution of the lectins was effected by 0.2 m thiodigalactoside in Buffer A. Fractions (2 ml) were collected and analyzed for protein content by the BCA method and for hemagglutinating activity against pronase-treated hamster erythrocytes. This purification scheme was used for bothA. archeri and A. lawnosa extracts. This was done by quantitative hapten inhibition using immobilized asialo α1-acid glycoprotein as a model glycoprotein. Briefly, asialo α1-acid glycoprotein dissolved in 50 mm sodium carbonate buffer, pH 9.6, containing 0.02% sodium azide (Buffer A) at 4 μg/ml was applied (50 μl) to each well of a 96-well microtiter plate and incubated for at least 2 h at 4 °C. The plate was then rinsed three times with 50 mm sodium phosphate buffer, pH 7.4, containing 0.05% Tween 20 (Buffer B). The remaining sites on the plate were coated by incubation with 350 μl of Buffer A containing 1% bovine serum albumin at 25 °C for 1 h. A 50-μl aliquot of biotinylated purified A. archeri lectin at about 50 pmol/ml in 50 mm Tris buffer, pH 7.4, containing 150 mmNaCl was mixed with aliquots of the different glycans at 4 °C for 2 h and then applied to each well and incubated at room temperature for 4 h. The plate was washed three times with Buffer A and streptavidin-horseradish peroxidase conjugate was added to each well. Finally, the plate was washed four times with Buffer B, and 100 μl of the substrate solution (ABTS, 0.3 mg/ml, dissolved in 50 mm citrate buffer, pH 5.0, containing 0.012% H2O2) was added and incubated at 25 °C for 10–20 min. The reaction was terminated by the addition of 100 μl of 5% SDS and the absorbance read at 405 nm. The carbohydrate content was measured by the phenol-H2SO4 method with glucose as the standard. Samples of purified lectins were analyzed by atomic absorption spectroscopy, either directly or after dialysis against 10 mm EDTA, pH 7.2, followed by dialysis against 150 mm NaCl in the presence of Chelex 100 resin (Bio-Rad). The metal-free lectins were tested for hemagglutinating activity as described above before incubation for 1 h in the absence or presence of different divalent cations. Solutions of purified A. archeri or A. lawnosa lectins (2.8 mg/ml) and lactose containing [14C]lactose were made in 100 mmTris-HCl, pH 6.8. Each chamber of the dialysis cells received 100 μl of the protein or monosaccharide solution. After equilibration for 50 h at 4 °C, aliquots (80 μl) were removed from each chamber and counted on a Beckman scintillation counter after being mixed with 10 ml of aqueous scintillation fluid. Purified Aplysina archeri lectin (AAL)1was coupled to AffiGel 10 (Bio-Rad) as suggested by the manufacturer in the presence of 100 mm lactose. The resin containing 4.5 mg of lectin/ml of resin was packed into a column (3-ml bed volume). The packed column was washed with 25 ml of Buffer A (50 mmHepes, pH 6.8, 100 mm NaCl, and CaCl2 and MgCl2, each at 10 mm). Labeled glycopeptides were applied to the column in volumes of 100 μl or less and allowed to interact for at least 1 h at ∼22 °C. The columns were eluted with Buffer A followed by 10 mm and 100 mm thiodigalactose in Buffer A. Fractions (0.5 ml) were collected and assayed for radioactivity. The recovery of labeled material was always higher than 92% of the amount applied to the column. In an effort to identify lectins with novel affinity properties, we studied 22 species from 12 families of tropical sponges for the presence of hemagglutinating activity against a panel of normal or protease-treated red blood cells from different species. The results are summarized in Table I. A total of 10 species from 8 families showed some sort of agglutinating activity against some of the red blood cells tested. Three of those species also showed lytic activity against some of the red blood cells used in this system. The remaining 12 sponge species analyzed showed only lytic activity against all or some of the red blood cells tested. No species selectivity was noticed with the exception of Niphates erecta extract that showed hemagglutinating activity only against red blood cells from rat. In addition, there was no evidence of red blood cells susceptible to hemagglutination that could be used as a model system in the search for lectin activities.Table ICharacterization of hemagglutinating or lytic activity from crude saline sponge extracts against different red blood cellsSpecies testedTiter againstaNR, normal rabbit red blood cells; TR, trypsin-treated rabbit red blood cells; NH, normal hamster red blood cells; PH, pronase-treated hamster red blood cells; TB, trypsin-treated bovine red blood cells; Nrat, normal rat red blood cells; O, A, B, refer to human red blood cells from blood groups O, A, and B, respectively; AT, trypsin-treated human blood group A red blood cells; L, lysis of the red blood cells was observed. The numbers refer to the highest serial dilution where macroscopic agglutination was visible after 60 min.NRTRNHPHTBNratOAATBCliona dilitrix31Petrosia weinberegi21L1LPlakortis angulospiculatusLLLLEctyoplasia ferrox133211LIothrocota birotulataLLLLLLLLLAgelas sceptrumLLLAgelas coniferaLLAgelas disparLLLAgelas clathroidesLLPseudoaxinella lunaaecharta12515Niphates erecta1Callispongia vaginalisLLLLLCallispongia fallaxL45Verongula rigidaLAplysina lawnosa8757835764Aplysina cauliformis3838411766Aplysina archeri3536633116Pseudocleratina crassa11312Ircinia felixLLIrcinia campanaLLIrcinia strobilinaLSpheciospongia vespariumLLLLa NR, normal rabbit red blood cells; TR, trypsin-treated rabbit red blood cells; NH, normal hamster red blood cells; PH, pronase-treated hamster red blood cells; TB, trypsin-treated bovine red blood cells; Nrat, normal rat red blood cells; O, A, B, refer to human red blood cells from blood groups O, A, and B, respectively; AT, trypsin-treated human blood group A red blood cells; L, lysis of the red blood cells was observed. The numbers refer to the highest serial dilution where macroscopic agglutination was visible after 60 min. Open table in a new tab Interestingly, all the members of the Aplysina sp. showed the highest titers of hemagglutinating activities in addition to being able to agglutinate red blood cells from all of the species tested. This finding prompted us to purify and characterize the lectin activity(ies) of A. archeri and A. lawnosa, from which a sufficient amount of biological material could be collected. The dark brown-violet crude saline extract from A. archeriwas first fractionated by precipitation with (NH4)2SO4 with more than 75% of the total hemagglutinating activity found in the cut between 15 and 30% saturation (NH4)2SO4 that contains less than 20% of the total protein (Fig.1 A), giving a purification of almost 4-fold above the crude extract. The clear, dialyzed solution (15–30% cut) was then applied to the affinity chromatography as described under "Experimental Procedures." After extensively washing the nonadsorbed proteins, the hemagglutinating activity was specifically eluted with 200 mm thiodigalactose. Eluting the column with 1m NaCl or 1m MgCl2did not release more hemagglutinating activity or material absorbing at 280 nm (Fig. 1 B). In another experiment, the column was eluted with 500 mm EDTA releasing a sharp peak containing the hemagglutinating activity; further elution with thiodigalactose did not release any more hemagglutinating activity from the column (data not shown). It is important to mention that the specific activity of the material eluted with EDTA was consistently lower than the material eluted specifically with thiodigalactose. When A. lawnosaextracts where applied to the affinity column, the pattern of elution was identical (data not shown) to that of A. archeri. Further purification of the lectins was accomplished by gel filtration on a calibrated Sephacryl S200 column (1.5 × 90 cm). The hemagglutinating activity from either species eluted from the column as a sharp symmetric peak at a volume corresponding to an apparent molecular mass of 63,000 Da (Fig. 2), with no additional peaks exhibiting hemagglutinating activity. The fractions with the highest specific activity were pooled and concentrated by ultrafiltration. The purified lectins were analyzed by SDS-PAGE as shown in Fig.3. Each purified lectin appeared as a single band corresponding to an apparent molecular mass of 16,000 Da, regardless of the presence or absence of reducing agents, which according to the native molecular mass, suggests that the lectins are organized in the native state as homotetramers. By chromatofocusing of the purified lectins, both of them eluted as sharp bands at pH 4.1 and 4.5 for A. archeri and A. lawnosa, respectively (Fig. 4) The carbohydrate content of the purified lectins amounted to 3.5 and 5% for A. archeri and A. lawnosa agglutinins, respectively. The hemagglutinating activity of both lectins was abolished by demetalization; this effect was reversible, as addition of CaCl2, MgCl2 to the metal-free lectins fully restored the activity. By contrast, manganese was less effective in restoring the hemagglutinating activity (about 45%), and zinc was without effect. Analysis by atomic spectroscopy of the metal content of the purified native lectins revealed that they contain significant amounts of calcium and magnesium. The contents were as follows: 0.5–0.8 atoms of Ca2+ and 0.3–0.4 atoms of Mg2+/subunit. As the total of calcium and magnesium is close to 1 mol/mol subunit of molecular mass 16 kDa, it suggests that these metals may be in the same site. These metal contents were diminished by 80–90% by prior dialysis against EDTA. The number of carbohydrate binding sites on the purified lectins was determined by equilibrium dialysis, using [14C]lactose (which is also able to displace the lectins from the affinity support used for the purification). The data are shown in Fig.5, plotted according to Scatchard. The number of binding sites obtained from that curve was found to be 3.8 ± 0.4 and 3.6 ± 0.3 for A. archeri andA. lawnosa lectins, respectively. As both lectins are likely to be tetramers under those conditions of ionic strength and pH, our data indicate one binding site per subunit of 16 kDa. To characterize the carbohydrate binding specificity of purifiedA. archeri lectin, hapten inhibition experiments were performed. An examination of the results shown in Fig.6 and summarized in TableII reveals that d-galactose and d-fucose were almost equal inhibitors on a molar basis;d-glucose and d-mannose were inactive up to 5000 nmol. d-GalNAc and d-GalNH2 inhibited 50% at 2500 and 1200, nmol, respectively, being 3.2 and 6.7 times less active than d-Gal. 2-Deoxy-d-Gal was three times more potent than d-Gal. Thus the site is specific for a terminal nonreducing d-Gal with the modifications at C-2 being important and that the OH at C-6 not essential. Introduction of a nonpolar substituent (methyl,p-nitrophenyl or 4-methylumbelliferyl) in either anomeric configuration proved a significant modification in increasing the affinity for the lectin. However, in all cases, the β-configuration was preferred. Thus, the most potent monosaccharide, methyl-β-d-Gal is 37 times more potent thand-Gal compared with only 3.7 times for methyl-α-d-Gal; andp-nitrophenyl-β-d-Gal is almost 7 times more effective than d-Gal compared with only 2 times for the α conformer. Methyl-β-d-thiogalactose was the second best inhibitor (50% inhibition at 12 nmol, 30 times more potent thand-Gal). With the more bulky substituent 4-methylumbelliferyl aglycon, the differences were less significant, as shown in Table II.Table IIInhibition by monosaccharides, oligosaccharides, and glycoproteins of red blood cell hemagglutination by purified A. archeri lectinnmolaMinimal quantities required for 50% inhibition of binding of AAL to asialo α1-acid glycoprotein-coated plate. NI, not indicated.Mono- and oligosaccharides d-Galactose372 d-Fucose398 Melibiose654 Raffinose725 Stachiose867 Methyl-β-d-galactose10 Methyl-α-d-galactose110 p-Nitrophenyl-β-d-galactose58 p-Nitrophenyl-α-d-galactose88 Methyl-β-d-thiogalactose12 4-Methylumbelliferyl-β-d-galactoside70 4-Methylumbelliferyl-α-d-galactoside88 Thiodigalactose2 Galβ1–4Glc372 Galβ1–4GlcNAc150 Galβ1–3GlcNAcβ1–3Galβ1–4Glc310 Galβ1–4GlcNAcβ1–3Galβ1–4Glc127 Galβ1–3GalNAc421 Galβ1–6GlcNAc210 Galβ1–6Gal518 Galβ1–3Gal892 Glcβ1–4Gal3000 Glcα1–2Gal2800 3-Fucosyllactose600 3′,3-Digalactosyllactose42 2-Deoxy-galactose120 SialyllactoseNI N-Acetylgalactosamine2500 d-Galactosamine1200 l-FucoseNI d-GlucoseNI d-GlucosamineNI N-AcetylglucosamineNIGlycoproteinsμg Fetuin4.2 Asialofetuin1.2 Thyroglobulin0.15 Agalactothyroglobulin9.3 α1-Acid glycoprotein4.3 Asialo α1-acid glycoprotein0.11 Agalacto α1-acid glycoprotein5.8a Minimal quantities required for 50% inhibition of binding of AAL to asialo α1-acid glycoprotein-coated plate. NI, not indicated. Open table in a new tab Those results indicate a preference for β-linked d-Gal. Of the disaccharides, those with terminal nonreducing β-linkedd-Gal were more active than those with terminal nonreducing α-linked d-Gal. Melibiose, raffinose, stachiose, Galα1–3Gal, and 3-fucosyllactose were about 2 times less active thand-Gal. Lactose and other disaccharides with Galβ1–3 and β1–4 linked were slightly more active than d-Gal. There appears to be a significant preference for a GlcNAc subterminal to galactose, because lactose is a somewhat weaker inhibitor than lactosamine. Confirming the requirement for a terminal nonreducing β-linked d-Gal was the fact that Glc-β1–4Gal and Glc-α1–2Gal were inactive. However 3-fucosyllactose was just 2 times less effective than lactose as inhibitor. The best disaccharide inhibitor was thiodigalactose, 180 times more potent thand-Gal. To gain further insight into the carbohydrate binding specificity of AAL we studied the behavior of diverse oligosaccharides and glycopeptides during affinity chromatography on immobilized AAL. Typical profiles are shown in Fig. 7, and the structures examined are given in TableIII. A non-inhibitory sugar such as sialyllactitol (oligosaccharide 11) eluted in the void volume, indicating no interaction at all with the immobilized lectin. Under the conditions used, lactitol (Galβ1–4Glcot) weakly bound to the column and eluted at fraction 7–8 of the buffer wash. By contrast,N-acetyllactosaminitol (Galβ1–4GlcNAcot) was bound to the column and eluted with the 10 mm thiodigalactoside wash, confirming the preference for a subterminal GlcNAc to the terminal β-linked galactose. Galβ1–3GalNAcot was retarded in a manner similar to lactose. Similarly, lacto-N-tetraose (oligosaccharide 4) was only retarded in the column eluting at fraction 7–8, whereas lacto-N-neotetraose (oligosaccharide 3), with a terminal Galβ1–4GlcNAc sequence, was eluted with 10 mmthiodigalactoside. Oligosaccharides lacking the terminal β-linked galactose residue did not bind to the column. Substitution of the subterminal GlcNAc with a fucose residue (oligosaccharides 7–8) or a sialic acid residue (oligosaccharide 10) did not affect the differential recognition but reduced the overall affinity as observed by comparing to the binding profile of compounds 3 and 4. Substitution of the reduced glucitol end group with Fucα1–3 (structure 9) did not affect the binding affinity compared with lacto-N-tetraose.Table IIIStructures of oligosaccharides and their behavior on AAL affinity-chromatography Open table in a new tab When analyzing the binding profile of branched oligosaccharides, it was noticed that they bind to the column more tightly than linear oligosaccharides, as shown for oligosaccharides 12 and 13 containing two Galβ1–4GlcNAc branches that were required to elute 100 mm thiodigalactose. In comparison, the mono-antennary hybrid-type oligosaccharide 14 was eluted with 10 mmthiodigalactoside. The bi-antennary hybrid oligosaccharide 15 required 100 mm thiodigalactose to elute from the column. Similarly, the bi-antennary complex oligosaccharide 16 also required 100 mm thiodigalactose to elute from the column. Introduction of a bisecting GlcNAcβ1–4 in a bi-antennary complex oligosaccharide (structure 17) reduces its binding affinity, as it was eluted from the column with 10 mm thiodigalactose. Cleavage of the linkage of the glycan to the asparagine does not have any effect on the binding, as shown by the profile exhibited by structures 16 and 18. The presence of a fucose in position α1–6 in the core sequence does not alter the binding profile, as seen with oligosaccharides 18 and 21. Interestingly, oligosaccharides 18, 19, and 20, containing different proportions of Galβ1–3GlcNAc and Galα1–4GlcNAc, which are distinguished on linear oligosaccharides (compounds 3 and 4) were also distinguished by the binding profile to the immobilized lectin column; this suggests that the exact linkage pattern determines the binding, contrary to what has been found for Tetracarpidium conophorum lectin (12Sato S. Anmasahum T. Hughes R. J. Biol. Chem. 1991; 266: 11485-11494Abstract Full Text PDF PubMed Google Scholar) but quite similar to the strict specificity for Galβ1–4GlcNAc exhibited by the marine sponge Halichondria okadai lectin (14Kawagishi H. Yamawaki M. Isobe S. Usui T. Kimura A. Chiba S. J. Biol. Chem. 1994; 269: 1375-1379Abstract Full Text PDF PubMed Google Scholar). Glycopeptides bearing three terminal Galβ1–4GlcNAc sequences (structures 22 and 23) showed high affinity for binding to AAL. By contrast, the tetra-antennary structure 24 was eluted from the column with 10 mm thiodigalactoside. Regarding the inhibition by glycoproteins, it was found that consistent with the specificity against terminal nonreducing β-linkedd-gal residues, the best inhibitors of all the glycoproteins tested were the asialo forms, as shown in Table II. No inhibitory effect was shown by the sialylated glycoproteins, confirming an absolute requirement for terminal β-galactosyl residues for interaction with AAL. Porcine thyroglobulin, which contains complex-type glycans terminated predominantly with exposed β-galactosyl residues (15Yamamoto K. Tsuji T. Itimura T. Osawa T. Biochem. J. 1981; 195: 701-713Crossref PubMed Scopus (111) Google Scholar), was a very effective inhibitor. The poor activity of asialofetuin could be related to the poor accessibility of its oligosaccharide chains. Further confirmation of the specificity is shown by the fact that treatment with β-galactosidase of the asialo form of thyroglobulin and α1acid glycoprotein almost abolished the inhibitory activity to the native form of the glycoproteins. The results presented in this report show that the presence of lectins in marine sponges is not as widespread as in other marine organisms (mollusks and crustaceans) or in plants. Interest in marine sponges as sources of novel reagents for cell biology has flourished due to the recent report on the isolation and characterization of the anti-HIV (human immunodeficiency virus) compound niphatevirin fromN. erecta (16O'Keefe B.R. Beutler J.A. Cardellina 2nd, J.H. Gulakowski R.J. Krepps B.L. McMahon J.B. Sowder 2nd, R.C. Henderson L.E. Pannell L.K. Pomponi S.A. Boyd M.R. Eur. J. Biochem. 1997; 245: 47-53Crossref PubMed Scopus (29) Google Scholar), a calcium channel blocker from Cliona celata (17Morel J.L. Drobecq H. Sautiere P. Tartar A. Mironneau J. Qar J. Lavie J.L. Hugues M. Mol. Pharmacol. 1997; 51: 1042-1052Crossref PubMed Scopus (16) Google Scholar), and ilimaquinone (18Acharya U. McCaffery J.M. Jacobs R. Malhotra V. J. Cell Biol. 1995; 129: 577-589Crossref PubMed Scopus (38) Google Scholar, 19Takizawa P.A. Yucel J.K. Veit B. Faulkner D.J. Deerinck T. Soto G. Ellisman M. Malhotra V. Cell