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Journal of Applied Phycology 14: 489–495, 2002. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

New affinity procedure for the isolation and further characterization of the blood group B specific lectin from the red marine alga Ptilota plumosa Alexandre H. Sampaio 1,*, David J. Rogers 2, Clive J. Barwell 2, Silvana Saker-Sampaio 1, Kyria S. Nascimento 1, Celso S. Nagano 1 and Wladimir R.L. Farias 1 1 BioMar – Marine Biochemistry Laboratory, Department of Fishing Engineering, BioMol Lab, Federal University of Ceará, P.O. Box 6033, Fortaleza, 60451-970, Ceará, Brazil; 2School of Pharmacy and Biomedical Sciences, University of Portsmouth, St Michael’s Building, White Swan Road, Portsmouth, PO1 2DT, UK; *Author for correspondence (e-mail: [email protected])

Received 18 September 2002; accepted in revised form 18 November 2002

Key words: Affinity chromatography, Agglutinin, Lectin, Ptilota plumosa, Ptilota lectins, Red marine alga, Sugar inhibition Abstract The red marine alga Ptilota plumosa has been shown to contain an anti-human blood group B lectin. We report here a new isolation procedure by affinity chromatography on Sephadex G-200 and characterisation of the isolated lectin. The M r, determined by gel filtration, was 52,500. SDS-PAGE revealed a single protein band with M r 17,440, indicating the native lectin was a trimer of subunits with the same M r, as reported for the lectins from two other Ptilota species, P. filicina and P. serrata. Analysis of amino acid composition showed slightly more basic than acidic amino acids. This was in contrast to the P. filicina and P. serrata lectins previously found to contain a higher proportion of acidic than basic amino acids. Haemagglutination inhibition tests showed the P. plumosa lectin was inhibited by galactose, glucose and their derivatives with p-nitrophenyl-␣-D-galactoside most strongly inhibitory. All glycoproteins tested failed to inhibit the lectin. The amino acid composition, human blood group-B specificity and lack of inhibition by glycoproteins indicate the lectin from P. plumosa possesses unique characteristics among marine algal lectins. Introduction Lectins are proteins that bind reversibly to carbohydrates, agglutinate cells and/or precipitate polysaccharides and glycoproteins. Lectins are widely distributed in nature and can be found in plants, algae, fungi, animals (vertebrates and invertebrates), microorganisms and viruses. However, there is a limited amount of information about algal lectins in comparison with that for higher plants and invertebrates. Marine algal lectins show similarities to lectins from terrestrial plants. However the results obtained on amino acid sequences (Calvete et al. 2000; Hori et al. 2000) and their N-terminal sequence (Kawakubo et al. 1999; Nagano et al. 2002) show no similarity with other sequences deposited in public databases.

Blunden et al. (1975) first reported agglutinating activity in extracts of the red marine alga P. plumosa which exhibited highly selective agglutination against human blood group B erythrocytes (Rogers et al. 1977). The lectin was inhibited most strongly by p-nitrophenyl-␣-D-galactoside. It did not agglutinate group B cells in the presence of EDTA but activity was restored by divalent cations (Rogers et al. 1977). The aim of the present study was to characterise the P. plumosa lectin after isolation by affinity chromatography.

492 Material and methods Algal collection and preparation of extracts Ptilota plumosa was collected by scuba-diving from Bull Bay, Anglesey, Wales, UK. Material was cleaned of epiphytes and transported in dry ice and frozen at −20 °C. The frozen alga was ground to a fine powder in liquid nitrogen, then extracted for 18 h, by stirring, at 4 °C with PBS containing 1 mM CaCl 2, 1:3, w/v. The suspension was filtered through nylon mesh. This step was repeated twice and combined filtrates centrifuged at 15,000 × g for 30 min at 4 °C. The supernatant was removed and used for further studies. Purification of lectin The aqueous extract was loaded onto a Sephadex G-200 column (1.6 × 18 cm), equilibrated and eluted with PBS containing 1 mM CaC1 2 at 10 mL h −1 until the column effluent showed absorbances at 280 nm of less than 0.05. Adsorbed proteins were eluted with 50 mL 0.1 M D-glucose in PBS containing 1 mM CaCl 2. Fractions were tested for haemagglutinating activity using blood group B papain-treated erythrocytes. Active fractions were pooled, dialysed against PBS containing 1 mM CaCl 2 and concentrated in a 50 mL stirred Ultra Filtration Cell (Amicon, Ltd.), fitted with a P10 membrane (size exclusion limit 10,000 Da) using a positive pressure of 20 psi. Finally the samples were dialysed against distilled water, freeze-dried and stored at −30 °C until required. Homogeneity and molecular mass determination Flat bed gel electrophoresis (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis-SDSPAGE) was carried out on the purified lectin at pH 7.0 using LKB Multiphor II electrophoresis equipment with a gel containing 10% (w:v) acrylamide. Samples and standards (Sigma) were prepared in 20% SDS with 1% 2-mercaptoethanol and heated at 95 °C for 2 min. A standard picrate-Coomassie-blue method was used for staining the gel following electrophoresis. Protein markers and their M r were: BSA (66,000), ovalbumin (45,000), carbonic anhydrase (29,000), trypsinogen (24,000), soybean trypsin inhibitor (20,100) and ␣-lactalbumin (14,200). The M r of the native lectin was determined by size exclusion chromatography on a Bio Gel P-100 column (60 × 1.6 cm) calibrated with BSA (66,000), ovalbumin

(45,000), carbonic anhydrase (29,000), myoglobin (18,800) and cytochrome C (12,400). The mobile phase was PBS. Preparation of human red blood cells Human red blood cells were obtained from the Wessex Regional Transfusion Centre, Southampton, UK. Native erythrocytes were prepared by washing the red cells three times with PBS, then resuspending at a final concentration of 5% (v/v) in PBS. Papain treated erythrocytes were prepared by suspending washed, packed, native cells in an equal volume of papain 0.1%; (v/v), incubating at 37 °C for 30 min and then washing three times with PBS and adjusting to a 5% (v/v) suspension in PBS. Haemagglutination and haemagglutination inhibition tests Haemagglutination tests were performed by standard methods using 5% (v/v) native or papain-treated erythrocytes. Inhibition studies were performed using 4 haemagglutinating units of lectin with a range of simple sugars and glycoproteins using established methods (Sampaio et al. 1998a). Amino acid and N-terminal amino acid analysis The amino acid analysis of the purified lectin was carried out on an Applied Biosystems 420H Amino Acid Analyser with automatic hydrolysis and derivatization, employing a C18 reverse phase narrow bore cartridge. The N-terminal sequence was analysed on an Applied Biosystems ABI 477A Protein Sequencer. Protein determination Protein was determined by the method of Bradford (Bradford 1976), using bovine serum albumin (Sigma) as standard. Eluate of columns was monitored spectrophotometrically at 280 nm.

Results The lectin in P. plumosa extracts (PPL) was purified by affinity chromatography on a column of Sephadex G-200. All haemagglutinating activity present in the aqueous extract was bound to the resin then eluted with 0.1 M D-glucose in the eluting buffer (Figure 1).

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Figure 1. Affinity chromatography on Sephadex G-200 of Ptilota plumosa lectin. Aqueous extract prepared with PBS containing 0.001 M CaCl 2 was applied to the column (1.6 × 18 cm) equilibrated and eluted with the same buffer, at a flow rate of 10 mL h −1. The adsorbed lectin was eluted with 50 mL D-glucose in buffer. Fractions of 6 mL were collected and assayed for haemagglutinating activity using blood group B papain-treated erythrocytes. HU haemagglutinating units. (‰—‰) absorbance 280 nm, (䡬—䡬) haemagglutinating activity.

Table 1. Purification of the lectin from Ptilota plumosa Fraction

Protein (mg)

Activity (HU)

Specific activity (HU mg −1)

Purification

Yield (%)

Aqueous extract Affinity column

115 2.1

25,070 97,188

218 46,280

1 212

100 388

Data from 50 g freeze-dried alga. Human blood group B papain-treated erythrocytes were used as indicator cells. HU = haemagglutinating units.

When the affinity-purified material was chromatographed on a column of Bio Gel P-100 a single peak was obtained (not shown). Purification was 212 fold with the specific activity increasing from 218, in the crude extract, to 46,282 in the purified material (Table 1). The minimum agglutination capacity was 0.02 ␮g mL −1. PPL was shown to be specific towards human blood group B erythrocytes. When native erythrocytes were used only B cells were agglutinated by the crude extract and by purified lectin. When papaintreated erythrocytes were used, the crude extract produced very low agglutination with blood group A and O erythrocytes while the purified lectin showed agglutination only towards B cells (Table 2). Haemagglutination inhibition studies with the purified lectin showed that p-nitrophenyl-␣-D-galactoside was the most powerful inhibitor, with a minimum inhibitory concentration of 0.78 mM (Table 3). p-Nitrophenyl-␣-D-glucoside and p-nitrophenyl-␤-D-fucoside were also strong inhibitors at 1.56 mM. Sub-

Table 2. Agglutination of human erythrocytes by Ptilota plumosa lectin Blood group of erythrocytes

Native cells A B O Papain-treated cells A B O

Haemagglutinating units mL −1 crude extract

pure lectin

0 4 0

0 32 0

2 128 2

0 4,096 0

stances with ␣-anomeric linkages showed inhibitory activity at lower concentrations than those with ␤-anomeric linkage. PPL was inhibited by D-glucose, Dgalactose or D-fucose and some of their derivatives. None of the large range of glycoproteins tested inhibited PPL at the maximum concentration tested (2.5 mg mL −1).

494 Table 3. Substances inhibitory to the purified lectin from Ptilota plumosa Substance

Minimum inhibitory concentration*

p-Nitrophenyl-␣-D-galactoside p-Nitrophenyl-␣-D-glucoside p-Nitrophenyl-␤-D-fucoside D-Glucose D-Fucose Methyl-␣-D-galactoside p-Nitropheyl-␤-D-galactoside p-Nitrophenyl-␤-D-glucoside D-Galactose L-Fucose o-Nitrophenyl-␣-D-galactoside o-Nitrophenyl-␤-D-fucoside 2-Deoxy-D-glucose Methyl-␤-D-galactoside D-Arabinose Melibiose Raffinose ␣-Lactose o-Nitrophenyl-␤-D-galactoside Lactulose Glucosamine-HCl Rhamnose N-Acetyl-glucosamine

0.78 mM 1.56 mM 1.56 mM 3.12 mM 3.12 mM 3.12 mM 3.12 mM 3.12 mM 6.25 mM 6.25 mM 10.0 mM 12.5 mM 12.5 mM 12.5 mM 12.5 mM 12.5 mM 25.0 mM 25.0 mM 25.0 mM 25.0 mM 50.0 mM 50.0 mM 50.0 mM

*Minimum concentrations required for inhibition of 4 haemagglutinating units of the lectin. Blood group B papain-treated erythrocytes were used as indicator cells. o-nitrophenyl-N-acetyl-␣-D-galactoside, o-nitrophenyl-N-acetyl-␤-D-galactoside, p-nitrophenylN-acetyl-␣-D-galactoside, p-nitrophenyl-N-acetyl-␤-D-galactoside were not inhibitory at concentrations up to 12.5 mM. Galactosamine-HCl, D-galacturonic acid, galactose-6-phosphate, 2-deoxy-D-galactose, N-acetyl-galactosamine, glucuronic acid, mannose, N-acetyl-␤-mannosamine, muramic acid, N-acetylneuraminic acid were not inhibitory at concentrations up to 50 mM. Fucoidan, bovine submaxillary gland mucin, asialo bovine mucin, egg albumin, fetuin, asialofetuin, ␣-acid glycoprotein, lactoferrin, ovomucoid, porcine stomach mucin thyroglobulin were not inhibitory at concentrations up to 2.5 mg mL −1.

SDS-PAGE in the presence of 2-mercaptoethanol revealed one band of protein corresponding to a M r of 17,440 ± 650 (n = 3) (Figure 2). The homogeneity of PPL was also indicated by a single symmetrical peak obtained by size exclusion chromatography on Bio Gel P-100 in PBS (not shown) with apparent M r of the native lectin of 52,540 ± 510 (n = 5). Mass spectrometry analysis of PPL, indicated a M r ranging between 17,540 to 22,320 (unpublished results). The amino acid composition of PPL is shown in Table 4. The lectin was found to have slightly more basic

Figure 2. SDS-PAGE of Ptilota plumosa lectin. Lane 3 purified P. plumosa lectin. Lanes 1 and 2 correspond to molecular weight markers: bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate-dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa), ␣-lactalbumin (14.2 kDa). Table 4. Amino acid composition of purified lectin from Ptilota plumosa Amino acid

Mol %

Amino acid

Mol %

Asparagine Glutamine Serine Glycine Histidine Arginine Threonine Alanine Proline

8.1 7.5 3.2 9.2 1.5 3.8 5.6 10.7 5.2

Tyrosine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine Tryptophan Lysine

3.2 9.9 1.2 ND 2.7 7.5 3.2 ND 17.5

ND = not detected

amino acids than acidic ones. Tryptophan and cysteine were not detected.

Discussion Some characteristics of PPL have been reported previously (Rogers and Blunden 1980) using crude extracts and semi-purified fractions. All characterisation studies discussed here were carried out using purified lectin. In contrast to the lectins isolated from P. filicina, (PFL) (Sampaio et al. 1998a) and P. serrata (PSL) (Sampaio et al. 1999), PPL agglutinated human B cells strongly, whereas very little agglutination against A and O cells was detected. This study confirms that P. plumosa contains an anti-B blood-group

495 specific lectin (Rogers et al. 1977; Rogers and Blunden 1980). The haemagglutination inhibition patterns of Ptilota lectins fall into two distinct categories. PFL and PSL were inhibited by D-galactose and its derivatives and some glycoproteins while PPL was inhibited by D-galactose, D-glucose and their derivatives, but not by any of the large number of glycoproteins tested (Table 3). The inhibitory substances for PPL show a distinct difference from those inhibitory to PSL and PFL (Sampaio et al. 1998a, 1999). The most strongly inhibitory substance for PPL was p-nitrophenyl-␣-D-galactoside. The results of sugar inhibition clearly show that PPL has ␣-anomeric specificity, since methyl-␣-D-galactoside or methyl-␣-D-glucoside were more inhibitory than their ␤-anomeric forms. As ␣-D-galactose is known to be the terminal immunodominat sugar of the human blood group B antigen, D-galactose might be expected to inhibit the activity of PPL more strongly than methyl-␣-D-galactoside. In fact it did not. The explanation could be that, in solution, D-galactose forms a mixture of ␣and ␤-forms. By comparing the galactosides methyl␣-D-galactoside with methyl-␤-D-galactoside, p-nitrophenyl-␣-D-galactoside with p-nitrophenyl-␤-Dgalactoside, o-nitrophenyl-␣-D-galactoside with o-nitrophenyl-␤-D-galactoside, melibiose (␣-galactosyl sugar) with lactose or lactulose (␤-galactosyl sugars), and p-nitrophenyl-␣-D-glucoside with p-nitrophenyl-␤-glucoside, it is evident that the ␣-anomer is bound more readily than the corresponding ␤-anomer. This indicates the importance of a C-1 ␣-anomeric link in the stabilisation of the protein-sugar complex. Interestingly, D-glucose was of equal inhibitory capacity to D-galactose. Both structures are very similar differing only in positioning of the hydroxyl group at C-4 of the pyranose ring. In fact, they are epimers at C-4. Like PFL and PSL, PPL seems to possess a hydrophobic region adjacent to the carbohydrate binding site specific for ␣-anomeric aromatic glycans, since p-nitrophenyl-galactosides inhibited more strongly than methyl-galactosides (Wu et al. 1981). The importance of a hydroxyl group at C-2 was revealed by the results shown by substances with different groups in C-2, such as galactosamine hydrochloride, 2-deoxy-D-galactose, N-acetylgalactosamine and nitrophenyl-N-acetylgalactosaminides. These all failed to inhibit PPL at the maximum concentration tested. C-5 seems not to be important in the binding of the lectin since arabinose and rhamnose were shown to be inhibitory.

However, the lack of inhibition shown by D-galactose-6-phosphate and galacturonic acid does not support this statement. PPL was inhibited by L-fucose but showed no inhibition by fucoidan, a polysaccharide composed of sulphated L-fucose. Also, no inhibition of PPL was observed by a large range of glycoproteins tested. (Table 3). This observation is very interesting, since the great majority of algal lectins have been shown to be inhibited by glycoproteins and not by simple sugars. All these observations clearly indicate that the lectin in P. plumosa possesses unique characteristics among marine algal lectins. The molecular weight of native PPL determined by size exclusion chromatography on Bio Gel P-100 was 52,540. The molecular weight of subunits of PPL was determined by SDS-PAGE after heating the native lectin in the presence of SDS and 2-mercaptoethanol. By this procedure oligomeric proteins are normally dissociated breaking the protein into its constituent subunits, fully open polypeptide chains. SDS-PAGE of PPL yielded a single component with M r 17,440 (Figure 2). This indicates that the lectin is a trimer composed of three identical or similar subunits. The same pattern was observed for the lectins of PFL (Sampaio et al. 1998a) and PSL (Sampaio et al. 1999), indicating that the lectins present in the Ptilota species are trimeric proteins. Although homotrimeric lectins are unusual, there are some reports of trimeric lectins, such as, the lectin purified from the land plant Sarothammus welwitschii (Sampietro and Vattuone 1994). This is a trimer of identical subunits of 21,500 with a native molecular weight of 64,000. The haemagglutinin glycoprotein of influenza virus is also a trimer (Wilson et al. 1981). Moreover, the lectin present in the sea sponge Geodia cydonium is a trimeric protein (Müller et al. 1983). Further studies of the molecular weights of the subunits on Ptilota lectins were carried out by mass spectrometry. The results obtained by this technique, indicated M r ranging between 16,190 to 22,690 for PFL (Sampaio et al. 1998a), 16,920 to 19,700 for PSL (Sampaio et al. 1999) and 17,540 to 22,380 for PPL. The values obtained by mass spectrometry are close to those obtained by SDS-PAGE for all three lectins. In a previous report we have found that PFL and PSL share similar amino acid compositions (Sampaio et al. 1998a, 1999). They share with other marine algal lectins, high amounts of acidic amino acids and hydroxyl amino acids and low amounts of basic amino acids (Shiomi et al. 1981; Fabregas et al. 1988; Okamoto et al. 1990; Sampaio et al. 1998b). How-

496 ever, PPL was shown to have slightly more basic amino acids than acidic ones. Also, cysteine and tryptophan were not present in PPL. Hori et al. (1986) reported amino acid differences between four agglutinins isolated from the marine alga Boodlea coacta. Boonin D was shown to have a slight difference from the other three agglutinins. This may be responsible for the different properties of binding to fetuin shown by these lectins and may explain the differences in sugar inhibition observed for PSL or PFL and PPL. Attempts were made to determine the sequence of the N-terminal amino acids of PPL. As observed with PSL, PPL was shown to have blocking groups in the N-terminal primary structure since no amino acids could be detected. Such behaviour is not uncommon in lectins. For example, two GalNAc- specific lectins from the mushroom Phaeolepiota aurea (Kawagishi et al. 1996) were reported to have their N-terminal amino acids blocked. Future work should attempt to elucidate the complete primary structure of all three Ptilota lectins and their glycosylation. This would contribute to understanding the differences in sugar inhibition and blood group specificity for the lectins.

Acknowledgements This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Cearense de Amparo à Pesquisa (FUNCAP), Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Programa de Apoio ao Desenvolvimento Científico Tecnológico (PADCT). A.H. Sampaio is senior investigator of CNPq/Brazil.

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