Journal of Applied Phycology 11: 149156, 1999. 1999 Kluwer Academic Publishers. Printed in the Netherlands.

149

Occurrence of highly yielded lectins homologous within the genus Eucheuma

Marine Greens Laboratory Co., Mori, Iyo 7993125, Japan Department of Materials Science, The University of Shiga Prefecture, Hassaka, Hikone 5228533, Japan 3 Faculty of Applied Biological Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 7398528, Japan 4 Present address; Yamaki Co., Kominato, Iyo 7993194, Japan 5 Present address; Sumitomo Chemical Co., Takatsukasa, Takarazuka 6650051, Japan ( Author for correspondence; fax +81-89-983-1540; e-mail a.kawakubo@tau.bekkoame.ne.jp)
Received 15 October 1998; revised and accepted 9 December 1998

Key words: Eucheuma amakusaensis, Eucheuma cottonii, Kappaphycus alvarezii, hemagglutinin, lectin, isolectins, carbohydrate-binding specicity, mitogenic activity, N-terminal amino acid sequence

Abstract We previously reported that the red alga Eucheuma serra contains large amounts of mitogenic isolectins (ESA1 and ESA-2), the hemagglutinating activities of which were strongly inhibited by glycoproteins bearing high mannose-type N-glycans. We therefore further examined two other species, E. amakusaensis and E. cottonii. Several lectins were isolated easily by a combination of extraction with aqueous ethanol, precipitation with cold ethanol, gel ltration, and ion exchange chromatography from both species, respectively. The puried lectins were designated as EAA-1, EAA-2, EAA-3, ECA-1 and ECA-2 after the specic names of both algae. The yields of EAAs and ECAs were as high as 2.8 and 2.7 mg g1 of dry tissue, respectively, indicating that both species would also be good sources for high lectin yields. The ve puried lectins shared the same properties in hemagglutinating activity, mitogenic activity, and hemagglutination-inhibition test in which glycoproteins bearing high mannosetype N-glycans were the most inhibitory. They also had almost identical molecular weight and 20 N-terminal amino acid sequence to each other and to those of ESAs, and only differed in the isoelectric point, indicating that they are isolectins to each other. The study thus demonstrated that several species of Eucheuma contain high yields of lectins homologous between species, suggesting that the genus as a whole may be considered as a valuable source of lectin proteins.

Introduction Since the survey of hemagglutinins in marine algae by Boyd et al. (1966), there have been many reports on distribution, isolation, and partial characterization of the compounds in marine algae. However, there is little information on their detailed biochemical and biological properties. This is partly due to the low contents of lectins in algal extracts, which make it difcult to obtain sufcient amounts of isolated lectins.

Several Eucheuma species have been found to contain hemagglutinating activity in the extracts (Chiles & Bird, 1989; Bird et al., 1993). Recently we also found that the extracts from ve other species of Eucheuma contain the activity, with E. serra, E. amakusaensis and E. cottonii having the highest activity; two isolectins were easily obtained in the pure forms with extremely high yields (total of 1 g from 100 g dry tissue) from one species, E. serra (Kawakubo et al.,

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150 1997). This was the rst example of lectins isolated with high yields from marine algae. The result also raised the possibility of the presence of such highly yields of lectins in two other species, E. amakusaensis and E. cottonii. E. serra lectins had strong mitogenic activity for mouse and human lymphocytes, and afnity for glycoproteins bearing high-mannose type N-glycans, promising the application as medical and biochemical reagents (Kawakubo et al., 1997). Species of Eucheuma have often been cultivated as edibles or sources of carrageenan. However, the supply of E. serra from the coast of Japan is limited. In contrast, E. amakusaensis and E. cottonii can be obtained continuously in relatively large amounts, because these species are often cultivated and harvested in tropical areas. The aim of this study was to describe the isolation and characterization of lectins from both E. amakusaensis and E. cottonii, with E. serra lectins used for comparison. N-acetylgalactosamine, N-acetylglucosamine, asialofetuin, 1 -acid glycoprotein, fetuin, thyroglobulin, yeast mannan, mucin, ovalbumin, -globulin (bovine) and IgM (mouse). All chemicals were obtained from commercial sources. Protein and sugar contents Protein contents were measured using a Bio-Rad DCProtein assay kit with bovine serum albumin as a standard. Sugar contents were determined by the phenol sulfuric acid method with D-glucose as a standard. Isolation of lectins Isolation of lectins from both E. amakusaensis and E. cottonii was performed by methods similar to that from E. serra (Kawakubo et al., 1997). The lectins were extracted from E. amakusaensis and E. cottonii with 20% aqueous ethanol and recovered as precipitates with 83% ethanol. The precipitates were puried by gel ltration chromatography on a Superdex 75 pg column (26600 mm). The puried lectins designated EAA and ECA respectively, were further subjected to ion exchange chromatography on a TSKgel DEAE 5-PW column (7.5 75 mm). The details of the chromatographic conditions were already described (Kawakubo et al., 1997). The eluate was monitored for absorption at 280 nm and for hemagglutinating activity with trypsin-treated sheep erythrocytes. Active fractions were pooled and dialyzed against distilled water. The lectins from E. serra named ESA-1 and ESA-2 were prepared as described by Kawakubo et al. (1997). Biochemical properties Molecular weights of puried lectins were determined by both gel ltration on a Superdex 75 HR column (10 300 mm) and SDS-PAGE (Laemmli, 1970). Isoelectric points were determined conventionally with isoelectric focusing on a 7% polyacrylamide gel at 460 V for 20 h using 2% Ampholine (pH 4.06.5) as carrier ampholite. Trypsin-treated sheep erythrocytes were used to determine the effects of temperature, pH and metal ions on hemagglutinating activity. The methodology is given by Kawakubo et al. (1997).

Materials and methods Materials Specimens of Eucheuma amakusaensis Okamura and E. cottonii Doty [Kappaphycus alvarezii Doty (Trono, 1993)] were collected on the Pacic coasts of Kochi, Japan, in July and November, respectively, of 1993. They were washed with sea water, freeze-dried, ground to powder and stored at 20 C until used. Hemagglutinating activity and hemagglutination-inhibition test Hemagglutinating activity was determined with trypsin-treated sheep erythrocytes according to the method of Hori et al. (1986). The activity was given as a titer, which was a reciprocal of the highest two-fold dilution exhibiting positive agglutination. Hemagglutination-inhibition tests were performed with trypsin-treated sheep erythrocytes according to the method of Hori et al. (1986). The inhibitory activity was given as the minimum inhibitory concentration (g mL1 ). The sugars and glycoproteins used were L-arabinose, 2-deoxy-Dglucose, D-fructose, galactose, D-glucose, D-glucose6-phosphate, lactose, maltose, mannitol, D-mannose, D-melibiose, D-rafnose, L-rhamnose, D-ribose, D-xylose, D-galacturonic acid, D-glucuronic acid,

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151 Mitogenic activity The mitogenic activity of the puried lectins on mouse lymphocytes was determined by the MTT method (Mosmann, 1983). A mouse lymphocyte suspension was obtained from the spleen cells of female BALB/c mice, 17 weeks old, by a conventional method. The details were given previously (Kawakubo et al., 1997). Amino acid composition and N-terminal amino acid sequence determination Amino acid composition was determined on an amino acid analyzer (Hitachi Model 835) after acid hydrolysis of sample (Kawakubo et al., 1997). The Nterminal amino acid sequencing was carried out using the puried lectins which were electroblotted onto a polyvinylidene diuoride (PVDF) membrane after SDS-PAGE. The sequence determination was made by automated Edoman degradation with an Applied Biosystems Model 477A or 476A gas-phase protein sequencer equipped with a 120A PTH-analyzer online system. high, respectively, as 280 and 272 mg from 100 g powdered alga, values approximately one-third of that for E. serra lectins. It should be noted that the lectins of both species were puried several-fold more actively from the extracts, this being a difference from the case with E. serra, where the specic activities hardly increased through the isolation procedures. As a result, the specic activities of the puried lectins were almost identical with those of the E. serra lectins reported previously (Kawakubo et al., 1997). Hemagglutinating activity and carbohydrate-binding specicity The ve puried lectins, EAA-1, EAA-2, EAA-3, ECA-1, and ECA-2 had the agglutinating activities toward both trypsin-treated and untreated sheep erythrocytes as well as trypsin-treated rabbit ones. The activities of these lectins were stable in the wide pH range from pH 2.5 to pH 10.5 and were not changed by incubation at 65 C for 1 h (data not shown). The activities were not affected by treatment with EDTA, indicating that these were divalent cation-independent. The ve lectins also revealed identical results in the hemagglutination inhibition test (Table 2). Their activities were not inhibited by any of the mono- and disaccharides tested, whereas they were inhibited by a number of glycoproteins. Among these, IgM (mouse), thyrogloblin and yeast mannan were the most inhibitory. Thus, the ve lectins shared almost the same properties as E. serra lectins with respect to hemagglutinating activity and the hemagglutination inhibition test. Biochemical property of lectins The biochemical properties of EAA-1, EAA-2, EAA3, ECA-1 and ECA-2 were also very similar to each other as well as those from E. serra. They had the same molecular weight and showed similar behaviors on heat and pH stabilities and divalent cation requirement. The molecular weights of EAA-1, EAA2, EAA-3, ECA-1, and ECA-2 were estimated to be 25 000 from gel ltration on a Superdex 75 HR column and 29000 from SDS-PAGE, respectively. No carbohydrate was detected in any of the lectins. These results implied that all the lectins were monomeric proteins. EAA-1, EAA-2, EAA-3, ECA-1, and ECA-2 gave a single band of pI 4.95, 5.20, 5.50, 5.05 and 5.10 on isoelectric focusing, respectively. Thus, they are

Results Isolation of E. amakusaensis and E. cottonii lectins Lectins from both E. amakusaensis and E. cottonii were efciently extracted with aqueous ethanol and precipitated with 83% ethanol. Each precipitate gave an active peak on Superdex 75 pg gel ltration (Figure 1). The active peak coincided well with a major protein peak, indicating that proteins in each precipitate consisted exclusively of lectin molecules. Each active peak gave a single band on SDS-PAGE (Figure 2). The active fractions from both species were further separated into three or two active peaks, respectively, by ion-exchange chromatography on TSKgel DEAE5PW (Figure 3). The puried lectins obtained were designated EAA-1 (retention time: 19.8 min), EAA-2 (23.5 min) and EAA-3 (26.7 min) for E. amakusaensis and ECA-1 (15.6 min) and ECA-2 (19.5 min) for E. cottonii. Thus, lectins from E. amakusaensis and E. cottonii were isolated easily by almost the same method as for E. serra lectins. The results of purications are summarized in Table 1. Considerably more than 1000 mg protein was extracted with aqueous ethanol from 100 g dry tissue of both E. amakusaensis and E. cottonii, a value slightly higher than for E. serra. The total yields of EAAs and ECAs were as

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Table 1. Summary of purication of E. amakusaensis and E. cottonii lectins. Powdered alga (100 g) was used as starting material. Hemagglutinating activity was determined with trypsin treated sheep erythrocytes and given by titer (see text) Purication Hemagglutinating activity Total titer Yield (%) Protein Total Yield (mg) (%) Specic activity (titer mg1 ) Sugar Total (mg)

Yield (%)

E. amakusaensis Extraction Ethanol precipitation Gel ltration EAAs Ion exchange EAA-1 EAA-2 EAA-3 E. cottonii Extraction Ethanol precipitation Gel ltration ECAs Ion exchange ECA-1 EAA-2

3 440 000 2 949 000 2 812 000 434 000 1 310 000 1 280 000

100 86 82 13 38 37

1320 660 290 39 120 121

100 50 22 3 9 9

2 600 4 500 9 700 11 100 10 900 10 600

3090 2520 31 0 0 0

100 82 1 0 0 0

3 680 000 3 880 000 3 640 000 1 310 000 1 966 000

100 105 99 36 53

1656 512 274 118 154

100 31 17 7 9

2 200 7 600 13 300 11 100 12 700

2200 2200 23 0 0

100 100 1 0 0

Figure 1. Gel ltrations of 83% ethanol precipitates recovered from 20% aqueous ethanolic extracts of E. amakusaensis and E. cottonii. : Absorbance at 280 nm, - - -- - -: Hemagglutinating activity.

isolectins exhibiting only slight differences in isoelectric point. Amino acid compositions were also almost the same among the ve lectins, being rich in Glx, Asx, Gly and Ser (data not shown). In addition to these results, the N-terminal amino acid sequences of these lectins up to the 20th residue were found to be identical not only to each other, but also to those of E. serra lectins (ESA-1 and ESA-2) (Figure 4). Only a replacement of Gln 6 with Lys 6 was observed for EAA-2 and EAA-3.

Mitogenic activities

All of the lectins from E. amakusaensis and E. cottonii showed mitogenic activity for mouse lymphocytes (data not shown). The activities were dose-dependent, showing the optimum concentrations of 3 g mL1 , which were also almost the same as those of E. serra lectins (ESA-1 and ESA-2) (Kawakubo et al., 1997).

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Figure 2. SDS-PAGE of the puried lectins from both E. amakusaensis and E. cottonii. Lanes: 1, a mixture of reference proteins; 2, a 20% ethanol extract from E. amakusaensis; 3, a 20 83% ethanol precipitate from E. amakusaensis; 4, EAAs; 5, EAA-1; 6, EAA-2; 7, EAA-3; 8, a 20% ethanol extract from E. cottonii; 9, a 20 83% ethanol precipitate from E. cottonii; 10, ECAs; 11, ECA-1; 12, ECA-2.

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Figure 3. Ion exchange chromatographies of the active fractions EAAs and ECAs obtained by gel ltrations from E. amakusaensis and E. cottonii. The eluates were monitored for absorbance at 280 nm, () and for hemagglutinating activity ().

Figure 4. N-terminal amino acid sequences of lectins from E. amakusaensis (EAA-1, EAA-2 and EAA-3), E. cottonii (ECA-1 and ECA-2), and E. serra (ESA-1 and ESA-2). = Indicates different amino acid residue.

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Table 2. Hemagglutination-inhibition tests of the lectins from E. amakusaensis and E. cottonii with carbohydrates and glycoproteins. The inhibitory activity is expressed as the minimum inhibitory concentration required to inhibit completely the hemagglutinating activity of a titer, 4. indicates no inhibitory activity at the concentration of 100 mM carbohydrates. Monosaccharides and disaccharides tested are given in the text. Minimum ihhibitory concentration (g mL1 ) EAA-1 EAA-2 EAA-3 ECA-1 ECA-2 1000.0 700.0 250.0 87.5 62.5 31.3 7.8 3.9 3.9 3.9 1000.0 700.0 250.0 87.5 62.5 31.3 15.6 3.9 3.9 3.9 1000.0 700.0 250.0 87.5 62.5 31.3 15.6 3.9 3.9 3.9 1000.0 700.0 250.0 87.5 62.5 31.3 15.6 3.9 3.9 3.9 1000.0 700.0 250.0 87.5 62.5 31.3 15.6 3.9 3.9 3.9

Carbohydrate and glycoprotein Monosaccharide Disaccharide 1 -Acid glycoprotein BSA Mucin -Globulin (bovine) Fetuin Asialo fetuin Ovalbumin IgM (mouse) Thyrogloblin Yeast mannan

Discussion

The total hemagglutinating activities in E. amakusaensis and E. cottonii were both recovered at levels as high as 90% of those from the extracts through the isolation procedures. Interestingly, the extent of recovery is quite similar to that for E. serra . The extracts from the two former species comprised relatively large amounts of proteins other than the lectins, whereas more than 80% of the extracted proteins were composed of lectins in E. serra. Thus, the results might imply that all the highly yield Eucheuma lectins had common structural properties, different from other proteins, so that they were efciently isolated by the same purication procedures. The yields of the lectin proteins from both E. amakusaensis and E. cottonii, as well as those from E. serra, were much higher than those of other algal lectins isolated so far. They are 10 to 50 times higher than for Solieria robusta (Hori et al., 1988) and about 100 to 1000 times higher than other algal lectins reported (Shiomi et al., 1979, 1981; Kamiya et al., 1980, 1982; Hori et al., 1987; Chiles, 1990). Although it is not evident at present why some species of the genus Eucheuma have such high yields of lectins, it is clear that these algae are convenient sources to obtain large amounts of the lectins, since they are cultured throughout the world and E. cottonii is mass-cultured articially in tropical areas.

The Eucheuma lectins studied had similar biochemical characters to those from Solieria robusta reported previously (Hori et al., 1988). They have common preferential afnity for glycoproteins bearing high mannose-type N-glycans, identical molecular weights and mitogenic activities, and slightly different isoelectric points. The N-terminal amino acid sequences (Figure 4) show high homology between species; however, they do not resemble those of any higher plant lectins (e.g. concanavalin A, Sharon & Lis, 1990; Amaryllidaceae lectins, Van Damme et al., 1992) or animal lectins, their specicity involving carbohydrate chains with high mannose content. This indicates the potential value of a wide investigation of the distribution of lectins in marine algae, including complete sequencing of the proteins involved. As the three species of Eucheuma produce lectins with almost identical properties and very high yields, it seems probable that these lectins share some important role in the algae. It is therefore also important to establish the physiological properties of these lectins.

Acknowledgements We thank Dr M. Ohno of Kochi University, Japan, for the preparation of algal materials. This work was supported by Bio-oriented Technology Research Advancement Institution, Japan.

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156 References
Bird KT, Chiles TC, Longley RE, Kendrick AF, Kinkema MD (1993) Agglutinins from marine macro algae of the southeastern United States. J. appl. Phycol. 5: 213218. Boyd WC, Almodovar LR, Boyd LG (1966) Agglutinins in marine algae for human erythrocytes. Transfusion (Philadelphia) 6: 82 83. Chiles TC (1990) Gracilaria tikvahiae agglutinin partial purication and preliminary characterization of its carbohydrate specicity. Carbohydr. Res. 207: 319326. Chiles TC, Bird KT (1989) A comparative study of animal erythrocyte agglutinins from marine algae. Comp. Biochem. Physiol. 94: 107111. Hori K (1994) Marine algal lectins a new category. Kagaku to Seibutsu 32: 586594. (in Japanese) Hori K, Ikegami S, Miyazawa K, Ito K (1988) Mitogenic and antineoplastic isoagglutinins from the red alga Solieria robusta. Phytochemistry 27: 20632067. Hori K, Miyazawa K, Ito K (1990) Some common properties of lectins from marine algae. Hydrobiologia 204/205: 561566. Hori K, Matsuda H, Miyazawa K, Ito K (1987) A mitogenic agglutinin from the red alga Carpopeltis abellata. Phytochemistry 26: 13351338. Hori K, Miyazawa K, Ito K (1986) Preliminary characterization of agglutinins from seven marine algal species. Bull. Jap. Soc. Sci. Fish. 52: 323331. Kamiya H, Ogata K, Hori K (1982) Isolation and characterization of a new agglutinin in the red alga Palmaria palmata (L.) O. Kuntze. Bot. mar. 35: 537540. Kamiya H, Shiomi K, Shimizu Y (1980) Marine biopolymers with cell specicity III agglutinins in the red alga Cystoclonium purpureum: isolation and characterization. J. nat. Prod. 43: 136 139. Kawakubo A, Makino H, Ohnishi J, Hirohara H, Hori K (1997) The marine red alga Eucheuma serra J. Agardh, a high yielding source of two isolectins. J. appl. Phycol. 9: 331338. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680685. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. immunol. Methods 65: 5563. Rogers DJ, Hori K (1993) Marine algal lectins: new developments. Hydrobiologia 260/261: 589593. Sharon N, Lis H (1990) Legume lectins a large family of homologous proteins. FASEB J. 4: 31983208. Shiomi K, Kamiya H, Shimizu Y (1979) Purication and characterization of an agglutinin in the red alga Agardhiella tenera. Biochim. Biophys. Acta 576: 118127. Shiomi K, Yamanaka H, Kikuchi T (1981) Purication and physicochemical properties of hemagglutinin (GVA-1) in the red alga Gracilaria verrucosa. Bull. Jap. Soc. Sci. Fish. 47: 10791084. Trono GC, Jr. (1993) Eucheuma and Kappaphycus: taxonomy and cultivation. In Ohno M, Critchley AT (eds), Seaweed cultivation and marine ranching, Japan International Cooperation Agency, Yokosuka: 7588. Van Damme EJM, Goldstein IJ, Vercammen G, Vuylsteke J, Peumans WJ (1992) Lectins of members of the Amaryllidaceae are encoded by multigene families which show extensive homology. Physiol. Plant. 86: 245252.

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