Fruit-Specific Lectins from Banana and Plantain
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Planta 2000) 211: 546±554 Fruit-speci®c lectins from banana and plantain Willy J. Peumans1, WenlingZhang 1, Annick Barre2, Corinne HouleÁ s Astoul2, Peter J. Balint-Kurti3, Paula Rovira4, Pierre Rouge 2, Gregory D. May3, Fred Van Leuven5, Paolo Trua-Bachi4, Els J. M. Van Damme1 1Laboratory of Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3001 Leuven, Belgium 2Institut de Pharmacologie et Biologie Structurale, UPR CNRS 9062, 205 Route de Narbonne, F-31077 Toulouse Cedex, France 3Plant Biology Division, The Samuel Roberts Noble Foundation Inc., 2510 Sam Noble Parkway, Ardmore, OK 73402, USA 4Unite d'Immunophysiologie Mole culaire, CNRS LA 1961, De partement d'Immunologie, Institut Pasteur, 25 Rue du Dr. Roux, F-75724 Paris Cedex 15, France 5Center for Human Genetics, Katholieke Universiteit Leuven, Herestraat 49, B-3001 Leuven, Belgium Received: 1 December 1999 / Accepted: 31 January 2000 Abstract. One of the predominant proteins in the pulp Introduction of ripe bananas Musa acuminata L.) and plantains Musa spp.) has been identi®ed as a lectin. The banana Recent advances in the biochemistry, molecular cloning and plantain agglutinins called BanLec and PlanLec, and structural analysis of plant lectins allowed us to respectively) were puri®ed in reasonable quantities using subdivide this heterogeneous group into seven families a novel isolation procedure, which prevented adsorption of structurally and evolutionarily related proteins Van of the lectins onto insoluble endogenous polysaccha- Damme et al. 1998). In the past, most of the research rides. Both BanLec and PlanLec are dimeric proteins has been concentrated on the four classical families of composed of two identical subunits of 15 kDa. They plant lectins, namely the legume lectins, type-2 ribo- readily agglutinate rabbit erythrocytes and exhibit spec- some-inactivating proteins, chitin-binding lectins con- i®city towards mannose. Molecular cloning revealed taining hevein domains and monocot mannose-binding that BanLec has sequence similarity to previously lectins. There is a growing interest in the family of the described lectins of the family of jacalin-related lectins, so-called jacalin-related lectins and now there are ®rm and according to molecular modelling studies has the indications that these lectins can play an important role same overall fold and three-dimensional structure. in plant defense. Jacalin-related lectins were originally The identi®cation of BanLec and PlanLec demonstrates discovered in jack fruit Artocarpus integrifolia) seeds, the occurrence of jacalin-related lectins in monocot which contain large amounts of a Thomson-Frieden- species, suggesting that these lectins are more wide- reich or T-antigen-speci®c agglutinin, called jacalin spread among higher plants than is actually believed. Sastry et al. 1986). Later, similar lectins were identi®ed The banana and plantain lectins are also the ®rst in other Artocarpus species and in the osage orange documented examples of jacalin-related lectins, which Maclura pomifera). All the Artocarpus lectins and the are abundantly present in the pulp of mature fruits but Maclura pomifera agglutinin are very similar with are apparently absent from other tissues. However, after respect to their molecular structure a4b4), amino acid treatment of intact plants with methyl jasmonate, sequence and carbohydrate-binding speci®city Young BanLec is also clearly induced in leaves. The banana et al. 1989, 1991). Due to their apparent con®nement to lectin is a powerful murine T-cell mitogen. The relevance a small taxonomic group, the jacalin-related lectins were of the mitogenicity of the banana lectin is discussed in considered to be a minor lectin family. However, this terms of both the physiological role of the lectin and the idea was abandoned after the identi®cation of a impact on food safety. mannose/maltose-speci®c lectin in rhizomes of the hedge bindweed Calystegia sepium), which shares a high Key words: Fruit lectins ± Jacalin ± Lectin ± sequence similarity with the precursor of jacalin Van Mannose ± Musa ± Plantain Damme et al. 1996; Peumans et al. 1997). The discovery of the Calystegia sepium agglutinin or Calsepa) in a typical representative of the family Convolvulaceae not only demonstrated the occurrence of a jacalin-related The nucleotide sequence reported in this paper has been submitted lectin outside the Moraceae but also revealed the to the Genbank/EMBL Data library under the accession number AF001527 existence of two subgroups within this lectin family. Jacalin and Calsepa have a dierent molecular structure Abbreviations: BanLec = banana lectin; Calsepa = Calystegia sepium agglutinin; HCA = hydrophobic cluster analysis; Hel- [2 kDa + 13 kDa]4 versus [16 kDa]2), and in addition, tuba = Helianthus tuberosus agglutinin; PlanLec = plantain lectin exhibit a totally dierent speci®city [Galb1,3)GalNAc Correspondence to: W. Peumans; versus mannose]. Furthermore, jacalin and Calsepa E-mail: [email protected]; Fax: +32-16-322976 strongly dier from each other regarding their biosyn- W. J. Peumans et al.: Banana and plantain fruit lectins 547 thesis and topogenesis. Jacalin is synthesized as a for 15 min. The supernatant was ®ltered through ®lter paper preproprotein, which undergoes a complex co- and Whatmann 3MM) and loaded onto a column of S Fast Flow post-translational processing involving the removal of a 5 cm diameter, 10 cm long; 100 ml bed volume; Pharmacia) equilibrated with 20 mM acetic acid. Afterwards, the column was signal peptide, N-glycosylation, removal of an washed with 1 l of formate buer 20 mM Na-formate, pH 3.8) and N-terminal propeptide and excision of a four-amino- the bound proteins eluted in a single step with 200 ml of 1 M NaCl acid residue linker between the mature a and b chains. in formate buer. Accordingly, one can reasonably assume that jacalin The lectin was isolated from the protein fraction desorbed from follows the secretory pathway and eventually accumu- the S Fast Flow column by anity chromatography on immobi- lates in storage-protein vacuoles. In contrast, mature lized mannose. After loading the protein mixture onto a column of mannose-Sepharose 4B 2.6 cm diameter, 10 cm long; 50 ml bed Calsepa corresponds to the entire open reading frame of volume) equilibrated with formate buer containing 0.2 M NaCl, the gene and hence undergoes no co- or post-transla- the column was washed with the same buer until the A280 fell tional processing. Due to the absence of a signal peptide below 0.01, and the bound proteins eluted with 100 ml of 20 mM it is assumed that Calsepa is synthesized and located in acetic acid. Then the pH of the lectin solution was adjusted to 3.8 the cytoplasm. This hypothesis was recently con®rmed and solid NaCl was added to a ®nal concentration of 0.2 M. After by immunolocalisation studies of the lectin data not standing overnight in the cold, the lectin solution was cleared by centrifugation 9000g for 15 min) and the anity-chromatography shown). Calsepa was originally considered as a special step repeated. At the end of the second anity chromatography the member of the family of jacalin-related lectins. However, lectin was eluted with 0.1 M mannose in formate buer, dialyzed evidence is now accumulating that the cytoplasmic against appropriate buers and frozen at )20 °C until use. The mannose-speci®c jacalin-related lectins are much more total yield of anity-puri®ed lectin was about 50 mg. widespread than the vacuolar galactose-speci®c homo- The same procedure was followed to isolate the lectin from the logues, which are apparently con®ned to a few genera of pulp of ripe plantains. Starting from 4 kg of peeled plantains about 200 mg anity-puri®ed lectin was obtained. the Moraceae family. A lectin similar to Calsepa has been found, indeed, in tubers of the Jerusalem artichoke Hemagglutination tests. Agglutination assays were carried out in Helianthus tuberosus, Asteraceae; Van Damme et al. small glass tubes in a ®nal volume of 50 ll containing 40 llofa1% 1999). Recently, we have shown that a mannose-binding suspension of red blood cells and 10 ll of lectin solutions. To jacalin-related lectin is also induced in salt-stressed rice determine the agglutination titer, the lectin was serially diluted with Oryza sativa) plants Zhang et al. 2000). In addition, 2-fold increments. Agglutination was assessed visually after 1 h at room temperature. Human and rabbit blood cells were treated with evidence has been presented that the polypeptides of the trypsin, as described previously Peumans et al. 1997). so-called myrosinase-binding proteins from Brassica The carbohydrate-binding speci®city of the lectin was deter- napus seeds and seedlings contain domains with a high mined by inhibition of agglutination of trypsinized rabbit erythro- sequence identity to the jacalin-related lectins Geshi cytes. To 10-ll aliquots of serially diluted stock solutions of the and Brandt 1998). Dierential screening of cDNA inhibitors glycoproteins, mono- and oligosaccharides, and methyl libraries representing banana Musa acuminata) pulp at monoglycosides) 10 ll of a lectin solution 10 lg/ml) was added. After preincubation for 1 h at 25 °C, 30 ll of a 1% suspension of dierent ripening stages also yielded a cDNA clone with trypsinized rabbit erythrocytes was added and the agglutination sequence similarity to jacalin Clendennen and May evaluated after 1 h. The sugars tested were: glucose, galactose, 1997). To check whether the putative jacalin-related galactosamine, N-acetylglucosamine, N-acetylgalactosamine, man- lectin from banana BanLec) corresponds to a previous- nose, lactose, melibiose, fucose, arabinose, ribose, fructose, treha- ly described mannose-binding banana agglutinin Kosh- lose, sorbose, xylose, sucrose, maltose and sorbitol. All sugars te et al. 1990) the pulp lectin was re-investigated and the tested were of the D-con®guration except for L-fucose and L- sorbose. Glycoproteins tested were thyroglobulin, ovomucoid, corresponding cDNA completely sequenced and ana- fetuin, asialofetuin, mucin and asialomucin.