A structural basis for the difference in specificity between the two jacalin-related lectins from mulberry (Morus nigra) bark q Pierre Roug e, a, * Willy J. Peumans, b Annick Barre, a and Els J.M. Van Damme c a Signaux et Messages Cellulaires chez les V eg etaux, UMR-CNRS 5546, P^ ole de Biotechnologie v eg etale, Chemin de Borde-Rouge, Castanet Tolosan 31326, France b Department of Applied Plant Sciences, Katholieke Universiteit Leuven, Leuven B-3001, Belgium c Department of Molecular Biotechnology, Ghent University, Coupure Links 653, Ghent B-9000, Belgium Received 10 March 2003 Abstract The activity and specificity of a galactose-specific and a mannose-specific jacalin-related lectin from the bark of the black mulberry (Morus nigra) tree has been re-investigated using different experimental approaches. Both lectins definitely behave as polyspecific lectins recognizing galactose, mannose, and glucose even though MornigaG and MornigaM interact preferentially with galactose and mannose, respectively. The exceptionally extended size of the carbohydrate-binding site of both lectins apparently accounts for their polyspecific character. Parallel studies with other mannose-specific jacalin-related lectins confirmed that their exclusive specificity towards mannose/glucose relies on a reduced size of their carbohydrate-binding site. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Black mulberry lectins; Jacalin-related lectins; Polyspecific lectins; Carbohydrate-binding site Hitherto, seven different families of structurally and evolutionary related carbohydrate-binding proteins (also known as lectins, agglutinins, or hemagglutinins) have been identified in plants [24]. The first members of the family of the jacalin-related lectins were identified in seeds of the Osage orange (Maclura pomifera) [1] and jackfruit (Artocarpus integrifolia) [16]. Until 1996, jaca- lin-related lectins were considered a small homogeneous family of galactose/T-antigen-binding agglutinins oc- curring exclusively in a small subgroup of the plant family Moraceae. However, this idea had to be aban- doned after the identification of mannose-specific lectins with a high sequence similarity to jacalin in the hedge bindweed (Calystegia sepium; Convolvulaceae) [19,23], Jerusalem artichoke (Helianthus tuberosus; Asteraceae) [25], jackfruit (Artocarpus integrifolia; Moraceae) [21], rice (Oryzasativa; Gramineae) [3,28], and banana (Musa acuminata; Musaceae) [4]. In addition several mannose- specific lectins have been identified that are built up of polypeptides consisting of two or more tandemly ar- rayed jacalin domains like the lectins of the Japanese chestnut (Castanea crenata; Fagaceae) [18] and Parkia platycephala (Fabaceae) [13], and the myrosinase-bind- ing proteins of Brassica napus (Brassicaceae) [9]. Re- cently, a lectin from the gymnosperm Japanese cycad (Cycas revoluta) has been identified as a new member of the jacalin-related lectins [27]. On the basis of the available data the family of jacalin- related lectins is sub-divided into galactose- and mannose-specific lectins. The galactose- and mannose- specific jacalin-related lectins differ not only in their specifity but also in the molecular structure of the pro- tomers and their sub-cellular location. Jacalin and its galactose-specific homologues are built up of ÔcleavedÕ protomers consisting of a heavy (a) and a light (b) polypeptide chain and are located in storage protein vacuoles [20]. In contrast, the mannose-specific jacalin- related lectins consist of ÔintactÕ protomers and are lo- cated in the cytoplasm [20]. Comparative structural and specificity studies done with jacalin and the mannose- binding homologues from jackfruit (artocarpin or Biochemical and Biophysical Research Communications 304 (2003) 91–97 www.elsevier.com/locate/ybbrc BBRC q Abbreviations: Heltuba, Helianthus tuberosus agglutinin; Morni- gaG, galactose-specific Morus nigra agglutinin; MornigaM, mannose- specific Morus nigra agglutinin. * Corresponding author. Fax: +33-5-61-17-59-94. E-mail address: Pierre.Rouge@ipbs.fr (P. Roug e). 0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00538-2