Analyses of Carbohydrate Recognition by Legume Lectins: Size of the Combining Site Loops and their Primary Specificity Vivek Sharma* and Avadhesha Surolia Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012, India Recognition of cell-surface carbohydrates by lectins has wide implications in important biological processes. The ability of plant lectins to detect subtle variations in carbohydrate structures found on molecules, cells and organisms have made them a paradigm for protein-carbohydrate rec- ognition. Legume lectins, one of the most well studied family of plant proteins, display a considerable repertoire of carbohydrate speci®cities owing perhaps to the sequence hypervariability in the loops constituting their combining site. However, lack of a rigorous framework to explain their carbohydrate binding speci®cities has precluded a rational approach to alter their ligand binding activity in a meaningful manner. This study reports an extensive analysis of sequences and structures of several legume lectins and shows that despite the hypervariability of their com- bining regions they exhibit within a signi®cant pattern of uniformity. The results show that the size of the binding site loop D is invariant in the Man/Glc speci®c lectins and is possibly a primary determinant of the monosaccharide speci®cities of the legume lectins. Analyses of size and sequence variability of loops reveal the existence of a common theme that subserves to de®ne their binding speci®cities. These results thus pro- vide not only a framework for understanding the molecular basis of carbohydrate recognition by legume lectins but also a rationale for rede- sign of their ligand binding propensities. # 1997 Academic Press Limited Keywords: plant lectins; protein-carbohydrate interactions; sequence analysis; binding site hypervariability; speci®city patterns *Corresponding author Introduction Speci®c recognition of carbohydrates by lectins is critical for cell-cell interactions such as those re- quired during fertilization, embryogenesis, lym- phocyte-homing, tissue development and host- pathogen interactions (Bookbinder et al., 1995; Lasky, 1992; Sastry & Ezekowitz, 1993; Chrispeels & Raikhel, 1991; Sharon & Lis, 1989, 1995). The un- derstanding of lectin-carbohydrate interactions at the molecular level (Sharon, 1993; Rini, 1995), therefore, has implications for intervention in sev- eral areas of biology and medicine (Liener et al., 1986; Harada et al., 1987; Beuth & Pulverer, 1994). Lectins from leguminous plants constitute a large family of homologous proteins displaying remark- able divergence in their carbohydrate speci®city (Sharon & Lis, 1990). Their three-dimensional structures are characterised by two anti-parallel b- sheets and the presence of tightly bound Ca 2  and Mn 2  . The crystal structures of nine legume lectins have so far been determined. Five of these (ConA, PSL, Favin, LenL and LOL I) are speci®c towards Man/Glc and the remaining four (GS IV, ECorL, PNA and SBA) are Gal/GalNAc-speci®c (Table 1). Lectins have been previously classi®ed on the basis of their blood group speci®city and subsequently on the potency with which a monosaccharide inhi- bits their agglutinating and glycoconjugate precipi- tating activity. Several speci®city groups have been described, mannose (or glucose), galactose, N- acetylglucosamine, N-acetylgalactosamine, L-fucose and N-acetylneuraminic acid (Goldstein & Hayes, 1978; Sharon & Lis, 1990). The lectins within each Present address: V. Sharma, Department of Biochemistry and Biophysics, Texas A&M University, College Stateion, Texas 77843, U.S.A. Abbreviations used: Abbreviations for the lectins are as in Table 2. Unless otherwise indicated all sugars are of D-con®guration. J. Mol. Biol. (1997) 267, 433±445 0022±2836/97/120433±13 $25.00/0/mb960863 # 1997 Academic Press Limited