Structural basis for the carbohydrate specificities of moraceae lectins

Abstract

Lectins are multivalent carbohydrate-binding proteins of non-immune origin. They have received considerable attention due to their ability to recognize diverse sugar structures with high specificity. Lectins are involved in various biological processes such as cell-cell recognition, host-pathogen interactions, cell targeting, cancer metastasis, and differentiation. They exist in almost all forms of life, but plant lectins are the most thoroughly characterized.

Plant lectins exhibit five different structural folds. Jacalin and artocarpin are two tetrameric lectins present in jackfruit (Artocarpus integrifolia) seeds. Each subunit of jacalin consists of two chains-a long chain of 133 amino acids and a short chain of 20 amino acids-whereas artocarpin is a single-chain protein of 149 residues. Crystal structures revealed both lectins to be highly homologous with a -prism I fold. Despite structural similarity, they differ considerably in carbohydrate specificity: jacalin is galactose-specific, while artocarpin is mannose-specific at the monosaccharide level.

The structure of the jacalin-Me- -galactose complex and uncomplexed artocarpin provided an explanation for this difference. The present work aimed to understand the structural basis of carbohydrate specificity of these lectins, particularly at the oligosaccharide level, and its implications for biological properties.

Crystallization experiments were carried out using the hanging-drop vapour diffusion technique. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 generator, with some datasets collected using synchrotron radiation. Data were processed using DENZO and SCALEPACK (HKL suite). Structures were solved using AMoRe (molecular replacement), refined with CNS, and validated using FRODO, INSIGHT II, HBPLUS, NACCESS, ALIGN, and PROCHECK.

Jacalin Studies Jacalin binds Thomsen-Friedenreich antigen (Gal 1-3GalNAc, T-antigen), expressed in >85% of human carcinomas, making it of diagnostic value.

The jacalin-T-antigen complex was determined at 1.62 Å resolution. Binding involved primarily the GalNAc moiety, with Gal interacting via water molecules.

Additional complexes solved (resolutions 1.6-2.8 Å) revealed three binding components: primary site, secondary site A, and secondary site B.

Binding mode depends on sugar linkage: -substituents interact hydrophobically with secondary site A, while -substituents bind at secondary site B through water bridges.

These structures explain jacalin’s binding to glycoproteins and provide qualitative support for thermodynamic data.

Artocarpin Studies Artocarpin (65 kDa tetramer) complexes with Me- -mannose, mannotriose, and mannopentose were solved.

Binding site comprises three loops forming primary and secondary subsites.

Primary site interactions are hydrogen-bond dominated; secondary site interactions are van der Waals.

Mannotriose and mannopentose interact through multiple mannose residues.

Comparison with heltuba (another mannose-specific jacalin-like lectin) showed that variation in loop length at the secondary site confers oligosaccharide specificity.

Promiscuity in Jacalin Surface plasmon resonance and crystallography revealed weak binding of jacalin to mannose.

Isothermal titration calorimetry confirmed Me- -mannose binds >10× weaker than Me- -galactose.

Structural analysis explained binding energetics in terms of primary and secondary site interactions.

Publications Pratap, J.V. et al. (2002). Crystal structures of artocarpin, a Moraceae lectin with mannose specificity, and its complex with methyl- -D-mannose. J. Mol. Biol. 317, 237-247.

Jeyaprakash, A.A. et al. (2002). Crystal structure of the jacalin-T-antigen complex and comparative study of lectin-T-antigen complexes. J. Mol. Biol. 321, 637-645.

Jeyaprakash, A.A. et al. (2003). Structural basis of carbohydrate specificities of jacalin: An X-ray and modeling study. J. Mol. Biol. 332, 217-228.

Jeyaprakash, A.A. et al. (2004). Structural basis for carbohydrate specificities of artocarpin: Variation in loop length as a strategy for ligand specificity. J. Mol. Biol. 338, 757-777.