Abstract:The trisaccharide b-dGlcNAc(1 32)a-d-Man(1 33)d-Man is a fragment of a biantennary glycan that is recognized by a-d-Man-specific lectins such as concanavalin A (ConA), Lathyrus ochrus lectin, lentil lectin, and can adopt several conformations upon binding. To probe the importance of loss of flexibility of a saccharide during binding with ConA this trisaccharide has been synthesized in a conformationally constrained form where a methylene acetal bridge mimics a GlcNAc ± O-6''´´´Man ± O-4 intramolecular hydrogen bond. Microcalorimetry measurements revealed that the conformationally constrained compound has a more favorable entropy term but this term is offset by a smaller enthalpy term. NMR spectroscopic studies have shown that the cyclic compound is indeed considerably less flexible than the linear compound and both compounds adopt mainly one conformation. SYBYL software together with energy parameters appropriate for carbohydrates was used for a systematic conformational search. The linear compound is very flexible. A clustering method determined seven main conformational families. Six possible conformational families were identified for the cyclic compound when considering the orientations of the bGlcNAc(1 32)Man and abMan-(1 33)Man glycosidic bonds. The central mannose residue was docked in the binding site of ConA and the complex was refined. The results are compared with crystal structures of legume lectin ± oligosaccharide complexes and with the NMR and thermodynamic data.
Lectins (or lectin domains) represent a specific class of carbohydrate binding proteins distinct from enzymes and antibodies. Different lectin families are found in a wide range of organisms including viruses, bacteria, plants and animals. Their biological activities are diverse and include roles in the innate immunity, bacterial and viral infection, sorting and trafficking of glycoproteins, development and differentiation as well as defense mechanisms in plants [1].The lectins from legume plants belong to one of the best studied lectin families [2]. Members of this family were initially identified in the seeds of legume plants, but an increasingly larger number is found in the vegetative parts. They show strong similarities on the level of their amino acid sequences and tertiary structures, and exhibits a wide range of carbohydrate specificities and quaternary structures. Recently, family members were discovered in nonlegume plants [3]. Furthermore, the ER-Golgi intermediate (ERGIC) proteins that in animals play a role in glycoprotein transport though the golgi apparatus (but are absent in plants) belong to the legume lectin family [4].Concanavalin A (con A) was the first lectin for which the crystal structure was determined [5,6]. Since this early work, the crystal structures of 28 members of the legume lectin family have been presented either The crystal structure of Pterocarpus angolensis lectin is determined in its ligand-free state, in complex with the fucosylated biantennary complex type decasaccharide NA2F, and in complex with a series of smaller oligosaccharide constituents of NA2F. These results together with thermodynamic binding data indicate that the complete oligosaccharide binding site of the lectin consists of five subsites allowing the specific recognition of the pentasaccharide GlcNAcb(1-2)Mana(1-3)[GlcNAcb(1-2)Mana(1-6)]Man. The mannose on the 1-6 arm occupies the monosaccharide binding site while the GlcNAc residue on this arm occupies a subsite that is almost identical to that of concanavalin A (con A). The core mannose and the GlcNAcb(1-2)Man moiety on the 1-3 arm on the other hand occupy a series of subsites distinct from those of con A.
AbbreviationsCon A, concanavalin A; ITC, isothermal titration calorimetry; LOL, Lathyrus ochrus lectin.
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