Specificity of Amaranthus leucocarpus syn. hypocondriacus lectin for O-glycopeptides

Amaranthus leucocarpus syn. hypochondriacus lectin (ALL) has been shown to be specific for N-acetyl-D-galactosamine (GaINAc). In this work, we determined a value of 1.0x10(-2) M for the association constant of ALL for GaINAc, calculated using fluorescence spectroscopy assays. Using neoglycopeptides obtained by in vitro O-glycosylation, we determined the main features of O-glycopeptides recognized by ALL using molecular dynamics simulations, capillary electrophoresis, and ELISA. Neo-glycopeptides were obtained by in vitro 0-glycosylation reaction using microsornal preparations of murine thymocytes, human gastric fundus and colonic mucosa. ELISA assays were performed with peroxidase-labeled murine monoclonal IgG2, kappa light chain (5134) antibodies against ALL. Among the in vitro neoglycopeptides, only those of TTSAPTTS containing GaNAc at Thr in #2 and #6 reacted with ALL. Neither the TTSAPTTS glycopeptide, containing a unique GaINAc residue at Thr in #2, nor others (with more than two GaINAc residues) interacted with the lectin. Computational docking assays of the lower energy conformers for interactions between glycopeptides and lectins confirmed that ALL recognized GaNAc residues when they are spaced out in glycan structures, whereas GaINAc residues arranged in clusters prevented interaction with the lectin, indicating that ALL is specific for a special GaINAc-containing motif found in different O-glycoproteins. (C) 2004 Elsevier B.V. All rights reserved.