Characterization of the secondary binding sites of Maclura pomifera agglutinin by glycan array and crystallographic analyses

Abstract

The Maclura pomifera agglutinin (MPA) recognizes the T-antigen disaccharide Galβ1,3GalNAc mainly through interaction of the α-GalNAc moiety with its primary site, but the interactions of the two flanking subsites A and B with aglycones and substituents other than Gal, respectively, are not well understood. We therefore characterized the specificity of MPA in more detail by glycan microarray analysis and determined the crystal structures of MPA without ligand and in complexes with Galβ1,3GalNAc and p-nitrophenyl α-GalNAc. In both sugar complexes, pairs of ligands created inter-tetramer hydrogen-bond bridging networks. While subsite A showed increased affinity for hydrophobic aglycones, it also accommodated several sugar substituents. Notably, a GalNAc-O-tripeptide, a Tn-antigen mimic, showed lower affinity than these compounds in surface plasmon resonance (SPR) experiments. The glycan array data that showed subsite B accepted compounds in which the O3 position of the GalNAc was substituted with various sugars other than Gal, but substitutions at O6 led to inactivity. Additions to the Gal moiety of the disaccharide also had only small effects on reactivity. These results are all compatible with the features seen in the crystal structures.

Fig. 1

Glycan array data for MPA. The blue bars indicate compounds related to Galβ1,3GalNAcα. Symbols for the sugars are given below, and α-glycosidic bonds are shown in red. Binding activities are scaled to that of the compound with the highest binding (fluorescence). The diagram is based on the CFG spreadsheets which can be accessed at http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2_234_01202006

Fig. 2

Structures of the MPA tetramer, monomer, and β chain region. (A) Crystal structure of the tetramer with the disaccharide Galβ1,3GalNAcα. (B) Monomer with the β chain. (C) Hydrogen bonding between α and β subunits in the MPA monomer. Note that the β numbers refer to the strands of the β structure, not to residues in the β subunit

Fig. 3

Hydrogen-bonding diagrams of the disaccharide complex. (A) Hydrogen-bonding network of Galβ1,3GalNAcα with MPA. (B) Hydrogen bonding of the disaccharide with water and symmetrically related MPA monomers (M5 and M7)

Fig. 4

Comparison of binding site regions. (A) The Galβ1,3GalNAcα complex. (B) The p-nitrophenyl α-GalNAc complex. (C) Apo-MPA. Three water molecules (W16, W3, and W6) occupy the binding site and form hydrogen bonds with Tyr122, Asn125, and Gly1 of the MPA α chain. The conformation of Tyr 122 from A is shown in green

Fig. 5

Summary of the MPA ligands from the glycan microarray. The compounds are shown in three groups based on the T antigen (with its hydrogen bonds to the amino acid residues), α-GalNAc, and α-Gal derivatives, respectively