Electron Microscopy and X-ray Diffraction Studies of Lotus tetragonolobus A Isolectin Cross-linked with a Divalent Lewisx Oligosaccharide, an Oncofetal Antigen

Abstract

The interactions of lectins with multivalent carbohydrates often leads to the formation of highly ordered cross-linked lattices that are amenable to structural studies. A particularly well ordered, two-dimensional lattice is formed from fucose-specific isolectin A from Lotus tetragonolobus cross-linked with difucosyllacto-N-neohexaose, an oligosaccharide possessing the Lewisx determinant, which is an oncofetal antigen. A combination of electron microscopy, x-ray diffraction, simulation of electron micrographs, and molecular model building was used to determine the relative positions of the tetrameric lectin and bivalent carbohydrate within the lattice. X-ray diffraction from unoriented pellets was used to determine the lattice dimensions and analysis of electron micrographs was used to determine the lattice symmetry. Molecular models of the lattice were constructed based on the known structure of the jack bean lectin concanavalin A and the high degree of sequence homology between the two lectins. Using the symmetry and dimensions of the lattice and its appearance in filtered electron micrographs, molecular models were used to determine the orientation of the lectin in the lattice, and to define the range of lectin-oligosaccharide interactions consistent with the structural data. The present study provides the first description of a highly ordered, two-dimensional, cross-linked lattice between a tetravalent lectin and a bivalent carbohydrate.