Electron Microscopy and X-ray Diffraction Studies ofLotus tetragonolobus AIsolectin Cross-linked with a Divalent LewisxOligosaccharide, an Oncofetal Antigen

Wen Cheng, Esther Bullitt, Lokesh Bhattacharyya, C. Fred Brewer, Lee Makowski
1998 Journal of Biological Chemistry  
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 Lewis x determinant, which is an oncofetal antigen. A combination of electron microscopy, x-ray diffraction, simulation of
more » ... 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. The carbohydrate moieties of glycoproteins and glycolipids have been shown to be involved in a variety of biological recognition processes including cell-cell and cell-substratum interactions, immunity, apoptosis, and metastasis of tumor cells (1-5). The composition and structures of the oligosaccharides correlate with cell differentiation and transformation (cf. Ref. 6). For example, the expression of oligosaccharides possessing specific Lewis blood group antigenic determinants is developmentally regulated and altered as a result of differentiation and oncogenic transformation (7). Some antigens such as the Lewis x (Le x ) 1 antigen are transiently expressed during ontogeny and reappear in tumors, and have hence been termed oncofetal antigens (cf. Ref. 5). The molecular recognition properties of the oligosaccharide chains of glycoproteins and glycolipids are often characterized in terms of their interactions with lectins (8). A number of mammalian lectins are involved in receptor-mediated endocytosis of glycoproteins (cf. Ref. 2), while others have been implicated in cellular recognition processes including apoptosis (4) and metastasis (9, 10). The biological signal transduction properties of lectins appear to be due to their ability to bind and cross-link specific glycoprotein and glycolipid receptors on cells. For example, lectin-induced cross-linking of receptors often leads to mitogenic responses in cells (11) , in the arrest of bulk transport in ganglion cell axons (12), in the induction of mating reactions in fungi (13), in the molecular sorting of glycoproteins in the secretory pathways of cells (14) , and in the apoptosis of activated human T-cells (4). Furthermore, lectin-induced crosslinking of transmembrane glycoproteins leads to changes in their interactions with cytoskeletal proteins and concomitant alterations in the mobility and aggregation of other surface receptors (15, 16) . Studies have shown that lectins undergo two general types of cross-linking interactions with multivalent carbohydrates, designated type 1 and type 2 complexes (17). In a type 1 complex, binding between a divalent lectin and a divalent carbohydrate results in one dimensional cross-linking (e.g. helical). In a type 2 complex, binding between a multivalent lectin and multivalent carbohydrate, where the valency of either the lectin or carbohydrate is greater than 2, results in two-dimensional (planar or tubular) or three-dimensional cross-linking (crystalline). Importantly, type 2 interactions can lead to the formation of homogeneous cross-linked complexes, even in the presence of mixtures of the molecules (cf. Ref. 17). Hence, type 2 interactions are an important source of binding specificity between lectins and glycoconjugate receptors. In order to understand the molecular basis of these crosslinking interactions, x-ray crystallography has been used to investigate several lectin-carbohydrate cross-linked complexes. These include two type 1 complexes involving dimeric animal lectins cross-linked with divalent oligosaccharides (18, 19) . Of type 2 complexes that have been determined, the three-dimensional crystal structures of the tetrameric soybean agglutinin (SBA) cross-linked with four biantennary carbohydrates have
doi:10.1074/jbc.273.52.35016 pmid:9857034 fatcat:m25x6bk5srbmdkxkbuvcn2zsca