The Tn determinant (GalNAc-alpha-O-Ser/Thr), normally cryptic in mucin-type O-glycans, is a useful tumor-associated marker. Expressed early in transformed cells during carcinogenesis, the density of the Tn antigen is directly correlated to carcinoma aggressiveness. In collaboration with E. Osinaga's group (Fac. Medecina, Montevideo) and the Unité de Chimie Organique, we have carried out structural and binding studies of Tn-specific proteins (anti-tumor antibodies, plant lectins) in complex with synthetic glycopeptides, in order to understand the molecular basis of Tn-specificity.
Protective immune responses against microbial pathogens are frequently based on anti-carbohydrate antibodies produced against polysaccharides located on their cell surface. In collaboration with the Unite de Cholera and Vibrions, we have determined the crystal structure of a murine Fab fragment from a protective anti-cholera antibody specific for the lipopolysaccharide (LPS) antigen of Vibrio cholerae, Ogawa serotype, in its unliganded form and in complex with synthetic fragments of the LPS. This structural analysis helps to account for the serotype specificity of protective antibodies and provides a rational basis towards the development of a synthetic carbohydrate-based anti-cholera vaccine.
The horse Equ c 1 glycoprotein, a member of the lipocalin superfamily, is a major allergen since it induces an IgE-mediated reaction in horse-allergic patients. To gain further insight into the nature of IgE epitopes, we have determined the crystal structure of the horse allergen at 2.3 Å resolution and carried out binding studies of surface mutants of Equ c 1 with specific monoclonal antibodies, to delineate the IgE-binding targets on the protein surface. In particular, a single specific monoclonal antibody was shown to totally inhibit polyclonal IgE recognition, revealing the restricted nature of the allergenic epitopes.
The bacterial cellulosome is a macromolecular agregate that is very efficient in degrading natural cellulose. All cellulosomal glycosidases catalyze a similar reaction (hydrolysis of the glycosidic linkage), but display a wide diversity of 3D structures and modes of action. During the last ten years, we have carried out structural studies of cellulosomal glycosidases in collaboration with P. Beguin (Unité de Microbiologie et Environement). Studied enzymes belong to different protein families, including both (alpha/alpha)6 barrel topology (family 8 and family 9 endoglucanases, family 48 cellobiohydrolases) and (beta/alpha)8 barrel topology (family 5 endoglucanases and family 10 xylanases). These studies provided important clues to understand glycosyl hydrolysis and cellulose degradation, and illustrated how conserved structural motifs have evolved to fulfill different biochemical functions.
The quaternary organization of the cellulosome depends on specific interactions between dockerin domains, which are double EF-hand (calcium-binding) subunits carried by the catalytic components of the complex, and cohesin domains, which are individual receptor subunits linearly arranged within a non-catalytic scaffolding polypeptide. The same modular principle of cohesin-dockerin complexes, but with a different specificity, is thought to mediate the attachment of cellulosomes to the cell membrane. We are currying out structural (X-ray, NMR) and calorimetric studies of the cohesin-dockerin association to gain further insights on cellulosomal assembly. In particular, we have determined the crystal structure of a single cohesin subunit at 1.7 Å resolution, and showed that the hydrophobic effect is the major driving force of the cohesin-dockerin interaction.
Technological advances in synchrotron radiation sources and cryocrystallography have made possible the structural studies of macromolecular crystals at atomic and subatomic resolution. Such studies considerably improve the stereochemical analysis of protein structure and the precise role of solvent in protein stability and protein-ligand interactions. In collaboration with V. Lamzin at the EMBL-Hamburg outstation, we have determined the crystal structure of cellulosomal endoglucanase CelA in complex with substrate at 0.94 Å resolution, and collected multiple-wavelength anomalous diffraction data of the protein at 1 Å resolution. These studies showed that an extended network of hydrogen bonding interactions involving solvent molecules stabilizes the enzyme-bound substrate in a strained conformation. Carbohydrate distortion is shown to play a direct role in catalysis, by facilitating the formation of an oxocarbenium ion-like transition state.