Engineering Two Human Proteins with β-Trefoil Fold for Therapeutic Applications

The expression of glycoproteins containing immature truncated O-glycans such as the Thomsen-Friedenreich antigen (Ser/Thr-O-Galβ1–3GalNAc; T-antigen) and the Lewis antigen (sialyl-T-antigen) is a characteristic feature observed on almost all malignant epithelial cells. Those antigens can be recognized by lectins, a group of highly specific carbohydrate-binding proteins whose three-dimensional structure has been studied in our laboratory by X-ray crystallography. BEL β-trefoil is a lectin found in mushrooms that contains three binding sites for the T-antigen, its antiproliferative activity was demonstrated in various human tumor cell lines and it has also been employed for the targeting of antitumor drugs. Unlike other lectins with these properties, BEL β-trefoil presents a structural fold that is also found in human proteins, unlocking the opportunity to use protein engineering tools to design new anticancer therapeutics. This thesis explores the possibility of modifying existing human proteins to recognize the carbohydrate antigens present on the surface of cancer cells, in order to reduce the potential immunogenicity risk that foreign lectins could have and allowing their future application in drug-delivery targeting. To reach this purpose, two human proteins structurally similar to BEL β-trefoil were modified following different strategies. Human acidic fibroblast growth factor (FGF1) was modified in an attempt to create a new carbohydrate binding site, while a truncated form of human N-acetylgalactosaminyltranferase-6 (GalNAc-T6) was produced to exploit its affinity to N-acetylgalactosamine for this new purpose. Biophysical methods such as spectrofluorimetry and isothermal titration calorimetry were used to analyze the ability of the engineered proteins to bind the T-antigen monosaccharides. The binding dissociation constant (Kd) of the protein-carbohydrate interaction was determined. The stability of each protein was also studied through their thermodynamic parameters of unfolding using differential scanning calorimetry. Crystallization screenings were set up using a broad variety of precipitants in order to produce crystals to be used to study the three-dimensional structure of the engineered proteins using X-ray diffraction. The crystals that were grown were taken to the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to carry out the diffraction experiments. In conclusion, this work provides a new and interesting insight for the production of optimized protein therapeutics applied in drug-delivery methods for cancer treatment. The present biophysical data are the prerequisite for future studies regarding the biological properties of the engineered proteins and clinical parameters for their potential use in medicine.