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Creation, evaluation and characterization of the ideal bioprobe for the cancer antigen Tn by improved selection of mutant lectins from phage display libraries

Final Report Summary - TNSPECIFIC_LECT_EVOL (Creation, evaluation and characterization of the ideal bioprobe for the cancer antigen Tn by improved selection of mutant lectins from phage display libraries)

Glycan-protein interactions are present in a myriad of biological events in living organisms. Glycan epitopes, which are sources of encoded information, are recognized by protein partners that translate those messages into functional activity. Glycans, present in the free form or as glycoconjugates (glycoproteins and glycolipids), are interpreted by lectins and other glycan-binding receptors. In the human body, these interactions occur in both health and disease, playing a key role in cell growth regulation, cell adhesion, cell migration or host-pathogen contacts, among other processes. Changes in normal glycosylation are often a sign of a disease state or condition. Cancer cells are frequently seen to have glycans expressed at different levels or aberrant structures than those observed in normal cells. The Tn antigen (GalNacα1-O-Ser/Thr) is one of such epitopes. While expressed in most carcinomas, during embryogenesis, on pathogenic parasites, and on HIV, its expression is rare in normal adult tissue, creating a considerable interest in monitoring the expression of the Tn antigen for diagnostic purposes as well as for the development of Tn-based vaccines for cancer. Our original project plan - to alter the binding specificity of bacterial lectin GafD (a β-GlcNAc binding protein) to bind the aimed Tn antigen (figure 1) using phage display - resulted in the isolation of 8 leads, whose phage ELISAs shown to have a broad but less specific binding preferences. All these receptors were cloned in an expression vector, expressed in E. coli and purified before starting the second phase of the project at the European Host. Surprisingly, the behavior of these leads (seen in “phage display presentation”) wasn’t seen after the proteins were individually expressed. Isothermal Titration Calorimetry and Surface Plasmon Resonance studies confirmed reduced binding affinity towards the original β-GlcNAc binding by 2 leads, where no binding could be seen by the others, indicating that the reading and analysis by phage ELISA could have just been an artifact that gravely interfered with the choice of which leads to pursue.

With our objective to obtain a novel probe for a biomarker with important biomedical relevance, we oriented our efforts towards another target: a sialic acid receptor probe. Sialic acids (SIAs) are common monosaccharides that are widely expressed as outer terminal units on all vertebrate cell surfaces, and play key roles in cell–cell and cell–microenvironment interactions. The unique structural features of these molecules, which includes a negative charge owing to a carboxyl group, allows it to play a role in cellular functions, such as transport of positively charged compounds, cell-to-cell repulsion, influencing conformation of glycoproteins on cell membranes, and even masking antigenic determinants on receptor molecules. SIAs as a tumor marker should be examined from the perspective of aberrant glycosylation in cancer cell membranes owing to activation of new glycosyl transferases that are characteristic of tumor cells, and the role played by sialic acid in tumor cell metastasis including increased capacity to adhere to vascular endothelium, and decreased capacity of cancer cells to be destroyed by host defence mechanisms. The high sensitivity of SIAs as tumor markers has been reported in a variety of cancerous conditions. SIA measurements have a strong value in monitoring cancer patients during diagnosis and treatment, thus making effective SIA receptors a necessity (in great demand).

While still at the outgoing phase, and as an alternative strategy, a bioinformatics approach was used to identify a glycan-binding protein (CBM40_CPF0721) predicted to have SIA binding properties. During the time at CERMAV (the European host), CBM40_CPF0721 was expressed and purified extensively. A dimeric version of the protein was also engineered, proving particularly useful for hemagglutination assays and as an analite for glycan array of the Consortium of Functional Glycomics. It was seen to show a strong preference towards α(2,3)-sialyllactosamine (figure 2). To our knowledge, there is no other protein as selective towards a sialylated epitope as CBM40_CPF0721. Another successful example of a SIA binder was published by Connaris et al. (JBC, 2009, 284, 7339) although showing specificity to α(2,3)-, α(2,6)- and α(2,8)-linked sialosides. The characterization of our novel sensor will be completed after the three-dimensional structure is unveiled. After successfully crystalizing the protein, this will be analyzed at to the European Synchroton Research Facility (ESFR) the 18th of November to be studied under by X-ray diffraction.

Our work successfully resulted in the presentation of a novel and highly specific α2-3 SIA binding probe, addressing the problematic with its unavailability.

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