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)
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.