Abstract 1982 Targeting Sialic Acids with Streptococcal Adhesin Proteins

Glycans terminating with sialic acid (sialoglycans) are important for many biological functions and are ubiquitous in both bacterial and mammalian cells. When found on mammalian cell surfaces one function is to act as ligands for sialic-acid-binding immunoglobulin-like lectins which suppresses autoimmunity. Likely linked to the attenuation of the immune response, aberrant glycosylation has been linked to initial oncogenic transformation and promotes tumor cell metastasis. This could make sialoglycan detection on cell surfaces a way to distinguish cancer cells. However, few techniques make glycan detection straightforward and high throughput. In ongoing work, we applied biochemical techniques to bacterial sialoglycan-binding proteins to build a library of probes specific for cancer-associated sialoglycans. The first target sialoglycans are 6-sulfo sialyl LewisX (6S-sLeX) and sialyl LewisX (sLeX), which are over-represented in colon and breast cancers. The starting scaffolds are Siglec-like binding region (SLBR) from Streptococcus sanguinis strain SK678 (SLBRSK678) and Streptococcus gordonii strain UB10712 (SLBRUB10712). SLBRSK678 and SLBRUB10712 are broadly selective and bind several Neu5Acα2-3gal containing glycans. The first round of engineering started with solving high-resolution crystal structures of parent SLBRs, Streptococcus gordonii strain Challis (Hsa) and Streptococcus gordonii strain M99 (GspB) bound with several sialoglycans. After structure evaluations and sequence comparison analysis with SLBRUB10712 and SLBRSK678, we created SLBRUB10712E285R/Q354D and SLBRSK678E298R/Q367D, both of whom were predicted to be more selective for a single sialoglycan, 6S-sLeX. Change in selectivity was tested with an ELISA assay where an almost 2-fold decrease in binding to other sialoglycans was measured. This indicates that SLBRUB10712E285R/Q354D and SLBRSK678E298R/Q367D are more selective for 6S-sLeX than their wild-type forms. We also solved a 2.1-angstrom resolution structure of SLBRUB10712E285R/Q354D using X-ray crystallography. In the second round, we continued with SLBRSK678 due to its greater mutability. We developed new SLBR probes with sLeX as our target. Using the same mutation creation method, we created five new mutations for SLBRSK678, V297T, V297Y, V297D, and T309H. The valine and threonine residues were chosen because they interact closely and form hydrogen bonds with sialoglycans. They were swapped out for residues that would help facilitate binding to sLeX while closing the binding site off to larger sialoglycans such as 6S-sLeX. These are currently being tested with an ELISA assay against wildtype SLBR and the first-round mutants. We predict that these probes will be better binders for sLeX, adding to our collection of probes. These findings are the starting point that will lead us toward a library of detection probes that could be used as a new screening technique for better detection and identification of sialoglycans. This project is supported, in part, by National Institutes of Health under award numbers T32AI007281, T32GM14492, and T32HL00773.