Abstract Title Harnessing the anti-neoplastic potential of novel mannose-binding lectins on human urothelial and breast cell carcinomas. Authors and Affiliation: Julie Marchioni, BS; Alec Skomo, BS; and Prerna Sharma, PhD* Geisinger Commonwealth School of Medicine, Scranton PA 18509

Background Mannose-binding lectins (MBLs) are a class of carbohydrate-binding proteins that are important in modulation of viral infections, inflammation, and cancer progression through interactions with glycosylated regions. High grade cancer cell lines have their membranes decorated with oligomannosidic sugars that can be harnessed to develop anticancer drugs. The model MBL, cyanovirin-N (CVN-N) has shown promise as an antiviral. However, its widespread use is limited due to difficult isolations, poor stability, and limited applicability to cancer targets. This led us to identify novel MBLs and test their anti-neoplastic effects on human cancer cell lines. Methods The novel proteins were expressed with the pET23a/BL21(DE3) Escherichia coli expression system and purified using Nickel-NTA chromatography. The purified proteins were assessed for their structure, folding, thermal, and chemical stability using circular dichroism (CD) spectroscopy. Mannose-binding studies using model glycoproteins were performed with electrophoretic mobility shift assay, EMSA. Functional studies to assess the antineoplastic effects were performed on human urinary bladder carcinoma, T24, and breast cell adenocarcinoma, MDA-MB-231, using MTS and WST-1-based assays. Results We identified two novel fungal lectins, LC-3 (17 kDa) and LC-4 (13 kDa) with proposed mannose binding ability by data mining using BLAST search. Their predicted secondary structure aligned well with the model structure of CVN-N. The far UV-CD spectra of LC-3 and LC-4 proteins displayed a well folded structure with single negative peaks at 215 and 219 nm respectively. Both the proteins were found to be thermostable up to 90°C. Chemical denaturation studies of LC-3 with guanidinium hydrochloride reveal a Cm of 2.75 M and a ΔG of unfolding to be 7241.7 J/mol. Chemical denaturation studies for LC-4 were significant for a ΔG of unfolding of 1458.4 J/mol and for cooperativity of unfolding of two sub-domains. Positive shifts with Fetuin A, a sialylated ligand, and RNase B, a mannosylated ligand were observed for both proteins in EMSA. Significant inhibition of cell proliferation by both the proteins were observed in low micromolar concentrations (1-5 µM) for the mannose expressing cell lines, T24 and MDA-MB-231, when compared to control groups.

Conclusion The identified fungal lectins, LC-3 and LC-4 are thermochemically stable and have high specificity for mannosylated ligands. The high yield, thermochemical stability, and significant anti-proliferative effects of LC-3 and LC-4 against urinary bladder and breast cell carcinomas make them a potential candidate for clinical applications.