In-Silico and Functional Characterisation of Nematode Galectin-3 and Galectin-9

Elizabeth Mullens; Gemma Zerna; Farah Ahmady-Nield; Travis Beddoe; David Piedrafita; Sarah Preston

Abstract

The search for novel treatments for chronic inflammatory conditions has led to an increased interest in gastrointestinal nematodes and the molecules they produce to evade the human immune system. Galectins are carbohydrate-binding proteins that are potent, multifunctional signalling proteins for the immune system, and form a large component of the excretory/secretory molecules nematodes produce during infection. The aim of this research was to determine if Necator americanus (New World Hookworm) and Trichuris trichiura (Human Whipworm), produced functional galectin homologues of human galectin-3 and -9 which interacted with host cells. Protein databases for N. americanus and T. trichiura were analysed for significant sequence and structural similarity to human galectin-3 and -9. Four proteins were expressed using Escherichia coli (E. coli) and purified by lactose affinity purification. Recombinant Hookworm-galectin-3 (rHW-gal-3) was capable of agglutinating horse red blood cells (RBCs) at >59.5 µg/mL, and increased proliferation in the human epithelial HCA-7 CRC colon carcinoma cell line, at test concentrations >1.25 µL/mL, compared to untreated cells (p-value < 0.05). Recombinant Whipworm-galectin-9 (rWW-gal-9) binding was visualised on HCA-7 CRC cells but had no-effect on proliferation. Recombinant Whipworm-gal-3 (rWW-gal-3) and Hookworm-gal-9 (rHW-gal-9) did not bind or significantly alter the proliferation of HCA-7 CRC cells but possessed carbohydrate binding evidence through lactose affinity purification. In-vitro results suggest that synthetic nematode galectin molecules, selected through in-silico comparison to human galectin-3 and galectin-9, had carbohydrate binding activity, and that some were capable of binding and interfering with host cell processes. This research furthers our understanding of nematode-host interaction.

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