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Recombinant mucin-type fusion proteins with Gal1,3Gal substitution as Clostridium difficile toxin A inhibitors

Cherian, RM;Jin, C;Liu, J;Karlsson, NG;Holgersson, J;

The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Gal1,3Gal1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated as C-PGC2, was purified by affinity and gel filtration chromatography from large scale cultures. Liquid chromatography-mass spectrometry was used to characterize O-glycans released by reductive -elimination, and new diagnostic ions to distinguish Gal1,3Gal- from Gal1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA compared to the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit the TcdA binding to rabbit erythrocytes, making it a high efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited moderate neutralization capability of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells; the former with and the latter without cell surface Gal1,3Gal1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA inhibitory effect and therapeutic potential in C. difficile-associated diseases.