title: Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors.
abstract: The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Galα1,3Galβ1,4GlcNAc 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 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 β-elimination, and new diagnostic ions to distinguish Galα1,3Gal- from Galα1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than 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 TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Galα1,3Galβ1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases.
authors.affiliation: Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden reejamaria@gmail.com.
authors.forename: Reeja Maria
authors.initials: RM
authors.lastname: Cherian
authors.affiliation: Department of Medical Biochemistry, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Jin
authors.affiliation: Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Jining
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Medical Biochemistry, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Niclas G
authors.initials: NG
authors.lastname: Karlsson
authors.affiliation: Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Jan
authors.initials: J
authors.lastname: Holgersson
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D016360:Clostridioides difficile / Q000502:physiology*; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000378:metabolism*; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics*
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D011993:Recombinant Fusion Proteins; C041348:tcdA protein, Clostridium difficile