Recombinant Mucin-Type Fusion Proteins with a Gal alpha 1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

• The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Gal alpha 1,3Gal beta 1,4GlcNAc 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 beta-elimination, and new diagnostic ions to distinguish Gal alpha 1,3Gal- from Gal alpha 1,4Gal-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 Gal alpha 1,3Gal beta 1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Immunologi inom det medicinska området (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Immunology in the medical area (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Klinisk medicin -- Infektionsmedicin (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Clinical Medicine -- Infectious Medicine (hsv//eng)

Nyckelord

hamster ovary cells

carbohydrate interactions

binding domain

b

determinants

high avidity

receptor

enterotoxin

metronidazole

recognition

antibodies

Immunology

Infectious Diseases

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)