title: Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein.
abstract: The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.
authors.affiliation: Section of Gastroenterology, Boston University School of Medicine, Massachusetts.
authors.forename: C
authors.initials: C
authors.lastname: Pothoulakis
authors.forename: J T
authors.initials: JT
authors.lastname: LaMont
authors.forename: R
authors.initials: R
authors.lastname: Eglow
authors.forename: N
authors.initials: N
authors.lastname: Gao
authors.forename: J B
authors.initials: JB
authors.lastname: Rubins
authors.forename: T C
authors.initials: TC
authors.lastname: Theoharides
authors.forename: B F
authors.initials: BF
authors.lastname: Dickey
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism* / Q000494:pharmacology; D016360:Clostridioides difficile; D004768:Enterotoxins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007082:Ileum / Q000378:metabolism; D037102:Lectins / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D011817:Rabbits; D011869:Radioligand Assay; D051381:Rats; D014316:Tritium; D014357:Trypsin / Q000494:pharmacology
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D037102:Lectins; D008562:Membrane Glycoproteins; C041348:tcdA protein, Clostridium difficile; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014357:Trypsin; D019204:GTP-Binding Proteins