Characterization of rabbit heal receptors for clostridium difficile toxin A: Evidence for a receptor-coupled G protein

Charalabos Pothoulakis, J. Thomas LaMont, Richard Eglow, Ning Gao, Jeffrey B. Rubins, Theoharis C. Theoharides, Burton F. Dickey

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66 Scopus citations

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 μCi/μg, 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 × 10-8 and 3.5 × 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-γS with rabbit ileal BB, and preincubation of BB with the GTP analogues GTPγS 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.

Original languageEnglish (US)
Pages (from-to)119-125
Number of pages7
JournalJournal of Clinical Investigation
Volume88
Issue number1
DOIs
StatePublished - 1991

Keywords

  • Clostridium difficile toxins
  • Enterotoxin
  • G protein
  • Membrane glycoprotein
  • Toxin receptor

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