Pergamon Bioorganic & MedicinalChemistry, Vol. 4, No. 11, pp 1919-1928, 1996 Copyright © 1996 Elsevier ScienceLtd Printed in Great Britain. All rights reserved PII: S0968-0896(96)00174-5 0968-0896/96 $15.00 +0.00 Unexpected Carbohydrate Cross-binding by Escherichia coli Heat-labile Enterotoxin. Recognition of Human and Rabbit Target Cell Glycoconjugates in Comparison with Cholera Toxin o Karl-Anders Karlsson, a* Susann Teneberg, a Jonas Angstr6m, a Anders Kjellberg, a Timothy R. Hirst, b J6rgen Bergstr6m, a and Halina Miller-Podraza a "Department of Medical Biochemistry, Institute of Medical Biochemistry and Microbiology, GOteborg University, Medicinaregatan 9/t, S-413 90 GOteborg, Sweden hThe Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ, U.K. Abstract--The bacterial protein enterotoxins, cholera toxin (CT) of Vibrio cholerae and heat-labile toxin (LT) of Escherichia coli, induce diarrhea by enhancing the secretory activity of the small intestine of man and rabbit (animal model). This physiological effect is mediated by toxin binding to a glycolipid receptor, the ganglioside GM1, Gal[33GalNAc[34(NeuAcct3)Gal[34Glc[31Cer. However, LT, but not CT, was recently shown by us to bind also to paragloboside, Gal134GlcNAc[33GalIMGlc131Cer, identified in the target cells. By molecular modeling of this tetrasaccharide in the known binding site of LT, the saccharide-peptide inter- action was shown to be limited to the terminal disaccharide (N-acetyllactosamine). This sequence is expressed in many glyco- conjugates, and we have therefore assayed glycolipids and glycoproteins prepared from the target tissues. In addition to paragloboside, receptor activity for LT was detected in glycoproteins of human origin and in polyglycosylceramides of rabbit. However, CT bound only to GM1. Two variants of LT with slightly different sequences, human (hLT) and porcine (pLT), were identical in their binding to target glycoproteins and polyglycosylceramides, but different regarding paragloboside, which was positive for pLT but negative for hLT. This difference is discussed on basis of modeling, taking in view the difference at position 13, with Arg in pLT and His in hLT. Although N-acetyllactosamine is differently recognized in form of paragloboside by the two toxin variants, we speculate that this sequence in human glycoproteins and rabbit polyglycosylceramides is the basis for the common binding. Much work remains, however, to clear up this unexpected sophistication in target recognition. Copyright © 1996 Elsevier Science Ltd Introduction Microbes and microbial toxins causing various diseases of animals and man require attachment sites for the association to specific target cells and to avoid elution by secretions at mucous membranes. Such sites are mainly carbohydrate epitopes, probably due to their abundance at host cell surfaces and their often tissue- specific appearance. Recent advances in assaying and identification of receptor sequences in combination with access to a growing number of crystal structures of microbial receptor-binding proteins, mainly viral hemagglutinins and bacterial toxins, also in complex with saccharide, have allowed interesting conclusions in several cases concerning infection mechanisms (recent review, see ref. 1), of potential interest for therapy. Bacterial toxins are mostly medium-sized secreted proteins accessible for preparation and detailed analysis of binding specificity. Bacteria causing diarrhea are common globally, including Vibrio cholerae secreting cholera toxin (CT), and Escherichia coli secreting the heat-labile toxin (LT). 2 These toxins are composed of one biologically active A subunit of about 27 kDa, and five receptor-binding B subunits of about 12 kDa each. The B subunits show great sequence homology (about 80%) and the two toxins probably induce the diarrhea by identical mechanisms in the epithelium of the small intestine. Both toxins have long been known to bind with high affinity to a glycolipid, the ganglioside GM1, Gal133GalNAc134(NeuAca3)- Gal134Glc131Cer. The epitope is apparently selectively expressed in glycolipid form, since attempts to bind CT to non-glycolipids have been unsuccessful. It has also been shown by synthetic neoglycoconjugates, added to toxin-insensitive cell cultures, that this receptor saccha- ride epitope has to be located close to the membrane to mediate the toxin effect, probably for penetration of the toxin A subunit into the cell. 3 However, it was earlier shown for LT, but not for CT, that the small intestine of the rabbit (animal model) carried additional sites of attachment, assumed to be glyco- protein, resulting in binding of 10 times more toxin than expected from the amount of GM1 present? Recently, we reported the isolation and identification of a glycolipid of rabbit small intestine, which was receptor-active for LT but not for CT, namely paraglo- boside or neolactotetraosylceramide, Gal134GlcNAc- 133Gal134Glc[31Cer? This finding was surprising, since this compound lacked sialic acid, which earlier was shown to be essential for the binding by CT, and also had a different core structure compared to GM1. Modeling of the tetrasaccharide in the binding site of the B subunit of LT of porcine origin (pLT) was highly suggestive of a critical hydrogen bond between the 1919