Glycobiology Advance Access originally published online on September 12, 2009
Glycobiology 2010 20(1):41-54; doi:10.1093/glycob/cwp141
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Mammalian cell ganglioside-binding specificities of E. coli enterotoxins LT-IIb and variant LT-IIb(T13I)
3 Infectious Disease Division, Department of Veterans Affairs Western New York Healthcare System, State University of New York at Buffalo School of Medicine, Buffalo, NY 14215
4 The Witebsky Center for Microbial Pathogenesis and Immunology, The Department of Microbiology and Immunology, The University at Buffalo, SUNY, 3435 Main St, Buffalo, NY 14214
5 Research Service, VA Medical and Regional Office Center, White River Junction, VT 05009-0001
6 Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755
7 Department of Chemistry, University of New Hampshire, Durham, NH 03824
8 Department of Periodontics/Oral Health and Systemic Disease and Department of Microbiology and Immunology, University of Louisville, Schools of Medicine and Dentistry, Louisville, KY 40292, USA
1 To whom correspondence should be addressed: Tel: +1-716-862-6529; Fax: +1-716-862-6526; e-mail: berenson{at}buffalo.edu
Received on July 7, 2009; revised on August 19, 2009; accepted on September 4, 2009
LT-IIb, a type II heat-labile enterotoxin of Escherichia coli, is a potent immunologic adjuvant with high affinity binding for ganglioside GD1a. Earlier study suggested that LT-IIb bound preferentially to the terminal sugar sequence NeuAc
2-3Galβ1-3GalNAc. However, studies in our laboratory suggested a less restrictive binding epitope. LT-IIb(T13I), an LT-IIb variant, engineered by a single isoleucine-threonine substitution, retains biological activity, but with less robust inflammatory effects. We theorized that LT-IIb has a less restrictive binding epitope than previously proposed and that immunologic differences between LT-IIb and LT-IIb (T13I) correlate with subtle ganglioside binding differences. Ganglioside binding epitopes, determined by affinity overlay immunoblotting and enzymatic degradation of ganglioside components of RAW264.7 macrophages, indicated that LT-IIb bound to a broader array of gangliosides than previously recognized. Each possessed NeuAc2-3Galβ1-3GalNAc, although not necessarily as a terminal sequence. Rather, each had a requisite terminal or penultimate single sialic acid and binding was independent of ceramide composition. RAW264.7 enterotoxin-binding and non-binding ganglioside epitopes were definitively identified as GD1a and GM1a, respectively, by enzymatic degradation and mass spectroscopy. Affinity overlay immunoblots, constructed to the diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIb bound NeuAc- and NeuGc-gangliosides with nearly equal affinity. However, LT-IIb(T13I) exhibited enhanced affinity for NeuGc-gangliosides and more restrictive binding. These studies further elucidate the binding epitope for LT-IIb and suggest that the diminished inflammatory activity of LT-IIb(T13I) is mediated by a subtle shift in ganglioside binding. These studies underscore the high degree of specificity required for ganglioside–protein interactions.
Key words: enterotoxins / ganglioside / glycosphingolipid / macrophage / sialic acid
2 These authors contributed equally to this work.