Ganglioside GD1a Is an Essential Coreceptor for Toll-like Receptor 2 Signaling in Response to the B subunit of Type IIb Enterotoxin

doi:10.1074/jbc.M611722200

Ganglioside GD1a Is an Essential Coreceptor for Toll-like Receptor 2 Signaling in Response to the B subunit of Type IIb Enterotoxin^*

^{^‡}Center for Oral Health and Systemic Disease and Departments of Periodontics and Microbiology/Immunology, University of Louisville Health Sciences Center, Louisville, Kentucky 40292, ^{^§}Department of Microbiology, University of Illinois, Urbana, Illinois 61801, ^{^¶}Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, Buffalo, New York 14214, and ^{^∥}Infection and Immunity Group, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom

1 To whom correspondence should be addressed: University of Louisville Health Sciences Center, 501 South Preston St., Rm. 206, Louisville, KY 40292. Tel.: 502-852-5276; Fax: 502-852-4052; E-mail: g0haji01{at}louisville.edu.

Abstract

Innate recognition and signaling by Toll-like receptors (TLRs) is facilitated by functionally associated coreceptors, although the cooperativity mechanisms involved are poorly understood. As a model we investigated TLR2 interactions with the GD1a ganglioside binding subunit of type IIb Escherichia coli enterotoxin (LT-IIb-B₅). Both LT-IIb-B₅ and a GD1a binding-defective mutant (LT-IIb-B₅(T13I)) could modestly bind to TLR2, but only the wild-type molecule displayed a dramatic increase in TLR2 binding activity in the presence of GD1a (although not in the presence of irrelevant gangliosides). Moreover, fluorescence resonance energy transfer experiments indicated that LT-IIb-B₅ induces lipid raft recruitment of TLR2 and TLR1 and their clustering with GD1a, in contrast to the GD1a binding-defective mutant, which moreover fails to activate TLR2 signaling. LT-IIb-B₅-induced cell activation was critically dependent upon the Toll/IL-1 receptor domain-containing adaptor protein, which was induced to colocalize with TLR2 and GD1a, as shown by confocal imaging. Therefore, GD1a provides TLR2 coreceptor function by enabling the ligand to recruit, bind, and activate TLR2. These findings establish a model of TLR2 coreceptor function and, moreover, suggest novel mechanisms of adjuvanticity by non-toxic derivatives of type II enterotoxins dependent upon GD1a/TLR2 cooperative activity.

Footnotes

↵² The abbreviations used are: TLR, toll-like receptor; LPS, lipopolysaccharide; LT-IIb-B₅, pentameric B subunit of type IIb E. coli enterotoxin; IL, interleukin; ODN, oligodeoxynucleotide; TIRAP, Toll/IL-1 receptor domain-containing adaptor protein; TIRAP-BP, TIRAP-blocking peptide; mAb, monoclonal antibody; CHO, Chinese hamster ovary; HEK, human embryonic kidney; PPMP, d,l-threo-l-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol·HCl; MCD, methyl-β-cyclodextrin; FRET, fluorescence resonance energy transfer; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; FITC, fluorescein isothiocyanate; MHC, major histocompatibility complex; GD1a, Neu5Acα3Galβ3GalNAcβ4(Neu5Acα3)Galβ4Glc-ceramide; GD1b, Galβ3GalNAcβ4(Neu5Acα8Neu5Acα3)Galβ4Glc-ceramide; GM1, Galβ3GalNAcβ4(Neu5Acα3)Galβ4Glc-ceramide.
↵^* This work was supported by United States Public Health Service, National Institutes of Health Grants AI052344 (to R. I. T.), DE13833 (to T. D. C.), and DE015254 and DE017138 (to G. H.) and by the Wellcome Trust and the Heart Research Fund, United Kingdom (to K. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.
- Received December 21, 2006.
The American Society for Biochemistry and Molecular Biology, Inc.