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Glycobiology Advance Access originally published online on August 8, 2007
Glycobiology 2007 17(10):1127-1137; doi:10.1093/glycob/cwm081
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© The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Affinities of Shiga toxins 1 and 2 for univalent and oligovalent Pk-trisaccharide analogs measured by electrospray ionization mass spectrometry

Elena N Kitova2, Pavel I Kitov2, Eugenia Paszkiewicz2, Jonghwa Kim2, George L Mulvey3, Glen D Armstrong3, David R Bundle2 and John S Klassen1,2

2 Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2 G2, Canada
3 Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Alberta T2 N 4N1, Canada

1 To whom correspondence should be addressed: Tel: +1 780 492 3501; Fax: +1 780 492 8231; e-mail: john.klassen{at}ualburta.ca.

Received on June 15, 2007; revised on July 20, 2007; accepted on July 22, 2007

The binding stoichiometry and affinities of the Shiga toxins, Stx1 and Stx2, for a series of uni- and oligovalent analogs of the Pk-trisaccharide were measured using the direct electrospray ionization mass spectrometry (ES-MS) assay. Importantly, it is shown that, for a given ligand, Stx1 and Stx2 exhibit similar affinities. The binding data suggest a high degree of similarity in the spatial arrangement and structural characteristics of the Pk binding sites in Stx1 and Stx2. The results confirm that both toxins recognize the {alpha}-D-Galp(1->4)-ß-D-Galp(1->4)-ß-D-Glcp carbohydrate motif of the cell surface glycolipid Gb3. This, taken together with the results of the chemical mapping study, suggests that the nature of the Pk binding interactions with Stx1 and Stx2 are similar. The affinities of Stx1-B5 and Stx2 for the multivalent ligands reveals that site 2 of Stx2, which shares the same spatial arrangement as site 2 in Stx1, is the primary Pk binding site and that site 1 of Stx1 and of Stx2 can also participate in Pk binding.

Key words: association constants / electrospray ionization mass spectrometry / protein-oligosaccharide complexes / Shiga toxins / stoichiometry


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