Design of a covalently bonded glycosphingolipid microarray

Abstract

Glycosphingolipids (GSLs) are well known ubiquitous constituents of all eukaryotic cell membranes, yet their normal biological functions are not fully understood. As with other glycoconjugates and saccharides, solid phase display on microarrays potentially provides an effective platform for in vitro study of their functional interactions. However, with few exceptions, the most widely used microarray platforms display only the glycan moiety of GSLs, which not only ignores potential modulating effects of the lipid aglycone, but inherently limits the scope of application, excluding, for example, the major classes of plant and fungal GSLs. In this work, a prototype “universal” GSL-based covalent microarray has been designed, and preliminary evaluation of its potential utility in assaying protein-GSL binding interactions investigated. An essential step in development involved the enzymatic release of the fatty acyl moiety of the ceramide aglycone of selected mammalian GSLs with sphingolipid N-deacylase (SCDase). Derivatization of the free amino group of a typical lyso-GSL, lyso-GM1, with a prototype linker assembled from succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester and 2-mercaptoethylamine, was also tested. Underivatized or linker-derivatized lyso-GSL were then immobilized on N-hydroxysuccinimide- or epoxide-activated glass microarray slides and probed with carbohydrate binding proteins of known or partially known specificities (i.e., cholera toxin B-chain; peanut agglutinin, a monoclonal antibody to sulfatide, Sulph 1; and a polyclonal antiserum reactive to asialo-GM2). Preliminary evaluation of the method indicated successful immobilization of the GSLs, and selective binding of test probes. The potential utility of this methodology for designing covalent microarrays that incorporate GSLs for serodiagnosis is discussed.

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Acknowledgements

This work was supported by The Copenhagen Center for Glycomics at the University of Copenhagen, the Carlsberg Foundation, The Benzon Foundation, The Velux Foundation, The Danish Research Councils, NIH/NCI 5U01 CA128437 and 5U01 CA111294, EU FP7-HEALTH-2007-A 201381, and the University of Copenhagen Programme of Excellence. Early phases of the work were supported by an NIH grant (R21 RR20355) to S.B.L.

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Correspondence to Steven B. Levery.

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Arigi, E., Blixt, O., Buschard, K. et al. Design of a covalently bonded glycosphingolipid microarray. Glycoconj J 29, 1–12 (2012). https://doi.org/10.1007/s10719-011-9359-9

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Keywords

  • Glycosphingolipid
  • Glycolipid
  • Ganglioside
  • Glycan
  • Lectin
  • Antibody
  • Microarray
  • Glycan array