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-G(M1), 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-G(M2)). 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.

Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions

“…A glycolipid array with 50 glycolipids non-covalently printed on a polyvinylidene difluoride membrane [119] could determine the presence of autoantibodies in human serum and cerebrospinal fluid. For GSL interactions, an array with covalently immobilized GSLs on Nhydroxysuccinimide-or epoxide-activated glass array slides is available [120]. A mucin 1 (MUC1) Oglycopeptide array could identify glycopeptide epitopes of cancer-specific antibodies [121], and an Nglycan-derived array successfully determined the glycan-binding specificity of anti-HIV antibodies.…”

Recent advances in the mass spectrometric analysis of glycosphingolipidome – A review

“…Enzymes such as endoglycoceramidase (EGcase) [103,130,131], or sphingolipid ceramide N-deacylase (SCDase) [132,133] can also be used to add reactive sites to glycosphingolipids. EGcase cleaves the glycosidic bond between the ceramide and the glycan moiety to form the oligosaccharide with a free reducing end (aldehyde form).…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…Underivatized or linker-derivatized lyso-GSL immobilized on N-hydroxy-succinimide-or epoxideactivated glass slides (Arigi et al, 2012) GM1-related carbohydrates TOF (DHB) Functional interaction analysis of GM1-related carbohydrates and Vibrio cholerae toxins Lactose on carbochips SAMDI…”