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Analysis of Glycosphingolipid Glycans by Lectin Microarrays.

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates on cell membrane. Identification of unknown GSL-glycan structures is still a major challenge. To address this challenge, we developed a novel strategy for analysis of GSL-glycans from cultured cells based on a lectin microarray that could directly detect and reveal glycopatterns of GSL extracts without the need for glycan release. There were six steps to perform analysis of GSL-glycans: (i) extraction of GSLs from cell pellets, (ii) quantification of GSL-glycans using orcinol-sulphuric acid reaction, (iii) lyso-GSLs were prepared by using the sphingolipid ceramide N-deacylase, (iv) fluorescence labeling of lyso-GSLs, (v) detection by a lectin microarray, (vi) data acquisition and analysis. Simultaneously, a supplementary verification analysis for GSL-glycans was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. An optimized experimental condition, which consist of the blocking buffer, incubation buffer, and appropriate GSL concentration, was investigated by analyzing glycopatterns of a standard ganglioside (GM1a) via a lectin microarray. The results of GSL-glycans from human hepatocarcinoma cell lines (MHCC97L, MHCC97H, and HCCLM3) showed there were 27 lectins (e.g. WFA, MAL-II, and LTL) to give significantly differential signal compared with normal human liver cell line (HL-7702), indicating up and/or down regulations of corresponding glycopatterns such as α1-2 fucosylation and α2-3 sialylation, and changes of certain glycostructures such as Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glc:Cer and GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ1-4Glc:Cer. The lectin microarray analysis of lyso-GSLs labeled by fluorescence has proven to be credible, which could provide the glycopatterns and detailed linkage information of GSL-glycans.