Analysis of Glycosphingolipid Glycans by Lectin Microarrays

Abstract

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates of cell membranes. 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 can directly detect and reveal glycopatterns of GSL extracts without the need for glycan release. There were six steps to perform the analysis of GSL−glycans: (i) extraction of GSLs from cell pellets, (ii) quantification of GSL−glycans using orcinol−sulfuric acid reaction, (iii) preparation of lyso-GSLs by using 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. Optimized experimental conditions, which consisted of the blocking buffer, incubation buffer, and appropriate GSL concentration, were investigated by analyzing the glycopatterns of a standard ganglioside (GM1a) via lectin microarray. The analysis of GSL−glycans from human hepatocarcinoma cell lines (MHCC97L, MHCC97H, and HCCLM3) showed that there were 27 lectins (e.g., WFA, MAL-II, and LTL) to give significantly different signals compared with a 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 can provide the glycopatterns and detailed linkage information on GSL−glycans.

Biological Functions and Analytical Strategies of Sialic Acids in Tumor

“…Lectins are sugar-binding proteins that can specifically recognize glycans on glycoconjugates. Sambucus nigra lectin (SNA) and Maackia amurensis lectin (MAL) are commonly used to Cells 2020, 9, 273 7 of 18 recognize the α2,6-linked and α2,3-linked sialic acid residues, respectively [116][117][118], in lectin blot [119], lectin microarray [120][121][122], histochemistry [123], fluorescent image and flow cytometry [56,124]. Using lectin MAL-1, α2,3-linked sialic acids were found to promote gastric cancer cell metastasis [125].…”

The Structure of Leukocyte Sialic Acid-Containing Membrane Glycoconjugates is a Differential Indicator of the Development of Diabetic Complications

“…The Maackia amurensis lectin (MAA) and Sambucus nigra lectin (SNA) are commonly used to recognize the α2,3-linked (Neu5Acα2,3Gal) and α2,6-linked (Neu5Acα2,6Gal/GalNAc) sialic acid residues, respectively [20,68,69]. Sialospecific lectins apply in lectin microarray [70], histochemistry [71], in lectin blot [72,73], fluorescent image and flow cytometry [74] (Figure 1). At the same time, the combination of lectins with monoclonal antibodies can be used to obtain complete information on the antigenic repertoire of cells both in normal and in case of pathologies [73].…”

“…It is very important to understand the morphofunctional state of the cell is to be able to detect these differences. Numerous methods are used for this purpose [70]. Aggregatometry is one of the assays used to evaluate the functional properties of platelets, leukocytes and erythrocytes in the dynamics, monitor antiplatelet therapy, study the mechanisms of aggregation.…”

Hierarchical Fluorescence Imaging Strategy for Assessment of the Sialylation Level of Lipid Rafts on the Cell Membrane

“…However, there is lack of direct visualization tools for glycosylation in a raft-specific manner. Current analytical methods for raft-residing glycans are basically destructive ones, which depend on mass spectrometry , or lectin microarray based techniques.…”