Analysis of Glycosphingolipid Glycans by Lectin Microarrays
Haoqi DuHanjie YuTianran MaFuquan YangLiyuan JiaChen ZhangJiaxu ZhangLili NiuJiajun YangZhiwei ZhangKun ZhangZheng Li
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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.Keywords:
Glycoconjugate
Glycosphingolipid
Fucosylation
Glycosphingolipid
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Abstract Glycolipid analysis of the Caribbean sponge Amphimedon compressa has shown it to contain two novel glycosphingolipids, amphiceramide A ( 1a ) and B ( 2a ), which possess an unusual Δ 6 ‐phytosphingosine. The saccharide chain of amphiceramide A is composed of a β‐glucose residue glycosylated at the 6‐position by an N ‐acetyl‐β‐glucosamine and has never been found before in a natural product. The saccharide chain of amphiceramide B consists of an allolactose [Gal(1β→6)Glc] residue β‐linked to the ceramide and is found here for the first time in a natural glycosphingolipid. In addition, the sponge contains a new molecular species, acetamidoglucosyl ceramide ( 3a ), and the known glucosyl ceramide 4a (halicerebroside A) and melibiosyl ceramide 5a (amphimelibioside C). (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)
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Fucosylation
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Fucosylation is one of the most common modifications involving oligosaccharides on glycoproteins or glycolipids. Fucosylation comprises the attachment of a fucose residue to N-glycans, O-glycans and glycolipids. O-Fucosylation, which is a special type of fucosylation, is very important for Notch signalling. The regulatory mechanisms for fucosylation are complicated. Many kinds of fucosyltransferases, the GDP-fucose synthesis pathway and GDP-fucose transporter are involved in the regulation of fucosylation. Increased levels of fucosylation have been reported in a number of pathological conditions, including inflammation and cancer. Therefore, certain types of fucosylated glycoproteins such as AFP-L3 or several kinds of antibodies, which recognize fucosylated oligosaccharides such as sialyl Lewis a/x, have been used as tumour markers. Furthermore, fucosylation of glycoproteins regulates the biological functions of adhesion molecules and growth factor receptors. Changes in fucosylation could provide a novel strategy for cancer therapy. In this review, the biological significance of and regulatory pathway for fucosylation have been described.
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Glycoconjugate
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Leukocyte adhesion deficiency/congenital disorder of glycosylation IIc (LAD II/CDG IIc) is a genetic disease characterized by a decreased expression of fucose in glycoconjugates, resulting in leukocyte adhesion deficiency and severe morphological and neurological abnormalities. The biochemical defect is a reduced transport of guanosine diphosphate-L-fucose (GDP-L-fucose) from cytosol into the Golgi compartment, which reduces its availability as substrate for fucosyltransferases. The aim of this study was to determine the effects of a limited supply of GDP-L-fucose inside the Golgi on core fucosylation (a1,6-fucose linked to core N-acetylglucosamine [GlcNAc]) of N-linked glycans in LAD II fibroblasts. The results showed that, although [3H]fucose incorporation was generally reduced in LAD II cells, core fucosylation was affected to a greater extent compared with other types of fucosylation of N-linked oligosaccharides. In particular, core fucosylation was found to be nearly absent in biantennary negatively charged oligosaccharides, whereas other types of structures, in particular triantennary neutral species, were less affected by the reduction. Expression and activity of a1,6-fucosyltransferase (FUT8) in control and LAD II fibroblasts were comparable, thus excluding the possibility of a decreased activity of the transferase. The data obtained confirm that the concentration of GDP-L-fucose inside the Golgi can differentially affect the various types of fucosylation in vivo and also indicate that core fucosylation is not dependent only on the availability of GDP-L-fucose, but it is significantly influenced by the type of oligosaccharide structure. The relevant reduction in core fucosylation observed in some species of oligosaccharides could also provide clues for the identification of glycans involved in the severe developmental abnormalities observed in LAD II.
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Previous studies have suggested that glycosphingolipids may be involved in a number of physiological functions of the small intestinal mucosa. Regional variations in many of these processes exist along the length of this organ. In the present studies, the glycosphingolipid and ceramide composition of the proximal, middle and distal thirds of the rat small intestine were characterized and compared. Mono- and trihexosylceramide were the major neutral glycolipids and hematoside (GM3), the principal ganglioside of this organ. Monohexosylceramide was the major glycolipid of the proximal segment, whereas trihexosylceramide predominated in the distal segments. The total content of neutral glycolipids, ceramide and gangliosides as well as the content of the individual glycosphingolipids and ceramide were highest in the distal segment, intermediate in the middle and lowest in the proximal segment. Additionally, regional variations were noted in the fatty acid composition of the major glycosphingolipids. These differences in the composition of glycolipids and ceramide along the length of the intestine may be responsible, at least partially, for the regional functional specialization seen in this organ.
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Sialic acid is a negatively charged monosaccharide attached to non-reducing end of N- and O-linked carbohydrate chains of glycoconjugates. The claimed biological functions of sialic acid include its participation in cell to cell recognition and interaction as well as affecting the function of receptors by providing binding sites for ligand. Increased sialic acid concentration have been observed in several diseases e.g. malignancies, diabetes, inflammatory disorders, rheumatoid arthritis and alcoholism.The aim of the present work was to determine if the amount of sialic acid attached to glycoconjugates of amniotic fluid changes during pregnancy.The sialic acid content in 47 samples of amniotic fluid derived from pregnant women with gestational age between 13 and 42 was studied by sialic acid specific lectins immunosorbent assay. The patient samples were divided into seven groups.Time dependent changes in the degree of sialylation of glycoconjugates in amniotic fluid during pregnancy, particularly in advanced pregnancy were observed. Moreover, the highest sialic acid content on glycoconjugates in pregnancies complicated by premature rupture of membranes and is prolonged pregnancy were also detected.Sialic acid content determination in amniotic fluid could be a potentially useful marker of inflammation process of amniochorion during pregnancy.
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