Analysis of the Neutral Glycan Fractions of Glycosyl-phosphatidylinositols by Thin-Layer Chromatography

doi:10.1006/abio.1993.1158

Analytical Biochemistry

Volume 210, Issue 1, April 1993, Pages 106-112

https://doi.org/10.1006/abio.1993.1158 Get rights and content

Abstract

The radiolabeled neutral glycan fractions of both glycosyl-phosphatidylinositol (GPI) protein anchors and related glycolipids were analyzed by thin-layer chromatography on silica gel 60, using butanol:ethanol:water (4:3:3, v/v) or a combination of 1-propanol:acetone:water (9:6:5, v/v and 5:4:1, v/v) as solvents. Dextran acid hydrolysates were used as standards, and oligomers up to 11 glucose units could be resolved. A comparison of 18 GPI-glycan standards revealed that their migration was dependent mainly on the size of the oligosaccharide. Isomers were generally not resolved, with the exception of Manα1-6Manα1-4AHM and Manα1-3Manα1-4AHM. Structures containing galactofuranose or GalNAc were well resolved from structures containing only Galp and/or Manp. The utility of this method for the microsequencing of radiolabeled neutral glycans derived from two GPI glycolipids, using exoglycosidases and chemical treatments, is demonstrated. This method is a simple and useful complement to the existing chromatographic techniques.

References (0)

Cited by (30)

Controlled expression of branch-forming mannosyltransferase is critical for mycobacterial lipoarabinomannan biosynthesis
2010, Journal of Biological Chemistry
Citation Excerpt :
PIMs were purified by 1-butanol/water (2:1, v/v) partitioning and analyzed by HPTLC. Acetolysis was performed following a published protocol (28). Monosaccharides were released by 2 m trifluoroacetic acid at 100 °C for 2 h. Released carbohydrates were analyzed by a Nanospace high performance liquid chromatography system (Shiseido, Tokyo, Japan) equipped with Screbead II (2.0 × 250 mm) (Shiseido) as an anion exchange column, 50 mm sodium acetate in 0.1 m NaOH as a mobile phase, and a pulsed amperometric detector.
Lipomannan (LM) and lipoarabinomannan (LAM) are phosphatidylinositol-anchored glycans present in the mycobacterial cell wall. In Mycobacterium smegmatis, the mannan core of LM/LAM constitutes a linear chain of 20–25 α1,6-mannoses elaborated by 8–9 α1,2-monomannose side branches. At least two α1,6-mannosyltransferases mediate the linear mannose chain elongation, and one branching α1,2-mannosyltransferase (encoded by MSMEG_4247) transfers monomannose branches. An MSMEG_4247 deletion mutant accumulates branchless LAM and interestingly fails to accumulate LM, suggesting an unexpected role of mannose branching for LM synthesis or maintenance. To understand the roles of MSMEG_4247-mediated branching more clearly, we analyzed the MSMEG_4247 deletion mutant in detail. Our study showed that the deletion mutant restored the synthesis of wild-type LM and LAM upon the expression of MSMEG_4247 at wild-type levels. In striking contrast, overexpression of MSMEG_4247 resulted in the accumulation of dwarfed LM/LAM, although monomannose branching was restored. The dwarfed LAM carried a mannan chain less than half the length of wild-type LAM and was elaborated by an arabinan that was about 4 times smaller. Induced overexpression of an elongating α1,6-mannosyltransferase competed with the overexpressed branching enzyme, alleviating the dwarfing effect of the branching enzyme. In wild-type cells, LM and LAM decreased in quantity in the stationary phase, and the expression levels of branching and elongating mannosyltransferases were reduced in concert, presumably to avoid producing abnormal LM/LAM. These data suggest that the coordinated expressions of branching and elongating mannosyltransferases are critical for mannan backbone elongation.
Characterization of a Low Molecular Weight Glycolipid Antigen from Cryptosporidium parvum
2003, Journal of Biological Chemistry
Citation Excerpt :
Butanol-extracted glycolipids were further purified by octyl-Sepharose (OS) chromatography (1 cm inner diameter × 10 cm length) as described by McConville et al. (31). The fractions having the strongest orcinol/H2SO4 reaction (1 μl of each 1-ml fraction was spotted on a silica gel HPTLC plate (EM Science, Gibbstown, NJ)) (32) were pooled (fractions 86–92), dried under vacuum, and dissolved in 40% 1-propanol. An aliquot of OS-purified glycolipid was further fractionated by preparative silica gel HPTLC using a solvent system of 10:10:3 (v/v/v) chloroform, methanol, 1.0 m NH4OH (31).
Cryptosporidium parvum, an Apicomplexan parasite of the mammalian gut epithelium, causes a diarrheal illness in a wide range of hosts and is transmitted by contamination of food or water with oocyst-laden feces from an infected animal. We have identified a glycosylinositol phospholipid from the sporozoite stage of the parasite that is frequently recognized by serum antibodies from human cryptosporidiosis patients. The humoral immune response is dominated by IgG₁ subclass antibodies but can also include IgA and IgM antibodies. The glycosylinositol phospholipids were purified by butanol extraction of a Triton X-114-soluble fraction followed by octyl-Sepharose column chromatography and preparative high performance TLC and were shown to include at least 5 species. By using mass spectrometry and radiolabeled neutral glycan analysis, we found that the structure of the dominant glycosylinositol phospholipid antigen contained a C18:0 lyso-acylglycerol, a C16:0-acylated inositol, and an unsubstituted mannose₃-glucosamine glycan core. Other diacyl species were also identified, most notably a series of glycosylinositol phospholipids having an acyl-linked C20:0 to C28:0 lipid on the inositol ring. Less abundant species having three acyl-linked fatty acids and species with an additional 1–3 hexoses linked to the mannose core were also observed. We are currently working to determine the role that these glycolipids may play in the development of disease and in the clearance of infection.
Biosynthesis of Entamoeba histolytica proteophosphoglycan in vitro
2003, Molecular and Biochemical Parasitology
A complex glycoconjugate proteophosphoglycan (PPG) is present on the surface of the pathogenic protozoan parasite Entamoeba histolytica but not in the non-pathogenic Entamoeba dispar. It is thought to be an important molecule involved in pathogenesis. In order to study its biosynthesis, an in vitro cell-free system was developed. The specificity of the system was demonstrated by various criteria including immunoprecipitation by a specific monoclonal antibody. The in vitro synthesized molecule was found to be susceptible to mild acid hydrolysis, digestion by phosphoinositol-specific phospholipase C and nitrous acid deamination, the salient features for a PPG-like molecule. The in vitro product was not synthesized when heat-treated cellular-extract was used in the assay or when the cell extract was prepared from Entamoeba invadens, a species that lacks these glycoconjugates. Analysis of the glycan side chains of the in vitro synthesized product by thin layer chromatography revealed side chains of variable sizes including a fraction greater than six glycan units. The crude membranes used in the cell-free system were further fractionated by sucrose density gradient centrifugation. The fraction containing the PPG synthesizing activity when used in the assay resulted in a 10-fold increase in specific activity. Development of this cell-free system will facilitate further studies on the nature of intracellular organelles and the pathways that are involved in PPG biosynthesis.
Glycosyl phosphatidylinositol myristoylation in African trypanosomes. New intermediates in the pathway for fatty acid remodeling
2000, Journal of Biological Chemistry
Citation Excerpt :
After each treatment, lipids were extracted by n-butanol-water partition. The treatments were modifications of published procedures (14, 15). Glycolipid θ′, generated in the absence of Mn2+, was purified by TLC, eluted with CHCl3/CH3OH/H2O (10:10:3), dried, and resuspended in 25 μl of ice-cold 48% (v/v) aqueous HF for dephosphorylation.
Glycosyl phosphatidylinositol (GPI) anchors in the bloodstream form of Trypanosoma brucei are unusual in that their two fatty acids are myristate. The myristates are added in the final stages of GPI biosynthesis in a remodeling reaction. Remodeling occurs first at the sn-2 position of glycerol, involving removal of a longer fatty acid and subsequent attachment of myristate. The second myristate is then incorporated into thesn-1 position, but the mechanism has been unclear due to the unavailability of a reliable cell-free system supporting complete remodeling. Here, we first refined the cell-free system (by removing Mn²⁺ ions), thereby allowing efficient production of the dimyristoylated GPI precursor. Using this improved system, we made three new discoveries concerning the pathway for fatty acid remodeling. First, we discovered a monomyristoylated GPI (known as glycolipid θ′) as an intermediate involved in remodeling at the sn-1 position. Second, we found an alternative pathway for production of glycolipid θ, the first lyso intermediate in remodeling. The alternative pathway involves an inositol-acylated GPI known as glycolipid lyso-C′. Finally, we found that there is significant breakdown of GPIs during remodeling in the cell-free system, and we speculate that this breakdown has a regulatory role in GPI biosynthesis.
The major surface antigens of Entamoeba histolytica trophozoites are GPI-anchored proteophosphoglycans
2000, Journal of Molecular Biology
Trophozoites of the parasitic protozoa, Entamoeba histolytica, synthesize a cell surface lipoglycoconjugate, termed lipophosphoglycan, which is thought to be an important virulence factor and potential vaccine candidate against invasive amebiasis. Here, we show that the E. histolytica lipophosphoglycans are in fact glycosylphosphatidylinositol (GPI)-anchored proteophosphoglycans (PPGs). These PPGs contain a highly acidic polypeptide component which is rich in Asp, Glu and phosphoserine residues. This polypeptide component is extensively modified with linear glycan chains having the general structure, [Glcα1–6]_nGlcβ1-6Gal (where n=2–23). These glycan chains can be released after mild-acid hydrolysis with trifluoroacetic or hydrofluoric acid and are probably attached to phosphoserine residues in the polypeptide backbone. The PPGs are further modified with a GPI anchor which differs from all other eukaryotic GPI anchors so far characterized in containing a glycan core with the structure, Gal₁Man₂GlcN-myo-inositol, and in being heterogeneously modified with chains of α-galactose. Trophozoites of the pathogenic HM-1:IMSS strain synthesize two distinct classes of PPG which have polydisperse molecular masses of 50–180 kDa (PPG-1) and 35–60 kDa (PPG-2) and are modified with glucan side-chains of different average lengths. In contrast, the non-pathogenic Rahman strain synthesizes one class of PPG which is only elaborated with short disaccharide side-chains (i.e. Glcβ1-6Gal). However, the PPGs are abundant in all strains (8×10⁷ copies per cell) and are likely to form a protective surface coat.
Erythropoietin induces glycosylphosphatidylinositol hydrolysis. Possible involvement of phospholipase C-γ<inf>2</inf>
1999, Journal of Biological Chemistry
We showed that erythropoietin induced rapid glycosylphosphatidylinositol (GPI) hydrolysis and tyrosine phosphorylation of phospholipase C (PLC)-γ₂ in FDC-P1 cells transfected with the wild-type erythropoietin-receptor. Erythropoietin-induced tyrosine phosphorylation of PLC-γ₂was time- and dose-dependent. By using FDC-P1 cells transfected with an erythropoietin receptor devoid of tyrosine residues, we showed that both effects required the tyrosine residues of intracellular domain on the erythropoietin receptor. Erythropoietin-activated PLC-γ₂ hydrolyzed purified [³H]GPI indicating that GPI hydrolysis and PLC-γ₂ activation under erythropoietin stimulation were correlated. Results obtained on FDC-P1 cells transfected with erythropoietin receptor mutated on tyrosine residues suggest that tyrosines 343, 401, 464, and/or 479 are involved in erythropoietin-induced GPI hydrolysis and tyrosine phosphorylation of PLC-γ₂, whereas tyrosines 429 and/or 431 seem to be involved in an inhibition of both effects. Thus, our results suggest that erythropoietin regulates GPI hydrolysis via tyrosine phosphorylation of its receptor and PLC-γ₂activation.

View all citing articles on Scopus

View full text

Regular ArticleAnalysis of the Neutral Glycan Fractions of Glycosyl-phosphatidylinositols by Thin-Layer Chromatography

Abstract

Regular Article
Analysis of the Neutral Glycan Fractions of Glycosyl-phosphatidylinositols by Thin-Layer Chromatography