glycosphingolipid lecture

Dr. Subroto Chatterjee

GLYCOSPHINGOLIPID LECTURE

The most common characteristic component of glycosphingolipids is the aliphatic amino alcohol discovered by Thudichum.Thudichum named it Sphingosine after the enigmatic Sphinx from Egypt having a head of Pharaoh and body of a lion.

WHAT ARE

GLYCOSPHINGOLIPIDS?

STRUCTURE OF LACTOSYLCERAMIDE

STRUCTURE OF GLYCOSPHINGOLIPIDS

LYSOSOMAL STORAGE DISORDERSSeveral metabolic basis of inherited

diseases in man occur due to the lack/deficiency of enzymes which catabolize glycosphingolipids

LYSOSOMAL STORAGE DISORDERS

Lysosomal Storage Disorders PrevalenceAdrenoleukodystrophy (ADL) Approx. 1 in 20,000 or 13,600 people

in USANiemann Pick (Type A,B, and C) Type A & B: Approx. 1 in 250,000

Type C: Approx. 1 in 150,000

Gaucher

Approx. 1 in 200 for general populationHigh as 1 in 10 in Jewish people with East. European ancestry

Krabbe Approx. 1 in 100,000 peopleMetachromatic leukodystrophy (MLD)

Approx. 1 in 625,000 people

Tay-Sachs Approx. 1 in 27 Jewish people in USAApprox. 1 in 250 for general population

Fabry

Approx. 1 in 40,000 malesApprox. 1 in 117,000 people for general population

PREVALENCE OF GLYCOSPHINGOLIPIDS DISORDER

FunctionMethods for Determining FunctionExtraction and PurificationQuantitationStructural DeterminationLocalization ImagingMetabolism

GLYCOSPHINGOLIPID LECTURE TOPICS

1) Superoxide generation and CAM expression2) Inhibition of nitric oxide production in endothelial

cells3) As mediators of growth factors contributing to cell

proliferation4) As receptors for toxins and bacteria

FUNCTION OF GLYCOSPHINGOLIPIDS

1) Generation of superoxide in arterial smooth muscle cells and expression of cell adhesion molecules.

FUNCTION OF GLYCOSPHINGOLIPIDS IN ATHEROSCLEROSIS AND

VASCULAR BIOLOGY

LACTOSYLCERAMIDE MEDIATES TNF Α-INDUCED ICAM-1 EXPRESSION IN

ENDOTHELIAL CELLS

LACTOSYLCERAMIDE STIMULATES SUPEROXIDE GENERATION IN HUMAN

ENDOTHELIAL CELLS

LACTOSYLCERAMIDE STIMULATES SUPEROXIDE GENERATION IN HUMAN ENDOTHELIAL CELLS

2) Inhibition of nitric oxide production in endothelial cells.


VASCULAR BIOLOGY

EFFECT OF LACCER ON ENDOTHELIUM DEPENDENT VASO-RELAXATION AND

PORCINE CORONARY ARTERY

EFFECTS OF LacCer ON eNOS mRNA LEVELS IN HCAECs.

3. As mediators of growth factors contributing to cell proliferation and angiogenesis


VASCULAR BIOLOGY

5) Serve as receptors to various toxins, e.g. cholera toxin and other bacteria

FUNCTION OF GLYCOSPHINGOLIPIDS

METHODS FOR DETERMINING GSL

FUNCTIONCellular assays

ProliferationAdhesionAngiogenesisMigrationApoptosis

METHODS FOR DETERMINING GSL

FUNCTION

1) Bligh and Dyer2) Folch Partitioning

GSL EXTRACTION

EXTRACTION OF GLYCOSPHINGOLIPIDS FROM HEART TISSUE

Analysis of Glycosphingolipids Subroto ChatterjeeOctober 7, 2013

• Modified Bligh & Dyer Methodo extraction in

chloroform:methanol 2:1

o homogenized by hand

o lipids extracted from organic phase

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Folch Partitioning

Alkaline methanolysis The Glycolipid fraction from the silicic acid column is

treated with mild base to remove contaminating phospholipids. This treatment does not affect glycolipids or gangliosides unless they contain an O-acyl group. The following quantities are used for 1-10mg of glycolipid fraction. Add 1ml of chloroform and 1ml of 0.6 N NaOH in methanol to the dry fraction and allow the mixture to react at room temperature for 1 hour. Then add 1.2 ml of 0.5 N HCL in methanol, 1.7ml of water, and 3.4ml of chloroform, mix well, centrifuge, and remove the lower layer containing the glycolipids. Was the lower layer three times with methanol:water (1:1) and then evaporate it to dryness in vacuo.

PURIFICATION GLYCOSPHINGOLIPIDS

A) Mixture of compounds in lane C through F

B) Monohexosyl ceramide, gal- and glc-ceramide

C) Dihexosyl ceramide, gal(1->4) glc-ceramide and gal(1->4)gal-ceramide

D) Trihexosyl ceramide, gal(1->4)gal(1->4)glc-cermaide

E) Tetrahexosyl ceramide galNAc(1->3)gal(1->4)gal(1->4)glc-ceramide

F) On a si l ica gel H plate developed with chloroform-methanol-water (100:42:6) and visualized with alpha-napthol spray

HPTLC ANALYSIS OF NEUTRAL GSL

HPTLC ANALYSIS OF GANGLIOSIDES

Thin-layer chromatogramA) disaloganglioside, NANA

(2->3)gal(1->3)galNAc(14)[NANA(2->3)]gal(1->4)glc-ceramide

B) Monosialoganglioside, galNAc(1->4)[NANA(2->3)gal(1->4)glc-ceramide

C) On a silica gel G (0.25mm) plate developed two times with chloroform-methanol-2.5N NH4OH (65:45:9) and visualized with alpha-naphtol spray

Gas chromatography of sugars TMSI derivatization

MS analysis TMSI derivatization

HPLC analysis perbenzoylation (McCluer) endoglycoceramidation (Butters)

HPTLC and densitometric scanningHPLC analysis

deacylation (Zama)MS analysis

Without derivatization

QUANTITATION OF GLYCOSPHINGOLIPIDS

TRIMETHYLSILYLATION AND GAS-LIQUID CHROMATOGRAPHY OF METHYL

GLYCOSIDES

Mass spectrometry of intact TMS derivatives of glycolipids gives information about the sugar groups, the fatty acid and the sphingosine portion of glycosphingolipids. Bis (trimethylsiyl) trifluroroacetamide (100microliter) and pyridine (50 microliter) are added to 20-200microgram of the purified glycospingolipid in a small capped vial and heated at 60 degrees F for about 30 minutes. An aliquot containing 10-20 microgram of the TMS glycolipid is evaporated to dryness under nitrogen in a mass spectrometer direct probe tube. The samples are volati l ized in the mass spectrometer ion source at temperatures ranging 100 degrees to 180 degrees depending on the size of the oligosaccharide unit.

The following information can be obtained by comparison of the resulting mass spectra with those of reference samples: (1) whether the terminal residue is a hexose or hexosamine; (2) the number of and nature of N-acetylneuramine acid groups (i.e., terminal or branched); (3) whether N-acetyl and/or N-glycolylneuraminate is present; (4) information regarding the number of glycosyl residues present and the fatty acid and sphingosine composition. It is essential, because of the limitations of this technique (e.g., the inabil ity to distinguish between hexoses), that it be used in conjunction with other techniques, such as permethylation analyses, and studies with specific glycosidases.

MASS SPECTROMETRY OF INTACT TMSI DERIVATIVES OF GLYCOLIPIDS

Principle: Since neutral glycosphingolpids do not

possess a characteristic chromophore that permits their quantitation by UV detection, they can be derivatized with benzoylchloride to form stable per-O,N benzoylated products. These products can be quantified by UV l ight at 280nm or at a higher sensit ivity at 230nm.

GSL(200ng) plus N-acetylpsychosine (an internal standard) are dried in N2. Perbenzoylation is carried out by adding 500uL of 10% benzoylchloride in pyridine for 16hr at 37 C. The samples are N2 dried and washed thrice with 1.8mL of methanol: water(saturated with sodium carbonate). The hexane layer is washed and finally dried in N2. The derivatives are dissolved in CCL4(100uL) and a suitable aliquot injected in to the HPLC column(Zipax,E.I DuPont column 2.1 mmx500nm).

PERBENZOYLATIONMcCluer: Methods in Enzymology 138: 1987

HPLC of male (C57BL/6J) mouse kidney perbenzoylated glycosphingolipids on a Zipax column with detection at 230nm

3) Endoglycoceramidase use to excise the oligosaccharides for quantification by HPLC.Wing, D. R., et al. "High-performance liquid

chromatography analysis of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide." Analytical biochemistry 298.2 (2001): 207-217.


INCREASED GLYCOSPHINGOLIPID LEVELS IN SERUM AND AORTAE

OF APOLIPOPROTEIN E GENE KNOCKOUT MICE

2) deacylase treatment and quantification of lyso GSL Lipid Extraction

Modified Bligh and Dyer N-deacylation

Dried sphingolipid standards and samples were deacylated using sphingolipid ceramide N-deacylase (SCDase). To each dried sample and standard, 27uL 25mM sodium acetate buffer (pH 5.5) was added

To this solution, 5uL SCDase was added Samples and standards were enzymatically digested for 19h The reaction was stopped with 200uL chloroform-methanol (1:1, v/v) The organic layer was removed and dried under N2 gas.

Derivatization Samples were derivatized with 15uL OPA solution.

HPLC Agilent 1260 Infinity using a Zorbax SB-C18 reversed-phase column Solvent System: isocratic (methanol:water acidified with 0.2%

trifluoroacetic acid at 88:12, v/v) Flow Rate: 0.75 mL/min Ex λ: 340nm Em λ: 360nm

QUANTITATION OF GLYCOSPHINGOLIPIDS USING

DEACYLASE

Mass Spectrometry Without DerivatizationPreparation

Lipid extract spotted directly onto MALDI Opti-TOF plate with DHB matrix for positive/negative ion mode analysis

GSL standards spottedAnalysis

Spectral ID for GSL ESTD precursor and product ions Comparison of ESTD to sample spectra Normalization to ISTD and protein concentration



Normalize to concentration of

ISTD


Normalize to concentration of

ISTD

Calculate molar concentration (nmol/mg) using standard MW (968) and protein concentration

from Bradford

A) Sequential digestion with exoglycosidases

B) Deacylation to determine fatty acid composition

C) Tandem mass spectrometryQTOF

D) NMR

STRUCTURAL DETERMINATION

Sequential digestion with exoglycosidases



Deacylation to determine fatty acid composition- Dr. Chatterjee “TMSI Slide”


NMR-Allen Bush lecture

Galactose oxdiase oxidizes D-galactosyl and N-acetyl-D-galactosaminyl residues at nonreducing terminals of glycoproteins and glycolipids to carbon-6 aldehydes.

These aldehydes are then reduced back to galactose/N-acetylgalactosamine with tritiated borohydride. When intact cells are treated with the enzyme, only surface-exposed glycoconjugates are oxidized subsequently reduced, because the enzyme is unable to penetrate the cell plasma membrane.

Because sialic acids often are linked to penultimate galactosyl residues, more efficient labeling is achieved by the simultaneous use of neuraminidase.

LOCALIZATION OF GSL USING GALACTOSEOXIDASE

LOCALIZATION OF GSL

A) cell surface labeling using galactoseoxidase

B) Immunohistochemistry

LOCALIZATION OF GSL

ELECTRON MICROSCOPY

METABOLIC LABELING OF GSL

use of radioactive serine, glucose, galactose , palmitate , acetate.

Use of galactose oxidase to label cell surface GSL

A : Gangliosides of mouse embryo secondary cells

B : Neutral GSLs of mouse embryo secondary cells

AUTORADIOGRAM OF GLYCOSPHINGOLIPIDS

1,5 – Standards of the neutral GSL fraction of human kidney were applied in channels 1 and 5.

2: 1-14C-N-acetyl-D-glucosamine

3: 1-14C-D-galactose

4: 1-14C-D-glucosamine

A. THIN-LAYER CHROMATOGRAMB.RADIOAUTOGRAM

1,5 – Human brain gangliosides

2: 1-14C-D-glucosamine3: 1-14C-D-galactose4: 1-14C-D-glucose

A. THIN-LAYER CHROMATOGRAMB. AUTORADIOGRAM

GSL GLYCOSYLTRANSFERASE ASSAYS

The Lactosylceramide Synthase Reaction

An essential feature of this galactosyltransferase is the requirement for manganese ions and detergent for optimal activity. This is because this is a Golgi –bound enzyme but is also present in the cell membrane.

Radiolabeled UDP-galactose serves as the galactose donor and GlcCer as the acceptor. The pH optima is 7.8 and Tris or Cacodylate buffer is preferred.

Following incubation for 2hr at 37 C, the reaction is terminated with C:M 2:1.The upper aqueous layer is discarded. The lower phase is N2 dried mixed with unlabeled LacCer and separated by HPTLC. The LacCer band id identified by exposing the HPTLC plate to iodine vapors. Gel area corresponding to LacCer is scraped and radioactivity s measured. The data is expressed as pmol LacCer synthesized /mg protein /2hr

GSL GLYCOSYLTRANSFERASE ASSAYS

MEASUREMENT OF GLYCOSYLTRANSFERASE MASS

Measurement of mass by:

A) western immunoblot assay

B) ELISA assay

D-PDMP ALTERS THE EXPRESSION OF VARIOUS GLYCOSYLTRANSFERASES AND COMPONENTS OF

SIGNALING PATHWAYS LEADING TO CELL PROLIFERATION AND ANGIOGENESIS IN A MOUSE

MODEL OF RENAL CANCER.

DELIVERY OF INHIBITORS OF GSL GLYCOSYLTRANSFERASE IN

EXPERIMENTAL ANIMAL MODELS:

Use of biopolymers, drug eluting stents



Use of a novel-antiproliferative compound coated on a biopolymer to mitigate platelet-derived growth factor-induced proliferation in human aortic smooth muscle cells: comparison with sirolimus

Yong-Dan Tang, Ambarish Pandey, Antonina Kolmakova, Xin-Tong Wang, Subbu S. Venkatraman, Subroto Chatterjee, Freddy Y.C. Boey: Glycoconjugate Journal 11/2008; 26(6):721-32.



1. Stent coating with PLGA and loading of DPDMP on the polymer Spraying fixture and enclosure Pre-spray materials preparations Metal stent preparation Airbrush or spray coating Stent sterilization and packaging for animal trial



2. In vitro degradation and drug release Preparation of PLGA films and coated stents Drug quantification Degradation study

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REFERENCES Bligh, EG, and WJ Dyer. "Canadian Journal of Biochemistry and

Physiology." Canadian Journal of Biochemistry and Physiology . 37.8 (1959): 911-917. Print. http://www.nrcresearchpress.com/doi/abs/10.1139/o59-099

Chatterjee, S. Methods in Enzymology Chatterjee, S. and Martin, S Methods in Enzymology Wei, H and Chatterjee, S. Methods in Enzymology Mccluer , S. Methods in Enzymology Wing, D. R., et al. "High-performance l iquid chromatography analysis

of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide." Analytical biochemistry 298.2 (2001): 207-217.

Zama, K, Y Hayashi, S Ito, Y Hirabayashi, T Inoue, K Ohno, N Okino, and M Ito. "Simultaneous quantification of glucosylceramide and galactosylceramide by normal-phase HPLC using O-phthalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase." Glycobiology . 19.7 (2009): 767-775. Print. doi: 10.1093/glycob/cwp047

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