Analysis of Glycosphingolipids

Report
GLYCOSPHINGOLIPID
LECTURE
Dr.
Subroto
Chatterjee
WHAT ARE GLYCOSPHINGOLIPIDS?
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.
STRUCTURE OF LACTOSYLCERAMIDE
STRUCTURE OF GLYCOSPHINGOLIPIDS
LYSOSOMAL STORAGE DISORDERS
Several metabolic basis of inherited diseases in
man occur due to the lack/deficiency of enzymes
which catabolize glycosphingolipids
LYSOSOMAL STORAGE DISORDERS
PREVALENCE OF GLYCOSPHINGOLIPIDS
DISORDER
Lysosomal Storage Disorders
Prevalence
Adrenoleukodystrophy (ADL)
Niemann Pick (Type A,B, and C)
Approx. 1 in 20,000 or 13,600 people in USA
Gaucher
Approx. 1 in 200 for general population
High as 1 in 10 in Jewish people with East.
European ancestry
Krabbe
Metachromatic leukodystrophy (MLD)
Tay-Sachs
Approx. 1 in 100,000 people
Fabry
Approx. 1 in 40,000 males
Approx. 1 in 117,000 people for general
population
Type A & B: Approx. 1 in 250,000
Type C: Approx. 1 in 150,000
Approx. 1 in 625,000 people
Approx. 1 in 27 Jewish people in USA
Approx. 1 in 250 for general population
GLYCOSPHINGOLIPID LECTURE TOPICS

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
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



Function
Methods for Determining Function
Extraction and Purification
Quantitation
Structural Determination
Localization
Imaging
Metabolism
FUNCTION OF GLYCOSPHINGOLIPIDS
1) Superoxide generation and CAM expression
2) Inhibition of nitric oxide production in endothelial cells
3) As mediators of growth factors contributing to cell
proliferation
4) As receptors for toxins and bacteria
FUNCTION OF GLYCOSPHINGOLIPIDS IN
ATHEROSCLEROSIS AND VASCULAR BIOLOGY
1) Generation of superoxide in arterial
smooth muscle cells and expression of
cell adhesion molecules.
LACTOSYLCERAMIDE MEDIATES TNF ΑINDUCED ICAM-1 EXPRESSION IN
ENDOTHELIAL CELLS
LACTOSYLCERAMIDE STIMULATES
SUPEROXIDE GENERATION IN HUMAN
ENDOTHELIAL CELLS
LACTOSYLCERAMIDE STIMULATES
SUPEROXIDE GENERATION IN HUMAN
ENDOTHELIAL CELLS
FUNCTION OF GLYCOSPHINGOLIPIDS IN
ATHEROSCLEROSIS AND VASCULAR BIOLOGY
2) Inhibition of nitric oxide production in
endothelial cells.
EFFECT OF LACCER ON ENDOTHELIUM
DEPENDENT VASO-RELAXATION AND PORCINE
CORONARY ARTERY
EFFECTS OF LacCer ON eNOS
mRNA LEVELS IN HCAECs.
FUNCTION OF GLYCOSPHINGOLIPIDS IN
ATHEROSCLEROSIS AND VASCULAR BIOLOGY
3. As mediators of growth factors
contributing to cell proliferation and
angiogenesis
FUNCTION OF GLYCOSPHINGOLIPIDS
5) Serve as receptors to various toxins, e.g.
cholera toxin and other bacteria
METHODS FOR DETERMINING
GSL FUNCTION
Cellular assays
Proliferation
Adhesion
Angiogenesis
Migration
Apoptosis
METHODS FOR DETERMINING
GSL FUNCTION
METHODS FOR DETERMINING
GSL FUNCTION
GSL EXTRACTION
1) Bligh and Dyer
2) Folch Partitioning
EXTRACTION OF GLYCOSPHINGOLIPIDS
FROM HEART TISSUE
• Modified Bligh &
Dyer Method
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extraction in
chloroform:methanol
2:1
homogenized by hand
lipids extracted from
organic phase
Folch Partitioning
PURIFICATION GLYCOSPHINGOLIPIDS
 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.
HPTLC ANALYSIS OF NEUTRAL GSL
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 silica gel H plate
developed with chloroformmethanol-water (100:42:6)
and visualized with alpha napthol spray
HPTLC ANALYSIS OF GANGLIOSIDES
Thin-layer chromatogram
A) disaloganglioside, NANA (2>3)gal(1->3)galNAc(14)
[NANA(2->3)]gal(1->4)glcceramide
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
QUANTITATION OF GLYCOSPHINGOLIPIDS
 Gas chromatography of sugars
 TMSI derivatization
 MS analysis
 TMSI derivatization
 HPLC analysis
 perbenzoylation (McCluer)
 endoglycoceramidation (Butters)
 HPTLC and densitometric scanning
 HPLC analysis
 deacylation (Zama)
 MS analysis
 Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
 Gas chromatography of sugars
 TMSI derivatization
 MS analysis
 TMSI derivatization
 HPLC analysis
 perbenzoylation (McCluer)
 endoglycoceramidation (Butters)
 HPTLC and densitometric scanning
 HPLC analysis
 deacylation (Zama)
 MS analysis
 Without derivatization
TRIMETHYLSILYLATION AND GAS-LIQUID
CHROMATOGRAPHY OF METHYL GLYCOSIDES
QUANTITATION OF GLYCOSPHINGOLIPIDS
QUANTITATION OF GLYCOSPHINGOLIPIDS
QUANTITATION OF GLYCOSPHINGOLIPIDS
QUANTITATION OF GLYCOSPHINGOLIPIDS
QUANTITATION OF GLYCOSPHINGOLIPIDS
QUANTITATION OF GLYCOSPHINGOLIPIDS
 Gas chromatography of sugars
 TMSI derivatization
 MS analysis
 TMSI derivatization
 HPLC analysis
 perbenzoylation (McCluer)
 endoglycoceramidation (Butters)
 HPTLC and densitometric scanning
 HPLC analysis
 deacylation (Zama)
 MS analysis
 Without derivatization
MASS SPECTROMETRY OF INTACT TMSI
DERIVATIVES OF GLYCOLIPIDS
 Mass spectrometr y of intact TMS derivatives of glycolipids gives
information about the sugar groups, the fatty acid and the sphingosine
por tion 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 dr yness under nitrogen in a mass
spectrometer direct probe tube. The samples are volatilized in the mass
spectrometer ion source at temperatures ranging 100 degrees to 1 80
degrees depending on the size of the oligosacc haride 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 -glycol ylneuraminate 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 inability to distinguish between hexoses), that it be used in
conjuncti on with other techniques, such as permethylation analyses, and
studies with specific glycosidases .
QUANTITATION OF GLYCOSPHINGOLIPIDS
 Gas chromatography of sugars
 TMSI derivatization
 MS analysis
 TMSI derivatization
 HPLC analysis
 perbenzoylation (McCluer)
 endoglycoceramidation (Butters)
 HPTLC and densitometric scanning
 HPLC analysis
 deacylation (Zama)
 MS analysis
 Without derivatization
PERBENZOYLATION
 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 light at 280nm or at a
higher sensitivity 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).
McCluer: Methods in Enzymology 138: 1987
HPLC of male (C57BL/6J) mouse kidney perbenzoylated
glycosphingolipids on a Zipax column with detection at
230nm
QUANTITATION OF GLYCOSPHINGOLIPIDS
 Gas chromatography of sugars
 TMSI derivatization
 MS analysis
 TMSI derivatization
 HPLC analysis
 perbenzoylation (McCluer)
 endoglycoceramidation (Butters)
 HPTLC and densitometric scanning
 HPLC analysis
 deacylation (Zama)
 MS analysis
 Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
 Gas chromatography of sugars
 TMSI derivatization
 MS analysis
 TMSI derivatization
 HPLC analysis
 perbenzoylation (McCluer)
 endoglycoceramidation (Butters)
 HPTLC and densitometric scanning
 HPLC analysis
 deacylation (Zama)
 MS analysis
 Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
 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
QUANTITATION OF GLYCOSPHINGOLIPIDS
 Gas chromatography of sugars
 TMSI derivatization
 MS analysis
 TMSI derivatization
 HPLC analysis
 perbenzoylation (McCluer)
 endoglycoceramidation (Butters)
 HPTLC and densitometric scanning
 HPLC analysis
 deacylation (Zama)
 MS analysis
 Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
 Gas chromatography of sugars
 TMSI derivatization
 MS analysis
 TMSI derivatization
 HPLC analysis
 perbenzoylation (McCluer)
 endoglycoceramidation (Butters)
 HPTLC and densitometric scanning
 HPLC analysis
 deacylation (Zama)
 MS analysis
 Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
USING DEACYLASE
 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
 Gas chromatography of sugars
 TMSI derivatization
 MS analysis
 TMSI derivatization
 HPLC analysis
 perbenzoylation (McCluer)
 endoglycoceramidation (Butters)
 HPTLC and densitometric scanning
 HPLC analysis
 deacylation (Zama)
 MS analysis
 Without derivatization
QUANTITATION OF GLYCOSPHINGOLIPIDS
Mass Spectrometry Without Derivatization
 Preparation
 Lipid extract spotted directly onto MALDI Opti-TOF plate with
DHB matrix for positive/negative ion mode analysis
 GSL standards spotted
 Analysis
 Spectral ID for GSL ESTD precursor and product ions
 Comparison of ESTD to sample spectra
 Normalization to ISTD and protein concentration
QUANTITATION OF GLYCOSPHINGOLIPIDS
QUANTITATION OF GLYCOSPHINGOLIPIDS
Normalize to
concentration of ISTD
QUANTITATION OF GLYCOSPHINGOLIPIDS
Normalize to
concentration of ISTD
Calculate molar concentration
(nmol/mg) using standard
MW (968) and protein
concentration from Bradford
STRUCTURAL DETERMINATION
A) Sequential digestion with
exoglycosidases
B) Deacylation to determine fatty acid
composition
C) Tandem mass spectrometry
QTOF
D) NMR
STRUCTURAL DETERMINATION
Sequential digestion with exoglycosidases
STRUCTURAL DETERMINATION
Deacylation to determine fatty acid
composition
- Dr. Chatterjee “TMSI Slide”
STRUCTURAL DETERMINATION
NMR
 - Allen Bush lecture
LOCALIZATION OF GSL USING
GALACTOSEOXIDASE
 Galactose oxdiase oxidizes D-galactosyl and N-acetylD-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
A) cell surface
labeling using
galactoseoxidase
LOCALIZATION OF GSL
B) Immunohistochemistry
ELECTRON
MICROSCOPY
METABOLIC LABELING OF GSL
use of radioactive serine, glucose,
galactose , palmitate , acetate.
Use of galactose oxidase to label cell
surface GSL
AUTORADIOGRAM OF GLYCOSPHINGOLIPIDS
 A : Gangliosides of
mouse embryo
secondary cells
 B : Neutral GSLs of
mouse embryo
secondary cells
A. THIN-LAYER CHROMATOGRAM
B.RADIOAUTOGRAM
 1 ,5 – Standards
of the neutral GSL
fraction of human
kidney were
applied in
channels 1 and 5.
 2: 1-14C-N-acetylD-glucosamine
 3: 1-14C-Dgalactose
 4: 1-14C-Dglucosamine
A. THIN-LAYER CHROMATOGRAM
B. AUTORADIOGRAM
1,5 – Human
brain
gangliosides
2: 1-14C-Dglucosamine
3: 1-14C-Dgalactose
4: 1-14C-Dglucose
GSL GLYCOSYLTRANSFERASE ASSAYS
The Lactosylceramide Synthase
Reaction
GSL GLYCOSYLTRANSFERASE ASSAYS
 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 buf fer 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
MEASUREMENT OF
GLYCOSYLTRANSFERASE MASS
Measurement of mass by:
A) western immunoblot assay
B) ELISA assay
D - P DMP A LT ERS T H E E X P RESSION O F VA RI OUS
G LYC OSYLTRA NSFE RASES A N D C OM P ONENT S O F S I G NA LI NG
PAT HWAYS L E A DI NG TO C E LL P ROLI FERATI ON A N D
A N G IOG ENE SIS I N A M O U SE M O D EL O F R E N A L C A N C ER .
DELIVERY OF INHIBITORS OF GSL
GLYCOSYLTRANSFERASE IN EXPERIMENTAL
ANIMAL MODELS:
Use of biopolymers, drug eluting stents
DELIVERY OF INHIBITORS OF GSL
GLYCOSYLTRANSFERASE IN EXPERIMENTAL
ANIMAL MODELS:
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.
DELIVERY OF INHIBITORS OF GSL
GLYCOSYLTRANSFERASE IN EXPERIMENTAL ANIMAL
MODELS:
 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
DELIVERY OF INHIBITORS OF GSL
GLYCOSYLTRANSFERASE IN EXPERIMENTAL ANIMAL
MODELS:
 2. In vitro degradation and drug release
 Preparation of PLGA films and coated stents
 Drug quantification
 Degradation study
REFERENCES
 Bligh, EG, and WJ Dyer. "Canadian Journal of Biochemistr y and
Physiology." Canadian Journal of Biochemistr y 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 Mar tin, S Methods in Enzymology
 Wei, H and Chatterjee, S. Methods in Enzymology
 Mccluer , S. Methods in Enzymology
 Wing, D. R., et al. "High -per formance liquid chromatography
analysis of ganglioside carbohydrates at the picomole level af ter
ceramide glycanase digestion and fluorescent labeling with 2 aminobenzamide." Analytical biochemistr y 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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