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J Biol Chem, Vol. 274, Issue 29, 20725-20732, July 16, 1999
§
From the Research Institute, Hospital for Sick Children, Toronto,
Ontario M5G 1X8, Canada and the Departments of
Biochemistry and Laboratory Medicine and Pathobiology, University of
Toronto, Toronto, Ontario M5S 1A8, Canada
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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A new method to cleave the double bond of
sphingolipids has been developed. Using limited concentrations of
KMnO4 and an excess of NaIO4, in a
neutral aqueous tert-butanol solvent system gave nearly
quantitative yields of the oxidized product. A variety of natural
glycosphingolipids (GSLs): GlcC, GalC, SGC, LC, Gb3C, Gb4C, Gg4C, Gb5C, and
GM1C, gave the corresponding acids:
2-hydroxy-3-(N-acyl)-4-(O-glycosyl)-oxybutyric acids, i.e. "glycosyl ceramide acids"
(GSL·CCOOH) in excellent yields (80-90%). Deacyl GSLs
(dGSLs) were oxidized to acids containing the oligosaccharides devoid
of hydrocarbon chains, i.e. "ceramide oligosaccharides"
(dGSL·NRR1CCOOH, where R = R1 = H; R = H, R1 = CH3CO;
or R = R1 = Me). The efficacy of this method was
demonstrated by transforming natural GSLs: GlcC, GalC, GalS, SGC, LC,
Gb3C, and Gb4C into neoglycoproteins via
coupling glycosyl ceramide acids (except GalS, which was coupled directly) to bovine serum albumin (BSA). Mass spectroscopic analysis of
GalC-BSA conjugates, (GalC·CONH)nBSA and
(GalS·NHCO)nBSA gave a value of 9 ± 1 and 16 ± 2 for n. Neoglycoconjugates derived from
GlcC, GalC (type I and II and the behenic analog), SGC, LC, and
Gb3C were recognized by the recombinant human
immunodeficiency virus coat protein gp120 (rgp120). The GalS conjugate
showed significantly reduced binding, and the Gb4C
conjugate showed no binding. Thus, rgp120/GSL-BSA interaction requires
a terminal galactose and/or glucose residue. Terminal
N-acetylgalactosamine containing GSLs are not bound. The
ceramide acid conjugates provide a more effective scaffold for
presentation of glycone for rgp120 binding than those derived from
dGSLs. The retention of receptor specificity of the glycoconjugates was
validated by retention of the expected binding specificity of VT1 and
VT2e for Gb3C and Gb4C conjugates,
respectively. These studies open a new vista in the generation of
glycoconjugates from GSLs and further emphasize the role of aglycone in
glycolipid recognition.
Glycosphingolipids form a unique amphipathic subclass of
glycoconjugates present on the external leaflet of most eukaryotic plasma membranes (1, 2). A variety of functions have been ascribed to
GSL,1 including intercellular
recognition (3-6), growth regulation (7, 8), differentiation (9-12),
microbial adhesion (13-16), and receptors for bacterial toxins (17,
18). The sphingolipid metabolites of GSLs have also been implicated as
an important new class of intracellular second messengers (19-22). In
many instances, characterization of GSL function has involved
purification and subsequent chemical modifications (23, 24). For
example, GSL function has been investigated by coupling the free amine
of dGSLs to various molecular units: fatty acids (25, 26),
cross-linkers, and fluorescent probes (27-29). Additionally, the
oxidative cleavage of the double bond of sphingosine has been
investigated with similar objectives (24, 30-32).
The carboxylate group of the oxidized GSLs can be coupled to an amino
matrix where the immobilized glycan can be used in the affinity
purification of antibodies (30, 33), glycosyl hydrolases, and
transferases (34). Also, the oxidized GSLs can be coupled to proteins
or tailor-made polymers to yield neoglycoproteins or multivalent, high
affinity glycopolymers. Although several synthetic schemes have been
contrived for the generation of such multivalent glycoforms, they
inevitably require the synthesis of oligosaccharide monomers containing
suitable functional groups for polymerization and/or coupling (35-38).
The availability of a facile method to cleavage the sphingosine double
bond of a GSL or dGSL should circumvent the necessity of synthesizing
such a glycan precursor. The availability of such carboxylate/amino
containing derivatives provides a robust route to transform natural
GSLs into various neoglycoconjugates.
Synthesis of ceramide acids from natural GSLs was investigated by
Hakomori and co-workers using systems such as ozone in dichloromethane (30), KMnO4-crown ether complex in benzene (31) and
KMnO4 in acetone (32). Our attempts to use
KMnO4-crown ether-benzene system to oxidize dGSLs (GalS or
Gb3S)2 gave the
ceramide oligosaccharides in very low yields, whereas using
KMnO4 in acetone gave some products (30%) with TLC
migration patterns (RF values) similar to the
products described in this paper. Our investigations suggested that the
heterogeneity of the reaction mixture, due to solids like
KMnO4 and manganese oxide (MnO2), affected the
yield of the desired product.
Considering our particular need to oxidize dGSLs and the limited
availability of natural GSLs, we required a microscale (<0.5 mg)
procedure that gave the ceramide acid or ceramide oligosaccharide as
the major (single) product. This study elaborates an oxidation method
that fulfills these criteria.
To illustrate the biological potential of the method, we took advantage
of the binding specificity of gp120 of HIV for GSLs (39-41).
Materials
Solvents, specifically dichloromethane, tert-butyl
alcohol, iso-propyl alcohol (isoPrOH),
1,2-dichloro ethane, pyridine, diethyl ether, benzene, methanol,
chloroform, and acetone, were purchased from either Caledon
(Georgetown, Ontario, Canada) or Aldrich, and ethanol (EtOH) from
Commercial Alcohols Inc. (Brampton, Ontario, Canada). Reagents were
purchased from the following suppliers: trifluoroacetic anhydride,
K2CO3, sodium cyanoborohydride
(NaBH3CN), and triethylamine (Et3N) were from
Caledon; 37% aqueous formalin solution, 0.5 N H2SO4 solution, trichloroacetic anhydride,
acetic anhydride, and N-hydroxysuccinimide were from
Aldrich; ANALAR KMnO4, ANALAR NaHSO3, and 30%
H2O2 were from BDH (Toronto, Ontario, Canada);
dimethyl sulfoxide (Me2SO), oleic anhydride
(C36H66O3), erucic anhydride (C44H82O3), 4-chloro-1-naphthol,
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were from Sigma;
meta-NaIO4 was from Fisher Scientific
(Unionville, Ontario, Canada); and goat anti-human IgG horseradish
peroxidase conjugate and goat anti-rabbit IgG horseradish peroxidase
conjugate were from Bio-Rad. Chromatographic materials (silica gel,
TLC, HPTLC, and aluminum-backed nanosilica plates (Alugram NanoSIL GI
UV254, Macherey & Nagel) were supplied by Caledon, and
hydroxyapatite was from Bio-Rad. Reverse phase C-18 cartridges were
obtained from Waters (Mississauga, Ontario, Canada), and molecular
sieves (4 Å) from Fisher. Centricon-30 centrifugal concentrators were purchased from Amicon®.
Solvents were dried by storing over activated (~120 °C for 16 h) molecular sieves. Crown ether (10 g) was recrystallized from a
hexane (4-5 ml) solution at LC, Gb3C, and Gb4C were purified from human
kidney (43), Gb5C was purified from sheep blood (44), and
GM1C was purified from bovine brain (45) as detailed
previously. GalC, GlcC, and SGC were purchased from Sigma.
Gg4C was prepared by acid hydrolysis of GM1C
with 1 M acetic acid at 80 °C for 1 h (46).
Deacylated derivatives Gb3S and GalS (phychosine) were
prepared by saponification of Gb3C and GalC at 102 °C
with 1 M methanolic NaOH for 3 h (44).
Methods
Synthesis of Dimethylated Derivatives
Gb3S·NNMe2, GalS·NNMe2
Formaldehyde (40 µl of 37% H2CO solution,
500 µmol) and a methanolic solution of NaCNBH3 (100 µl,
0.3 M stock) were added to a solution of dGSL (1 mg, 2 µmol for GalS or 1.25 µmol for Gb3S) in methanol (0.5 ml) (47). After stirring the reaction mixture for 16 h at room
temperature (25 °C), methanol was removed under N2 and
the remaining solid was then dissolved, by sonication, in 5 ml of
distilled water. The resulting suspension was passed through a C-18
reverse phase cartridge, washed with 20 ml of water, and eluted with 20 ml of methanol. The estimated yield was >90% by TLC. Methylated
compounds have reduced mobility on TLC; the RF
for GalS and GalS·NNMe2 are 0.80 and 0.75 (in
C:M:H2OS; 60:35:8), respectively.
Positive ion mass spectroscopy data (m/z) were as follows:
GalS·NNMe2, FAB, 489, (M+H);
Gb3S·NNMe2, ES, 814 (M+H), 836 (M+Na).
Synthesis of Trihaloacetyl Derivatives: GalS·NTfa,
GalS·NTca
Acylating reagents, imidazole-Tfa (Tfa (trifluoroacetate) = CF3CO) imidazole-Tca (Tca (trichloroacetate)=
CCl3CO), were prepared by adding (divided in three portions
and added at 15-min intervals) a DCM (2 ml) solution of the anhydride
(e.g. a 2-ml solution of (Cl3CO)2O,
0.85 g, 2.7 mmol) to an imidazole (0.41 g, 6.0 mmol) suspension in
DCM (3 ml). The reaction mixture was stirred for 2 h and was
assumed to be approximately 0.5 M imidazole-trihaloacetyl derivative.
The imidazole derivative (e.g. imidazole-Tca, 20 µl, 10 µmol) was added to a suspension of GalS (3 mg, 6 µmol) in DCM (3 ml), and the mixture was stirred at room temperature. Monitoring the reaction by TLC (C:M:H2OS; 70:30:2) showed many
orcinol-positive products, suggesting some acylation of OH groups. Once
all the GalS was consumed, DCM was removed under N2 and a
solution of Et3N:M:H2O; 2:6:10 (0.5 ml/mg of
GSL) was added and analyzed by TLC (C:M:H2OS;
70:30:2) after stirring at room temperature for 3 h. Once
all the orcinol-positive species collapsed to a single band, the
reaction mixture was dried under N2, redissolved in DCE,
loaded onto a silica column (0.5 × 6 cm, in DCE) and eluted with
C:M; 98:2 (batch elution, 15 ml) and then with C:M:H2O;
80:20:2 (10, 3-ml fractions). The estimated yield by TLC was
>90%.
Synthesis of GalOC and GalEC
Homologues
To a solution of GalS (2 mg, 4 µmol) in dry pyridine (2 ml),
an excess of the anhydride (approximately 5 mg, 9 or 8 µmol for oleic
or erucic anhydrides, respectively) was added and stirred at 37 °C
for 18 h. Pyridine was removed under N2, and the
residue was treated with 1 M methanolic NaOH (2 ml) for
5 h at 25 °C, neutralized with 1 M HCl (2 ml),
diluted with water (5 ml), extracted with Et2O (three
times, 5 ml each), and the combined extracts were dried. Dried crude
material was dissolved in DCM (1 ml) and loaded onto a silica column
(0.5 × 10 cm in C:M; 98:2). The free fatty acids were eluted with
DCE:isoPrOH; 85:15 (20 ml), and the product was
eluted with C:M:H2O; 80:20:2 (six 4-ml fractions). The
estimated yield by TLC was >95%.
Synthesis of Peracetylated Derivatives
Method A--
Method A was suitable for natural, NAc, and
NNMe2 derivatives. A mixture of 1:2 acetic
anhydride:pyridine was added to a dried sample of GSL or dGSL (final
concentration of 1 mg/ml) and stirred at 37 °C for 2 h. The
reactions were monitored by TLC (DCE:isoPrOH, 80:15)
and, upon completion, dried under N2.
Method B--
Method B was suitable for the preparation of NTca
and NTfa derivatives. A mixture of 2:1 trifluoroacetic
anhydride:glacial acetic acid was added to a dried sample of
N-trihaloacetyl GSL derivative (final concentration of 1 mg/ml) and stirred at 25 °C. The reactions were monitored every 30 min by TLC (DCE:isoPrOH, 80:15) and, upon
completion, dried under N2.
The peracetylated crude material was dissolved in DCE (1 ml) and loaded
on a silica column (for 3 mg, 0.5 × 5 cm in DCE) and eluted with
DCM:M (25:Y), where Y was varied from 100 µl, in 100-µl increments
(for each solvent composition, six 4-ml fractions were collected). The
mobility of the peracetylated derivatives during column chromatography
varies significantly with small changes in the activity of silica gel.
Concomitant changes to the polarity of the eluent may be necessary.
Purity (important for the oxidation to proceed smoothly) of the product
was checked by TLC, where the plates were developed with
I2 vapor.
Oxidation Method
Reagents--
Reagents consisted of a 2:1 mixture of
t-BuOH:H2O and solutions of NaIO4
(0.4 M) and KMnO4 (0.05 M).
Quenching Solution--
Quenching solution was a 5:1 mixture of
0.25 M NaHSO3 solution and 0.5 N
H2SO4 solution.
Peracetylated neutral glycolipids (GSL(OAc)n,
0.5 mg or dGSL(OAc)n, 0.3 mg, depending on GSL;
amounts correspond to 1-0.3 µmol) were dissolved in
t-BuOH/H2O (500 µl), and NaIO4 (30 µl, 10 µmol) and KMnO4 (15 µl, 0.75 µmol) solutions
were added in the given sequence. The resulting purple mixture was
stirred at room temperature and monitored by TLC every 4 h (for
(GSL(OAc)n, 90:15:1,
C:M:H2OS and for
dGSL(OAc)n, 80:20:2,
C:M:H2OS). When clean precursors were used,
catalytic regeneration of KMnO4 proceeded smoothly until
the reaction was terminated. Otherwise, the purple color diminished
with concomitant formation of MnO2. In such cases,
additional aliquots (5 µl) of KMnO4 solution was added.
The reaction was quenched by the addition of 1.5 ml of quenching
solution and 1 ml of water, and the resulting colorless solution was
extracted with Et2O (three times, 5 ml each). Occasionally (due to insufficient quenching) the ether extract turned yellow, and in
such cases, the combined extracts were washed with 1 ml of quenching
solution. The ether extract was washed with water (two times, 1 ml
each) and dried under N2 at 25 °C. Residual water in the crude product was removed by
adding absolute EtOH (1 to 2 ml) and evaporating under N2
(Figs. 1 and 2).
For some peracetylated ceramide oligosaccharides, e.g. those
with hydrophobic substitutions like Tfa or Tca (e.g.
(OAc)5GalS·NTfaCCOOH), the work-up procedure
described above is applicable. However, the ceramide oligosaccharides
with smaller hydrophobic substitutions such as acetyl or dimethyl
(e.g. (OAc)5GalS·NRCCOOH, R = Ac or Me2) and charged ceramide acids
((OAc)4SGC·CCOOH), partitioning into
Et2O was inefficient. In such cases, the reaction was
quenched by adding an excess of solid NaHSO3 (50 mg). The
colorless (occasionally pale yellow) suspension was dried on a rotary
evaporator and extracted (three times, 5-7 ml) with
C:M:H2O; 80:20:2 and the combined extracts was passed
through a silica column (0.5 × 4 cm in C:M:H2O;
80:20:2) to remove most of the salts (Fig. 2).
Deprotection of the ceramide acids or the ceramide oligosaccharides was
carried out by treating dried material (0.5 mg) with triethylamine
solution (1 ml of Et3N:M:H2O; 2:6:10) at
37 °C for 2-3 h. The mixture was then
dried under N2 and the residue redissolved in methanol
(Figs. 3 and 4).
Mass Spectroscopic Analyses
The ES spectra were recorded on a Sciex API III spectrometer,
FAB on a VG ZAB-SE and MALDI-TOF on a Voyager-Elite spectrometer (sinapinic acid matrix, linear mode, delayed extraction) using standard
conditions.
Synthesis and Analysis of BSA Glycoconjugates
To maximize the number of glycosyl units coupled to BSA,
reactions were carried out with 1:30 mol ratio of BSA to glycosyl ceramide acid. Prior to coupling, peracetylated ceramide acids were
deprotected with Et3N solution (see oxidation protocol) and dried under N2. Residual acetate was removed by adding an
acidic aqueous ethanolic solution (to 1 mg of ceramide acid was added 1 ml of 0.01 M HCl in 9:1, EtOH:H2O) and dried on
a rotary evaporator. During coupling, a dried sample of ceramide acid
(1 mg of deprotected acid) or dGSL was added a solution of BSA (1 mg/ml
in 40 mM phosphate buffer, pH 7.4) and NHS (approximately 2 mg, 20 nmol) and the pH of the resulting mixture was adjusted (with 0.1 M NaOH) to between 7 and 8. EDAC was then added in three
portions (total of 6 mg, 30 nmol) at 2-h intervals and stirred at room
temperature for 12 h. Low molecular weight species (reagents, etc.)
were removed by washing (three cycles) in a Centricon-30 (which had
been first washed with 1% BSA and then with water). During each cycle,
the reaction mixture was concentrated to 0.5 ml and was diluted with 2 ml of 40 mM phosphate buffer (Fig. 7).
Ligand Binding
Neoglyoconjugates were adsorbed on to nitrocellulose membranes
either directly or transferred after SDS-PAGE. The membranes were
blocked with 2.5% milk powder in TBS100 (10 mM
TBS, 100 mM NaCl) for 1 h at room temperature, rinsed
three times with TBS100, and incubated with rgp120 (1 µg/ml) in TBS50 (10 mM TBS, 50 mM NaCl) for 3 h at room temperature. The blots were washed as above with TBS100 and incubated with human HIV serum (1:50) in
2.5% milk powder in TBS100 or rabbit anti-rgp120 (1:500)
in TBS100 for 3 h. After rinsing as above, blots were
incubated with the secondary antibody (1:1000) in TBS100
for 45 min. Finally, the blots were rinsed as above and the binding was
visualized by treating with 4-chloro-1-naphthol (48) (Fig. 8).
The verotoxin binding assays were performed under conditions similar to
the TLC overlay assay in which conjugates adsorbed to the
nitrocellulose membrane were substituted for lipids on a TLC plate (48)
(Fig. 8).
Oxidation Method--
The chemical steps involved in the oxidation
of natural GSLs is depicted in Scheme 1.
This method provides the means to transform a natural GSL into a
variety of neoglycoconjugates. Our objective was to develop a protocol
that can be carried out with microgram quantities of GSLs, using
readily available reagents. We reasoned that a procedure using "off
the shelf" KMnO4 could be readily implemented, as
compared with ozonolysis. The oxidation step in Scheme 1 is based on
the principles developed by Lemieux and von Rudloff (49); however, it
is modified for GSL substrates. Oxidation of protected GSLs and dGSLs
using limited amounts of KMnO4, with an excess of
NaIO4 in an aqueous tert-butanol solvent system
gave high yields (>80%) of
2-hydroxy-3-(N-acyl)-4-(O-glycosyl)-oxybutyric acid (glycosyl ceramide acid). In the case of dGSLs, the acids generated by oxidation are referred to as ceramide oligosaccharides, to
emphasize the formation of oligosaccharide free of hydrocarbon chains.
Synthesis of such oligosaccharides from hexose precursors would require
highly specialized synthetic organic chemistry expertise and
considerable time.
Our investigations with KMnO4 as an oxidant for GSLs showed
(discussed below) that maintaining a homogeneous reaction mixture was
important. We found that a tert-BuOH-water solvent system satisfied this criterion, i.e. by dissolving both the ionic
KMnO4 and NaIO4 and hydrophobic peracetylated
GSLs. It is important that pure peracetylated GSLs are used in the
oxidation (see "Experimental Procedures"). It was observed that
impurities formed during peracetylation impede the catalytic
regeneration of KMnO4, presumably by precipitating NaIO4.
The quantities of the reagents used in the reaction have been adjusted
such that they can accommodate at least a 50% variation in the
concentration of GSLs. Therefore, the procedure can be directly
employed for similar quantities of different GSLs. The minimum number
of equivalents of NaIO4 and KMnO4 used with
respect to the GSL are 10 and 0.75, respectively, and the measured pH of the initial reaction mixture was approximately 7. Although, in
principle, catalytic amounts of KMnO4 should have been
adequate, using closer to 1 eq (i.e. in comparison to GSL
precursor) decreased the reaction time significantly. Attempts to
employ greater than 1 eq of KMnO4 resulted in the formation
of brown manganese dioxide precipitates, which, in turn, led to lower yields.
Prior to the development of this new procedure, our attempts to use the
previously described KMnO4-crown ether-benzene system (31)
gave very low yields. For example, oxidation of 1 mg of Gb3S gave undetectable product. Further investigations
suggested that the heterogeneous nature of the reaction mixture, from
excess solid KMnO4 and MnO2 precipitate, was an
important factor contributing to low yields. Analysis of the
precipitate demonstrated that it trapped ~25% of the starting
material. Fig. 5 depicts HPTLCs of products obtained from a microscale oxidation of GalC(OAc)5
using KMnO4-crown-ether and the
KMnO4-NaIO4-tert-BuOH methods.
Analyzing the product by HPTLC with a basic solvent system showed that
the sample from the new method gave a strong band at the origin (due to
the formation of the salt). The crown ether method gave no such band.
Oxidation using KMnO4-acetone (32) system gave some product
(approximately 30%) that showed similar characteristics on TLC
analysis as the products from
KMnO4-NaIO4-tert-BuOH method. Here
again, the formation of MnO2 precipitate might have led to the lowering of the yield, again suggesting the importance of maintaining a homogeneous reaction mixture.
Glycosyl Ceramide Acids--
Fig. 1 shows the TLCs of the
protected ceramide acids,
GSL(OAc)n·CCOOH of the natural
GSLs (GalC, LC, Gb3C, Gb4C, Gg4C,
Gb5C, and GM1C). For the higher homologue GSLs
(Gb4C and above), the desired products are formed in almost
quantitative yields. In the case of shorter chain GSL (Gb3C
and shorter), TLC analysis (Fig. 2, lanes 7-10) shows only
two additional bands with higher RF values. Of
these bands, the one closer to the solvent front is the starting material and the one beneath, which usually appears as a doublet (diasteriomers), corresponds to an oxidation intermediate.
An interesting feature of the reaction is the appearance of more than
one band for each of the protected ceramide acids
GSL(OAc)n·CCOOH, which upon
deprotection collapse to fewer bands (Fig. 3). We hypothesize that the
multiple bands seen for
GSL(OAc)n·CCOOH are in part due to
fatty acid heterogeneity and to the additional cleavage of double bonds
present in unsaturated fatty acid chains. The heterogeneity in the
fatty acid is apparently reflected in the RF to
a higher degree than variation in the number of sugar residues. This
hypothesis was tested by synthesizing GalC derivatives containing oleic
(cis-9 octadecenoic, C18) or erucic (cis-13
docosenoic, C22) acyl chains. After oxidation each of these
monounsaturated fatty acid containing homologues gave two products in
approximately 6:1 ratio (Fig. 1B). The products from erucic
homologue showed higher RF value than the
products from the oleic homologue. On the assumption that the product
obtained from the oxidation of the sphingosine double bond,
(i.e. the ceramide acid) will have a greater hydrophobic
character than the product from the oxidation of both double bonds
(GalC(OAc)5·CCOOHnCOOH,
n = 9 or 13), the major band is assigned to the former
case. This study also suggests that the oxidation has selectivity
toward the sphingosine double bond.
Glycosyl Ceramide Oligosaccharides--
Ceramide oligosaccharides,
having two reactive groups for conjugation, enables the synthesis of
more types of glycoconjugates than ceramide acids. Fig. 2 shows the
TLCs of the protected ceramide oligosaccharides derived from GalS and
Gb3S. The Tfa or the Tca groups can be removed to generate
an amine function at an appropriate stage of glycoconjugate synthesis.
Oxidation of dGSL(OAc)n precursors proceeded at
a slightly faster rate (than GSL(OAc)n) and gave
good yields (80%) of the oligosaccharides. The oxidation was cleaner
for precursors with NAc, NTfa, and NTca groups on sphingosine. The
protected and deprotected oligosaccharides from dimethyl derivatives
show two orcinol-positive bands (Figs. 2 and 4). We also found that
treatment of GalS(OAc)5·NTfaCCOOH with
Et3N/M/H2O for a long duration (18 h at
37 °C) gave two orcinol-positive bands with
RF values comparable to ceramide oligosaccharides (Fig. 4). However, only the lower band stained with
ninhydrin, consistent with the ceramide oligosaccharide
GalS·NH2CCOOH. Absence of an amine function
on the second compound suggests a partial Hoffmann-type deamination
(50) side reaction during the deprotection procedure.
The positive ion FAB mass spectrum of ceramide acid
LC·CCOOH is depicted in Fig.
6. Two types of ceramide acids can be
identified; mass peaks at m/z = 860 (M+Na, M = L26C·CCOOH), 846, 832, 818, and 804 correspond to ceramide acids having saturated fatty acids, and the peak
at m/z = 764 (n = 18) has a
hydroxylated fatty acid. Also, the spectrum shows a series of peaks
resulting from the loss of a terminal sugar (loss of one glycal
fragment, 162); e.g. peaks at m/e = 684 arise from the molecular ion peak at 846 (M+Na) and the one at
m/e = 602 is from the ion at m/e = 764 (M+Na).
Applications--
We have shown that this oxidation method is
reliable and can be used to transform small quantities of natural GSLs
to novel bioactive glycosphingolipid derivatives. By way of preliminary illustration of this potential, we have coupled ceramide acids of GlcC,
GalC (type I, type II, and the behenic homologue), GalS, SGC, LC,
Gb3, and Gb4 to BSA and studied their
interaction with HIV coat protein rgp120 (40, 51, 52) and verotoxins
(17, 53). The pathway of HIV entry into T-lymphocytes is initiated by
the binding of gp120 to the cellular CD4 receptor. This induces a
conformational change in gp120 and causes a second interaction with a
chemokine receptor. These events eventually lead to the fusion of viral
and cellular membranes (54, 55). However, GSLs, specifically GalC and
SGC, have been implicated in the HIV infection of CD4-negative tissues
such as fibroblasts and neural and intestinal epithelial cells (40, 51,
56, 57). A class of synthetic GalC mimics inhibited the interaction of
rgp120 to suramin and inhibited the infection of HIV-1 in human
peripheral blood mononuclear cells (58). We used verotoxin-GSL
interactions as a means to verify the specificity of our
glycoconjugates (discussed later) (17, 53).
Deprotected ceramide acids were coupled to BSA using the
carbodiimide-NHS system. A molar ratio of 30:1 of the ceramide acid to
BSA was employed during coupling and the SDS-PAGE separation of the
conjugates is shown in Fig. 7. Although
the conjugates have a defined number (discussed later) of ceramide
acids per BSA, they appear as diffused bands. Also, the gels show two
sets of bands, presumably corresponding to monomeric (~80 kDa) and dimeric conjugates (close to the gel front).
The number of galactosyl ceramide acid units per BSA was estimated
using MALDI-TOF mass spectroscopic analysis (Fig. 6). Conjugates (GalC·CCO)nBSA and
(GalS·NH)nBSA gave broad peaks around 82 and
83 kDa, respectively. However, BSA treated with EDAC-NHS alone gave a
peak at 75 kDa, (compared with 66 kDa for BSA), probably due to the
coupling of small peptide impurities present in BSA. Therefore, 75 kDa
was considered as the mass for uncoupled BSA. Shifts of 6.1 and 7.2 kDa
for (GalC·CCO)nBSA and
(GalS·NH)nBSA conjugates correspond to a mean
value of 9 and 16 for n, respectively. On the assumption that the broader peaks of the conjugates (as compared with the uncoupled BSA) result from variation in n, the value of
n was calculated to be n = 16 ± 2 and
n = 9±1 for (GalS·NH)nBSA and
(GalC·CCO)nBSA, respectively. In
the case of (GalC·CCO)nBSA, a
(weighted) average molecular weight was used for the ceramide acid
GalC·CCOOH. The value of n obtained by this
analysis for (GalS·NH)nBSA is consistent with
a recent report, where 17 globo H antigens (monomers with an aldehydic
spacer) were coupled to BSA by reductive amination (59). Investigating
various coupling conditions showed that the amine function of dGSLs
couples readily, whereas the coupling of ceramide acids is more
difficult (data not shown).
A Western blot analysis, where the glycoconjugates were first separated
by SDS-PAGE, transferred to nitrocellulose membrane and probed for
rgp120 binding is shown in Fig. 7. Due to the diffuse nature of the
monomeric conjugates, the binding profile of rgp120 is also diffuse,
whereas the high molecular weight conjugates show stronger signals.
Qualitatively, GalC, SGC, and LC conjugates bind rgp120, whereas GalS
showed reduced binding, and Gb4C no binding. In the case of
GalC·CCOOH and GalS conjugates, since the substitution
per BSA is known, a quantitative interpretation of binding can be made.
Although nearly twice as many GalS units were coupled per BSA than
GalC·CCOOH, the ceramide acid conjugate is a better
ligand for rgp120.
Dose responses for the binding of BSA conjugates to rgp120 by dot blot
are shown in Fig. 8. Conjugates derived
from GlcC, GalC, GalOHC, GalBC, SGC, LC, and
Gb3C showed dose-dependent binding, whereas
Gb4C did not bind. The binding profiles of the GalS
conjugate consistently showed less binding than the ceramide acid
conjugates. These findings differ significantly from the binding
observed for intact GSLs adsorbed on TLC plates in which, consistent
with the literature (39, 40, 56, 57), only GalC and SGC bound rgp120.
We propose that the presentation of carbohydrate is different in these
two cases (i.e. the natural GSL on TLC versus
neoglycoconjugate), modulated by the aglycone.
Some natural GSLs, such as bovine GalC, have a significant fraction of
unsaturated fatty acids, which give rise to dicarboxylic acids after
oxidation. For such species, coupling could occur at the distal acid
group of the acyl chain or at the ceramide acid. To ensure that the
coupled ceramide acid is recognized by rgp120, a GalC homologue
containing only behenic acid was synthesized and transformed into the
corresponding conjugate. The behenic conjugate showed a binding profile
comparable to the conjugates derived from natural type I and type II GalC.
To authenticate the Gb4C and Gb3C conjugates, a
dose-response dot blot binding analysis (Fig. 8) of Gb4C,
Gb3C and GlcC BSA conjugates with verotoxins VT1 and
VT2e was performed. It is well established that VT1 is
highly specific for Gb3C, whereas VT2e binds to
both Gb3C and Gb4C (60). Neither toxin binds to
GlcC. The blots clearly showed that these conjugates bind to VTs with the expected selectivity, and their binding profiles are consistent with multiple binding sites of the toxin interacting with a
multidentate ligand.
Three conclusions can be made from these studies involving rgp120.
First, the binding specificity for the neoglycoconjugate is distinct
from that for the free GSL. Neither GlcC or Gb3C are bound,
but their BSA conjugates are efficiently recognized. The binding of
Gb3-BSA is of particular interest in light of the role of
Gb3 in HIV-induced cell fusion (61), suggesting that the "molecular environment" may modulate Gb3 binding.
Second, glycoconjugates derived from ceramide acids of GalC are more
effective receptors than those derived from the dGSL, GalS, indicating
the utility of this oxidation procedure. This could be exploited in the
design of soluble GSL mimetics. For example, ceramide acids having
optimized acyl units could be coupled to dendrimers or to defined
peptides. Third, our studies show that when GSLs are transformed into a neoglycoprotein scaffold, rgp120 recognizes epitopes with terminal galactose or glucose but does not bind ligands containing terminal N-acetylgalactosamine. This is consistent with the observed
binding profile of the variable loop-derived (V3) synthetic peptide
SPC3, which showed very low affinity toward terminal GalNAc containing Gb4 or GM2 but bound to SGC, LC,
GM3, and GD3 (51).
Our finding that GlcC-BSA is a good gp120 receptor is not explained by
current concepts that govern the specificity of protein/carbohydrate interaction. The binding of both GalC and GlcC conjugates with rgp120
suggests that, in this context, the 4-OH of these hexoses may not play
a direct role in binding. If we assume that rgp120 is a monomer and has
only one carbohydrate binding domain, a possible explanation for the
altered binding specificity might be carbohydrate-carbohydrate interaction within the GalC or GlcC conjugates. Since Gb3
and LC BSAs are bound, it may be that the binding domain of rgp120 recognizes a linear disaccharide unit (i.e. 1-4 type
arrangement) containing a Gal-Gal, Glc-Glc, or Gal-Glc sequence. In the
case of Gb3C and LC conjugates, such arrangement is
covalently established within each oligosaccharide unit. In GalC and
GlcC conjugates, hydrogen bonding between the glycosidic oxygen of one
sugar and the 4-OH (irrespective of whether axial or equatorial) of the second sugar could give a similar hexose-hexose arrangement,
particularly along the edges of the pyranose rings (rather than the
planes of the two rings). We observed that the rgp120 binding to GSL conjugates, particularly for the monohexose conjugates, was
significantly better at lower salt concentration (50 mM
versus 100 mM). Higher salt concentrations might
disrupt such carbohydrate-carbohydrate interactions mediated by direct
hydrogen bonding.
In summary, we have developed a new procedure for the microscale
oxidation of the sphingosine double bond, which gives a high yield of
the desired carboxylic acid. This procedure will allow a new systematic
approach to the generation of GSL mimics that can be used in the
investigation of GSL-protein interactions and design of possible
therapeutic agents.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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20 °C, washed with cold (20 °C) hexanes (1 ml) and dried at 40 °C under a stream of N2.
BSA (99%, essentially fatty acid-free) was purchased from Sigma and
further purified on a hydroxyapatite column (42). Recombinant gp120 (rgp120, 1 mg/ml in TBS; strain: LAV, baculovirus expression system) was purchased from Protein Sciences Corp. (Meriden, CT), and rabbit anti-gp120 polyclonal antibody (IgG, 1 mg/ml in phosphate-buffered saline) from Immuno Diagnostics, Inc. (Bedford, MA). Human sera from
HIV patients containing anti-gp120 antibodies was a gift from Dr. S. Read, Division of Infectious Disease, HSC.
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Fig. 1.
TLC (nanosil plates, visualized with orcinol)
showing the relative migration of peracetylated ceramide acids
(GSL(OAc)n·CCOOH). In
each case, 500 µg of GSL(OAc)n was oxidized
for 4 h and the dried organic extract (see "Experimental
Procedures") was dissolved (0.5 ml, DCM:M; 2:1) and analyzed in
10-µl aliquots. Bands beneath the horizontal
line correspond to ceramide acids and above the line
(indicated by arrows) to starting material and/or
intermediates and/or GSLs with dihydrosphingosine. Appearance of
multiple bands for ceramide acids is attributed to the heterogeneity
present in the acyl chain. A, lane 1,
GalC(OAc)5·CCOOH; lane
2, LC(OAc)8· CCOOH;
lane 3,
Gb3C(OAc)11·CCOOH;
lane 4,
Gg4C(OAc)13·CCOOH;
lane 5,
Gb4C(OAc)13·CCOOH;
lane 6,
Gb5C(OAc)15·CCOOH;
lane 7,
GM1C(OAc)17·CCOOH. Solvent
systems: lanes 1-6,
C:M:H2OS; 90:15:1; lane
7, C:M:H2OS; 80:20:2. B,
lane 1,
GalEC(OAc)5·CCOOH
(upper) and
GalC(OAc)5·CCOOH13COOH
(lower); lane 2,
GalOC(OAc)5·CCOOH
(upper) and
GalC(OAc)5·CCOOH9COOH
(lower); lane 3, acids from an
oxidation using a 1:1:1 mixture of GalEC(OAc)5,
GalOC(OAc)5 and natural GalC(OAc)5;
lane 4, natural
GalC(OAc)5·CCOOH. Solvent system:
C:M:H2OS; 90:15:1 arrow, as for
panel A.
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Fig. 2.
TLC (nanosil plates, visualized with orcinol)
showing the relative migration of peracetylated ceramide
oligosaccharides
(dGSL(OAc)n·CCOOH). In
each case, 300 µg of dGSL(OAc)n was oxidized
for 4 h and the dried organic extract (see "Experimental
Procedures") was dissolved (0.3 ml DCM:M; 2:1) and 10-µl aliquots
analyzed. In lanes 7-10, the starting material
(lanes 7 and 9) and the reaction
products after premature termination at 2 h (lanes
8 and 10) are shown. Lane
1, GalS(OAc)5·NAcCCOOH;
lane 2,
Gb3S(OAc)11·NAcCCOOH;
lane 3,
GalS(OAc)5·NNMe2CCOOH;
lane 4,
Gb3S(OAc)11· NNMe2CCOOH;
lane 5,
GalS(OAc)5·NTfaCCOOH; lane
6, GalS(OAc)5· NTcaCCOOH;
lane 7, GalS(OAc)5·NAc;
lane 8,
GalS(OAc)5·NAcCCOOH; lane
9, Gb3S(OAc)11·NAc;
lane 10,
Gb3S(OAc)11·NAcCCOOH. Solvent
system: lanes 1-6,
C:M:H2OS; 60:30:2; lanes
7-10, DCE:isoPrOH; 80:15. Comparison of
lanes 7-8 and 9-10 shows that, when
prematurely quenched, the reaction contains only three components:
ceramide oligosaccharide (at the origin), unreacted precursor (closer
to the solvent front in 8, but closer to the origin in
10), and the intermediate.
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Fig. 3.
TLC (nanosil plates, visualized with orcinol)
showing the relative migration of deprotected ceramide acids,
GSL·CCOOH, synthesized by treating the material used in
Fig. 1 with Et3N:M:H2O;
2:6:10 (4 h at 37 °C). Lane 1, GalC;
lane 2, GalC·CCOOH; lane 3, LC;
lane 4, LC·CCOOH; lane 5,
Gb3C; lane 6,
Gb3C·CCOOH; lane 7,
Gg4C; lane 8,
Gg4C·CCOOH; lane 9,
Gb4C; lane 10,
Gb4C·CCOOH; lane 11,
Gb5C; lane 12,
Gb5C·CCOOH; lane 13,
GM1C; lane 14,
GM1C·CCOOH. Solvent system: lanes
1-4, C:M:H2OS; 65:25:4;
lanes 5-14, C:M:H2OS;
60:40:9.
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Fig. 4.
TLC (nanosil plates, visualized with orcinol)
showing the relative migration of deprotected ceramide
oligosaccharides, dGSL·CCOOH, synthesized by treating the
material used in Fig. 2 with
Et3N:M:H2O; 2:6:10 (4 h
at 37 °C). Lane 1,
GalS·NNMe2CCOOH; lane 2,
GalS·NAcCCOOH; lane 3,
GalS·NTfaCCOOH; lane 4,
GalS·NH2CCOOH made by treating the sample in
lane 3 with Et3N:M:H2O
for 16 h at 37 °C; lane 5, sample in
lane 4 stained with ninhydrin; lane
6, GalS (stained with ninhydrin); lane
7, GalS; lane 8, Gb3C;
lane 9, Gb3S; lane
10, Gb3S·NAcCCOOH; lane
11, Gb3S·NNMe2CCOOH.
Solvent systems: lanes 6 and 7,
C:M:H2OS; 80:20:2.; other
lanes, C:M:acetone:acetic acid:H2O;
5:5:5:2:2.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Scheme 1.
The steps involved in the conversion of
natural GSLs into the corresponding ceramide acids and
oligosaccharides. R1 = H;
R2 = acyl for natural GSLs or
R2 = CH3CO, CF3CO, etc.,
for dGSLs.
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Fig. 5.
HPTLCs (visualized with orcinol) showing the
analysis of crude products obtained from the oxidation of
GalC(OAc)5 using KmnO4-crown ether-benzene
system. Lanes 1, 3, and 5,
products from KMnO4-NaIO4-t-BuOH
oxidation; lanes 2, 4, and
6, products from KmnO4-crown ether-benzene
oxidation. The bands which appear at the origin in lanes
3, 4, and 6 are not orcinol-positive.
Solvent systems: lanes 1 and 2,
C:M:H2O (28% NH4OH); 80:20:2; lanes
3 and 4, C:M:H2O (1 M
HCl); 80:20:2; lanes 5 and 6,
C:M:H2O; 80:20:2.
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Fig. 6.
Mass spectra of ceramide acid and BSA
conjugates. A, FAB mass spectrum (positive ion mode) of
LC·CCOOH. B and C are MALDI-TOF
mass spectra of the conjugates (GalS·NHCO)nBSA
and (GalC·C CONH)nBSA,
respectively. Double-headed arrows indicate
approximate peak widths at half-height.
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Fig. 7.
SDS-PAGE showing the relative migration of
BSA conjugates (Coomassie stain, left) and a Western
blot type analysis of rgp 120 binding (right).
Conjugates, 1,
(GlcC·CCONH)nBSA; 2,
(GalC·CCONH)nBSA; 3,
(GalHOC·CCONH)nBSA;
4,
(GalBC·CCONH)nBSA;
5, (GalS·NHCO)nBSA; 6,
(SGC·CCONH)nBSA; 7,
(LC·CCONH)nBSA; 8,
(Gb3C·CCONH)nBSA;
9,
(Gb4C·CCONH)nBSA.
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Fig. 8.
Dose response for rgp120 and verotoxin
BSA neoglycoconjugate binding. The highest concentration for each
conjugate is 2 µg of protein (corresponding to less than 200 ng of
glycosylceramide acid). All conjugates are numbered according to Fig.
7. Upper panel, dot blot binding of
glycoconjugates (0.5 dilutions) and rgp120, where the bound rgp120 was
detected by human or rabbit antisera. The rgp120 binding of conjugates
2-5 is quantitated in the inset by pixel integration. Data
in solid lines are for human antiserum and, in
the dotted lines, for rabbit antiserum.
Open symbols, GalS conjugates; solid
symbols, GalC·C COOH conjugates.
Lower panel, dose-response analysis involving
glycoconjugates (0.2 dilutions) and verotoxins. Controls for goat
anti-rabbit (GAR) and goat anti-human (GAH) serum
alone showed no binding.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Jianyao Wang, Toronto Carbohydrate Research Center, University of Toronto for recording the mass spectra; B. Boyd and A. Nutikka for the purification of GSLs; and S. Skandakumar for assistance with some aspects of syntheses.
| |
FOOTNOTES |
|---|
* This work was supported by Canadian Medical Research Council Grant MT13073 and National Institutes of Health Grant R01 DK52098.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed.
2 Glycosphingolipids (GSLs) are annotated with "C" (for ceramide) and deacyl GSLs (dGSLs) annotated with "S" (for sphingosine), e.g. galactosyl ceramide, GalC, and lysogalactosyl ceramide, GalS. Other GSLs are abbreviated as follows: glucosyl ceramide (GlcC), sulfogalactosyl ceramide (SGC), lactosyl ceramide (LC), globotriaosyl ceramide (Gb3C), globotetraosyl ceramide (Gb4C), gangliotetraosyl ceramide (Gg4C), Forssmann antigen (Gb5C), and gangliosides (GM1C, GM2C, GM3C, GD3C, etc.). Type I and type II GalC refer to nonhydroxylated (GalC) and hydroxylated (GalHOC) fatty acid containing fractions from bovine brain. GSLs with specific acyl chains are denoted: GalBC, GalOC, GalEC, and L26C, where superscripts denote behenic, oleic, erucic, or a C26-fatty acid-containing ceramide. Notations following the chem point indicate modifications to the ceramide or the sphingosine: galactosyl N-acetylsphingosine (GalS·NAc); galactosyl N,N-dimethyl sphingosine (GalS·NNMe2); galactosyl N-trifluoroacetylsphingosine (GalS·NTfa); galactosyl N-trichloroacetylsphingosine (GalS·NTca). Glycosyl ceramide acids are denoted GSL·CCOOH, where superscript "c" indicates ceramide acid, e.g. GalC·CCOOH, GM1·CCOOH, etc. Glycosyl ceramide oligosaccharides are denoted dGSL·CCOOH, e.g. GalS·NAcCCOOH, Gb3S·NNMe2CCOOH, etc. Dicarboxylic acids (from the oxidation of double bonds on acyl chain and sphingosine), e.g. GalC·CCOOHnCOOH, where n is the length of the acyl chain. Acetylated derivatives are denoted with (OAc)n, where n = number of OH groups on glycan +1 for sphingosine, e.g. GalC(OAc)5, SGC(OAc)4, GalC(OAc)5·CCOOH, SGC(OAc)4·CCOOH, GalC(OAc)5·NAcCCOOH, etc. BSA conjugates, e.g. (GalC·CCONH)nBSA and (GalS·NHCO)nBSA, are from GalC·CCOOH and GalS, respectively. In TLC solvent systems, H2OS denotes 0.88% KCl solution.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: GSL, glycosphingolipid; dGSL, deacyl GSL; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; HPTLC, high performance thin layer chromatography; DCM, dichloromethane; t-BuOH, tert-butyl alcohol; isoPrOH, iso-propyl alcohol; DCE, 1,2-dichloroethane; Py, pyridine; Et2O, diethyl ether; Bz, benzene; M, methanol; C, chloroform; A, acetone; NHS, N-hydroxysuccinimide; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; FAB, fast atom bombardment; HIV, human immunodeficiency virus; ES, Electro Spray.
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ABSTRACT
INTRODUCTION
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