Structural Basis for the Resistance of Tay-Sachs Ganglioside GM2
to Enzymatic Degradation*
Yu-Teh
Li
§,
Su-Chen
Li
,
Akira
Hasegawa¶,
Hideharu
Ishida¶,
Makoto
Kiso¶,
Anna
Bernardi
,
Paola
Brocca**,
Laura
Raimondi
, and
Sandro
Sonnino**
From the
Department of Biochemistry, Tulane
University School of Medicine, New Orleans, Louisiana 70112, the
¶ Department of Bio-Organic Chemistry, Gifu University, Gifu
501-11, Japan, the
Department of Organic and Industrial
Chemistry, University of Milan, Via Venezian 21, 20133 Milano, Italy,
and the ** Department of Medical Chemistry and Biochemistry, University
of Milan, Via Fratelli Cervi 93, 20090 Segrate (Mi), Italy
 |
ABSTRACT |
To understand the reason why, in the
absence of GM2 activator protein, the GalNAc and the NeuAc in GM2
(GalNAc
1
4(NeuAc
2
3)Gal
1
4Glc
1-1'Cer) are refractory
to
-hexosaminidase A and sialidase, respectively, we have recently
synthesized a linkage analogue of GM2 named 6'GM2 (GalNAc
1
6(NeuAc
2
3)Gal
1
4Glc
1-1'Cer). While GM2 has
GalNAc
1
4Gal linkage, 6'-GM2 has GalNAc
1
6Gal linkage
(Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa,
A. (1997) Carbohydr. Res. 302, 223-227). We have studied
the enzymatic susceptibilities of GM2 and 6'GM2, as well as that of the
oligosaccharides derived from GM2, asialo-GM2 (GalNAc
1
4Gal
1
4Glc
1-1'Cer) and 6'GM2. In addition, the conformational properties
of both GM2 and 6'GM2 were analyzed using NMR spectroscopy and
molecular mechanics computation. In sharp contrast to GM2, the GalNAc
and the Neu5Ac of 6'GM2 were readily hydrolyzed by
-hexosaminidase A
and sialidase, respectively, without GM2 activator. Among the
oligosaccharides derived from GM2, asialo-GM2, and 6'GM2, only the
oligosaccharide from GM2 was resistant to
-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid
oligosaccharide head group, 6'GM2 has an open spatial arrangement of
the sugar units, with the GalNAc and the Neu5Ac freely accessible to
external interactions. These results strongly indicate that the
resistance of GM2 to enzymatic hydrolysis is because of the specific
rigid conformation of the GM2 oligosaccharide.
 |
INTRODUCTION |
Tay-Sachs disease is caused by the impaired catabolism of
ganglioside GM21
(GalNAc
1
4(Neu5Ac
2
3)Gal
1
4GlcCer). It has been
shown that the terminal GalNAc in GM2 is resistant to
-hexosaminidase A (Hex A), and that a specific protein cofactor, GM2
activator, is required to assist the hydrolysis (1-4). The disease,
therefore, can be caused by the deficiency of either Hex A (5-7) or
GM2 activator protein (8-10). The reason for the resistance of GM2 to
Hex A is still an enigma.
We have shown that, in addition to the GalNAc, the enzymatic
hydrolysis of the Neu5Ac in GM2 also requires GM2 activator protein (11). In GM2, both the GalNAc and the Neu5Ac are linked to the Gal to
form GalNAc
1
4(Neu5Ac
2
3)-Gal
-, a branched trisaccharide (GM2-epitope). We have rationalized that the resistance of the GalNAc
and the Neu5Ac in GM2 to enzymatic hydrolysis may be because of the
specific rigid structural conformation of the GM2-epitope and
that GM2 activator may interact with the GM2-epitope to make the GalNAc
and the Neu5Ac accessible to Hex A and sialidase, respectively (11). To show that this is the case, we have recently synthesized a GM2
analog, 6'GM2 (GalNAc
1
6(Neu5Ac
2
3)-Gal
1
4GlcCer),
in which the GalNAc is linked
1
6 to the Gal (12). Here, we report that, in sharp contrast to GM2, the GalNAc and the Neu5Ac in 6'GM2 were
readily hydrolyzed by Hex A and clostridial sialidase, respectively, independent of GM2 activator protein. NMR analysis and molecular mechanics computation revealed that the trisaccharide head group of
6'GM2 is much more flexible than that of GM2. Our results provide the
structural basis for the resistance of the GalNAc and the Neu5Ac in GM2
to enzymatic degradation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
GM2 was isolated from the brain of a Tay-Sachs
patient according to the published method (13). Asialo-GM2 (GA2) was
prepared from GM2 by mild acid hydrolysis (14). Hex A (33.3 units/mg) from human liver was prepared according to our previous report (1).
6'GM2 was chemically synthesized (12). The oligosaccharides OM2, 6'OM2,
and OA2 were prepared by the removal of ceramide moieties from GM2,
6'GM2, and GA2 using ceramide glycanase (15). The following were
obtained from commercial sources: precoated silica gel-60 TLC plates,
Merck (Darmstadt, Germany); GM3, Matreya; sialyllactose, lactose, type
X clostridial sialidase, (CD3)2SO, and
D2O, Sigma; and Chelex-100 resin, Bio-Rad.
Enzymatic Hydrolysis--
For enzymatic hydrolysis of the GalNAc
from glycolipids or from the oligosaccharides derived from glycolipids,
each reaction mixture contained 8 nmol of the substrate and the
specified amount of Hex A, as indicated in each figure, in 100 µl of
10 mM sodium acetate buffer, pH 4.6. Incubations were
carried out at 37 °C for the preset time. For hydrolysis of the
Neu5Ac from the glycolipids, 8 nmol of GM2 or 6'GM2 was incubated with
10 units of clostridial sialidase in the presence or absence of 5 µg
of GM2 activator protein (11) in 100 µl of 20 mM sodium
acetate buffer, pH 5.5. Incubation was carried out at 37 °C for
16 h.
TLC Analysis--
After incubation, each reaction mixture was
evaporated to dryness and analyzed by silica gel-60 thin layer
chromatography. The solvent system used for developing the glycolipids
was chloroform/methanol/water (60:35:8) and that for the
oligosaccharides was n-butyl alcohol/acetic acid/water
(2:1:1). The plate was sprayed with the diphenylamine reagent (16) and
heated at 110 °C for 15 min to reveal oligosaccharides and
glycoconjugates. Quantitative results on the % hydrolysis of
substrates were obtained from the scanning of the TLC plates using Scan
Jet 2C/ADF scanner (Hewlett Packard) and the NIH Image 1.41 program.
NMR Sample Preparation--
Five mg of 6'GM2 were dried under
vacuum and were dissolved in 0.5 ml of (CD3)2SO
or (CD3)2SO/D2O (20:1) under a
stream of nitrogen. To overcome signal broadening of OH groups, such as the Neu5Ac OH8, Gal OH2, and GalNAc OH6, the sample was passed through
a Chelex-100 column (0.5 × 2.5 cm, pH 6) before analysis. This
procedure allowed the detection of the Neu5Ac OH8 proton as a typical
sharp signal at low fields (6.15 ppm), but caused the loss of other OH
signals. To complete the assignment, about 4 mg of 6'GM2 in 2 ml of
water was dialyzed against 2 mM EDTA, followed by water,
and then passed through a Chelex-100 column at pH 7.0. This treatment
furnished the GalNAc OH6 and Gal OH2 resonance at 5.16 and 4.35 ppm,
respectively, while it caused the loss of the previously assigned OH2
of Glc.
NMR Spectroscopy--
1H-NMR and 13C-NMR
spectra were obtained at 500 and 125 MHz, respectively, on a Bruker
AM500 spectrometer and analyzed on a X32 Bruker satellite station
equipped with standard Bruker UXNMR software. 1H-1H and 1H-13C
two-dimensional spectra were acquired as 2048 × 512 and 2048 × 256 matrices, respectively, with 64 scans per
t1 point and processed after zero filling in F1
dimension and appropriate window function multiplication. Proton inter-
and intra-residual contacts were evaluated by NOE experiments in the
rotating frame ROESY (17) with a spin lock pulse strength of 2.6 kH
applied at one end of the spectrum to avoid scalar transfer (18, 19).
Temperature was varied in the range of 305-323 K and mixing time
between 100-200 ms. The experiments were conducted on both the
D2O-exchanged and not-exchanged samples as well as the
samples before and after the treatments. Cross-peak volumes were
transformed into proton-proton distances r, under the
hypothesis of the 1/r6 NOE dependence and using
internal calibration on Glc, Gal, GalNAc H1/H3, and H1/H5 distances,
and on Glc H1/H2 when accessible. Distances from four experiments were
averaged to give the reported values. To achieve reliable comparisons
between the oligosaccharide structure of 6'GM2 and GM2, parallel NMR
analyses were conducted on GM2 and 6'GM2.
Computational Methods--
All calculations were performed using
the MacroModel/Batchmin package (20) (version 5.5) on an O2 SGI
workstation and the AMBER* sugar force field (21) augmented by modified
neglect of diatomic differential overlap (MNDO)-derived parameters for the anomeric torsion of sialic acid (22). To simplify the computational problem, all calculations on 6'GM2 were run on the methyl derivatives. The calculations were carried out using the generalized born/surface area (GB/SA) water solvation model (23) of MacroModel. This model
treats the solvent as an analytical continuum starting near the van der
Waals surface of the solute and uses a dielectric constant of 78 for
the bulk water and 1 for the molecule. Extended nonbonded cut-off
distances were used. Thus, all calculations were run with a van der
Waals cut-off of 8.0 Å and an electrostatic cut-off of 20.0 Å.
The conformational searches were carried out using the pseudo
systematic variant (SUMM) (24) of the Monte Carlo (MC)/energy minimization procedure (25). The search proceeded by a pseudo systematical alteration of the torsion angles of a starting structure (a minimum energy conformation), thus, a new geometry was generated and
energy minimized. This process produced a new minimum energy conformation, which in turn was tested for duplication with previously found conformations. The procedure, therefore, alternated random changes of coordinates (MC), which allowed wide sampling of the potential energy surface, and energy minimization. This MC/energy minimization procedure has been proven to be among the most effective methods for finding nearly all conformations of flexible molecules with
low energy. All the anomeric torsion
,
C and
, the
C5-C6
torsion, the side chain
C6
C7 and C8
C9
torsion of the Neu5Ac residue, and the C
N bonds were used as
explicit MC variables.
Energy minimization was performed using the Truncated Newton Conjugate
Gradient (TNCG) procedure (26) and was terminated either after 200 iterations or when the energy gradient RMS fell below 0.1 kJ/mol Å.
All conformers that differed from the global minimum energy
conformation by no more than 50 kJ/mol were saved, and comparison was
made only on the heavy atoms to avoid duplicate conformations. Thus,
only the global minimum for each conformer was retained, independent of
the OH conformations. After addition of explicit H atoms on the sugars,
they were further subjected to energy minimization to reduce the energy
gradient RMS to 0.01 kJ/mol Å. Fifty four conformers with minimum
energy that differed from the global minimum energy conformation by no
more than 10 kJ/mol were found. Among them, 15 were contained within 4 kJ/mol and 37 within 8 kJ/mol from the global minimum.
 |
RESULTS AND DISCUSSION |
Hydrolysis of GM2 and 6'GM2 by Hex A and Clostridial
Sialidase--
The enzymatic susceptibilities of GM2 and 6'GM2 were
examined, and the results are shown in Fig.
1. While the
GalNAc
1
4(Neu5Ac
2
3)Gal
- in GM2 is resistant to Hex A
(Fig. 1A, a), the
GalNAc
1
6(Neu5Ac
2
3)Gal
- in 6'GM2 is readily hydrolyzed by
Hex A without the assistance of GM2 activator protein (Fig.
1A, b). We subsequently examined the
susceptibility of the Neu5Ac in 6'GM2 to clostridial sialidase, and the
result is shown in Fig. 1B. The hydrolysis of the Neu5Ac in
GM2 by clostridial sialidase required the assistance of GM2 activator,
whereas the Neu5Ac in 6'GM2 could be readily hydrolyzed by clostridial
sialidase in the absence of GM2 activator protein. These results
clearly indicate that the mutation of the GalNAc linkage in GM2 from
GalNAc
1
4Gal to GalNAc
1
6Gal affects not only the enzymatic
susceptibility of the GalNAc but also that of the Neu5Ac which is
linked to C3 of the Gal in both gangliosides.

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Fig. 1.
Thin layer chromatograms showing the
susceptibility of the GalNAc and the Neu5Ac in GM2 and 6'GM2 to
enzymatic hydrolysis. A, hydrolysis of the GalNAc from
GM2 and 6'GM2. Hex, Hex A; h, hour; m,
minute; 6'M2, 6'GM2. For the hydrolysis of GM2, 0.25 unit of
Hex A was used. For the hydrolysis of 6'GM2, only 0.06 unit of Hex A
was used because this ganglioside was completely hydrolyzed by 0.25 unit of Hex A in less than 5 min. B, hydrolysis of the NeuAc
from GM2 and 6'GM2. CS, clostridial sialidase;
Act, GM2 activator protein. Detailed conditions are
described under "Experimental Procedures."
|
|
The quantitative comparison on the hydrolyses of GM2 and 6'GM2 by human
Hex A in the presence or absence of GM2 activator protein is presented
in Table I. Under our assay conditions, GM2 was not hydrolyzed by Hex A in the absence of GM2 activator. The
resistance of GM2 to Hex A has been widely recognized. However, in the
presence of the activator protein, GM2 became susceptible to Hex A. In
contrast, 6'GM2 was readily hydrolyzed by Hex A without GM2 activator
protein. To obtain a comparable level of the hydrolysis for both
substrates, it was necessary to use only 0.06 units of Hex A for 6'GM2
hydrolysis and 0.25 units of the enzyme for GM2 hydrolysis (see Table
I). This suggests that 6'GM2 is a better substrate for Hex A. Moreover,
in the presence of the activator protein, the hydrolysis of 6'GM2
increased only 5-10%. These results indicate that GM2 activator
protein does not stimulate the two substrates equally. Thus, GM2
activator protein has a specific stimulatory effect on the hydrolysis
of GM2 by Hex A.
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Table I
Hydrolysis of GM2 and 6'GM2 by human Hex A in the presence of GM2
activator
Each assay contained 8 nmol of GM2 or 6'GM2 with or without 1 µg of
GM2 activator in 100 µl of 10 mM sodium acetate buffer, pH 4.6. For
the hydrolysis of GM2, 0.25 unit of Hex A was used. For the hydrolysis
of 6'GM2, 0.06 unit of Hex A was used. The mixtures were incubated at
37 °C for a preset time as indicated, and the percent hydrolysis of
the substrate was determined from the TLC plate using a Scan Jet 2C/ADF
scanner (Hewlett-Packard).
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|
Susceptibility of the Oligosaccharides Derived from GM2, 6'GM2, and
GA2 to Hex A--
To eliminate any possible influence by the ceramide
moiety of the glycolipids on the enzymatic hydrolysis of the GalNAc
residue, the ceramide residue was removed from the glycolipids, GM2,
6'GM2 and GA2, to yield the oligosaccharides OM2, 6'OM2 and OA2,
respectively. As shown in Fig. 2, both
6'OM2 and OA2 were readily hydrolyzed by Hex A alone. In contrast, OM2
was completely resistant to Hex A. Similarly, the Neu5Ac of 6'OM2, but
not that of OM2, was susceptible to clostridial sialidase (results not
shown). Inclusion of GM2 activator protein in the reaction mixture did
not alter the above results because this activator protein requires the
lipid moiety of the substrate to exert its stimulatory activity. These
results further support that the resistance of GM2 to Hex A and
clostridial sialidase is because of the rigid carbohydrate structure of
GM2-epitope.

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Fig. 2.
Thin layer chromatogram showing the
susceptibility of the GalNAc in OM2, OA2, and 6'OM2 to enzymatic
hydrolysis. OM2, OA2, and 6'OM2 are the oligosaccharides derived
from GM2, GA2, and 6'GM2, respectively. For each incubation, 0.1 unit
of Hex A was used. SL, sialyl lactose; Hex, Hex
A; h, hour; Lac, lactose. Detailed conditions are
described under "Experimental Procedures."
|
|
Conformational Analysis of GM2 and 6'GM2--
To understand the
conformation/function relationship of GM2 and 6'GM2, we have also
investigated the conformational properties of these two gangliosides by
high resolution NMR spectroscopy and molecular mechanics computation,
applying an MC conformational search. As shown in Fig.
3A, the pattern of
interresidual NOE interactions detected on GM2 is consistent with a
single prevalent spatial arrangement of the
GalNAc
1
4(Neu5Ac
2
3)Gal- trisaccharide. In particular,
because of the interaction of the GalNAc with the Neu5Ac residue (Fig.
3A), the GalNAc-Gal bond shows a limited flexibility. Such a
conformational restriction is confirmed by the MC calculation (Figs.
4 and 6A) and is in agreement
with the previous results on the conformation of GM2-oligosaccharide
(27).

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Fig. 3.
The ROESY interaction maps.
Two-dimensional ROESY sections are shown for GM2 (A) and
6'GM2 (B), composed by three regions of the two-dimensional
maps: the GalNAc-H1 (III-H1), the Neu5Ac-OH8
(A-OH8), and the GalNAc-NH(III-NH) regions.
I, Glc; II, Gal; III, GalNAc;
A, Neu5Ac. The interactions critical for the definition of
the oligosaccharide conformation are marked. Note the loss of the
GalNAc/Neu5Ac interactions in 6'GM2.
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Fig. 4.
The ,
maps of the GalNAc-Gal glycosidic bond. The
lowest energy conformations within 8 kJ/mol from the global minimum of
the MC conformational searches performed on the methyl derivatives of
the oligosaccharides of GM2
(GalNAc 1 4(Neu5Ac 2 3)Gal 1 4Glc OMe) and 6'-GM2
(GalNAc 1 6(Neu5Ac 2 3)Gal 1 4Glc OMe) are shown.
Glycosidic angles , maps of GM2 ( ) and 6'GM2 ( ) are
defined as follows: for GM2, = GalNAcH1-GalNAcC1-O1-GalC4, = GalNAcC1-O1-GalC4-GalH4; for 6'GM2, = GalNAcH1-GalNAcC1-O1-GalC6,
= GalNAcC1-O1-GalC6-GalC5. The GalNAc-Gal glycosidic bond of GM2
appears to populate a single well at , 30°, 20°, which is in
agreement with the experimental data shown in Fig. 3. In contrast, a
marked flexibility is observed around the GalNAc-Gal glycosidic bond of
6'GM2. The GalNAc anomeric torsion appears to be well defined
around 45°, but the Gal C6-O6 bond is freely switching from the two
conformations at 90° (global minimum shown in Fig.
6B) and +70° (1.2 kJ/mol above the global minimum
shown in Fig. 6C). Another conformer of the Gal C6-O6 bond
which has energy higher than 8 kJ/mol appears around +180°.
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In sharp contrast, a considerable spatial flexibility is found for the
GalNAc residue of 6'GM2. As shown in Fig. 3B, the NOE interactions across the GalNAc
1
6Gal linkage of 6'GM2 consist uniquely of the contacts between the anomeric proton of the GalNAc and
the two protons on the C6 of the Gal; the intensities of the two
interactions are almost equal. Another important interresidual interaction detected on the GalNAc of 6'GM2 is between the GalNAc-NH and the Glc-OH3 (Fig. 3B). Fig.
5 shows that these three contacts cannot
be achieved simultaneously in a single conformation. The MC
analysis suggests that they arise from the average of two
conformations of the GalNAc
1
6Gal linkage at
,
45°,
90°, and 45°, +70° (Fig. 4). Fig. 5 also shows how
the
90° conformers give rise to the GalNAcNH-GlcOH3 and
GalNAcH1-GalH6' contacts, whereas the
+70° conformers generate
the GalNAcH1-GalH6 cross-peaks. The distances calculated by Boltzmann
averaging (28) of the MC conformations in Fig. 5 agree well with the
experimental data (see legend to Fig. 5). The interresidual
interactions between the Neu5Ac and the Gal protons in 6'GM2 are
centered on the side chain of the Neu5Ac, showing the contacts of
Neu5Ac-H8 and -OH8 with Gal-H3 (Fig. 3B). These contacts are
not detected in the trisaccharide GalNAc
1
4(Neu5Ac
2
3)Gal- of
GM2, or in the same trisaccharide found in GM1, GD1a, and GalNAc-GD1a
(17, 27, 29-31). The conformations of these gangliosides are highly
restrained at the Neu5Ac-Gal glycosidic bond in an antiperiplanar
conformation with the side chain of the Neu5Ac interacting with the
GalNAc residue. This is not the case for 6'GM2. The interactions of
Neu5Ac-H8 and -OH8 with GalH3 in 6'GM2 suggest that the most populated
conformation of the Neu5Ac-Gal bond in this ganglioside is a gauche
conformation. The MC calculations corroborate and strengthen the
results of NMR analyses and also enable comparison of the lowest energy
conformations of the oligosaccharide moieties of GM2 (Fig.
6A) and 6'GM2 (Fig. 6,
B and C).

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Fig. 5.
The distances of GalNAc-H1/Gal-H6,
GalNAc-H1/Gal-H6', and GalNAc-NH/Glc-OH3 in the conformations for
6'GM2. The three distances are shown on a single three-axis graph.
Points in the graph correspond to the distances obtained from the MC
lowest energy conformations within 8 kJ/mol from the global minimum.
Point correspondence to the GalNAc-Gal bond 90° and 70°
conformations of Fig. 4 is also highlighted. The figure
shows the incompatibility of those distances with a single GalNAc-Gal
preferred conformation. The experimentally derived distances of the
three contacts are 2.8, 3.0, and 3.4 Å, respectively. The threshold
for the detection of NOE interactions is 4 Å. GalNAc-NH/Glc-OH3
(z axis) for the points corresponding to the 90°
conformations of the GalNAc-Gal bond is <4 Å (Fig. 4). They represent
conformations with the distances of GalNAc-H1/Gal-H6' that are
shorter than that of GalNAc-H1/Gal-H6. The Boltzmann mediated
distances (2.5, 2.7, and 3.3 Å, respectively) obtained by the MC
calculation are in good agreement with the experimental values.
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Fig. 6.
The spatial arrangements of the
oligosaccharide moieties in GM2 (A) and 6'GM2
(B and C). Representative
conformers are shown for GM2 and 6'GM2 as calculated by molecular
mechanics. Conformers are taken from the lowest energy conformations
within 8 kJ/mol. The computational results were validated by comparing
the calculated distances with the experimental (NOE-derived) values.
The calculated distances were obtained as r = ri 6 1/6 where
ri 6 is the Boltzmann average
of ri 6 of the individual
conformations within 10 kJ/mol from the global minimum. The
GalNAc 1 4(Neu5Ac 2 3)Gal 1- trisaccharide of GM2 shows one
preferred spatial arrangement which is defined by a number of
experimentally detected interactions (arrows). The
GalNAc 1 6(Neu5Ac 2 3)Gal 1- of 6'GM2, on the contrary, shows
a fairly "opened" spatial arrangement, with the GalNAc-Gal bond
sampling the two conformations as shown in Fig. 4. The conversion of
GM2 to 6'GM2 dramatically alters the spatial disposition of the Neu5Ac
residue because of the loss of its interactions with the GalNAc that
exists in the very compact GalNAc 1 4(Neu5Ac 2 3)Gal 1-
trisaccharide structure of GM2. The significantly more populated
conformation found in GM2 is at , 160°, 25°, whereas in
6'GM2 the lower energy is at , 70°, +10° (gauche
conformation).
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The conformational studies of 6'GM2 and GM2 reveal that the
modification of the GalNAc linkage in GM2 from
1
4Gal to
1
6Gal dramatically alters the dynamics of the sugar chain. The
overall effect of this modification is to give the oligosaccharide head group of 6'GM2 an "open" spatial arrangement (Fig. 6, B
and C) in which the GalNAc and the Neu5Ac residues are
freely accessible to external interactions. Indeed, it has been shown
that a flexible ligand has a lower affinity to its receptor than a
rigid ligand fitting perfectly to the binding site. However, a flexible
ligand with a high degree of mobility can increase the rate of
association (32).
The above results clearly show that the specific trisaccharide
structure of GM2-epitope in which the GalNAc
1
4 and the
Neu5Ac
2
3 are both linked to the penultimate Gal residue can
render the GalNAc and the Neu5Ac resistant to enzymatic hydrolysis. The
disruption of this rigid structure by mutating the GalNAc
1
4Gal
linkage to GalNAc
1
6Gal makes the GalNAc susceptible to Hex A and
the Neu5Ac to sialidase. A well known example where the linkage
mobility is closely associated with the ganglioside-protein
interactions is that of GD1a ganglioside. GD1a is a disialoganglioside
whose structure can be regarded as having an extension of the sugar chain of GM2 by adding an extra Neu5Ac
2
3Gal
- disaccharide to the C3 of the GalNAc. It is widely known that, of the two
Neu5Ac
2
3Gal
- linkages in GD1a, only the external one is
susceptible to sialidase. The inner Neu5Ac
2
3Gal
- linkage of
GD1a is part of the trisaccharide in GM2, which is very rigid because
of the presence of a "blocking" GalNAc residue. The external
Neu5Ac
2
3Gal
- linkage, absence of the "blocking" GalNAc
residue, is more flexible and fluctuates between two limiting
conformations (31). Thus, the susceptibility of the Neu5Ac in GD1a to
sialidase is associated with the dynamics of the Neu5Ac
2
3Gal
- linkage.
Although complex carbohydrates have been recognized as information rich
molecules, functions expressed by complex carbohydrate chains are still
not readily decipherable as those of proteins and nucleic acids. The
primary structure of a complex carbohydrate chain includes the sugar
sequence, the anomeric configurations of the sugar units, and the
linkages of the two adjacent sugar residues. It is not difficult to
understand that the anomeric configuration of a sugar residue can
profoundly affect physicochemical and biological properties of a sugar
chain. However, the effect of a single sugar linkage on the overall
physicochemical and biological properties of a sugar chain is still not
well appreciated. Our results show that the biological properties of a
sugar chain can be expressed through the specific linkage of the sugar
chain. Our investigation through chemical mutation of sugar linkages provides an explanation for the resistance of GM2 to enzymatic degradation.
 |
ACKNOWLEDGEMENTS |
We thank Drs. A. D. French, S. J. Landry, and K. Maskos for critical review of the manuscript and helpful discussions.
 |
FOOTNOTES |
*
This research was supported by the National Institutes of
Health Grant NS 09626 (to Y.-T. L.) and CNR 96.03182.CT04 (to S. S.).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: Dept. of Biochemistry,
Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA
70112. Tel.: 504-584-2459; Fax: 504-584-2739; E-mail:
yli{at}tmcpop.tmc.tulane.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
GM2, GalNAc
1
4(NeuAc
2
3)Gal
1
4Glc
1-1'Cer;
6'GM2, GalNAc
1
6(NeuAc
2
3)Gal
1
4Glc
1-1'Cer;
asialo-GM2
(GA2), GalNAc
1
4Gal
1
4Glc
1-1'Cer;
GM3, NeuAc
2
3Gal
1
4Glc
1-1'Cer;
OM2, GalNAc
1
4(NeuAc
2
3)Gal
1
4Glc;
6'OM2, GalNAc
1
6(NeuAc
2
3)Gal
1
4Glc;
OA2, GalNAc
1
4Gal
1
4Glc;
Hex A,
-N-acetylhexosaminidase A;
Lac, lactose;
SL, sialyl-lactose;
TLC, thin layer chromatography;
NOE, nuclear Overhauser
effect;
MC, Monte Carlo;
ROESY, rotating frame nuclear Overhauser
enhancement spectroscopy.
 |
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