From the Neurology Centre of Excellence for Drug Discovery,
GlaxoSmithKline, New Frontiers Science Park North, Third Ave., Harlow,
Essex CM19 5AW, United Kingdom
Received for publication, January 16, 2001, and in revised form, March 6, 2001
Myelin-associated glycoprotein (MAG) is expressed
on myelinating glia and inhibits neurite outgrowth from post-natal
neurons. MAG has a sialic acid binding site in its N-terminal domain
and binds to specific sialylated glycans and gangliosides present on
the surface of neurons, but the significance of these interactions in
the effect of MAG on neurite outgrowth is unclear. Here we present
evidence to suggest that recognition of sialylated glycans is essential
for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is
known to make a key contact with sialic acid. We show that mutation
of this residue reduces the potency of MAG inhibitory activity but that
residual activity is also a result of carbohydrate recognition. We then
go on to investigate gangliosides GT1b and GD1a as candidate MAG
receptors. We show that MAG specifically binds both gangliosides and
that both are expressed on the surface of MAG-responsive neurons.
Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a,
mimics the effect of MAG, in that neurite outgrowth is inhibited
through activation of Rho kinase. These data strongly suggest that
interaction with GT1b on the neuronal cell surface is a potential
mechanism for inhibition of neurite outgrowth by MAG.
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INTRODUCTION |
Expression of myelin-associated glycoprotein (MAG; siglec
4a),1 is restricted to
myelinating glial cells on myelin membrane adjacent to the axon and is
required for maintenance of myelin integrity (1-4). In
vitro, MAG inhibits outgrowth of postnatal neurons (5-8),
involving activation of Rho GTPase, a key signaling step for the
inhibitory effect of myelin on regeneration of neurons in
vivo (9). MAG is therefore thought to contribute to the inhibitory
properties of myelin, which is in part responsible for the lack of
regenerative capacity of the central nervous system after injury or
disease (10, 11).
Like other siglecs, MAG binds to sialic acid residues at the termini of
glycans on opposing cells through a sialic acid binding site located in
the N-terminal V-set Ig domain (12-22). MAG binds specifically to
terminal sialic acid residues in
2-3 linkage to galactose, which
occurs in glycans linked
1-3 to GalNAc or GlcNAc or
1-4 to
GlcNAc (12, 23-25). Use of sialic acid analogues has identified
specific groups on sialic acid essential for interaction with MAG (26),
consistent with interactions seen between sialic acid and conserved
amino acids in the siglec 1 crystal structure (21). It is also thought
that the core glycan structure on which the terminal sialic acid is
presented plays a role in recognition by MAG (23, 26, 27). The ability
of MAG to bind specific gangliosides bearing terminal
2-3-linked
sialic acid has been well documented. Gangliosides bind to MAG with the
relative potencies GQ1b
> GT1a
, GD1
> GD1a, GT1b
GM3, GM4, whereas GM1, GD1b, GD3, and GQ1b do not support
adhesion (23, 27, 28).
Although the binding of MAG to sialylated glycans and gangliosides is
well characterized, the functional importance of these interactions to
the inhibition of neurite outgrowth by MAG is unclear. MAG binding to
neurons is dependent on the presence of cell surface sialic acid (7).
Furthermore, addition of exogenous sugars or neuraminidase treatment of
neurons reduces the effect of MAG on neurite outgrowth (7), suggesting
that the interaction between MAG and sialylated cell surface receptors
results in inhibition of neurite outgrowth. However, mutation of
arginine 118, an amino acid which is predicted to form hydrogen bonds
with the carboxylate group of sialic acid (21), failed to inactivate
the protein (29). This led to the suggestion that a second, sialic
acid-independent, site on MAG interacts with an unknown
counter-receptor on neurons, triggering the intracellular signaling
cascade leading to inhibition of neurite outgrowth (29).
In this report, we investigate the roles of sialic acid and ganglioside
recognition by MAG in the inhibition of neurite outgrowth. We show
that, in the absence of arginine 118, the potency of MAG is
significantly reduced, but the residual inhibitory activity also
involves carbohydrate recognition. We then show that interaction of MAG
with ganglioside GT1b is a potential mechanism for the inhibitory
effect of MAG on neurite outgrowth. These results strongly suggest that
GT1b represents a potential receptor for MAG, mediating inhibition of
neurite outgrowth.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Unless otherwise specified, all reagents
were purchased from Sigma Chemical Co. (UK). Tissue culture media and
B27 supplement were from Life Technologies (Paisley, UK). BCA protein
quantification kit was purchased from Pierce (Chester, UK). Anti-MAG
antibody (MAB1567) and isotype control antibody were purchased
from Chemicon (Harrow, UK). SDS-polyacrylamide gel electrophoresis gels
were purchased from Bio-Rad. ECL reagents were from Amersham Pharmacia Biotech (UK). Neu5Ac, 3'-sialyllactose, and purified gangliosides were
purchased from Sigma. Anti-GT1b (clone GMR5, an IgM) and biotinylated
anti-GT1b and anti-GD1a (clone GMR17, an IgM) were purchased from
Seikagaku America (Falmouth, MA). Anti-GM1 (an IgG) was from Cambio
(Cambridge, UK) and A2B5 (an IgM that recognizes uncharacterized
ganglioside(s) (30, 31)) was from Roche Molecular Biochemicals
(Mannheim, Germany). TRITC-conjugated anti-mouse immunoglobulin was
from Dako (Cambridge, UK) and streptavidin-Texas red was from Amersham
Pharmacia Biotech. Y27632 was from Tocris (Bristol, UK).
Recombinant Protein Production--
The constructs MAG-Fc/pIg
and MAGR118A-Fc/pIg have been described elsewhere (29) and
were kindly provided by Prof. M. T. Filbin. The rat SIRP-Fc/pIg
construct consisted of the three extracellular N-terminal Ig-like
domains fused to human IgG1 and was provided by Dr. L. Vernon-Wilson.
Recombinant protein was produced by transient transfection of COS-7
cells as described previously (12).
ELISA--
ELISA was carried out using standard methods (19,
29). Briefly, a 96-well plate was coated with 10 µg/ml goat
anti-human IgG overnight at 4 °C. After washing, Fc proteins were
applied at varying concentrations and incubated at 37 °C for 2 h. Wells were washed and incubated with anti-MAG antibody at 10 µg/ml
for 1 h. This was followed by visualization using anti-mouse
horseradish peroxidase and O-phenylenediamine substrate.
Primary Neuronal Cell Culture--
The hippocampi of gestational
day 18 rat embryos were dissected out, incubated in trypsin (0.08%, 30 min at 37 °C), and dissociated mechanically (32). Hippocampal cells
were resuspended in neurobasal medium supplemented with B27,
anti-oxidants, 1 mM glutamine, 25 µM
glutamate, and 1 mM pyruvate, and plated at a density of
3000 cells/well into 96-well dishes that had previously been coated with poly-D-lysine followed by 10% FCS. Cerebellar granule
neurons were prepared from postnatal day 8 Harlan Sprague-Dawley rat
pups. Cerebella were enzyme-digested, triturated, and plated into
poly-L-lysine-coated 96-well plates at a density of 20,000 cells/well in Eagle's basal medium supplemented to contain 25 mM KCl, 10% FCS, and 50 µg/ml gentamicin (32).
Neurite Outgrowth Assays--
One hour after plating primary
neuronal cells, Fc proteins or anti-ganglioside antibodies at various
concentrations were added in equal volumes of PBS to triplicate wells.
All antibodies used in these assays were previously dialyzed against
cell culture medium. For preincubation experiments, 10 µg/ml
anti-MAG or control antibody or various concentrations of sugars were
incubated with MAGR118A-Fc for 1 h at room temperature
prior to addition to cells. For ganglioside preincubation experiments,
gangliosides were reconstituted at 25 mg/ml in chloroform:methanol
(1:1) and stored at
20 °C. Fresh stock solutions of 250 µg/ml
ganglioside in 1% fatty acid-free bovine serum albumin in PBS in glass
tubes were prepared by vigorous vortexing immediately before each
experiment. Gangliosides were diluted to 2 µg/ml into Fc protein
solution in sterile glass tubes and incubated for 1 h at room
temperature before being added to cultures as above. After 24 h
(cerebellar granule neurons) or 48 h (hippocampal cells), cells
were fixed with 4% paraformaldehyde for 1 h on ice, washed with
PBS, and stained using Coomassie Blue (11). Briefly, 50 µl of 0.1%
Coomassie Blue R-250 (in 40% methanol, 10% acetic acid) were added
per well and incubated for 30 s. Stain was tipped off and the
wells were washed three times with PBS. Assays were quantified using a
KS300 image analysis system (Imaging Associates, UK). For each cell
measured, the length from the edge of the cell to the end of the
longest neurite was measured for 100 cells/well for each treatment in
triplicate. Results are expressed as a percentage of the length of
neurites of cells treated with control-treated with PBS alone. The
length of neurites from control-treated neurons varied between
experiments (between 35 and 50 µm for hippocampal neurons after
48 h of culture and 20-40 µm for cerebellar neurons cultured
for 24 h). Therefore control-treated cells were included on every
96-well plate and results for each treatment in an experiment were
expressed as a percentage of the length of control-treated cells. This
allowed data from three independent experiments to be pooled. Data
points therefore represent mean and S.E. of data pooled from three
independent experiments.
Immunocytochemistry--
Primary neuronal cells were
plated onto 8-well chamber slides coated as described above. After
24 h (cerebellar granule neurons) or 48 h (hippocampal
neurons), cells were fixed as above and stained by standard
immunocytochemistry techniques. 10 µg/ml primary antibody (or isotype
control) or biotinylated cholera toxin in PBS/10% FCS was incubated
overnight at 4 °C. Secondary reagents were then incubated at room
temperature for 1 h as follows: for biotinylated anti-GT1b and
cholera toxin, 1:200 dilution of streptavidin-Texas Red; for anti-GD1a
and A2B5, 1:30 dilution of anti-mouse-TRITC.
 |
RESULTS |
Arginine 118 Is Required for Optimal Inhibition of Neurite
Outgrowth by MAG--
To assess their structural integrity, MAG-Fc and
MAGR118A-Fc recombinant proteins were tested in an ELISA
assay using the anti-MAG monoclonal antibody, which recognizes a
conformation-dependent epitope. MAG-Fc and
MAGR118A-Fc reacted identically in a
dose-dependent manner (Fig.
1a) indicating that both
proteins were correctly folded.

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Fig. 1.
Comparison of MAG-Fc and
MAGR118A-Fc. a, MAG-Fc and
MAGR118A-Fc are both recognized by anti-MAG monoclonal
antibody. Fc proteins were coated onto wells of a 96-well dish
previously coated with anti-human Fc antibody. After washing, 10 µg/ml anti-MAG monoclonal was added, followed by anti-mouse
horseradish peroxidase and O-phenylenediamine
substrate. Data points are means and standard errors from a typical
experiment. b, MAG-Fc and MAGR118A-Fc inhibit
neurite outgrowth from primary cultured hippocampal neurons.
Hippocampal neurons were plated at 3000 cells/well into a 96-well
plate. Cells were allowed to adhere before adding Fc proteins in equal
volumes of PBS. Cells were cultured for 48 h, fixed and stained
with Coomassie Blue. Data are expressed as percentage neurite length of
wells treated with PBS alone and are means and standard errors pooled
from three independent experiments. , MAG-Fc; ,
MAGR118A-Fc; human IgG; SIRP-Fc; MAGR118A-Fc
plus mouse IgG; MAGR118A-Fc plus anti-MAG monoclonal
antibody, human IgG; , SIRP-Fc; , MAGR118A-Fc plus mouse
IgG; , MAGR118A-Fc plus anti-MAG mAb.
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To investigate the role of arginine 118 in inhibition of neurite growth
by MAG, neurite outgrowth experiments were carried out in primary
cultured hippocampal neurons. Consistent with previous reports, MAG-Fc
potently inhibited neuronal outgrowth (Fig. 1b). SIRP-Fc
(consisting of the three extracellular N-terminal Ig-like domains of
rat SIRP (33) fused to the Fc region of human IgG1) had no effect on
neurite outgrowth from hippocampal neurons (Fig. 1b),
consistent with previous observations using cerebellar granule neurons
(34), therefore showing that inhibition of outgrowth by MAG-Fc was
specific for MAG and not a result of the presence of the Fc region.
MAGR118A-Fc also inhibited neurite outgrowth but with lower
potency compared with the unmutated form (Fig. 1b). This
inhibition was specific when compared with negative control Fc protein
SIRP-Fc, and was reversed with the anti-MAG antibody mAb 513 but not
mouse IgG1 isotype control. A similar effect was seen for neurite
outgrowth from cerebellar granule neurons (data not shown).
Neurite Outgrowth Inhibition by MAGR118A-Fc Is Blocked
by Preincubation with Mono- and Trisaccharides--
To investigate the
basis for the residual activity of MAGR118A-Fc, we carried
out neurite outgrowth assays using MAGR118A-Fc in the
presence of mono- and trisaccharides. The sugars tested did not affect
neurite length when added to concentrations up to 5 mM in
the absence of MAGR118A-Fc (data not shown). Inhibition of
neurite outgrowth by MAGR118A-Fc was blocked by
preincubation with
-methyl sialic acid (Neu5Ac) and the sialylated
trisaccharide 3'-sialyllactose (Neu5Ac
2-3Gal
1-4Glc) (Fig.
2); however, incubation with 0.1 mM 6'-sialyllactose did not significantly reverse
inhibition (data not shown). This suggests that MAGR118A-Fc
inhibits neurite outgrowth via recognition of sialylated glycans on the
surface of neurons.

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Fig. 2.
Neurite outgrowth activity of
MAGR118A-Fc is blocked by small sugars.
MAGR118A-Fc (300 nM) was preincubated with
increasing concentrations of -methyl sialic acid 3'-sialyllactose
prior to addition to cultured hippocampal neurons and outgrowth assays
were carried out as described for Fig. 1.
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MAG and MAGR118A Interact with Gangliosides GT1b and
GD1a--
Specific gangliosides are known to bind MAG via their
carbohydrate epitopes with relative potencies GQ1b
> GT1a
,
GD1
> GT1b, GD1a
GM1. The recent availability of
anti-ganglioside antibodies (35, 36) has provided the opportunity to
investigate the functional significance of these interactions. Although
MAG binds to the
-series gangliosides with higher affinity, their expression in brain is very low (0.5, 0.9, and 0.3 mg/kg for GQ1b
, GT1a
, and GD1
, respectively) compared with that of GD1a and GT1b
(abundance of GD1a is 1200 mg/kg), which are among two of the four
major brain gangliosides (27). If gangliosides are involved in neurite
outgrowth inhibition in response to MAG, GD1a and GT1b would be
predicted to be involved, because MAG affects all neuronal types tested.
To examine whether GD1a and GT1b could compete with endogenous neuronal
MAG receptors for MAG binding, we preincubated Fc protein with
gangliosides prior to addition into neurite outgrowth assays (37).
Preincubation of MAG-Fc with a mixture of brain gangliosides, purified
GT1b or GD1a but not asialo GM1 or GM1, blocked the inhibitory action
of MAG (Fig. 3a). The
inhibitory activity of MAGR118A-Fc was also partially
reversed by GT1b or GD1a preincubation (Fig. 3b).

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Fig. 3.
Neurite outgrowth activity of MAG and
MAGR118A is blocked by preincubation with purified
gangliosides GT1b and GD1a. MAG-Fc (60 nM,
a) or MAGR118A-Fc (300 nM,
b) were added to hippocampal neurons alone (Cont)
or following preincubation with 2 µg/ml of a mixture of gangliosides
(Mix) or individual purified gangliosides. Neurite outgrowth
assays were carried out as described for Fig. 1. aGM1,
asialo GM1.
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GT1b and GD1a Are Expressed on the Surface of Primary Neurons That
Respond to MAG--
Immunocytochemistry using anti-ganglioside
antibodies confirmed the expression of GT1b and GD1a on the surface of
surface of primary cerebellar and hippocampal neurons (Fig.
4). Both these antibodies showed most
intense staining associated with cell bodies. Weaker staining was
observed on some neurites. Ganglioside GM1 and the A2B5 antigen (an
uncharacterized epitope carried by several gangliosides) were also
found to be expressed on the surface of these cells (Fig. 4). A lack of
fluorescence for cells stained with secondary antibody alone confirmed
specificity (data not shown).

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Fig. 4.
Ganglioside expression on the surface of
primary neuronal cells. Hippocampal neurons (a)
or cerebellar neurons (b) were fixed and stained using
antibodies against GT1b or GD1a, the A2B5 antigen, or biotinylated
cholera toxin (CTOX), which recognizes GM1.
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GT1b Mediates Inhibition of Neurite Outgrowth--
The effect of
anti-ganglioside antibodies on neurite outgrowth was then assessed. If
MAG inhibits neurite outgrowth by binding to either GD1a or GT1b on the
surface of neurons, antibodies recognizing these gangliosides may be
expected to mimic the effect of MAG on neurite outgrowth. Anti-GT1b
antibody, but not isotype control or antibodies recognizing GD1a, GM1,
or the A2B5 antigen inhibited neurite outgrowth in a
dose-dependent manner (Fig.
5, a and b). The
specificity of this effect was demonstrated by preincubation of
anti-GT1b antibody with purified gangliosides. Preincubation with GT1b
but not with other gangliosides blocked the inhibition of neurite
outgrowth by the anti-GT1b antibody (Fig. 5c).

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Fig. 5.
Anti-GT1b antibody inhibits neurite
outgrowth. Antibodies were added to hippocampal neurons in equal
volumes of PBS and neurite outgrowth assays carried out as described
for Fig. 1. a, anti-GT1b, but not antibodies recognizing
other gangliosides, inhibits neurite outgrowth. Isotype control or
anti-ganglioside antibodies were added to a final concentration of 5 µg/ml. b, anti-GT1b inhibits neurite outgrowth in a
dose-dependent manner. Increasing concentrations of
anti-GT1b antibody were added to cells. c, inhibition of
neurite outgrowth by anti-GT1b is blocked by preincubation of antibody
with purified GT1b, but not other gangliosides. 1 µg/ml anti-GT1b
antibody was added to cells alone or following preincubation
with purified gangliosides as described for Fig. 3. Cont,
IgM isotype control.
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Inhibition of Neurite Outgrowth by MAG-Fc or Anti-GT1b Antibody Is
Reversed by a Rho Kinase Inhibitor--
Rho GTPase has been shown to
be a key signaling step for the inhibitory effect of myelin on
regeneration of neurons in vivo (9). Furthermore, inhibition
by MAG has been shown to be blocked by C3 exoenzyme, a specific
inhibitor of Rho (9). We investigated the effect of Y27632, a specific
inhibitor of Rho kinase, a downstream effector of Rho, on inhibition of
neurite outgrowth by MAG and anti-GT1b antibody. Y27632 blocked
inhibition of neurite outgrowth by both MAG and anti-GT1b antibody from
cerebellar and hippocampal neurons (Fig.
6).

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Fig. 6.
The Rho kinase inhibitor Y27632 blocks
inhibition of neurite outgrowth by MAG or anti-GT1b antibody.
a, Y27632 blocks neurite outgrowth inhibition by MAG. 60 nM Fc protein was added to untreated neurons or neurons
pretreated with 10 µM Y27632. b, Y27632 blocks
neurite outgrowth inhibition by anti-GT1b antibody. 1 µg/ml anti-GT1b
was added neurons as described in a. Neurite outgrowth
assays were carried out as described in Fig. 1. , hippocampal
neurons; , cerebellar granule neurons.
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 |
DISCUSSION |
Carbohydrate Recognition Is Essential for Inhibition of
Neurite Outgrowth by MAG--
The blockade of the inhibitory activity
of MAGR118A-Fc with sialic acid, 3'-sialyllactose, and
specific gangliosides shown in this study suggests that this mutated
form of MAG retains the ability to bind carbohydrate ligands and that
these sugars and gangliosides, when added exogenously, can compete with
endogenous neuronal receptors. Although these findings do not rule out
interactions between MAG and other neuronal cell surface molecules,
they strongly suggest that MAG inhibits neuronal outgrowth by
recognition of sialylated glycans on the neuronal cell surface.
From the crystal structure of siglec 1 (sialoadhesin) complexed
with 3'-sialyllactose, it is evident that amino acids other than
arginine 118 are involved in binding sialic acid (21). These are
conserved in MAG and include tryptophan 2, a key amino acid for sialic
acid recognition (21). Furthermore, components of the core glycan on
which sialic acid is presented are able to increase the binding
affinity of MAG for sialic acid either through direct contacts or more
favorable presentations of sialic acid, increasing the affinity of MAG
for sialic acid itself (23, 26). The reduced potency of inhibition of
MAGR118A-Fc compared with the unmutated protein is
therefore consistent with a reduced affinity of binding due to the loss
of arginine 118.
Inhibitors of neurite outgrowth in myelin may prevent aberrant
sprouting under steady-state conditions (38). The activity of these
molecules may require regulation to prevent unwanted neuronal
retraction. Linking carbohydrate recognition by MAG to biological
function raises the possibility that the effect of MAG on neurons
during steady-state or disease conditions could be regulated by changes
in lectin activity. Two mechanisms of regulation of lectin activity for
other siglecs have been identified. First, "cis " interactions between siglecs (including MAG) and sialylated glycans on
the same cell surface (and perhaps on siglecs themselves) have been
shown to block binding to opposing cells (13, 14, 29, 39, 40). In the
case of siglec 2 (CD22), blocking of lectin activity by cis
interactions on resting B-lymphocytes can be "unmasked" on
pharmacological or physiological activation, demonstrating the
potential for this type of regulation in physiological processes (39).
Second, the ability of siglecs to bind sialic acid may be regulated
intracellularly. In the case of siglecs containing immunoreceptor
tyrosine-based inhibition motifs in their cytoplasmic domains (siglecs
2, 3, 5, 6, and 7), inside-out regulation of the ability to bind
sialylated glycans occurs through the recruitment of SHP-1 and
SHP-2 (41). Regulation of sialic acid binding therefore appears
to be an important aspect of siglec biology and may represent for MAG a
mechanism of regulation of activity.
Gangliosides as Neuronal Receptors for MAG--
The sialic acid
binding site on MAG creates the potential to bind to many molecules
(protein and lipid) bearing the correctly presented terminal sialic
acid (23, 27, 28, 42, 43). Extensive studies on the ability of MAG to
bind the carbohydrate domains of gangliosides exists in the literature,
and if sialic acid recognition by MAG is essential for inhibition of
neurite outgrowth by MAG, then gangliosides are candidate MAG
receptors. A receptor for MAG that mediates inhibition of neurite
outgrowth would be expected to (a) directly interact with
MAG, (b) be expressed on the surface of cells that respond
to MAG, and (c) be able to trigger the appropriate
signaling cascade that results in neurite outgrowth. In this paper, we
have shown that the ganglioside GT1b fulfills all of these criteria,
and therefore, MAG-GT1b interaction is a possible mechanism for
inhibition of neurite outgrowth. The anti-GT1b antibody was raised
against purified ganglioside and specifically binds to GT1b but
not to other gangliosides tested (Fig. 5c and Seikagaku
America product information). However, it is possible that GT1b-like
carbohydrate epitopes exist on carrier molecules other than
gangliosides. Therefore, the possibility remains that both MAG and the
anti-GT1b mAb recognize molecules other than gangliosides.
A MAG receptor that mediates inhibition of neurite outgrowth may also
be expected to be present in neurites and growth cones. The
immunocytochemistry presented in this paper suggests that GT1b is
present on the surface of cells that respond to MAG, although the more
intense staining is localized to the cell bodies with much weaker
staining in neurites. However, the presence of GT1b has been
demonstrated biochemically on purified growth cone membranes (24.4% of
the sialic acid at the growth cone is carried by GT1b) (44). Therefore,
the absence of strong staining on neurites and growth cones is likely
to reflect the use of this particular IgM for staining. A similar
result was recently reported for an anti-GD1a IgG that showed more
robust GD1a localization than the previously available IgM (45).
GD1a has been shown to bind MAG with equal potency as GT1b (23);
however, our results show that cross-linking of cell surface GD1a does
not affect neurite outgrowth. This result may reflect the precise
epitope recognized by the anti-GD1a antibody. An alternative explanation may be that regulated expression of different gangliosides on the cell surface may in turn regulate the activity of MAG. The
-series gangliosides have been shown to bind MAG with considerably greater potencies than GD1a and GT1b (27) but have an extremely low
relative abundance in brain. It is not known whether the interaction of
MAG with these gangliosides also results in inhibition of neurite outgrowth or whether they are expressed at critical times to regulate the activity of MAG.
Studies on knockout mice support the theory that interaction between
MAG and gangliosides mediates the effects of MAG on neurons. The
phenotype of MAG-deficient mice closely resembles that of mice lacking
complex gangliosides. This includes decreased central myelination,
axonal degeneration in the central and peripheral systems, and
demyelination of peripheral nerves (1-4, 46).
Potential Mechanisms for Neurite Outgrowth Inhibition Mediated by
GT1b--
Gangliosides have been implicated in the modulation of many
neuronal functions (47). Glycosphingolipids (including gangliosides) are known to exist in domains (known as lipid rafts,
detergent-insoluble glycosphingolipid-enriched domains, caveoli, or
caveoli-like domains) on the cell surface (48). Also enriched within
these domains are GPI-linked molecules on the outer leaflet of the
membrane and signal transducing molecules on the intracellular side
(48, 49). These domains are known to exist on the surface of neuronal cells and have been implicated in processes, including signal transduction, cell adhesion, and lipid/protein sorting (50-52). On
neurons, ~60% of cell surface gangliosides are found in
sphingolipid-enriched domains (53), and the correct structure of these
domains and glycosylation of gangliosides within them is essential for
neuritogenesis (54, 55).
Within glycosphingolipid-enriched domains, glycosphingolipids are known
to make several types of interaction. Glycolipids segregate into
domains through hydrogen bonding between ceramide domains and
carbohydrate-carbohydrate interactions (49). Furthermore, gangliosides
have been shown to interact with proteins within lipid-enriched domains
(growth factor receptors, GPI-linked molecules, and signal transducers)
and several of these types of interactions have been shown to occur on
neurons (49, 56). GM1 interacts directly with the BDNF receptor TrkB,
and the quantity of GM1 at the cell surface directly modulates TrkB
activity (57-59). GM3 is localized to domains enriched in c-Src, RhoA,
and FAK and forms a close association with c-Src (54). GD3 interacts
directly with the GPI-linked protein TAG-1 and with the src-family
kinase Lyn in cerebellar granule neurons (55, 60). Interaction of gangliosides with extracellular molecules or ganglioside-specific antibodies can trigger intracellular signaling and cellular response, due to modulation of their endogenous interactions. In neurons, anti-GM3 antibody activates c-Src and inhibits melanoma cell growth (61, 62). In neuronal cells, anti-GD3 antibody treatment leads to Lyn
activation and phosphorylation of an 80-kDa protein (60), an event that
mimics cross-linking of TAG-1 (55).
Specific gangliosides have also been shown to be associated with
integrins at focal adhesions (63-64) and to modulate integrin-mediated adhesion (65, 66). In neuronal cells, binding to disialogangliosides by
tenascin-C, tenascin-R, or an anti-GD2 antibody causes inhibition of
protein kinase C and prevention of integrin-dependent
adhesion to fibronectin, resulting in inhibition of neurite
outgrowth (37, 67).
It is clear that gangliosides play an important role in modulating
intracellular signaling within lipid-enriched domains and at focal
adhesions and that interactions between gangliosides and extracellular
molecules can modulate cellular responses in a specific manner. GT1b
has been shown to interact with proteins (68). MAG binding to GT1b on
the surface of neurons may therefore modulate GT1b interactions within
the neuronal plasma membrane, resulting in inhibition of neurite outgrowth.
We thank Prof. Marie T. Filbin for the MAG-Fc
and MAGR118A-Fc constructs and Dr. Liz Vernon-Wilson for
SIRP-Fc. We also thank Jack Alden for help with protein production and
Drs. Paula Green, Gayle Chapman, and Rabinder Prinjha for helpful discussions.
The abbreviations used are:
MAG, myelin-associated glycoprotein;
siglec, sialic acid-binding Ig-related
lectin;
Neu5Ac,
-methyl sialic acid;
3'-sialyllactose, Neu5Ac
2-3Gal
1-4Glc;
TRITC, tetramethylrhodamine
isothiocyanate;
ELISA, enzyme-linked immunosorbent assay;
FCS, fetal calf serum;
PBS, phosphate-buffered saline;
mAb, monoclonal
antibody; ganglioside nomenclature is that of Suennerholm
(69)..
1.
|
Li, C. M.,
Tropak, M. B.,
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