From the Zentrum für Molekulare Neurobiologie, Universität Hamburg, Martinistr. 52, D-20246 Hamburg, Germany
Received for publication, February 27, 2001, and in revised form, March 23, 2001
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ABSTRACT |
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Among the recognition molecules that determine a
neuron's interaction with other cells, L1 and CD24 have been suggested
to cooperate with each other in neurite outgrowth and signal
transduction. Here we report that binding of CD24 to L1 depends on
Path-finding of growth cones and neurite outgrowth toward targets
are important events in the developing and regenerating nervous system
and in synaptic remodeling during learning and memory. Axonal guidance
depends on molecules at the cell surface and in the extracellular
matrix. The different and often changing combinations of molecularly
associated recognition molecules at the cell surface are important
determinants of the ways by which the cell surface communicates with
the cell interior, where cell surface signals are integrated to
influence cell behavior.
Two recognition molecules, L1 of the immunoglobulin superfamily and
CD24, a highly glycosylated mucin type glycoprotein, interact with each
other functionally (1, 2). L1 is a 200-kDa homophilic and heterophilic
adhesion molecule expressed by many postmitotic neurons in the central
nervous system (for reviews, see Refs. 3 and 4). It is one of the most
potent promoters of neurite outgrowth in vitro known so far.
Mutants of L1 in mice and men strongly underscore its importance during
embryonic development in vivo (3, 5, 6).
CD24 is linked to the surface membrane by a glycosyl
phosphatidylinositol anchor and is, therefore, unable to directly
interact with cytoplasmic proteins. It is also known as heat-stable
antigen or nectadrin with a peptide core of only 30 amino acids (for
references, see Ref. 1). Similar to L1, it is highly expressed by
neurons (7, 8). The apparent molecular weight of CD24 varies
considerably among cell types and also within each cell type, depending
on its developmental stage due to differences in glycosylation pattern (for references, see Refs. 1 and 7). These observations suggest that
post-translational modifications of CD24 play an important functional
role. CD24 acts as a co-stimulator for various physiological functions.
In the nervous system, CD24 has been reported to interact with L1 to
stimulate cell adhesion and to increase intracellular Ca2+
levels (1, 2). Interestingly, CD24 has been shown to inhibit neurite
outgrowth of neonatal retinal ganglion cells and dorsal root ganglion
neurons in culture (8) by yet unknown signal transduction mechanisms.
Based on these observations on the functional interplay and molecular
association between L1 and CD24, we decided to further study their
functional interdependence. Here we report that L1 is a sialic
acid-binding lectin for CD24 and that CD24 inhibits neurite outgrowth
of dorsal root ganglion neurons and promotes neurite outgrowth of
cerebellar neurons via interaction in trans-position with L1 at the
cell surface of the neurite outgrowth-competent cell. Our experiments
appear to underscore four important observations in neural cell
interactions: 1) the pivotal function of glycans as mediators of
interactions between neural recognition molecules in trans-association,
2) the characterization of L1 as a sialic acid-binding lectin and
identification of the siglec domain (9) in the first fibronectin type
III homologous repeat of the L1 family, 3) the implication of L1 as a
signal-transducing cell surface receptor for CD24-induced cell
type-specific effects on neurite outgrowth, and 4) the importance of
the intracellular signal transduction machinery of a particular
neuronal cell type that determines whether neurite outgrowth
enhancement or inhibition result from cell surface interactions.
Animals--
Outbred ICR wild type mice were used for
purification of L1 and CD24, and C57BL/6J mice were used for
subfractionation. ICR wild type, CD24-deficient (CD24 Antibodies--
Polyclonal antibodies against recombinant L1-Fc
fusion protein comprising the whole extracellular part of mouse
L1,1 rat monoclonal antibody
555 against mouse L1, or monoclonal rat antibody 79 reacting
with mouse CD24 were used (11).
Purification of L1 and CD24--
L1 was purified by
immunoaffinity chromatography from 2-3-day-old ICR mouse brains as
described (11). For purification of CD24, frozen brain tissue was
thawed on ice and homogenized with 10 volumes of cold acetone
( Synthetic Peptide--
The 30-mer comprising the total protein
backbone of mouse CD24 was obtained by solid phase peptide synthesis on
a Milligen 9050 continuous flow peptide synthesizer following
preparative HPLC. After purification, the peptide was more than 95%
pure as determined by analytical HPLC.
Biotinylation--
Proteins were diluted to a concentration of 1 mg/ml in phosphate-buffered saline (PBS), pH 8.0. 50 µl of NHS-biotin
(Calbiochem-Novabiochem) dissolved in PBS at a concentration of 5 mg/ml
was added to 1 mg of protein in 1 ml of PBS. After an incubation time
of 4 h at room temperature, reaction mixtures were desalted on
PD10 columns and stored at 5 °C.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analysis--
SDS-polyacrylamide gel electrophoresis was performed on
10 or 12.5% gels. Proteins were visualized by Coomassie or silver staining. Proteins separated by SDS-polyacrylamide gel electrophoresis were transferred electrophoretically onto nitrocellulose membranes (BA
85; Schleicher & Schüll). For antibody detection assays, nitrocellulose membranes were incubated for 1 h at room
temperature in blocking buffer (TBS or PBS containing 5% skim milk
powder), washed three times with TBS or PBS, and incubated at room
temperature for 1 h with monoclonal antibody 79 or 555 (1 µg/ml). After washing three times with TBS or PBS, filters were
incubated for 1 h with alkaline phosphatase or horseradish
peroxidase-conjugated secondary antibody in blocking buffer. Filters
were then washed five times with TBS or PBS. Bound antibodies were
detected using 37.5 µl of 5-bromo-4-chloro-3-indolyl-phosphate (50 mg/ml in 100% dimethylformamide) and 50 µl of 4-nitro blue
tetrazolium chloride (75 mg/ml in 70% dimethylformamide) in 10 ml of
AP buffer (0.1 M Tris, 50 mM MgCl2, 0.1 M NaCl, pH 9.5). Alternatively, detection was done by
ECL using Super Signal (Pierce). Glycans were characterized by lectin detection assays using the Glycan Differentiation Kit (Roche Molecular Biochemicals).
Desialylation of CD24 and L1--
100 µg of CD24 was diluted
in 250 µl of TBS containing 0.2% (w/v) CHAPS, 1 mM
CaCl2, 1 mM MgCl2, and 1 mM MnCl2 and then 250 µl of
neuraminidase-agarose (1 unit/360 µl) from Vibrio cholerae (Sigma) in the same buffer was added. The mixture was incubated at
37 °C in a head-over-head mixer for 2 h. After centrifugation, the supernatant was collected, and the free sialic acids were removed
by centrifugation using Centricon 10 tubes. To remove the sialic acid
from L1 and CD24 in solid phase, L1 and CD24 were coated to 96-well
microtiter plates at a concentration of 3 µg/ml at 4 °C overnight.
Upon washing with TBS, 1 milliunit of neuraminidase from V. cholerae (Roche Molecular Biochemicals) in 100 µl of enzyme incubation buffer (50 mM Tris, pH 7.2, containing 0.1%
bovine serum albumin, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2,
and 0.05% Tween 20) was added to each well and incubated at 37 °C
for 2 h.
Binding Assays--
Purified L1 at a concentration of 3 µg/ml
in TBS was coated into 96-well microtiter plates (Maxisorp from Nunc,
Wiesbaden, Germany) at 4 °C overnight. After washing with
TBS, 1% bovine serum albumin in TBS was added and incubated for 1 h at room temperature. After washing three times with TBST (TBS with
0.05% Tween 20), the different proteins diluted in buffer A (TBST
containing 1% bovine serum albumin, 1 mM
CaCl2, 1 mM MgCl2, and 1 mM MnCl2) were added and incubated for 1 h
at room temperature. After washing with TBS, the plates were incubated
with biotinylated affinity-purified monoclonal antibodies (40 ng/ml in
buffer A) and streptavidin coupled to horseradish peroxidase (200 ng/ml
in buffer A) for 1 h at room temperature. 100 µl of freshly
prepared staining solution (2% (w/v) ABTS in 100 mM sodium
acetate buffer, pH 4.2, and 0.001% (v/v) H2O2)
was added, and, depending on its intensity, the reaction was stopped
after 10 to 15 min by the addition of 0.6% (w/v) SDS. Finally, bound
conjugates were quantified by measuring the absorbance at 405 nm.
Neurite Outgrowth Assays for Dorsal Root Ganglion and Cerebellar
Neurons--
Dorsal root ganglion neurons from 0- and 5-day-old mice
and cerebellar neurons from 5-day-old mice were prepared as described (12). In brief, 96-well plates (Nunc) were pretreated with 0.01% poly-L-lysine (PLL) for 1-2 h at 37 °C, washed twice
with water, and air-dried. Proteins and protein mixtures as indicated
were substrate-coated at a concentration of 10 µg/ml onto the dried surfaces for 4 h at 37 °C in a humidified atmosphere. The
plates were washed three times with Ca2+- and
Mg2+-free Hank's balanced salt solution, and neurons were
plated at a density of 2000 cells/well (dorsal root ganglion neurons)
or 4000 cells/well (cerebellar neurons) in 100 µl of chemically
defined medium (13). For antibody blocking experiments, monoclonal
antibody 79 against CD24, affinity-purified polyclonal L1 antibody, and nonimmune rabbit IgG at a concentration of 100 µg/ml were added to
the wells 5 min after cells had been seeded. After 18 h, cells were fixed without a preceding washing step by the gentle addition of
25% glutaraldehyde to a final concentration of 2.5%. After fixation,
cultures were stained with toluidine blue, and morphological parameters
were quantified with an IBAS image analysis system (Kontron, Milan,
Italy). For morphometric analysis, only cells without contact with
other cells were evaluated. Neurites were defined as those processes
with a length of at least one cell body diameter. To determine the
total neurite length per cell, 50 cells in each of two wells were
analyzed per experiment. The data were analyzed by analysis of variance
and the Newman-Keuls test, with p < 0.05 and
p < 0.01 being considered significant or highly
significant, respectively. All graphs comprise data derived from at
least three independent experiments.
Subfractionation of Membranes from Mouse Brain--
Brains were
removed from 7-day-old C57BL/6J mice and homogenized in homogenization
buffer (0.32 M sucrose in buffer H: 50 mM
Tris/HCl, pH 7.4, 1 mM CaCl2, 1 mM
MgCl2, and 1 mM NaHCO3) using a
Potter homogenizer and applying 10 strokes. All steps were carried out
at 4 °C. The homogenate was centrifuged for 10 min at 700 × g, and the resulting supernatant was further centrifuged at
17,000 × g. The pellet was resuspended in
homogenization buffer and applied on top of a discontinuous sucrose
gradient consisting of 1.2, 1.0, 0.8, and 0.65 M sucrose in
buffer H. After centrifugation for 2 h at 100,000 × g, the turbid material from the different interfaces was
collected, diluted with buffer H, and pelleted by centrifugation for 30 min at 100,000 × g. Western blot analysis showed that
the membrane fraction collected from the 1.0/1.2 M sucrose
interface contained both L1 and CD24, whereas membranes collected from
the other interfaces did not contain L1 together with CD24. Myelin was
prepared according to Norton and Poduslo (14).
The pelleted membrane fraction from the 1.0/1.2 M interface
was resuspended in buffer H. To disrupt noncovalent protein
interactions, one volume of the resuspended membrane fraction was
supplemented with one volume of either 10 mM EDTA or 300 mM NaHCO3, pH 10, and incubated for 1 h on
ice. For the selective solubilization of proteins, cold Triton X-100
was added to the membrane suspension to a final concentration of 1%
and incubated for 1 h on ice. The samples were then centrifuged
for 30 min at 120,000 × g. To isolate detergent
insoluble lipid microdomains of low density (rafts) the Triton
X-100-insoluble membrane fraction was adjusted to 1.2 M
sucrose using 2.34 M sucrose in buffer H and overlaid with
1.1 and 0.32 M sucrose in buffer H. After centrifugation
for 2 h at 120,000 × g, the material at the
0.32/1.1 M sucrose interface was collected, diluted with
buffer H, and pelleted by centrifugation for 30 min at 120,000 × g.
For immunoprecipitation, the Triton X-100-insoluble membrane fraction
was first incubated with the polyclonal L1 antibody for 2 h at
4 °C and then with protein A/G beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 °C. The beads were pelleted and
washed five times with buffer H. Proteins bound to the beads were
eluted by boiling the beads at 100 °C in SDS-sample buffer.
Purification of L1 and CD24 from Mouse Brain--
Since L1 and
CD24 tend to bind to each other, particular attention was paid to the
isolation procedure to assure a maximal degree of purity. L1 was
directly isolated from homogenates of early postnatal mouse brain,
while CD24 was purified after extracting lipids from the brain tissue
homogenate by acetone. The purity of the L1 and CD24 preparations was
checked by Coomassie and silver staining as well as by Western blot
analysis. Immunoaffinity-purified L1 yielded two bands with apparent
molecular masses of 200 and 180 kDa as detected by Coomassie staining
(Fig. 1, lane 1),
silver staining (Fig. 1, lane 2), and
immunostaining with monoclonal L1 antibodies (Fig. 1, lane
3). The CD24 preparation yielded a smear characteristic of
highly glycosylated proteins in which three bands with apparent
molecular masses of ~33, ~30, and ~27 kDa could be detected,
although not very distinctly. When characterizing the three bands as
three glycoforms of CD24, we refer to the upper, middle, and lower
parts of the smear. The smear containing the three faint bands could be
visualized only by silver staining but not by staining with Coomassie
(Fig. 1, lanes 5 and 6). Three bands
could usually be recognized by monoclonal antibody 79 (Fig. 1,
lane 7). The L1 preparation did not contain any
immunoreactivity for CD24 (Fig. 1, lane 4); nor
did the CD24 preparation contain any L1 immunoreactivity (Fig. 1,
lane 8).
Different Glycoforms of CD24--
Glycans of purified CD24 and L1
were characterized by lectin affinity blotting. The three glycoforms of
CD24 showed a different extent of reactivity with Datura
stramonium agglutinin (DSA; Fig. 1, lane
9), which recognizes Gal Glycosylation-dependent Binding of CD24 to L1--
It
has been shown that CD24 binds to L1 (1, 2). To assess the importance
of glycan chains on the CD24 molecule for its binding to L1, we
investigated in a solid phase immunoassay whether the two molecules
interact with each other via their glycan chains. Binding of CD24 to
immobilized L1 was dose-dependent and saturable, whereas a
synthetic full-length CD24 polypeptide devoid of glycans did not bind
to immobilized L1 (Fig. 2). We also
analyzed whether the binding of CD24 to L1 depends on Inhibitory Effect of CD24 on Neurite Outgrowth from Dorsal Root
Ganglion Neurons--
To assess the functional importance of the
glycan chains on CD24 for neurite outgrowth, dorsal root ganglion
neurons from neonatal mice were maintained on different
substrate-coated proteins. Total neurite length per cell was
significantly reduced on the CD24 substrate in comparison with the
poly-L-lysine control (Fig. 3A, compare CD24 with PLL).
Neurons plated either onto the CD24 peptide or asialo-CD24 were
not inhibited in their neurite outgrowth when compared with the
poly-L-lysine control (Fig. 3A, compare CD24
peptide and asialo-CD24 with PLL). A monoclonal antibody against CD24
and polyclonal antibodies against L1 were able to reduce or inhibit the
negative influences on neurite outgrowth elicited by substrate-coated
CD24 but not on poly-L-lysine (Fig. 3A, compare
CD24 + anti-CD24 and CD24 + anti-L1 with CD24 and PLL), whereas
nonimmune IgG control antibodies had no effect (Fig. 3A,
compare CD24 + IgG with CD24), indicating that the L1 antibody binds to
L1 at the cell surface of dorsal root ganglion neurons and masks it for
binding to CD24 in trans-interaction.
When neurite outgrowth was assayed on L1-coated substrates, neurite
outgrowth was enhanced over the poly-L-lysine control (Fig.
3B, compare L1 with PLL). In the presence of L1 antibodies, neurite outgrowth was reduced to control levels (Fig. 3B,
compare L1 + anti-L1 with PLL and L1). Antibodies to CD24, which were able to neutralize the inhibitory effect of substrate-coated CD24, did
not affect the enhanced neurite outgrowth on L1 (Fig. 3B, compare L1 + anti-CD24 with L1). As control, nonimmune IgG did not
reduce the enhanced neurite outgrowth on substrate-coated L1 (Fig.
3B, compare L1 + IgG with L1).
Neurite outgrowth was considerably reduced on the mixture of L1 with
CD24 in comparison with that seen on the L1 substrate and was rather
similar to that observed on the CD24 substrate alone (Fig.
3B, compare L1 + CD24 with PLL, L1, and CD24). The CD24
peptide and asialo-CD24 had no effect on L1-enhanced neurite outgrowth
(Fig. 3B, compare L1 + CD24 peptide and L1 + asialo-CD24 with PLL and L1).
These experiments show that in mixture with L1, CD24 completely
abolishes the enhancing effect of L1 on neurite outgrowth and thus
predominates over L1. That the inhibitory effect of CD24 on L1-enhanced
neurite outgrowth is abolished when sialic acid is removed or glycans
on CD24 are absent indicates that this interaction depends on glycans
and, in particular, on sialic acid residues present on CD24.
CD24 Inhibits L1- but Not CD24-mediated Neurite Outgrowth of Dorsal
Root Ganglion Neurons--
To investigate the functional consequences
of the interaction between CD24 and L1 on neurite outgrowth, neurite
outgrowth from dorsal root ganglion neurons from neonatal wild type
mice was compared with neurite outgrowth from CD24- or L1-deficient mice. In these experiments, dorsal root ganglion neurons from knockout
mutant mice were maintained on different substrates, and total lengths
of neurites per cell were measured. Neurons from CD24-deficient animals
responded in the same manner as neurons from the wild type animals on
the different substrates; neurite outgrowth of CD24-deficient neurons
was substantially reduced on the CD24-coated substrate (Fig.
3C, compare CD24 with PLL), was not reduced on asialo-CD24
(Fig. 3C, compare asialo-CD24 with PLL and CD24), and was
significantly better on the L1-coated substrate when compared with the
poly-L-lysine control (Fig. 3C, compare L1 with
PLL and CD24). When neurons were cultured on the mixture of L1 with
CD24, neurite outgrowth was considerably reduced when compared with
that on the L1-coated substrate and was similar to neurite outgrowth on
the CD24 substrate alone (Fig. 3C, compare L1 + CD24 with L1
and CD24). Asialo-CD24, when coated in a mixture with L1, had no
influence on the L1-induced enhancement of neurite outgrowth (Fig.
3C, compare L1 + asialo-CD24 with CD24 and L1).
When neurons from L1 knockout mice were used, substrate-coated L1 no
more enhanced neurite outgrowth, and CD24 no more inhibited neurite
outgrowth (Fig. 3D, compare L1 and CD24 with PLL).
Similarly, the mixture of L1 and CD24 did not show any effects
different from those on the control substrate (Fig. 3D,
compare L1 + CD24 with PLL). These experiments indicate that L1 and not
CD24 is the molecule at the cell surface of neonatal dorsal root
ganglion neurons that mediates the inhibitory effect of CD24 on neurite outgrowth.
CD24 Promotes Neurite Outgrowth of Cerebellar Neurons--
To
investigate whether the inhibitory effect of CD24 was due to unique
functions of CD24 or whether CD24 exerts different effects on different
neuronal cell types, cerebellar neurons from 5-day-old mice were
maintained on different substrates. Cells cultured on the CD24-coated
substrate extended longer neurites than on the
poly-L-lysine control (Fig.
4A, compare CD24 with PLL).
Asialo-CD24 did not enhance neurite outgrowth (Fig. 4A, compare asialo-CD24 with PLL). On L1-coated substrates, cells had even
longer neurites than on CD24 or poly-L-lysine (Fig.
4A, compare L1 with CD24 and PLL). On the mixture of CD24
with L1, neurons extended neurites longer than on the
poly-L-lysine control, but neurite lengths were
significantly reduced when compared with L1 alone and comparable with
CD24 alone (Fig. 4A, compare L1 + CD24 with PLL, L1, and
CD24). Asialo-CD24 was not neurite outgrowth-promoting or -inhibiting
in substrate mixture with L1 (Fig. 4A, compare L1 + asialo-CD24 with L1). These results show that CD24 predominates in its
effect over that of L1 and that the different responses to neurite
outgrowth depend on the neuronal cell type.
CD24 Enhances L1-mediated but Not CD24-mediated Neurite Outgrowth
of Cerebellar Neurons--
Next, the interaction between CD24 and L1
and the response on the neurite outgrowth of cerebellar neurons was
further investigated using neurons from CD24- or L1-deficient mice. In
these experiments, neurite lengths of cerebellar neurons from wild type
and knockout mutant mice maintained on different substrates were
determined. Neurons from CD24-deficient animals and from wild type
animals showed comparable neurite outgrowth on the different
substrates; neurite outgrowth was substantially enhanced on the
CD24-coated substrate (Fig. 4B, compare CD24 with PLL) but
less extensive than on the L1 substrate (Fig. 4B, compare L1
with PLL and CD24), while the mixture of L1 and CD24 enhanced neurite
outgrowth to the level of the CD24 substrate only (Fig. 4B,
compare L1 + CD24 with L1 and CD24). In contrast, cerebellar neurons
from L1 knockout mice showed no enhanced neurite outgrowth on L1, CD24,
or a mixture of L1 and CD24 when compared with the
poly-L-lysine control (Fig. 4C, compare L1,
CD24, and L1 + CD24 with PLL). These experiments indicate that L1, but
not CD24, is involved in L1 and CD24 substrate-dependent enhancement of neurite outgrowth of cerebellar neurons.
Distinct Glycoforms of CD24 Interact with Neuronal L1--
The
interaction of L1 and CD24 was investigated biochemically by analyzing
the distribution of L1 and CD24 in membrane subfractions from 7-day-old
mouse brain obtained by sucrose gradient centrifugation. Western blot
analysis of the different membrane fractions showed that L1 was
exclusively found in a membrane fraction obtained from the 1.0/1.2
M sucrose interface (data not shown), which has been
reported to contain synaptosomal membranes. In this membrane fraction,
the three glycoforms of CD24 (27, 30, and 33 kDa) were detected by the
CD24 antibody (Fig. 5A). The
astrocyte marker glial fibrillary acidic protein was also present in
this fraction, whereas the oligodendrocyte marker MAG was not
detectable (Fig. 5A). In contrast, analysis of a crude
myelin fraction as a control showed that the preparation contained no
L1 and negligible amounts of CD24 and glial fibrillary acidic protein
but large amounts of MAG (Fig. 5A).
The co-distribution of the three glycoforms of CD24 with L1 was further
studied. If L1 and CD24 were associated by trans-interaction, it should
be possible to disrupt this association either with EDTA or sodium
bicarbonate at alkaline pH. For this reason, membranes were subjected
to further sucrose gradient centrifugation after those treatments.
Western blot analysis of residual membranes showed that the three
glycoforms were present in the mock-treated membranes. The EDTA-treated
membranes also showed all glycoforms, indicating that the membrane
association of CD24 does not depend on divalent cations. In contrast,
in the membranes treated with alkaline bicarbonate, only the 27-kDa
glycoform was detectable, whereas the 30- and 33-kDa glycoforms were
absent (Fig. 5B). The amount of L1 did not change
significantly in the differently treated membranes (Fig.
5B). From these results, we infer that the 30- and 33-kDa
glycoforms of CD24 and L1 are located in the trans-position, while the
27-kDa glycoform and L1 are located in the cis-position on the same membrane.
In parallel experiments, the membrane fraction containing L1 and CD24
was treated with 1% Triton X-100 at 4 °C, and detergent-insoluble material was separated from detergent-soluble proteins. Part of the
detergent-insoluble material was applied to a sucrose floatation gradient to isolate lipid raft microdomains (for a review, see Ref.
15). Western blot analysis of the different membrane subfractions showed that the 27-kDa glycoform is predominantly detergent-insoluble and thus operationally present in rafts (Fig. 5C), while the
30- and 33-kDa glycoforms of CD24 are completely detergent-soluble (Fig. 5C). The vast majority of L1 was detergent-soluble,
while only very small amounts were detergent-insoluble and thus
raft-associated (Fig. 5C).
Since L1 and the 30- and 33-kDa glycoforms of CD24 showed a predominant
co-distribution in the detergent-soluble fraction, we analyzed whether
L1 and these glycoforms of CD24 would interact. Immunoprecipitates with
L1 antibody were probed by Western blot analysis with CD24 antibody
revealing co-immunoprecipitation of the 30- and 33-kDa glycoforms with
L1 (Fig. 5D). The lectin M. amurensis agglutinin,
which specifically binds to In this study, we present evidence that the interaction between L1
and CD24- and CD24-induced neurite outgrowth depends on the presence of
Consensus sequences in sialic acid binding recognition molecules
of the immune and nervous systems have been recognized in the siglecs,
previously called the sialoadhesin family: sialoadhesin, CD33, the
myelin-associated glycoprotein MAG, Schwann cell myelin protein, and
CD22 (for a review, see Ref. 16, and for a new nomenclature, see Ref.
9). These consensus sequences contain a crucial arginine residue in the
F-strand of the immunoglobulin fold that is flanked by hydrophobic
amino acids (see Ref. 17 and references therein). Sialoadhesion, CD33,
and MAG, which prefer NeuAc2,3-sialic acid on CD24, which determines the CD24 induced and cell
type-specific promotion or inhibition of neurite outgrowth. Using
knockout mutants, we could show that the CD24-induced effects on
neurite outgrowth are mediated via L1, and not GPI-linked CD24, by
trans-interaction of L1 with sialylated CD24. This glycoform is
excluded together with L1 from raft microdomains, suggesting that
molecular compartmentation in the surface membrane could play a role in
signal transduction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) (10), and
L1-deficient (L1
/y) (5) mice were used for cell culture.
20 °C) using a Potter homogenizer. The residue was recovered by
filtration on a G3 glass filter under reduced pressure, and the
collected tissue pieces and powder were again homogenized in cold
acetone and filtered. All of the following steps were carried out at
4 °C. The freshly prepared acetone powder was washed twice in 25 ml
of Hepes-buffered saline (10 mM Hepes, 150 mM
NaCl, pH 7.4) by incubation for 15 min in a head-over-head mixer and
subsequent centrifugation at 20,000 × g for 15 min. The washed residue was homogenized and agitated for 2 h in 50 ml
of Hepes-buffered saline containing 1% (w/v) deoxycholate, 0.1 mM phenylmethylsulfonyl fluoride, and 0.02% (w/v)
NaN3. After centrifugation at 100,000 × g
for 60 min, the supernatant was collected and applied to an affinity
column prepared by coupling monoclonal antibody 79 to CNBr-activated
Sepharose B (Amersham Pharmacia Biotech). After loading, the column was
extensively washed with buffer A (10 mM Hepes, pH 7.4, 150 mM NaCl, 2% Triton X-100, 0.02% NaN3),
followed by washing with buffer B (10 mM Hepes, pH 7.4, 500 mM NaCl, 0.1% Triton X-100, 0.02% NaN3) and
with 50 ml of Hepes-buffered saline. Bound CD24 was eluted with buffer C (50 mM ethanolamine, pH 11.5, 150 mM NaCl,
0.2% (w/v) CHAPS).2 The
eluate was immediately neutralized by addition of 1 M
Tris/HCl, pH 6.7. The CD24-containing fractions were pooled and
dialyzed against Tris-buffered saline (TBS; 50 mM Tris/HCl,
pH 7.2, 150 mM NaCl) containing 0.2% CHAPS. The fractions
were concentrated by centrifugation using Centricon tubes 10 (Amicon,
10-kDa cut-off; Millipore Corp., Bedford, MA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of L1 and CD24 purified from mouse
brain. L1 and CD24 were separated on 10% SDS-polyacrylamide gels
and visualized by Coomassie (lanes 1 and
5) and silver staining (lanes 2 and
6) or transferred to nitrocellulose membranes. For
immunodetection of L1 or CD24, monoclonal antibodies 555 against L1
(lanes 3 and 8) and 79 against CD24
(lanes 4 and 7) were used, while for
lectin affinity blotting of purified CD24 (lanes
9-13) or asialo-CD24 (lane 14;
+SAase), digoxigenin-labeled agglutinins from D. stramonium (DSA; lane 9),
G. nivalis (GNA; lane 10),
M. amurensis (MAA; lane
11), S. nigra (SNA; lane
12), and peanut (PNA; lanes
13 and 14) were applied.
1,4-GlcNAc, whereas no reactivity was observed for Galanthus nivalis agglutinin
(GNA; Fig. 1, lane 10), which is
specific for terminal mannose residues. These results indicate the
absence of hybrid or oligomannosidic type oligosaccharides and the
presence of complex type oligosaccharides on CD24. The 30- and 33-kDa
glycoforms of CD24 were recognized by Maackia amurensis agglutinin (MAA; Fig. 1, lane 11) but
not by Sambucus nigra agglutinin (SNA; Fig. 1,
lane 12), showing that these glycoforms are
solely sialylated in
2,3- but not in
2,6-linkage,
whereas the 27-kDa glycoform does not appear to be sialylated. Peanut
agglutinin, which recognizes the unsubstituted terminal
Gal
1,3-GalNAc core unit of O-linked mucin type glycan
chains, binds to the 30- and 33-kDa glycoforms of CD24 only upon the
removal of sialic acid residues by neuraminidase and not without
removal. These observations indicate that only the 30- and 33-kDa
glycoforms of CD24 carry
2,3-linked sialic acid on the
O-glycan core unit Gal
1,3-GalNAc and not the 27-kDa
glycoform. However, it could not be determined whether the 30- and
33-kDa glycoforms also carry
2,3-linked sialic acid residues on
complex type N-glycans.
2,3-linked
sialic acid residues. Therefore, sialic acid-free CD24 was generated by
treatment with neuraminidase from V. cholerae, which removes
terminal
2,3-linked sialic acid residues. In its asialo form, CD24
no longer bound to L1 (Fig. 2). These observations indicate that the
binding of CD24 to L1 depends on its glycan chains and that sialic acid
residues on CD24 are essential for the binding to L1.
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Fig. 2.
Binding of CD24 to immobilized L1. L1
was coated to 96-well microtiter plates and incubated with different
amounts of CD24, asialo-CD24, or CD24 peptide. Binding was evaluated
through enzyme-linked immunosorbent assay using biotinylated monoclonal
antibody 79 against CD24 and streptavidin-horseradish peroxidase.
Values from experiments carried out with different batches of purified
L1 and CD24 are shown.
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Fig. 3.
Effects of CD24 on neurite outgrowth from
dorsal root ganglion neurons. A, neurons from wild type
mice were plated as single cell suspensions on PLL-treated tissue
culture plastic coated without (PLL) or with CD24 in the
absence or presence of monoclonal antibody directed against CD24
(CD24 + antiCD24), polyclonal antibodies against
L1 (CD24 + antiL1), nonimmune IgG
(CD24 + IgG), or asialo-CD24 or CD24-peptide.
B, neurons of wild type animals were plated either on
PLL-treated tissue culture plastic coated without (PLL) or
with L1 in the absence or presence of antibodies against L1
(CD24 + antiL1) or CD24 (CD24 + antiL1) or on mixtures of L1 and CD24 (L1 + CD24), asialo-CD24 (L1 + asialoCD24),
or CD24 peptide (L1 + CD24peptide). C,
neurons from CD24-deficient mice were plated on PLL-treated tissue
culture plastic coated without (PLL) or with CD24,
asialo-CD24, L1, or a mixture of L1 and CD24 (L1 + CD24) or asialo-CD24 (L1 + asialoCD24). D, neurons from L1-deficient mice
were plated on PLL-treated tissue culture plastic coated without
(PLL) or with L1, CD24, or a mixture of L1 and CD24
(L1 + CD24). A-D, cells were
maintained for 18 h before fixation and staining with toluidine
blue. The length of total neurites per cell was determined (mean ± S.D.). Error bars indicate S.D. from at least
three independent experiments. Bars marked by
asterisks and double asterisks are
significantly (p < 0.01) different from the control
(PLL) and L1, respectively.
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Fig. 4.
Effect of CD24 on neurite outgrowth from
cerebellar neurons. A, cerebellar neurons from wild
type mice were plated as single cell suspensions onto PLL-treated
tissue culture plastic coated without (PLL) or with CD24,
asialo-CD24, and L1 and on mixtures of L1 and CD24 (L1 + CD24) or asialo-CD24 (L1 + asialoCD24). Cerebellar neurons from CD24-deficient
(B) or L1-deficient (C) mice were seeded onto
PLL-treated tissue culture plastic coated without (PLL) or
with CD24, L1, and their mixture (L1 + CD24).
A-C, cells were maintained for 18 h before fixation
and staining with toluidine blue. Length of total neurites per cell is
shown (mean ± S.D.). Error bars indicate
S.D. from at least three independent experiments. Bars
marked by asterisks and double
asterisks are significantly (p < 0.01)
different from the control (PLL) and L1, respectively.
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Fig. 5.
Analysis of membrane subfractions from adult
mouse brain. A, membranes from the 1.2/1.0
M sucrose interface (1/1.2M) and a crude myelin
preparation (my) were subjected to SDS-polyacrylamide gel
electrophoresis and Western blot analysis using antibodies against
CD24, L1, glial fibrillary acidic protein, and MAG. B,
membranes from the 1.2/1.0 M sucrose interface were
incubated in the absence (1/1.2M) or the presence of either
10 mM EDTA (+EDTA) or 150 mM
NaHCO3 at pH 10 (+NaHCO3/pH 10), separated
on sucrose gradients, and subjected to Western blot analysis using
antibodies against CD24 and L1. C, after incubation of
membranes from the 1/1.2 M sucrose interface
(1/1.2M) in the presence of 1% Triton X-100, detergent-
insoluble material (Tx-insoluble) was separated from
detergent-soluble proteins (Tx-soluble), which were
subsequently subjected to methanol precipitation. Part of the Triton
X-100-insoluble fraction was used for sucrose floatation gradient
centrifugation, and the material floating to low density was collected
(raft). The different subfractions were run on
SDS-polyacrylamide gels and analyzed with antibodies against CD24 and
L1. D, membranes from the 1/1.2 M sucrose
interface (1/1.2M) were treated with 1% Triton X-100, and
detergent-soluble material (Tx-soluble) was isolated. For
immunoprecipitation, antibodies against L1 and protein A/G beads were
added to part of this fraction. The different fractions and the
immunoprecipitates ( L1-IP) were subjected to Western blot
analysis (WB) using an antibody against CD24. The
immunoprecipitate was also analyzed for binding of the lectin M. amurensis (MAA), which reacts specifically with
2,3-linked sialic acid residues.
2,3-linked sialic acid, recognized these
glycoforms of CD24 in the L1 immunoprecipitate (Fig. 5D),
indicating that these glycoforms carry
2,3-linked sialic acid
residues (see also Fig. 1, B and C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,3-linked sialic acid residues on CD24. Lectin blot analysis of
CD24 affinity-purified from mouse brain indicates that the three major
CD24 glycoforms differ in their glycans; the 30- and 33-kDa glycoforms,
but not the 27-kDa glycoform, carry glycans substituted with terminal
2,3-linked sialic acid residues, which are crucial for binding of
CD24 to L1. These observations and the fact that the 30- and 33-kDa
glycoforms co-distribute with L1 in a detergent-soluble compartment,
whereas the 27-kDa glycoform is found in the L1-depleted lipid raft
microdomain fraction (for a review, see Ref. 15) indicate that L1
specifically recognizes
2,3-linked sialic acid on CD24 and therefore
functions as a sialic acid-binding lectin.
(2,3)-Gal contain the characteristic
YXFR motif, while CD22, which only binds to
NeuAc
(2,6)-Gal, contains LXFR. The importance of a
conserved arginine in the binding of sialic acid has been reported for
several lectins, such as galectin-3 (18) and the siglecs (17), since
the guanidinium group of the arginine forms a salt bridge with the
carboxylate group of sialic acid. The sequence found in
NeuAc
(2,3)-Gal preferring siglecs is present in the first
fibronectin type III (FNIII) domain of L1. This short sequence motif
(YXFR) is present in all members of the L1 superfamily, including F3/F11/contactin, TAG-1/TAX-1/axonin, BIG-1/PANG, NB-2/NB-3, the close homologue of L1 (CHL1), Ng-CAM, Nr-CAM, neurofascin, and
neuroglian (see Ref. 19 and references therein). The L1 family appears
to be distinct from the original siglec family of adhesion molecules by
the feature that the FNIII but not the immunoglobulin-like domains
contain the consensus sequences for sialic acid binding.
Three-dimensional structure analysis of a neuroglian fragment
comprising the first and second FNIII domain indicate that the
YXFR sequence is part of a
-sheet (Fig.
6A) (20), which, together with
the flanking sequences, is highly conserved within the L1 family of
several animal species and comprises the consensus sequence
(L/I/M)XP(W/Y/F)(V/A/M)XYXFR (V/I)XAXNXXG (Fig. 6B). Mutations of the conserved alanine to glutamic
acid or threonine were found in L1 from patients with MASA
syndrome (mental retardation, aphasia, shuffling gait, and adducted
thumbs) (21, 22). However, no gross morphological abnormalities were detectable in L1- or CD24-deficient mice in the cerebellum
(23)3 or dorsal root
ganglia3 (for abnormalities in the sensory nerves of L1
deficient mice, see Ref. 24). Interestingly, some but not all
recognition molecules containing FNIII domains show similarity in the
sialic binding consensus sequence: the netrin receptors DCC/frazzled
and neogenin (25, 26); the receptor phosphate-tyrosine phosphatases
and
, which belong to the LAR subfamily of receptor
phosphate-tyrosine phosphatases (27); the Usher syndrome protein
usherin (28); the sidekick protein (29); and the Kallmann's syndrome
protein anosmin (30). All of these molecules play crucial roles in
neurogenesis and axonal path-finding. It is noteworthy that the major
cell adhesion site that is neuritogenic in anosmin was identified as a
32-amino acid sequence (31), which contains the putative sialic binding
site proposed here. A mutation of the conserved asparagine to lysine in
anosmin from a patient with Kallmann's syndrome (30) also localizes to
the putative sialic acid binding motif. The combined observations
indicate the importance of this putative sialic acid binding motif for
nervous system development.
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Fig. 6.
Three-dimensional structure of neuroglian
fragment and identification of a consensus sequence. A,
the crystal structure of tandem type III fibronectin domains (1. FNIII, 2. FNIII) comprising amino acids 610-814 of
Drosophila neuroglian (National Center for Biotechnology
Information accession number 1CFB) is shown. -Strands are
shown in blue, extended
-turns in
green, and the
-strand containing the motif in
red. The amino acids conserved among the proteins depicted
in B are shown in capital letters.
B, identical (dark gray) and similar
(light gray) amino acids and the highly conserved
arginine (box) are highlighted. The consensus
sequence comprising the
-strand and the flanking sequences is shown
at the bottom (aromatic (
), polar (p), and
hydrophobic (
) amino acids as shown). The position number within the
sequence and the SwissProt accession numbers are given. The alanine mutated in human L1 and the asparagine mutated
in human anosmin are marked by a circle.
The binding of 2,3-linked sialic acid residues on CD24 to L1 is
likely to play a functional role in neurite outgrowth. CD24 but not
asialo-CD24 inhibits neurite outgrowth of dorsal root ganglion neurons
and promotes neurite outgrowth of cerebellar neurons. Sialic acid
residues on L1 are not required for binding to CD24 and promotion of
neurite outgrowth, since desialylation of L1 did not interfere with
neurite outgrowth3 and since procaryotically expressed L1
fragments also promote neurite outgrowth, although less efficiently on
a molar basis than L1 isolated from eucaryotic systems (see Ref. 32 and
references therein). It is interesting that the mixture of L1 and CD24
substrate-coated proteins together reduces the levels of neurite
outgrowth from an L1-mediated strong enhancement of neurite outgrowth
of both dorsal root ganglion and cerebellar neurons to the level of
substrate-coated CD24 alone. It is thus possible that CD24 competes
with L1 in trans-interaction with L1 on neuronal membranes. It is also
conceivable that binding of CD24 to L1 leads to a conformational change
or to a steric hindrance of homophilic binding of L1 to the six
immunoglobulin-like domains of L1 that comprise the decisive domains
for cell binding and neurite outgrowth (32). That carbohydrates may be
important for protein-protein interaction has been proposed for P0 (33) or L1-NCAM interactions (11, 34). A striking dependence of glycans in
trans-interactions has recently been reported for notch, which
interacts with its ligands delta and jagged/serrate. These interactions are modulated through the glycosyltransferase fringe. It
has been proposed that a cell type-specific modification of glycosylation may provide a general mechanism to regulate
ligand-receptor interactions in the nervous system (for discussion, see
Ref. 35).
The effects on neurite outgrowth of dorsal root ganglion or cerebellar
neurons by substrate-coated CD24 are neutralized by antibodies to L1
and are not observed when neurons are taken from L1 knockout mice,
whereas neurons from CD24 knockout mice are not different in neurite
outgrowth from wild type mice. These complementary findings show that
substrate-coated CD24 generates its neurite outgrowth-inhibitory and
-promoting effects via a trans-interaction with L1 at the neuronal cell
surface and exclude the possibility that a homophilic interaction of
CD24 mediates the effects on neurite outgrowth. Our biochemical
analysis indicates that the 2,3-sialic acid-containing 30- and
33-kDa glycoforms are involved in this trans-interaction with L1, which
was not only seen in the developing, 7-day-old brain but also in adult animals.4 These glycoforms
can be removed from L1-containing membranes by alkaline treatment
and co-distribute with L1 in a detergent-soluble membrane
subfraction, and they are co-immunoprecipitated with L1 antibodies.
Interestingly, the
2,3-sialic acid-depleted 27-kDa glycoform cannot
be removed from membranes by treatment with alkaline buffer, and since
it does not co-distribute with L1 in lipid raft microdomains, it is
unlikely to interact in cis- or trans-positions with L1 (Fig.
7).
|
The observation that CD24 is highly expressed in the spinal cord where
corticospinal axons decussate (6) suggests that repellent interactions
between L1 and CD24 may play a role in axon guidance as has recently
been shown for the functional connection between the repellent
extracellular matrix glycoprotein semaphorin 3A and L1, which
associates with the semaphorin 3A receptor neuropilin-1 (36). A
striking reversion of the repellent activity of semaphorin 3A on
L1-expressing neurons was observed when L1 was offered as a
trans-acting partner together with semaphorin 3A. These observations and the ones in the present study support the view that the decision whether inhibition or enhancement of neurite outgrowth occurs could
depend on several possibilities; the signal transduction machinery is
different between different cell types and depends on the different
cis-interacting partners in the different cell types, resulting in a
different mixture of regulatory interdependences in the recruitment of
signal cascades. Such cis-interacting partners could be cytokine or
neurotrophin receptors (e.g. fibroblast growth factor
receptor (37) or neurotrophin tyrosine receptor kinases (12)). The
presence and specific triggering of these receptors by their ligands
could not only activate the tyrosine receptor kinases but also could
lead to different basal activity levels of cis-interacting partner
molecules, such as L1, in conjunction with trans-interacting partner
molecules, such as CD24. The dissection of the network of
glycan-dependent cis- and trans-interacting partners in
terms of their signal transduction machineries remains an exciting
topic for future investigations.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Stefan Kunz and Georg Orberger for help with initial experiments, Drs. Miriam Dahme and Peter Nielsen for the L1 and CD24 null mutants, Dr. Thomas Vorherr for peptide synthesis, Peggy Putthoff and Birte Rossol for excellent technical assistance, and Dr. Birgit Hertlein for helpful comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant Scha 185/27-1, 2.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. Tel.:
49-40-42803-6246; Fax: 49-40-42803-6248; E-mail:
melitta.schachner@zmnh.uni-hamburg.de.
Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M101790200
1 R. Kleene, H. Yang, M. Kutsche, and M. Schachner, unpublished results.
3 R. Kleene, H. Yang, M. Kutsche, and M. Schachner, unpublished observations.
4 R. Kleene, H. Yang, M. Kutsche, and M. Schachner, unpublished data.
![]() |
ABBREVIATIONS |
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The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TBS, Tris-buffered saline; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; PLL, poly-L-lysine.
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