* Department of Biological Sciences, Hunter College of the City University of New York 10021; Department of Cell Biology
and Neurology, New York University Medical School, New York 10016; and § Imperial Cancer Research Fund, Institute for
Molecular Medicine, University of Oxford, OX3 9DU, England
Inhibitory components in myelin are largely responsible for the lack of regeneration in the mammalian CNS. Myelin-associated glycoprotein (MAG), a sialic acid binding protein and a component of myelin, is a potent inhibitor of neurite outgrowth from a variety of neurons both in vitro and in vivo. Here, we show that MAG's sialic acid binding site is distinct from its neurite inhibitory activity. Alone, sialic acid-dependent binding of MAG to neurons is insufficient to effect inhibition of axonal growth. Thus, while soluble MAG-Fc (MAG extracellular domain fused to Fc), a truncated form of MAG-Fc missing Ig-domains 4 and 5, MAG(d1-3)-Fc, and another sialic acid binding protein, sialoadhesin, each bind to neurons in a sialic acid- dependent manner, only full-length MAG-Fc inhibits neurite outgrowth. These results suggest that a second site must exist on MAG which elicits this response. Consistent with this model, mutation of arginine 118 (R118) in MAG to either alanine or aspartate abolishes its sialic acid-dependent binding. However, when expressed at the surface of either CHO or Schwann cells, R118-mutated MAG retains the ability to inhibit axonal outgrowth. Hence, MAG has two recognition sites for neurons, the sialic acid binding site at R118 and a distinct inhibition site which is absent from the first three Ig domains.
IT is generally believed that inhibitory molecules in the
mammalian central nervous system (CNS),1 which are
particularly enriched in myelin, are largely responsible for the lack of axonal regeneration after injury to the
brain or spinal cord (Johnson, 1993 The inhibitory properties of MAG vary depending on
the age and the type of neuron. Thus, MAG inhibits outgrowth from adult dorsal root ganglion (DRG) neurons
and from cerebellar, retinal, spinal, hippocampal, and superior cervical ganglion neurons of all postnatal ages
(Mukhopadhyay et al., 1994 Studies with the MAG knockout mouse (MAG The identity of the neuronal molecule(s) that interacts
with MAG is not known, but some light was recently shed
on this question when MAG was shown to belong to a subgroup of the Ig super family, termed the sialoadhesins
(Kelm et al., 1994 In this study, we further elucidate the role of the sialic
acid-binding epitope of MAG on neurite outgrowth. Through
site-directed mutagenesis, residues involved in the sialic
acid-dependent binding of CD22 and sialoadhesin were
recently mapped to the GFCC Mutation of R118 in MAG
The Chameleon double-stranded site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to mutate the nucleotide sequence coding
for R118 in L-MAG to either an alanine or an aspartate as described by
the manufacturer. L-MAG in the antisense orientation in pBluescript
(Stratagene, La Jolla, CA) was used for the initial annealing step. The mutations were confirmed by dideoxy sequencing. The R118D- and R118A-mutated forms of MAG were then subcloned into the pSJL plasmid for expression in CHO and Schwann cells. To obtain Fc chimeras of the mutated
MAG proteins, a XhoI restriction fragment that cuts MAG cDNA at nucleotides 182 and at 1627, and that contains the mutated nucleotides, was
cut and purified from the R118-mutated MAG cDNAs in pBluescript and
subcloned into the pIG1 vector already harboring the extracellular domain of MAG.
Preparation of Fc Chimeras
The pIG1 vector containing the cDNA coding for the extracellular domain of wild-type sialoadhesin, wild-type MAG, mutated MAG, or MUC18
were prepared as described previously (Kelm et al., 1994 ELISA of MAG-Fc Chimeras
A 96-well ELISA was coated with anti-human Fc at 15 µg/ml in 0.1 M
NaHPO4 for 3 h at 37°C. The plates were washed with PBS and blocked
with 5% nonfat milk in PBS for 30 min at room temperature. The Fc chimeras were added at a concentration of 1 µg/ml and incubated overnight
at 4°C. The MAG 513 monoclonal antibody was added at various concentrations (0.1-333 ng/ml) and incubated for 3 h at 37°C. The plates were
washed and then blocked again with 3% normal goat serum in PBS for 30 min at room temperature. Peroxidase-conjugated sheep anti-mouse IgG
diluted 1:500 (Sigma Chemcial Co., St. Louis, MO) was added and color
was developed using O-phenylenediamine as a substrate. Absorbance was
measured in an ELISA reader at 490 and 630 nm.
Expression of MAG and Sialoadhesin by Transfected
CHO Cells
CHO cells deficient in the dhfr gene were transfected with the pSJL plasmid containing the L-MAG cDNA, R118D-MAG, R118A-MAG or, as a
control, MAG in a 3 The cDNA for sialoadhesin (Crocker et al., 1994 Expression of MAG by Transfected Schwann Cells
A spontaneously transformed Schwann cell line that does not express
MAG was used (Porter et al., 1987 Isolation of Neurons
Neurons were isolated as previously described (Doherty et al., 1990 Desialylation of Neurons
A single-cell suspension of the cerebellar neurons was washed and resuspended in PBS, ~2 × 106 cells were incubated with 50 mU of Vibrio cholerae sialidase (Calbiochem-Novabiochem, La Jolla, CA) in a final volume
of 0.5 ml, for 2 h at 37°C. The neurons were washed with DME and resuspended in SATO containing 2% FCS for the neurite outgrowth experiments, or in PBS for the binding assay.
Binding of Fc Chimeras to Neurons
Fc-chimeric proteins were adsorbed for 3 h at 37°C onto wells of microtiter plates that had been coated for 2 h at 37°C with anti-human IgG at 15 µg/ml in 0.1 M bicarbonate buffer, pH 9.6. Before the binding assay, neurons were vitally labeled with the fluorescent dye, calcein AM (Molecular
Probes, Inc., Eugene, OR), as previously described (DeBellard et al.,
1996a). Where indicated, MAG 513 monoclonal antibody (Boehringer-Mannheim Biochemicals, Indianapolis, IN) was included in the assay at a
concentration of 10 µg/ml and neurons were desialylated before being
used. 100 µl of a suspension of vitally labeled neurons, containing 1-2 × 105 cells, was added to each well and allowed to incubate for 1 h at room
temperature. The plates were washed three times with PBS applied to
each well under gravity by allowing free flow from an elevated reservoir
and the fluorescence measured in a FluorImager (Molecular Dynamics,
Inc., Sunnyvale, CA).
The Fc Chimera Neurite Outgrowth Assay
The Fc chimera neurite outgrowth assay was carried out as described previously (Doherty et al., 1995 Neurite Outgrowth on Transfected CHO and
Schwann Cells
Confluent monolayers of CHO cells expressing wild-type MAG, wild-type
sialoadhesin, mutated MAG or control cells were established over a 24-h
period in individual chambers of an eight-well tissue culture slide (Lab-Tek). Cocultures were established as described previously (Doherty et al.,
1990 Binding of Human Erythrocytes to CHO and
Schwann Cells
Confluent monolayers of CHO or Schwann cells expressing wild-type
MAG, mutated MAG, or control cells were established over a 24-h period
in individual chambers of an eight-well tissue culture slide (Lab-Tek).
Monolayers were treated for 1 h with 200 mU/ml V. cholerae at 37°C and
washed twice. The medium was changed to DME and erythrocytes, either
desialylated or not, were added. Unbound erythrocytes were washed off
and cells were examined under a phase-contrast microscope and photographed.
The Sialic Acid Binding Site on MAG Maps to R118 of
the First Ig Domain
All members of the sialoadhesin family are sialic acid-
binding proteins, although the specific sialic acid linkage
recognized by each may differ (Kelm et al., 1994
Before carrying out the neuron binding or neurite outgrowth assays, we assessed the recognition of R118A- and
R118D-MAG-Fc by a conformation-dependent MAG
monoclonal antibody, the 513 MAG antibody (Poltorak et
al., 1987
To determine if R118 is involved in neuronal adhesion
a binding assay was performed with wild-type and the
R118A- and R118D-mutated forms of MAG-Fc. For this
assay, the Fc-chimeras were immobilized on a 96-well
ELISA plate. Another five-domain Ig family member, MUC18, fused to Fc, was used as a control chimera. A single-cell suspension of neurons, vitally labeled with the fluorescent dye calcein AM, was allowed to bind to the immobilized Fc chimeric molecules. Using this assay previously,
we showed that as the concentration of immobilized
MAG-Fc increases, the number of neurons bound also increases until saturation is reached at about 5 µg/ml (DeBellard et al., 1996a). Confirming our earlier results, Fig. 2
b shows that at a saturating concentration of 10 µg/ml
MAG-Fc, a significant number of neurons are specifically
bound. MAG antibody blocks this binding (results not
shown), and there is no binding to the control chimera,
MUC18. In contrast, although the MAG conformation-dependent antibody, 513, binds the mutated forms of
MAG, neither immobilized R118A- nor R118D-MAG-Fc,
at saturating concentrations, will bind cerebellar neurons.
Binding remains at background levels, indistinguishable from binding to MUC18-Fc (Fig. 2 b). Since antibody
binding suggests that both mutated forms of MAG retain
their native conformation, the abolition of neuronal binding by either a nondisruptive or a disruptive substitution at
R118 indicates that this residue is critical for sialic acid-
dependent binding of MAG.
Inhibition of Neurite Outgrowth by Soluble MAG
Requires the R118 Binding Site
We have shown previously that when soluble MAG-Fc at
a concentration of 25 µg/ml, is added to cerebellar neurons
growing on a substrate of purified, immobilized L1-Fc (a
neurite promoting molecule), there is ~50% inhibition of
neurite outgrowth while MUC18-Fc, at an equivalent concentration, has no effect (Tang et al., 1997 Sialic Acid-dependent Binding can Be Dissociated
from Inhibition of Neurite Outgrowth
To determine if CD22 and sialoadhesin also bind to neurons, like MAG, in a sialic acid-dependent manner, we
performed a binding assay with each of these sialoadhesins. Each of the sialoadhesin Fc chimeras was immobilized
at a concentration of 10 µg/ml, on a 96-well ELISA plate
and vitally labeled neurons were allowed to bind as described above. Of the three sialoadhesins, binding of neurons to MAG-Fc and sialoadhesin is significant and equivalent. Furthermore, sialoadhesin binding, as with MAG
binding, is abolished by desialylation of the neurons prior
to the assay (Fig. 3 a). There is no neuronal binding to
CD22 (Fig. 3 a). These results are consistent with the fact
that sialoadhesin, but not CD22, recognizes sialic acid in a
linkage similar to the one recognized by MAG (NeuAc
As both sialoadhesin and MAG(d1-3)-Fc bind to neurons in a sialic acid-dependent manner similar to MAG-Fc,
we tested their effect on axonal regeneration. We compared neurite outgrowth from cerebellar neurons growing
on a substrate of immobilized L1-Fc in the presence of 25 µg/ml of MAG-Fc, MAG(d1-3)-Fc, sialoadhesin-Fc, or as
a control MUC18-Fc. As reported above, under these conditions, MAG-Fc inhibited neurite outgrowth by ~50%
while MUC18-Fc had no effect (Fig. 3 b). Surprisingly,
although they each bind to neurons via sialic acid, in a
linkage similar to MAG, neither sialoadhesin nor MAG
(d1-3)-Fc had any effect on neurite outgrowth (Fig. 3 b).
Neurite length was the same as when neurons were grown in the presence of the control MUC18-Fc or in the absence of Fc chimera. Therefore, although mutation of
R118 in MAG-Fc indicates that sialic acid-dependent
binding is required for MAG-Fc to inhibit axonal growth,
the results with both sialoadhesin and MAG(d1-3)-Fc demonstrate that sialic acid binding alone is insufficient to effect inhibition of axonal regeneration.
Isolation of Transfected CHO and Schwann Cells
Expressing High Levels of MAG
To assess the effect of mutation at R118 on inhibition of
neurite outgrowth by full-length MAG expressed by cells,
cDNAs coding for full-length, mutated MAG in the pSJL
vector were transfected into CHO cells and into Schwann
cells as previously described (Mukhopadhyay et al., 1994
For the Schwann cells, a spontaneously transformed
Schwann cell line, which does not express MAG and is
permissive for axonal growth was used (Porter et al., 1987
Mutated MAG Expressed by CHO or Schwann Cells
Fails to Mediate Sialic Acid-dependent Adhesion but
Retains Its Ability to Inhibit Neurite Outgrowth
Before carrying out the neurite outgrowth assay, we first
wanted to ensure that R118-mutated MAG expressed by
either CHO cells or Schwann cells had, like R118-mutated
MAG-Fc, lost the ability to bind sialic acid. To do this we
monitored the binding of human erythrocytes to these
cells, as the majority of cell-cell interactions of human
erythrocytes is sialic acid dependent. Previously we reported that MAG-expressing COS cells bind human erythrocytes in a sialic acid-dependent manner (Kelm et al.,
1994 To test the effect of mutated MAG expressed by CHO
and Schwann cells on neurite outgrowth, isolated cerebellar neurons were grown overnight on CHO cells expressing R118A- or R118D-MAG, or Schwann cells expressing
R118A-MAG. Neurite length was compared to outgrowth
from similar neurons grown on cells expressing unmutated MAG and on control transfected cells not expressing
MAG. As we reported previously (Mukhopadhyay et al.,
1994
Finally, we wanted to assess if sialoadhesin expressed by
CHO cells had any effect. Neurite outgrowth on CHO
cells expressing an abundance of sialoadhesin, either a truncated form of the molecule consisting of Ig domains 1-6 or
the entire molecule, consisting of 17 Ig domains (characterized by Western blot and indirect immunofluorescence, results not shown) was compared to neurite outgrowth on
MAG-expressing and control CHO cells. As before,
neurite outgrowth was inhibited by ~70% on the MAG-expressing CHO cells. However, when the same neurons
are grown on CHO cells expressing sialoadhesin, (either d1-6 or d1-17) there is no difference in neurite length compared to outgrowth on control cells (Fig. 7). Axonal outgrowth, therefore, is not inhibited on sialoadhesin-expressing CHO cells. These results demonstrate that the binding
of sialoadhesin expressed by CHO cells, like sialoadhesin-Fc, is insufficient to bring about inhibition. MAG, therefore,
must carry a specific site responsible for inhibition of neurite outgrowth, separate from its sialic acid-binding epitope.
Myelin-associated glycoprotein can either promote or inhibit axonal regeneration, depending on the age and type
of neuron (Johnson et al., 1989 Previously, we showed that soluble MAG-Fc is completely dependent on sialic acid binding to inhibit neurite
outgrowth (Tang et al., 1997
The idea of two distinct, neuronal recognition sites on
MAG is supported by a number of other observations.
First, high levels of sialic acid-dependent binding of human
erythroctyes to either MAG-expressing CHO or Schwann
cells can only be measured if the CHO or Schwann cells
are desialylated before the binding assay. This could result
from MAG expressed by these cells engaging in cis interactions and not being available for trans sialic acid binding
with opposing neurons. However, both MAG-expressing
CHO and Schwann cells inhibit neurite outgrowth without
being desialylated before the assay. Therefore, high levels
of MAG-sialic acid-dependent binding to neurons is not
required for inhibition by MAG when expressed by cells.
Second, the binding requirements of MAG 513 monoclonal antibody further support the suggestion of distinct sites on MAG. The epitope recognized by this monoclonal
antibody is conformation dependent (Poltorak et al., 1987 For both MAG-Fc and MAG expressed by CHO and
Schwann cells, we have shown that their sialic acid binding
capabilities are lost by mutation of R118. Although we
suggest here that the sialic acid binding of MAG is not
required to inhibit neurite outgrowth when expressed by
CHO or Schwann cells, we previously reported that inhibition by MAG expressed by CHO cells was partially reversed by desialylation of the neurons (DeBellard et al.,
1996a Arginine 118 in MAG was chosen because mutation of
an arginine conserved at the same location in two other
members of the sialoadhesins, CD22 (R130) and sialoadhesin (R97), have been shown to abolish sialic acid-dependent binding (Vinson et al., 1996 It is of interest that R118 in MAG is the first residue in
an Arg-Gly-Asp (RGD) motif, a motif known to be recognized by some integrins (Ruoslahti and Pierschbacher, 1987 What role does the sialic acid-binding epitope of MAG
play in the inhibition of neurite outgrowth? The simplest
interpretation of our results is that MAG requires this site
for neuronal binding, which then permits an interaction of
the inhibition epitope. This is certainly true for soluble
MAG-Fc but not so apparent for MAG expressed by
CHO cells where, taking all the information together, inhibition is somewhat independent of MAG's sialic acid binding. This would seem fitting since sialic acid residues attached in an O-linkage to glycoconjugates are ubiquitous
in the nervous system (Varki and Marth, 1995 A two-site model has also been proposed for the interaction of P-selectin with its ligand PSGL-1 (Pouyani and
Seed, 1995 In conclusion, we provide evidence that R118 plays an
important role in the sialic acid binding of MAG but that
sialic acid binding alone is insufficient to induce inhibition
of neurite outgrowth. We propose a model whereby MAG
recognizes neurons via two sites, a sialic acid binding site
and a neurite outgrowth inhibition site. A model based on
two separate sites on MAG leads to the possibility that a
mutated form of MAG can eventually be engineered to
bind the neuronal receptor but not inhibit neurite outgrowth. Such a form of MAG would act as an antagonist of
inhibition and could be used to reverse inhibition of axonal outgrowth by MAG in vivo and be of possible therapeutic value after injury to the CNS.
; Schwab et al., 1993
;
Keynes and Cook, 1995
). A monoclonal antibody, termed
IN-1, recognizes a component(s) in CNS myelin that is
likely to play a major role in this inhibition (Caroni and
Schwab, 1988a
,b; Schnell and Schwab, 1990
; Bregman et al.,
1995
). The antigen(s) recognized by this antibody has yet
to be described. Distinct from the inhibitory activity that
is blocked by the IN-1 antibody, recent studies strongly
suggest that myelin-associated glycoprotein (MAG), a well-characterized component of both CNS and peripheral
nervous system (PNS) myelin, may be an important component of this inhibitory activity (Filbin 1995
, 1996
). We
(Mukhopadhyay et al., 1994
; DeBellard et al., 1996a
) and
others (McKerracher et al., 1994
) have recently shown
MAG to be a potent inhibitor of axonal regrowth from a
variety of postnatal neurons in vitro. The ability of MAG
to induce growth cone collapse when presented to growing
neurites in a multivalent form (Li et al., 1996
) and the ability of a soluble form of MAG to inhibit axonal regeneration (Tang et al., 1997
) provide strong evidence that MAG
is indeed an inhibitory molecule and not merely a nonpermissive substrate.
; DeBellard et al., 1996a
)
whereas it promotes axonal growth from neonatal DRG
neurons (Johnson et al., 1989
; Mukhopadhyay et al., 1994
).
We observed a 50-90% inhibition on MAG-expressing cells,
depending on the type of neuron (Mukhopadhyay et al.,
1994
; DeBellard et al., 1996a
). McKerracher et al. (1994)
compared neurite length from neurons cultured on CNS
myelin as a substrate, to CNS myelin immunodepleted of MAG. Their parallel study demonstrated that MAG may
account for 60% of the inhibitory properties of CNS myelin.
/
)
(Li et al., 1994
; Montag et al., 1994
) also suggest that MAG
contributes to the inhibitory properties of myelin in vivo
and in vitro. We found that neurites from a variety of neurons were about twice as long when grown on myelin from
MAG
/
mice compared to MAG +/+ mice (DeBellard
et al., 1996b
). Previous studies, however, reported inconsistent results with MAG
/
myelin using either the neuronal cell line NG108 or primary neurons (Bartsch et al.,
1995
; Li et al., 1996
; Ng et al., 1996
). In vivo the ability of
MAG to inhibit regeneration has been clearly demonstrated in the PNS (Schafer et al., 1996
). Normally after
injury to the PNS, myelin is removed and Schwann cells
downregulate expression of myelin proteins before axonal
regeneration occurs (Fawcett and Keynes, 1990
). In the mutant C57BL/Ola mouse, myelin is not rapidly removed,
and regeneration takes place only very slowly, if at all
(Brown et al., 1991
, 1992
). By itself, the C57BL/Ola mouse
suggests a role for PNS myelin in inhibiting axonal regeneration, but in mice bred by crossing C57BL/Ola and
MAG
/
mice, myelin is MAG deficient and regeneration is much more robust (Schafer et al., 1996
). In the
CNS, however, the extent to which regeneration in vivo
is improved in the MAG
/
mouse compared to the
MAG +/+ mouse is controversial; while one group reports
both a significant improvement in the length regenerated
and an increase in the number of regenerating axons (Li et
al., 1996
), another reports no change (Bartsch et al., 1995
).
). All members of the sialoadhesins have
significant amino acid sequence similarity among their
first four Ig-like domains, and all bind sialic acid. The five
sialoadhesins identified so far are CD22, sialoadhesin, CD33, SMP (a protein very similar to MAG but found
only in avian species), and MAG (Filbin 1995
, 1996
). Although all bind sialic acid, the specificity of binding may
differ. For example, MAG prefers
2,3-linked sialic acid
attached to O-linked glycoconjugates, CD22 prefers
2,6-linked sialic acid attached to N-linked glycoconjugates, while
sialoadhesin recognizes
2,3-linked sialic acid attached to
either N- or O-linked glycoconjugates (Kelm et al., 1994
). We have shown that whether it promotes (neonatal DRG
neurons) or inhibits (cerebellar neurons) axonal regeneration, MAG binds to neurons in a sialic acid-dependent
manner (Kelm et al., 1994
; DeBellard et al., 1996a
). Although MAG binding to isolated gangliosides has been
demonstrated (Yang et al., 1996
), sialic acid-dependent binding to neurons is trypsin sensitive, indicating that
MAG binds to a neuronal sialo-glycoprotein rather than
to a sialo-glycolipid (DeBellard et al., 1996a
). More important, if neurons are desialylated before the neurite outgrowth assay, inhibition by soluble MAG is completely
lost while inhibition by MAG expressed by CHO cells is
only partially reversed (DeBellard et al., 1996a
; Tang et
al., 1997
). This strongly suggests that a neuronal sialo-glycoprotein mediates, directly or indirectly, the effects of
MAG on axonal regeneration.
C
face of the NH2-terminal domains, centered on Arg130 and Arg97, respectively
(Vinson et al., 1996
; van der Merwe et al., 1996
). Alignment of the first Ig domain of MAG with CD22 and sialoadhesin reveals that this arginine is conserved in MAG at
amino acid 118 (R118). Here, we show that if R118 in
MAG is mutated to either an alanine (R118A) or an aspartic
acid (R118D) the sialic acid-dependent binding of MAG,
when either expressed by cells or in a soluble form,
MAG-Fc, to cerebellar neurons is lost. Similarly, while MAG-Fc is a potent inhibitor of axonal regeneration
(Tang et al., 1997
) when mutated to either R118A- or
R118D-MAG-Fc the capacity to inhibit regeneration is
lost. Of note, however, when expressed by transfected
CHO or Schwann cells, these R118-mutated forms of MAG
are still potent inhibitors of axonal regeneration. Finally, although both sialoadhesin and a truncated form of MAG-Fc,
consisting of the first three Ig-domains fused to Fc, each
bind to neurons in a sialic acid-dependent manner, neither
molecule has an effect on neurite outgrowth. Taken together, these results suggest that R118 plays an important
role in the sialic acid-dependent binding of MAG to neurons. However, sialic acid binding by itself is insufficient to
effect inhibition of axonal regeneration. Following from
this they also indicate that there is a second site on MAG,
a "neurite inhibition" site, that is distinct from the sialic
acid-binding site and not present in the first three Ig-domains
of MAG.
Materials and Methods
). The plasmids
were transfected into COS cells and the Fc-chimeric proteins purified
from the media as described previously (Simmons, 1993
; Kelm et al., 1994
).
-5
orientation as previously described (Mukhopadhyay et al., 1994
). Expression of MAG protein was amplified and characterized by Western blotting and indirect immunofluorescence as previously described (Mukhopadhyay et al., 1994
). Transfected cells were
maintained in DME supplemented with 10% dialyzed FCS, proline (40 µg/liter), thymidine (0.73 mg/liter), and glycine (7.5 mg/liter) at 37°C in
5% CO2.
), both full-length sialoadhesin containing all 17 Ig-like domains or truncated sialoadhesin
consisting of domains 1-6, in the pcDNA1 Amp vector (British Biotechnology, Oxford, UK) were cotransfected with pcDNA3 as a source of
G418 resistance, into CHO cells. After selection in 200 µg/ml G418, colonies were pooled and live cells immunostained for sialoadhesin and subjected to FACS®. Cells expressing high levels of sialoadhesin were expanded and expression again assessed by Western blotting and by
immunofluorescence of intact cells.
; Owens et al., 1990
). The cDNAs for
MAG, R118D-MAG, or R118A-MAG in the pSJL plasmid were transfected into this Schwann cell line as previously described (Mukhopadhyay
et al., 1994
) and transfectants were selected in 200 µg/ml G418. Colonies
were combined and live cells were stained for surface MAG with the 513 MAG monoclonal antibody. A second antibody, phycoprobe-conjugated
anti-mouse IgG (1:100), was used and cells expressing high levels of MAG
were separated by FACS®. These cells were expanded and expression of
MAG characterized by Western blotting and by immunofluorescent staining as before (Mukhopadhyay et al., 1994
).
;
Mukhopadhyay et al., 1994
). Briefly, animals 1-7-d-old were used and the
cerebella were removed. Tissue from one to two animals was combined
and placed in 5 ml of 0.025% trypsin in PBS, triturated, and incubated for
a further 10 min at 37°C. Trypsinization was stopped by addition of 5 ml
DME containing 10% FCS, and cells were centrifuged at 1,000 rpm for 5 min. The cells were resuspended to a single-cell suspension in 2 ml of sato
(Doherty et al., 1990
), containing 2% FCS for neurite outgrowth on immobilized Fc chimeras or in 2 ml of sato (Doherty et al., 1990
) without serum for neurite outgrowth on monolayers of CAM-expressing cells. Cells were
counted with a Coulter counter (Coulter Electronics Ltd., Luton, England).
; Tang et al., 1997
). Briefly, L1-Fc (20-40 µg/
ml) was immobilized onto eight chamber slides (Lab-Tek, Naperville, IN)
already coated with poly-L-lysine (16.6 µg/ml), and then anti-human IgG
(15 µg/ml). Neurons and the Fc chimera at 25 µg/ml were added simultaneously to the immobilized L1-Fc at a concentration of 4 × 104 neurons
per well and cultured for 16 h. Where indicated MAG 513 monoclonal antibody (25 µg/ml) was incubated with the MAG-Fc or neurons were desialylated with 50 mU V. cholerae sialidase and the sialidase was included
during the culture period. Cultures were fixed, stained for GAP43, and
neurite length was measured as described above.
; Mukhopadhyay et al., 1994
) by adding ~5,000 cerebellar neurons to
the CHO cell monolayers. Culture medium was SATO. Where indicated,
10 mU of V. cholerae sialidase were included throughout the coculture period. After 16-18 h, the cocultures were fixed for 30 min with 4%
paraformaldehyde, and permeabilized with ice-cold methanol for 2 min.
The cells were then blocked for 20 min with DME containing 10% FCS
and incubated for 2 h with a rabbit polyclonal antibody against GAP43 (1:
4,000; from R. Curtis and G. Wilkins, Imperial College, London, UK). Cells were washed three times with PBS-BSA (2%), and then incubated for 30 min at room temperature with a biotinylated donkey anti-rabbit Ig
(1:300; Amersham Corp., Arlington, Heights, IL), washed three times,
and incubated with streptavidin-conjugated Texas red (1:300; Amersham
Corp.) for 45 min. After three more washes, the slides were mounted in
Permfluor (Baxter Healthcare Corp. Miami, FL) and viewed with a Zeiss
fluorescent microscope (Carl Zeiss, Inc., Thornwood, NY). The length of
the longest neurite for each GAP43-positive neuron was determined using
the Uncor image analysis program.
Results
). In keeping with the characteristics of this family of molecules, we
have shown that MAG binds to neurons in a sialic acid-
dependent manner (Kelm et al., 1994
; DeBellard et al.,
1996a
). Recently, Arg97 in sialoadhesin and Arg130 in CD22 were shown to be critical for the sialic acid binding
of each molecule (Vinson et al., 1996
; van der Merwe et al.,
1996
). If the first Ig domain of MAG is aligned with the
first Ig domain of sialoadhesin and CD22 (Fig. 1), then this
arginine appears conserved in all three proteins. In MAG,
the conserved amino acid corresponds to Arg118 (R118).
To determine if R118 in MAG is involved in sialic acid-
dependent binding to neurons, the nucleotides coding for
this amino acid were mutated to code either for alanine (R118A), a nondisruptive substitution, or for aspartate
(R118D), a disruptive substitution. The mutations were
created in both a soluble, chimeric form of MAG, MAG-Fc, and in the full-length MAG cDNA.
Fig. 1.
Alignment of the first Ig domains of sialoadhesin (Sn),
rat CD22 (CD22) and MAG (MAG). Predicted -strands are labeled as A, B, C, C
, C
, D, E, and F. Amino acids involved in
sialic acid binding of sialoadhesin and conserved in MAG are
highlighted. The arrow refers to Arg118 in MAG.
[View Larger Version of this Image (20K GIF file)]
). Fig. 2 a shows that MAG 513 monoclonal antibody binds to immobilized, unmutated-, R118A-, and
R118D-MAG-Fc in a dose-dependent manner. Furthermore, and more importantly, the characteristics of binding
of the antibody to both R118A- and R118D-MAG-Fc are
indistinguishable from that of the unmutated MAG-Fc
(Fig. 2 a). This demonstrates the affinity of the antibody
for MAG is unchanged by these mutations suggesting that
the conformation of MAG is maintained in R118A- and
R118D-MAG-Fc.
Fig. 2.
The effect of mutation of R118 in MAG-Fc on antibody
recognition, binding to neurons and inhibition of axonal growth.
(a) Binding of conformation-dependent MAG 513 monoclonal
antibody to mutated MAG-Fc. The Fc chimeras were immobilized at a concentration of 1 µg/ml on an ELISA plate already
coated with anti-Fc antibody. MAG 513 monoclonal antibody
was added at various concentrations and bound antibody was detected by ELISA. MAG-Fc (), R118A-MAG-Fc (
), R118D-
MAG-Fc (
), or MUC18-Fc (
). Results represent three experiments +/
SEM. (b) Binding of R118-mutated MAG to cerebellar neurons. Dissociated PND4 cerebellar neurons, vitally labeled with calcein AM, were allowed to bind to MAG-Fc (MAG), R118A- MAG-Fc (R118A), R118D-MAG-Fc (R118D), or MUC18-Fc
(MUC) immobilized at a concentration of 10 µg/ml, on a 96-well
dish. 200,000 neurons were added to each well and after incubation and washing, the number of cells bound was quantitated with
a FluorImager. Results are from three experiments, each with
10 samples, and represent the mean ± SEM. (c) The effect of
R118-mutated MAG-Fc on neurite outgrowth. Dissociated
PND4 cerebellar neurons were incubated with MAG-Fc, R118A-
MAG-Fc (R118A-Fc), R118D-MAG-Fc (R118D-Fc), or MUC18-Fc (MUC-Fc) at a concentration of 25 µg/ml before being plated
onto a substrate of L1-Fc immobilized on wells coated with anti-Fc antibody. Neurons were cultured for 18 h, fixed and immunostained for GAP 43, and neurite length was measured. Results
show the mean length of the longest neurite per cell (±SEM) for
100-200 individual neurons.
[View Larger Versions of these Images (12 + 15 + 13K GIF file)]
). To determine
if soluble R118A- or R118D-MAG-Fc also have an inhibitory effect on cerebellar neurons growing on immobilized
L1-Fc, each mutated MAG chimera was added to the cultures at a concentration of 25 µg/ml. As before, unmutated
MAG-Fc inhibited axonal outgrowth by ~50% while
MUC18-Fc had no effect, and neurite length was the same
as without Fc chimera (Fig. 2 c). When either R118A- or
R118D-MAG-Fc was added to the growing neurites at the same concentration, no effect was seen on neurite outgrowth. Neurite length was similar to that for neurons grown
with MUC18-Fc or in the absence of Fc chimera (Fig. 2 c).
Therefore, it appears that soluble MAG-Fc is entirely dependent on its ability to bind neurons in a sialic acid-
dependent manner to inhibit axonal growth.
2,3 Gal in O-linked glycans). When a truncated form of
MAG-Fc, consisting of the first three Ig-domains, MAG (d1-3)-Fc, was used as a substrate for binding, binding was
equivalent to the binding of the full-length, unmutated
MAG-Fc (Fig. 3 a). Furthermore, binding of MAG(d1-3)-Fc
is also completely sialic acid-dependent (Fig. 3 a). This is
not an unexpected finding as the sialic acid binding site on
MAG, R118, is in the first Ig-domain (see above).
Fig. 3.
Binding to and effect on neurite outgrowth from cerebellar neurons of sialoadhesin-Fc, CD22-Fc, and MAG(d1-3)-Fc.
(a) Dissociated PND4 cerebellar neurons, vitally labeled with calcein AM, were allowed to bind to MAG-Fc, MAG(d1-3)-Fc, sialoadhesin (SN)-Fc, CD22-Fc, or MUC18-Fc immobilized at a
concentration of 10 µg/ml, on a 96-well dish coated with anti-Fc
antibody. 200,000 neurons, either pretreated with sialidase (stippled bars) or not (solid bars) were added to each well and after
incubation and washing, the number of cells bound was quantitated with a FluorImager. Results are from three experiments,
each with 10 samples and represent the mean ± SE. (b) Dissociated PND4 cerebellar neurons were incubated with MAG-Fc,
sialoadhesin (SN)-Fc, MAG(d1-3)-Fc, or MUC18-Fc (MUC-Fc) at a concentration of 25 µg/ml before being plated onto a substrate of L1-Fc immobilized on wells coated with anti-Fc antibody. Neurons were cultured for 18 h, fixed and immunostained
for GAP 43, and neurite length was measured. Results show the
mean length of the longest neurite per cell (± SEM) for 100-200
individual neurons.
[View Larger Versions of these Images (22 + 14K GIF file)]
).
For the CHO cells, after selection in G418, gene amplification by the methotrexate/dhfr strategy and single-cell cloning, a number of colonies were screened for expression of
the mutated forms of MAG. Fig. 4 a shows immunostaining of a Western blot of the lysates from two such clones.
Although each of these cell lines expresses slightly less
MAG (per mg total protein) than the cells that express
unmutated MAG (compare lanes c and d with lane b),
the level of expression is within the range we previously
showed to effectively inhibit regeneration (Mukhopadhyay et al., 1994
). In addition, both mutated forms of
MAG have a molecular weight of ~100 kD, similar to that
of wild-type MAG expressed either in CHO cells or by sciatic nerve. This indicates that both R118A- and R118D-MAG are glycosylated to the same extent as wild-type MAG. To ensure that mutated MAG is reaching the surface of these transfected CHO cells, live cells were immunostained with the MAG 513 monoclonal antibody. Fig. 4
b shows: first, the MAG 513 antibody recognizes mutated
MAG when expressed by CHO cells, again demonstrating
the conformational integrity of the mutated proteins; and
second, MAG reaches the cell surface in cells expressing
unmutated MAG, R118A-MAG, and R118D-MAG. Furthermore, the intensity of staining is comparable for all
three cell lines, indicating that equivalent amounts of
MAG are reaching the surface.
Fig. 4.
Characterization of
the expression of mutated
forms of MAG by CHO cells.
(a) Immunodetection of MAG
(arrow) in lysates of control
transfected CHO cells (lane
a), MAG-expressing cells (lane b), R118A-MAG-expressing
cells (lane c), R118D-MAG-expressing cells (lane d), and
rat sciatic nerve (lane e). Each
lane was loaded with 50 µg of
protein except for lane e,
which was loaded with 30 µg.
Proteins were separated by
polyacrylamide gel electrophoresis (8%), transferred to
PVDF membrane, and immunostained for MAG with a
monoclonal rat anti-MAG
antibody (1:100), followed by
alkaline phosphatase-conjugated, goat anti-mouse (1:
1,000). The substrate was
5-bromo-4-chloro-3-indolylphosphate, and nitroblue tetrazolium was the chromogen. Bars refer to molecular weight standards from top to bottom, 198, 120, 88, 70, and 56 kD. (b) Surface detection of mutated-MAG on transfected CHO cells by immunofluorescent staining. Live CHO cells expressing R118A-MAG (panel a)
or R118D-MAG (panel b) were incubated with MAG 513 monoclonal antibody (5 µg/ml), and then fixed with 4% paraformaldehyde
and incubated with phycoprobe-conjugated, goat anti-mouse IgG (1:50). (c) Binding of human red blood cells to transfected CHO cells.
Confluent monolayers of MAG-expressing (A) and (B), R118A-MAG-expressing (C), and R118D-MAG-expressing (D) CHO cells were
desialylated by incubation with sialidase. A single-cell suspension of human red blood cells was added to each monolayer and allowed to
bind. For B, the red blood cells were desialylated before being added to the monolayer. Binding was assessed by appearance of rosettes
of human red blood cells bound to the monolayers under phase microscopy.
[View Larger Versions of these Images (83 + 75 + 39K GIF file)]
;
Owens et al., 1990
). We have shown previously that when
these Schwann cells are induced to express wild-type MAG
after transfection, neurite outgrowth is inhibited by ~40%
(Shen et al., 1996
). Here, after transfection and selection
in G418, resistant colonies were pooled, live cells were immunofluorescently stained using the 513 MAG monoclonal antibody and sorted by FACS® for cells expressing
high levels of MAG. These cells were expanded, expression of MAG was characterized by Western blotting and
immunofluorescence of intact cells, and these high expressors were used in the neurite outgrowth assay. It can be
seen from the immunostained Western blot in Fig. 5 a that
the Schwann cells expressing wild-type MAG and those
expressing R118A-MAG are expressing approximately
the same amount of MAG (per mg of total protein). Furthermore, both the wild type and the R118A-MAG expressed by these Schwann cells are ~100 kD in size and
therefore most likely glycosylated to the same extent as
MAG expressed in vivo. However, no expression of MAG
was detected in the cells transfected with the R118D-mutated MAG. This is surprising because a small proportion of these cells appeared to express high levels of R118D-MAG upon FACS® sorting. After expansion of the first
FACS®-sorted R118D-MAG transfected cells, the cells were
again FACS® sorted. Again a few of these cells appeared
to express high levels of the R118D-MAG at the cell surface. However, when these Schwann cells were expanded
and analyzed by Western blotting and immunostaining, again there was no detectable expression of R118D-MAG.
These results suggest that the Schwann cells expressing
high levels of this particular mutated form of MAG either
died or stopped expressing the protein with passage. The
latter possibility is most likely correct as there was no evidence of dying cells in the cultures. The reason why these
Schwann cells stopped expressing R118D-MAG is not
known as this protein would appear to be folded correctly based on the ability to bind the conformation-dependent
MAG antibody, 513 (Figs. 2 a and 4 b). For the Schwann
cells expressing wild-type MAG or R118A-MAG, Fig. 5 b
shows that each of these proteins reaches the cell surface
and is evenly distributed.
Fig. 5.
Characterization of the expression of mutated forms of
MAG by Schwann cells. (a) Immunodetection of MAG in lysates
of wild-type MAG-transfected Schwann cells (MAG), R118A-MAG-transfected Schwann cells (R118A), R118D-MAG-transfected Schwann cells (R118D) and control transfected Schwann
cells (Sch). Each lane was loaded with 20 µg of protein. Proteins
were separated by polyacrylamide gel electrophoresis (8%),
transferred to PVDF membrane, and immunostained for MAG
with a monoclonal rat anti-MAG antibody (1:100), followed by
alkaline phosphatase-conjugated, goat anti-mouse (1:1,000). Detection was by chemilluminescence. Bars refer to molecular
weight standards from top to bottom, 198, 120, 88, 70, and 56 kD.
(b) Detection of MAG on the surface of transfected Schwann
cells by immunofluorescent staining. Live Schwann cells expressing wild-type MAG (A) or R118A-mutated MAG (B) were incubated with MAG 513 monoclonal antibody (5 µg/ml), then fixed
with 4% paraformaldehyde and incubated with phycoprobe-conjugated, goat anti-mouse IgG (1:50). (c) Binding of human red
blood cells to transfected Schwann cells. Confluent monolayers of
MAG-expressing (A and B) and R118A-mutated MAG-expressing (C) Schwann cells were desialylated by incubation with sialidase. A single-cell suspension of human red blood cells was
added to each monolayer and allowed to bind. For B, the red
blood cells were desialylated before being added to the monolayer. Binding was assessed by appearance of rosettes of human
red blood cells bound to the monolayers under phase microscopy.
[View Larger Versions of these Images (42 + 85 + 48K GIF file)]
). However, high levels of binding of human erythrocytes to MAG-expressing CHO cells and MAG-expressing Schwann cells was only detected if the CHO or
Schwann cells were desialylated before the binding assay
(Figs. 4 c and 5 c).This finding implies that a large portion of MAG expressed by CHO or Schwann cells is engaged
in sialic acid-dependent binding in a cis manner (i.e., with
sialoglycoconjugates on the surface of these cells). Alternatively, the lack of binding before desialylation could be
the result of a charge effect of the CHO-sialic acid residues. There was no binding of human erythrocytes to either CHO or Schwann cells not expressing MAG, regardless of whether they were desialylated or not. Similarly,
neither R118A- nor R118D-MAG-expressing CHO, nor
R118A-expressing Schwann cells supported human erythrocyte binding, regardless of whether they were desialylated or not. These results indicate that the sialic acid-
dependent binding of MAG expressed by CHO or Schwann
cells is lost, as in MAG-Fc, by mutation of R118 (Figs. 4 c
and 5 c).
; DeBellard et al., 1996a
), axonal outgrowth from cerebellar neurons was inhibited by ~70% when grown on
MAG-expressing CHO cells and by ~40% when grown on
MAG-expressing Schwann cells, compared to control
transfected cells not expressing MAG (Fig. 6 a and b). Importantly, a similar degree of neurite outgrowth inhibition
still occurred when neurons were cultured on CHO or
Schwann cells expressing the mutated MAG. These results
indicate that even though mutation at R118 abolishes the
ability of MAG to engage in measurable sialic acid-dependent binding, the capacity to inhibit neurite outgrowth is
unaffected when mutated MAG is expressed in CHO or
Schwann cells.
Fig. 6.
The effect of R118-mutated-MAG expressed by CHO or
Schwann cells on neurite outgrowth from cerebellar neurons.
Dissociated PND4 cerebellar neurons were cultured for 18 h on
confluent monolayers of (a) unmutated MAG-expressing, (MAG),
R118A-MAG-expressing (R118A), R118D-MAG-expressing
(R118D), or control (C) CHO cells or (b) MAG-expressing
(MAG), R118A-MAG-expressing (R118A), or control Schwann
cells not expressing MAG (Sch). Cocultures were fixed and immunostained for GAP 43, and neurite length was measured. Results show the mean length of the longest neurite per cell (± SEM)
for 100-200 individual neurons.
[View Larger Versions of these Images (10 + 11K GIF file)]
Fig. 7.
The effect of sialoadhesin expressed by CHO cells on
neurite outgrowth. Dissociated PND4 cerebellar neurons were
cultured for 18 h on confluent monolayers of MAG-expressing
(MAG), sialoadhesin-expressing (SN) or control CHO cells (C).
Cocultures were fixed and immunostained for GAP43 and neurite length was measured. Results are the mean length of the
longest neurite per cell (± SEM) for 100-200 individual neurons.
[View Larger Version of this Image (12K GIF file)]
Discussion
; Mukhopadhyay et al.,
1994
; McKerracher et al., 1994
; DeBellard et al., 1996a
). In
addition, we have shown that regardless of whether neurite outgrowth is promoted or inhibited, MAG binds to all
neurons tested in a sialic acid-dependent manner (Kelm
et al., 1994
; DeBellard et al., 1996a
). As a sialic acid-binding protein and a member of the Ig superfamily, MAG has
been included in the Ig subfamily, the sialoadhesins (Kelm
et al., 1994
). In this study we show that the sialic acid-
dependent binding of MAG to neurons is abolished by
mutating R118 in the first Ig domain. It is highly unlikely
that the loss of binding by mutation of R118 results from a
loss of native conformation because both a nondisruptive
and a disruptive amino acid substitution at R118 have the
same effect. Although a nondisruptive substitution is not
predicted to alter conformation, the amino acid involved
directly in binding can be expected to be highly conserved;
therefore, even a nondisruptive substitution is predicted to
abolish or diminish function of such an amino acid. In addition, the MAG 513 monoclonal antibody still recognizes
mutated R118A- and R118D-MAG with the same affinity
as it recognizes unmutated MAG. Since the epitope on
MAG recognized by 513 is conformation dependent (Poltorak et al., 1987
), it is very unlikely that MAG is misfolded as a result of the mutations introduced at R118. Together these results strongly suggest that R118 in the first
Ig like domain is critical for the sialic acid-dependent
binding of MAG.
) while only a portion of the
inhibition exerted by MAG expressed by CHO cells is
sialic acid-dependent (DeBellard et al., 1996). Consistent
with these observations mutation of R118 abolishes completely the ability of MAG-Fc to inhibit neurite outgrowth.
On the other hand, R118-mutated MAG expressed by
either CHO or Schwann cells inhibits axonal regeneration
to the same extent as wild-type MAG, even though we
showed that R118-mutated forms of MAG expressed by
these cells have lost their sialic acid binding capabilities. In
contrast, two proteins, sialoadhesin and a truncated form
of MAG, MAG(d1-3)-Fc, that each bind neurons in a
sialic acid-dependent manner and each recognize the same sialic acid linkage as MAG (Kelm et al., 1994
), do not inhibit axonal growth. Together, these results indicate that
sialic acid binding to neurons is, in itself, insufficient to induce inhibition. Therefore, the intact extracellular domain
of MAG must carry an additional degree of specificity that
dictates inhibition. It follows that there must be two recognition sites on MAG for neurons, one that binds one or
more sialoglycoproteins, and another that is the neurite inhibition effecting site. As shown schematically in Fig. 8,
when R118-mutated MAG is expressed in CHO cells or Schwann cells, the absence of the sialic acid binding site
can be compensated for by another, as yet unidentified,
adhesion molecule(s) on these cells; this adhesion molecule allows the interaction of the MAG inhibition site with
neurons. In contrast, soluble MAG-Fc is dependent exclusively on its own sialic acid-dependent binding to allow
the neurite inhibition site to engage the neuron. If the
sialic acid-dependent binding is abolished in soluble
MAG-Fc, inhibition of neurite outgrowth by MAG-Fc is
lost (Fig. 8). Furthermore, because MAG(d1-3)-Fc can
bind neurons in a sialic acid-dependent manner but does
not inhibit axonal growth, this truncated form of MAG
carries only one of these sites, the sialic acid binding site.
The site responsible for inhibition must be carried entirely by domains 4 and 5 or require these domains, together
with domains 1-3, to effect inhibition (Fig. 8). An alternative interpretation of these results is that, to bring about
inhibition, full-length MAG-Fc recognizes a sialoglycoconjugate distinct from those recognized by MAG(d1-3)-Fc,
and sialoadhesin. This is unlikely as full-length MAG-Fc,
MAG (d1-3)-Fc, and sialoadhesin-Fc all recognize sialic acid residues in an
2,3-linkage attached to O-linked glycoconjugates (Kelm et al., 1994
).
Fig. 8.
The sialic acid binding site on MAG (R118) is critical
for inhibition of axonal regeneration by soluble MAG-Fc but not
MAG expressed by CHO cells. There are two recognition sites
on MAG, a sialic acid binding site (SA; square symbol) and a neurite outgrowth inhibition site (NOG; triangular symbol). When
MAG is expressed by CHO cells, both sites engage the neuron
and neurite outgrowth is inhibited (A). When MAG mutated at
its sialic acid binding site is expressed in CHO cells, another cell
adhesion molecule (CAM) on the CHO cell surface engages the
neuron along with the neurite outgrowth inhibition site and neurite outgrowth is still inhibited (B). When soluble MAG-Fc is
added to neurons, both the sialic acid and the inhibition sites engage the neuron and neurite outgrowth is inhibited (C). However, when MAG-Fc mutated at the sialic acid binding site is
added to neurons, it cannot bind to neurons and consequently the
inhibition site cannot engage and there is no inhibition of neurite
outgrowth (D). In contrast, when MAG(d1-3)-Fc is added to
neurons in binds via its sialic acid binding site but does not inhibit
axonal growth because the inhibition site is absent.
[View Larger Version of this Image (21K GIF file)]
).
However, this monoclonal antibody effectively blocks
sialic acid-dependent binding of MAG to neurons but still
recognizes R118A- and R118D-MAG. Therefore the epitope for the 513 monoclonal antibody must be very close to, but not coincident with R118, the sialic acid binding
site and the antibody must block sialic acid-dependent
binding by steric hindrance. In keeping with this observation, the 513 monoclonal antibody reverses inhibition of
neurite outgrowth by soluble MAG-Fc, which is dependent on MAG-sialic acid-dependent binding (Tang et al.,
1997
), but it has no effect on inhibition of neurite outgrowth by MAG expressed by CHO cells (Mukhopadhyay
et al., 1994
).
). A similar small, but significant reversal in inhibition of neurite outgrowth by R118-mutated MAG upon
neuronal desialylation was also observed (results not shown).
There are two possible explanations for this apparent discrepancy in findings. First, because this improvement in
outgrowth is small (rarely more than 25%) it may result
from removal of a steric effect brought about by the
charge on sialic acid. A small effect on neurite outgrowth
resulting from changes in charge may not be apparent in
the more robust neurite outgrowth observed on the control cells. Second, although sialic acid-dependent binding
of R118-mutated MAG could not be measured for either
MAG-Fc or MAG expressed by CHO or Schwann cells, it
is possible that sialic acid-dependent binding is not completely abolished by this mutation. That is to say, mutation
of R118 may decrease the affinity of MAG binding to
sialic acid to a level that cannot be measured in the binding assays available. On the other hand, when expressed by CHO or Schwann cells, mutated, R118-mutated MAG
may still be able to engage the neuron in low-affinity sialic
acid-dependent binding, which is sufficient to influence
neurite outgrowth. However, the inability of MAG(d1-3)
to inhibit axonal outgrowth despite binding to neurons in a
sialic acid-dependent manner argues that even if sialic
acid binding of MAG is required, alone it is insufficient to
inhibit axonal growth.
; van der Merwe et al.,
1996
). In those studies a number of point mutations were
introduced at different sites in the first Ig domain of each
molecule. The mutated proteins were assessed for sialic acid-binding and, using a panel of conformation-dependent monoclonal antibodies, for misfolding. For sialoadhesin, disruptive substitutions at six residues abolished
sialic acid-dependent binding with little effect on protein
folding. In a model of Ig domain 1 of sialoadhesin, based
on the crystal structure of CD8, these amino acids in the G, F, and C
-strands form a discrete cluster on one
-sheet, surrounded by residues that do not affect binding
upon mutation. When a conservative amino acid change
was introduced at these various sites, only substitution at
R97 led to a complete loss of binding (Vinson et al., 1996
).
Therefore, it was concluded that these six amino acids, with
R97 as a key residue, are critical for sialic acid-dependent
binding of sialoadhesin. Similar analysis concluded that
R130 in CD22 is a key amino acid in sialic acid-dependent binding (van der Merwe et al., 1996
). Therefore, it appears
that an arginine, conserved in the first Ig domain of at least
three sialoadhesin family members, is imperative for the
successful binding of these molecules to sialic acid.
).
However, studies with antibodies to RGD-containing peptides and with the peptides themselves, indicate that MAG
does not use this sequence in its interactions (Pedraza et al.,
1990
; Sadoul et al., 1990
). Antibodies to a 20-mer peptide
corresponding to a MAG-amino acid sequence spanning
the RGD sequence does not bind to native MAG but recognizes denatured MAG. This implies that the RGD sequence is not exposed at the surface of the molecule and
consequently is not available for interaction (Pedraza et
al., 1990
). Consistent with this observation, a synthetic
MAG peptide containing this RGD sequence failed to block binding to axons by MAG incorporated into liposomes (Sadoul et al., 1990
). From the studies with sialoadhesin, the six amino acids that comprise the sialic acid
binding site are not consecutive but are found on
-strands
G, F, and C (Vinson et al., 1996
). Three of these amino acids, including R118, are conserved in MAG (Fig. 1). It is
probable, therefore, that only R118 of the RGD sequence
of MAG is involved in binding and that this binding is distinct from integrin-mediated binding.
) and evolutionary selection of a second site for inhibition on MAG
would allow greater specificity to be exerted. The sialic
acid binding of MAG may promote, but may not be essential for, the interaction of the inhibition site. It is not
known if the two components recognized by the sialic acid-binding site and the neurite outgrowth inhibition site
of MAG are carried by a single neuronal protein or closely
associated proteins. This point can only be resolved when
the component(s) with which MAG interacts on neurons
has been identified.
; Sako et al., 1995
). P-selectin interacts with sialylated, fucosylated O-linked glycans attached to a variety
of ligands (Varki, 1994
). For interaction with the PSGL-1
ligand, however, P-selectin also requires a sulfated tyrosine for high affinity binding (Pouyani and Seed, 1995
;
Sako et al., 1995
). Consequently, two binding sites are proposed for the interaction of P-selectin with PSGL-1, one
that is carbohydrate-dependent and one that is amino acid (sulfated)-dependent. In contrast, another family member, E-selectin, also binds to PSGL-1 but only with low affinity and only via the carbohydrate epitope (Asa et al.,
1995
); E-selectin does not carry the sulfated-tyrosine-recognition epitope.
Received for publication 22 May 1997 and in revised form 8 July 1997.
Address all correspondence to Marie T. Filbin, Department of Biological Sciences, Hunter College of City University of New York, 695 Park Avenue, New York, NY 10021. Tel.: (212) 772-5270. Fax: (212) 772-5227. e-mail: filbin{at}genectr.hunter.cuny.eduWe would like to thank R. Persell for critically reading this manuscript.
This work was supported by a grant from the National Multiple Sclerosis Society, and a core facility Research Centers in Minority Institutions, National Institutes of Health grant. M.T. Filbin is the recipient of an Established Investigator Award from the American Heart Association, New York Chapter.
CNS, central nervous system; DRG, dorsal root ganglion; MAG, myelin-associated glycoprotein.
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