(Received for publication, January 22, 1996; and in revised form, February 1, 1996)
From the
The cell adhesion molecule L1 plays an important role in neural development. We have previously demonstrated that the second immunoglobulin-like domain (Ig2) of L1 contains both homophilic binding and neuritogenic activities (Zhao, X., and Siu, C.-H. (1995) J. Biol. Chem. 270, 29413-29421). Recently, two mutations (R184Q and H210Q) within the Ig2 region of the human L1 gene have been shown to be responsible for X-linked hydrocephalus and the related MASA (mental retardation, aphasia, shuffling gait, and adducted thumbs) syndrome. Glutathione S-transferase-Ig2 fusion proteins containing these mutations were used to evaluate their effects on L1. The homophilic binding activity of fusion proteins and their ability to promote neurite outgrowth from retinal cells were examined. The R184Q mutation led to a complete loss of both homophilic binding and neuritogenic activities, while the H210Q mutation resulted only in a partial loss. These results provide, for the first time, direct demonstration of the deleterious effects of hydrocephalus/MASA mutations on two intrinsic properties of L1.
The cell adhesion molecule L1 is expressed primarily in
postmitotic neurons and has been implicated in neural migration,
neurite outgrowth, and fasciculation during brain development (for a
review, see (1) ). L1 is a 200-kDa transmembrane glycoprotein
and a member of the immunoglobulin (Ig) superfamily of cell adhesion
molecules. It contains six Ig-like domains in the amino-terminal
region, followed by five fibronectin type III repeats, one
transmembrane domain, and a cytoplasmic domain(2, 3) .
L1 can undergo homophilic interactions with L1(4, 5) ,
as well as heterophilic interactions with other adhesion molecules,
such as NCAM()(6) ,
TAG-1/axonin-1(7, 8) , F3/F11 (9) ,
glia(10) , and components of the extracellular
matrix(11, 12) . In addition to cell adhesion,
substrate-coated L1 is a potent inducer of neurite outgrowth from
primary neurons(4, 5) .
The human L1 cDNA has been cloned(13) , and the gene has been mapped to chromosome Xq28(14) . Several recent reports show that a group of heterogeneous mutations in L1 are responsible for X-linked hydrocephalus and two related neurological disorders, MASA (mental retardation, aphasia, shuffling gait, and adducted thumbs) syndrome, and spastic paraplegia type 1(15, 16, 17) . Most of them are missense mutations, resulting in amino acid changes in the extracellular and cytoplasmic domains, while others are nonsense, deletion, or splicing mutations resulting in the truncation or secretion of L1. However, little is known about how these mutations give rise to these related neurological diseases. An investigation of the role of the mutated residues in L1 function is, therefore, crucial to our understanding of these defects.
We recently demonstrated that the Ig2 domain of L1 harbors both homophilic binding and neuritogenic activities(18) . Interestingly, two missense mutations have been localized to Ig2. One results in the replacement of Arg-184 with Gln. This mutation is found in patients with severe hydrocephalus, which is characterized by the absence of the corticospinal tract and stenosis of the aqueduct of sylvius(16) . The other mutation results in the substitution of His-210 with Gln and is detected in MASA patients with a milder phenotype(16) . Those who survived suffered from mental retardation. We have investigated the effects of these two mutations on the homophilic binding and neuritogenic activities associated with L1 Ig2. These activities are completely lost in the R184Q mutation, but are only partially affected in the H210Q mutation.
To investigate the effects of R184Q and H210Q mutations on
the homophilic binding and neuritogenic activities of L1, Ig2
containing these mutations were expressed in bacteria as GST fusion
proteins. GST-Ig2mt1 contained the R184Q mutation, and GST-Ig2mt2
contained the H210Q mutation. Purified proteins were analyzed by gel
electrophoresis (Fig. 1). Under reducing conditions, both
wild-type and mutant GST-Ig2 fusion proteins migrated with an M of 41,000. The purified and refolded proteins
were used in subsequent assays.
Figure 1: Construction and expression of mutant GST-L1 fusion proteins. A, schematic drawings of GST-mutant Ig2 fusion proteins. B, gel profiles of purified GST fusion proteins. Protein samples were separated on 10% SDS-polyacrylamide gel and stained with Coomassie Blue. Lane a, GST-Ig2; lane b, GST-Ig2mt1; lane c, GST-Ig2mt2; lane d, GST.
To facilitate the analysis of these
fusion proteins, LR73 cells were transfected with the L1 cDNA. LR73
cells do not express L1 and are especially suitable for the analysis of
cell adhesion molecules(19) . Sense or antisense L1 cDNAs were
inserted into the pRc/CMV (Fig. 2A) for expression in
LR73 cells. Immunoblot analysis revealed a major L1 band at 200
kDa and two cleavage products of smaller size in cells transfected with
the sense L1 cDNA but not in those with the antisense construct (Fig. 2A). Cell surface expression of L1 on
transfectants was confirmed by immunofluorescence staining (data not
shown). The Covasphere-to-cell binding assay demonstrated that the L1
molecules expressed in these cells were functional. About 50% of
L1-expressing cells bound L1-conjugated Covaspheres, while only 3% of
antisense L1 transfectants showed positive binding (Fig. 2, B and C). Binding of L1 Covaspheres to L1
transfectants was inhibited by anti-Ig1-2-3 Fab, but not by
anti-Ig4-5-6 Fab. This is consistent with our previous finding that
L1-L1 binding is dependent on the Ig2 domain(18) .
Figure 2: Binding of L1-conjugated Covaspheres to L1-transfected LR73 cells. A, construction of the pRc/CMV/L1 expression plasmid and L1 expression in transfected LR73 cells. Immunoblots of transfectants were stained with 10 µg/ml rabbit anti-Ig4-5-6 IgG: lane a, control LR 73 cells; lane b, antisense transfectants; lane c, L1 transfectants. B, epifluorescence micrographs showing the binding of L1-conjugated Covaspheres to L1-expressing LR73 cells (a) or antisense transfectants (b). C, effects of mutant GST-Ig2 fusion proteins on the binding of L1-conjugated Covaspheres to L1 transfectants. a, binding of L1 Covaspheres to antisense-L1-LR73 cells and L1-LR73 cells. L1-LR73 cells were also precoated with either anti-Ig1-2-3 Fab or anti-Ig4-5-6 Fab (100 µg/ml), and their effects were determined. In b, binding of L1 Covaspheres was carried out after the preincubation of L1-LR73 cells with various protein competitors at 50 µg/ml (stippled bars) or 100 µg/ml (solid bars). Identical cell samples were stained with anti-L1 antibody. The percentage of cells with bound Covaspheres was normalized to the percentage of L1-expressing cells.
To assess
the effects of the R184Q and H210Q mutations on the ability of Ig2 to
compete for L1 binding, the Covasphere-to-cell binding assay was
performed in the presence of these fusion proteins (Fig. 2C). Fifty percent inhibition was achieved at 50
µg/ml GST-Ig2, and binding was abolished at 100 µg/ml. In
contrast, GST-Ig2mt1 failed to inhibit the binding reaction at these
concentrations. Similar results were obtained with GST as the control
competitor. Interestingly, partial competition was observed with
GST-Ig2mt2. At 100 µg/ml, it inhibited the binding of Covaspheres
to cells by 70%.
To directly test the homophilic binding
activity of the mutant fusion proteins, binding of fusion
protein-conjugated Covaspheres to substrate-coated protein was carried
out. All fusion proteins adsorbed to Petri dishes with similar
efficiency. As a positive control, IgG directed against Ig1-2-3 of L1 (18) was adsorbed onto a Petri dish for Covasphere binding,
while substrate-coated GST was used as a negative control.
GST-Ig2-conjugated Covaspheres attached very well to substrate-coated
GST-Ig2. However, binding of GST-Ig2 Covaspheres to GST-Ig2mt2 was
reduced by 50%, but no significant binding to the GST-Ig2mt1
substrate was observed (Fig. 3A). In addition,
GST-Ig2mt1-conjugated Covaspheres attached to substrate-coated
GST-Ig2mt1 only at background level (Fig. 3B),
indicating that the Arg to Gln substitution led to the loss of its
homophilic binding activity. In contrast, GST-Ig2mt2 still retained
significant homophilic binding activity (Fig. 3C).
Thus, the H210Q mutation had a milder effect on the homophilic binding
activity of Ig2.
Figure 3: Binding of Covaspheres conjugated with mutant GST-Ig2 fusion proteins to different substrates. Round spots on Petri dishes were coated with 5 µl of different proteins (1 µM). Covaspheres conjugated with GST-Ig2 (A), GST-Ig2mt1 (B), or GST-Igmt2 (C) were allowed to adhere to the protein substrate for 30 min. The number of Covaspheres attached per unit area was estimated, and the results were normalized to the amount of Covaspheres bound to the substratum coated with anti-Ig1-2-3 IgG. Data represent the mean ± S.D. (n = 9).
Next, the effects of these fusion proteins on
neurite outgrowth were examined. The mean neurite length of neural
retinal cells cultured on L1-expressing LR73 cells was 3 times
longer than those cultured on the antisense transfectants (Fig. 4A). When individual fusion proteins were
included in this assay, GST-Ig2, being a strong competitor for L1
binding, reduced the mean neurite length to near background level,
while GST and GST-Ig2mt1 exhibited no significant inhibitory effects.
However, GST-Ig2mt2 was
80% as effective as GST-Ig2 in the
inhibition of neurite outgrowth from retinal cells (Fig. 4B).
Figure 4: Differential effects of mutant GST-Ig2 fusion proteins on neurite outgrowth. Retinal cells were labeled with DiI and then deposited on a monolayer of LR73 transfectants. After 18 h of coculture, cells were fixed and neurites extended from retinal neurons were measured. In competition experiments, cocultures were carried out in the presence of different competitors at 1 µM concentration. Data represent the mean ± S.D. of three experiments.
We have previously demonstrated that the L1 Ig2 fragment can serve as a potent substrate for neurite outgrowth from retinal cells(18) . The effects of the two hydrocephalus/MASA syndrome-related mutations on the neuritogenic activity of Ig2 were examined. In comparison with cells cultured on the GST substrate, GST-Ig2 stimulated a 2.8-fold increase in the mean neurite length of retinal cells (Fig. 5). In contrast, GST-Ig2mt1 failed to promote neurite outgrowth from these cells. GST-Ig2mt2, on the other hand, retained substantial neuritogenic activity, and only a 20% reduction in the mean neurite length was observed.
Figure 5: Neurite outgrowth promotion activity of mutant GST-Ig2 fusion proteins. Coverslips were coated with 80 µl of GST-Ig2 fusion protein at 1 µM concentration. Retinal cells were seeded on different protein substrates and cultured for 18 h. Mean neurite lengths for cells cultured on these protein substrates were determined. Data represent the mean ± S.D. of three experiments.
The above results, taken together, demonstrate that the
R184Q mutation abolishes both the homophilic binding activity and the
neuritogenic activity associated with the Ig2 domain of L1, whereas the
H210Q mutation results only in a partial loss of these two activities.
It is likely that Arg-184 plays a crucial role in the structure and
function of L1. Arg-184 is highly conserved in L1 among different
species, including mouse(2) , rat(3) ,
chicken(20) , and the Drosophila homolog(21) .
In addition, this residue lies within a region corresponding to the
predicted C` -strand of the Ig fold, suggesting that the C` region
of L1 Ig2 may participate directly in L1-L1 homophilic binding. It is
conceivable that the forward binding reaction may depend on
electrostatic interactions involving Arg-184. Interactions centered at
this region may then lead to other interactions at secondary sites
along the length of the extracellular segments of two apposing
molecules, further stabilizing the binding reaction. This possibility
has been observed in NCAM, which is also a member of the Ig
superfamily. NCAM homophilic interaction is centered around the C`
-strand and the C` E loop in the third Ig-like domain (22) . This region of the NCAM molecule is capable of
undergoing isologous interactions with the same region of an apposing
molecule (23) . The charged residues in this region also appear
to play a crucial role(22) .
In both L1 and NCAM, homophilic
binding is closely coupled to their ability to induce neurite outgrowth
from neuronal cells(18, 24) . Although several other
structural domains in L1 have been implicated in neurite outgrowth
promotion(25, 26) , the severity of the
neuropathological phenotype of patients with the R184Q mutation attests
to the importance of the homophilic binding and neuritogenic activities
centered around Arg-184. It is conceivable that homophilic binding may
generate neurite outgrowth signals by inducing conformational changes
in the molecule and alter its interactions with other membrane or
cytoplasmic components. Potential candidates involved in downstream
events of L1-dependent neurite outgrowth include fibroblast growth
factor receptor(27) ,
pp60(28) , and ankyrin(29) .
His-210 is predicted to lie within the F -strand of the Ig
fold, with its charged side chain pointing outward on the surface of
the molecule. Substitutions with another charged or polar residue are
likely to be tolerated. Indeed, this residue is less conserved in L1
homologs, and His-210 is replaced by Asn in mouse and rat (2, 3) and by Ser in the Drosophila homolog(21) . This is also supported by the less
deleterious effects of the H210Q mutation. Therefore, a close
correspondence exists between the in vitro activities of the
mutant proteins and the pathological phenotype caused by these two
mutations. Exactly how these two mutations affect neural development is
not known. Further studies will depend on the availability of
transgenic animals that express these mutant forms of L1.
To date,
23 mutations in L1 have been reported in hydrocephalus, MASA, and
spastic paraplegia type 1 patients. These mutations are evenly
distributed along the L1 molecule(1, 17) , indicating
that these structural domains may have important biological functions.
Alternatively, some of the amino acid substitutions may induce
conformational changes, causing the inactivation of functional domains
at a distance. Mutations outside the Ig2 domain may affect heterophilic
interactions of L1 with other matrix and membrane components. Recently,
the RGD sequence located in the sixth Ig-like domain of L1 has been
found to interact with the integrin (30) .
is present predominantly in the glia
of the central nervous system(31) , suggesting a role for the
L1 RGD sequence in neuron-glia interaction in the brain cortex and in
the cerebellum. L1 is also known to interact heterophilically with
axonin-1/TAG-1, F3/F11, and brain proteoglycans. It is evident that L1
has a very complex biology, and mutations affecting its interactions
with different binding partners may have a wide range of effects on
neuron migration and neurite outgrowth. Our future understanding of the
diverse biological roles of L1 in brain development will depend on the
identification of its binding sequences and the elucidation of their
mechanisms of interaction.