(Received for publication, August 28, 1995; and in revised form, November 10, 1995)
From the
The laminin binding properties of -dystroglycan purified
from rabbit skeletal muscle membranes were examined. In a solid phase
microtiter assay,
I-laminin (laminin-1) bound to purified
-dystroglycan in a specific and saturable manner with a
half-maximal concentration of 8 nM. The binding of
I-
-dystroglycan to native laminin and merosin (a
mixture of laminin-2 and -4) was also compared using the solid phase
assay. The absolute binding of
I-
-dystroglycan to
laminin (6955 ± 250 cpm/well) was similar to that measured for
merosin (7440 ± 970 cpm/well). However, inclusion of 1 mg/ml
heparin in the incubation medium inhibited
I-
-dystroglycan binding to laminin by 84 ±
4.3% but inhibited
I-
-dystroglycan binding to
merosin by only 17 ± 5.2%. Similar results were obtained with
heparan sulfate, while de-N-sulfated heparin, hyaluronic acid,
and chondroitin sulfate had no differential effect. These results were
confirmed by iodinated laminin and merosin overlay of
electrophoretically separated and blotted dystrophin-glycoprotein
complex. In contrast to the results obtained with skeletal muscle
-dystroglycan, both laminin and merosin binding to purified brain
-dystroglycan was significantly inhibited by heparin. Our data
support the possibility that one or more heparan sulfate proteoglycans
may specifically modulate the interaction of
-dystroglycan with
different extracellular matrix proteins in skeletal muscle.
Skeletal muscle dystrophin has been shown to co-purify with a
large oligomeric complex of proteins termed the dystrophin-glycoprotein
complex(1, 2, 3) , which is reduced in
abundance or abnormal in dystrophic muscle(4) . A highly
similar complex is associated with utrophin(5) , an autosomal
homologue of dystrophin (6) that is specifically localized to
the neuromuscular junction(7) . Both complexes are thought to
interact with the extracellular matrix by way of the 156-kDa
glycoprotein, now referred to as -dystroglycan(8) , based
on binding to the laminins (9, 10, 11) and
agrins(12, 13, 14, 15) , two
structurally related extracellular protein families with markedly
distinct functions (16, 17, 18) .
The
importance of the -dystroglycan/laminin interaction is inferred
from studies that have demonstrated that the absence or abnormality in
various laminin subunits also causes muscular
dystrophies(11, 19, 20, 21, 22) similar to those involving the dystrophin-glycoprotein
complex(4) . These findings, coupled with data documenting the
importance of the extensive post-translational modification of
-dystroglycan for laminin (10) and agrin
binding(15) , support further examination of the role that
-dystroglycan post-translational modification plays in binding to
proteins of the extracellular matrix. To begin addressing this
question, we purified
-dystroglycan from rabbit skeletal muscle
membranes. In characterizing the laminin binding properties of purified
-dystroglycan, we were surprised by the significant difference in
heparin sensitivity apparent between laminin (laminin-1) and merosin (a
mixture of laminin-2 and -4). We propose a mechanism by which this
difference may be important in modulating skeletal muscle
-dystroglycan interactions with different extracellular matrix
proteins.
Figure 1:
Binding of I-laminin to
purified skeletal muscle
-dystroglycan. Shown is the binding of
eight different concentrations (0.14-56 nM)
I-laminin to purified skeletal muscle
-dystroglycan
in the absence (
) or presence (
) of 1 mg/ml heparin.
Specific binding (
) is taken as the difference of values
obtained with and without heparin. Each point represents the average of
triplicate determinations. Nonlinear regression analysis yielded a
half-maximal concentration of 8
nM.
I-Laminin binding to purified skeletal muscle
-dystroglycan was examined with a solid phase assay previously
used to characterize the laminin binding properties of brain
-dystroglycan(28) . The concentration curve for
I-laminin binding to purified skeletal muscle
-dystroglycan (Fig. 1) was virtually identical to that
previously published for brain
-dystroglycan (see Fig. 7 of (28) ).
I-Laminin bound to purified skeletal
muscle
-dystroglycan in a saturable manner with a half-maximal
concentration of 8 nM (Fig. 1).
The binding of I-
-dystroglycan to immobilized laminin and merosin
was also compared using the solid phase microtiter assay. In
side-by-side experiments, the absolute binding of
I-
-dystroglycan to laminin (6955 ± 250
cpm/well) was similar to that measured for merosin (7440 ± 970
cpm/well).
I-
-Dystroglycan binding to both laminin
and merosin was similarly competed by increasing concentrations of
unlabeled laminin (IC
, 2 nM). As further evidence
of the specificity of the interaction, it was observed that
I-
-dystroglycan binding to equivalent amounts of BSA
(246 ± 38 cpm/well), collagen IV (407 ± 15 cpm/well), or
heparan sulfate proteoglycan (671 ± 51 cpm/well) was
substantially less than that observed for laminin (6955 ± 250
cpm/well) and merosin (7440 ± 970 cpm/well). In agreement with
our previous results using the
I-laminin
overlay(10) ,
I-
-dystroglycan binding to
laminin and merosin was similarly inhibited by increasing ionic
strength with an IC
for NaCl of 250 mM (not
shown).
I-
-Dystroglycan binding to laminin and
merosin was further compared by examining the concentration dependence
of heparin inhibition (0-2 mg/ml) using the microtiter assay.
Surprisingly,
I-
-dystroglycan binding to laminin was
significantly more sensitive to heparin over the range of 0.5-2
mg/ml in comparison with merosin (Fig. 2).
I-
-Dystroglycan binding to merosin was notably
insensitive to heparin at concentrations greater than or equal to 0.2
mg/ml (Fig. 2). Performance of the microtiter assay using wells
coated with purified
-dystroglycan and probing with iodinated
laminin and merosin yielded heparin inhibition curves virtually
identical to those illustrated in Fig. 2(not shown).
Figure 2:
The effect of heparin on I-
-dystroglycan binding to laminin and merosin.
I-
-Dystroglycan (125I
-DG) binding to laminin
(
) and merosin (
) in the presence of the indicated
concentration of heparin was measured using the solid phase microtiter
assay described under ``Experimental Procedures.'' Binding
data were normalized as a percent of control for individual experiments
performed in triplicate, and the graph represents the mean ±
S.E. of three independent experiments.
The
specificity of heparin in differentially inhibiting I-
-dystroglycan binding to laminin and merosin was
examined by comparing the relative effect of various glycosaminoglycans
(1 mg/ml) in the solid phase assay. The sodium salt of heparin
inhibited
I-
-dystroglycan binding to laminin by an
average of 84 ± 4.3% (Fig. 3). Ca
heparin and heparan sulfate also dramatically inhibited
I-
-dystroglycan binding to laminin, although heparan
sulfate appeared less effective than either heparin salt (Fig. 3). In contrast to the results obtained with laminin,
Na
heparin inhibited
I-
-dystroglycan binding to merosin by only 17
± 5.2%, which was similar to the inhibition effected by the
other glycosaminoglycans tested (Fig. 3). These data document
the specificity of heparan sulfate-like glycosaminoglycans in
differentially inhibiting
-dystroglycan binding to laminin versus merosin.
Figure 3:
Relative effect of various
glycosaminoglycans on I-
-dystroglycan binding to
laminin and merosin.
I-
-Dystroglycan (
I
-DG) binding to laminin and merosin in
the presence of 1 mg/ml Na
heparin (Hep),
Ca
heparin, de-N-sulfated heparin, heparan
sulfate (HS), hyaluronic acid (HA), or chondroitin
sulfates A (CSA), B (CSB), and C (CSC) was
measured using the solid phase microtiter assay described under
``Experimental Procedures.'' Binding data were normalized as
a percent of control performed in the absence of added
glycosaminoglycan. The data for Na
heparin represent
the mean ± S.E. of nine independent experiments, each performed
in triplicate. The data for heparan sulfate and chondroitin sulfate B
represent the mean of two independent experiments, each performed in
triplicate. All other data represent the mean of triplicate
determinations.
The binding of I-laminin and
I-merosin to
-dystroglycan was also compared by the
protein overlay assay using nitrocellulose transfers containing
electrophoretically separated dystrophin-glycoprotein
complex(10) .
I-Laminin and
I-merosin binding to
-dystroglycan in the
dystrophin-glycoprotein complex were similarly inhibited by the
inclusion of 10 mM EDTA or 0.5 M NaCl to the overlay
medium (Fig. 4A). However, addition of 1 mg/ml heparin
completely inhibited
I-laminin binding to
-dystroglycan but had little effect on
I-merosin
binding to
-dystroglycan (Fig. 4B). Comparison of
heparin's effect on nonradioactive laminin and merosin overlay of
dystrophin-glycoprotein complex, as detected with a polyclonal laminin
antibody(29) , yielded results similar to those presented in Fig. 4(Fig. 5). In contrast to our results with 156-kDa
skeletal muscle
-dystroglycan, merosin binding to 120-kDa brain
-dystroglycan was markedly inhibited by heparin (Fig. 5),
which agrees with previous findings on 120-kDa peripheral nerve
-dystroglycan(29) . In addition to confirming the results
obtained with the solid phase assay ( Fig. 2and 3), our overlay
results ( Fig. 4and Fig. 5) suggest that the more
extensive post-translational modification of skeletal muscle
-dystroglycan is necessary for the observed differences in heparin
sensitivity of its binding to laminin versus merosin.
Figure 4:
Effect of heparin on I-Laminin and
I-merosin overlay of
dystrophin-glycoprotein complex. Shown in A are identical
nitrocellulose transfers containing electrophoretically separated
dystrophin-glycoprotein complex overlaid with
I-laminin
or
I-merosin in the absence (Control) or
presence of 10 mM EDTA or 0.5 M NaCl. Shown in B are identical nitrocellulose transfers containing
electrophoretically separated dystrophin-glycoprotein complex overlaid
with
I-laminin or
I-merosin in the absence (Control) or presence of 1 mg/ml heparin
(+Heparin). The molecular weight standards (
10
) are indicated on the left.
Figure 5:
Effect of heparin on laminin and merosin
overlay of brain -dystroglycan. Identical nitrocellulose transfers
containing purified rabbit skeletal muscle dystrophin-glycoprotein
complex (DGC) and brain
-dystroglycan (
-DG)
were incubated with 1 µg/ml native laminin or merosin in the
absence (-HEP) or presence (+HEP) of 1
mg/ml heparin. Transfers were washed and then sequentially incubated
with affinity-purified polyclonal antibodies to laminin followed by
peroxidase-coupled secondary antibody. Laminin and merosin binding was
detected by chemiluminescence using SuperSignal CL-HRP (Pierce) as
substrate. Densitometric analysis indicated that heparin inhibited
merosin binding to skeletal and brain
-dystroglycan 26 and 63%,
respectively, while heparin inhibition of laminin binding to skeletal
and brain
-dystroglycan was 68 and 87%, respectively. The
molecular weight standards (
10
) are
indicated on the left.
The differential heparin sensitivity of skeletal muscle
-dystroglycan binding to laminin versus merosin (Fig. 2Fig. 3Fig. 4Fig. 5) is surprising in
light of the fact that both laminin and merosin bind
heparin(32, 33) . Variations in the purity or
integrity of commercial merosin preparations are likely not the reason
for this difference because experiments with four different merosin
lots from two vendors yielded similar results. Other than differential
heparin inhibition, laminin and merosin exhibited very similar
-dystroglycan binding properties. Furthermore, unlabeled laminin
was equally effective at inhibiting
I-
-dystroglycan
binding to laminin and merosin. Our data imply that laminin and merosin
have similar
-dystroglycan binding sites that overlap with
distinct heparin binding sites. Consistent with our data, it was shown
that laminin and merosin binding to heparan sulfate proteoglycan
(perlecan) were differentially inhibited by heparin with IC
values of 0.8 and >500 µg/ml, respectively(34) .
The overall sequence similarity between the A chain of laminin and the
M chain of merosin is 46.6%, with a 41.8% sequence identity in the
carboxyl-terminal G domain(18) , which is responsible for
laminin binding to
-dystroglycan(28) . While laminin and
merosin exert similar effects on cell attachment and neurite
outgrowth(33) , it has been noted that merosin promoted a
significantly greater level of neuronal cell migration than did
laminin(35) . Finally, that increased expression of laminin
fails to correct for genetic (22) merosin deficiency in the
dystrophic dy/dy mouse (20) is additional proof that laminin
and merosin are not functionally redundant. Thus, there is sufficient
sequence variability as well as precedent for a functional difference
between laminin and merosin like that implied by our observation of
differential heparin sensitivity in
-dystroglycan binding.
One
question raised (36) by the observations that skeletal muscle
-dystroglycan binds to both laminins (9, 10, 11) and agrins (12, 13, 14, 15) is whether and how
-dystroglycan might discriminate between different extracellular
matrix proteins, even when all are present in the same
tissue(37, 38) . This issue is particularly relevant
because laminin has been shown to inhibit agrin binding to
-dystroglycan(14) , while nerve and muscle agrins bind
-dystroglycan with similar affinity (15) yet exhibit
dramatically different potencies in clustering acetylcholine
receptors(39) . Our results support the possibility that unique
heparan sulfate-containing proteoglycans may differentially modulate
-dystroglycan interactions with various extracellular matrix
proteins. In support of this hypothesis, we have demonstrated that
heparin and heparan sulfate, but not de-N-sulfated heparin,
hyaluronic acid, or chondroitin sulfates, are effective in inhibiting
-dystroglycan binding to laminin (Fig. 3). However, the
high concentration of heparin necessary to inhibit
-dystroglycan
binding to laminin (Fig. 2) further suggests that a minor
subpopulation of heparin is responsible for our observed effects. As
reviewed by Rapraeger(40) , specifically sulfated microdomains
of heparan sulfate are important in the mechanism of action of
antithrombin and fibroblast growth factor. Furthermore, unique heparan
sulfate proteoglycans can exhibit restricted localization at the
neuromuscular junction (41) and have been implicated in the
development and/or repair of muscle (42, 43, 44) and
nerve(45, 46, 47, 48) . Finally,
laminin and other extracellular matrix proteins bind to structurally
distinct subpopulations of heparin with variable
affinities(49) . Taken together, these findings support the
possibility that one or more heparan sulfate proteoglycans may
specifically modulate the interaction of
-dystroglycan with
different extracellular matrix proteins in skeletal muscle.
Because
dystroglycan is encoded by a single gene(8) , the apparent size
difference between neuronal and skeletal muscle -dystroglycan is
likely due to differential post-translational
modification(3, 9) . While recent progress has been
made in the characterization of brain
-dystroglycan
post-translational modification(27) , little is presently
understood concerning the differences in post-translational
modification between different tissue forms of
-dystroglycan and
how they translate into variations in
-dystroglycan function.
However, since post-translational modification is important to the
laminin binding activity of
-dystroglycan(10) , it seemed
reasonable that merosin binding to neuronal and skeletal muscle
-dystroglycan may also be differentially inhibited by heparin. In
support of this possibility, Gee et al.(28) demonstrated that heparin inhibited
I-laminin binding to brain
-dystroglycan with an
IC
of less than 0.1 µg/ml, while our previous (10) and present (Fig. 2) results indicate that heparin
inhibited skeletal muscle
-dystroglycan with an IC
of
250 µg/ml. We have further demonstrated that heparin does indeed
inhibit both laminin and merosin binding to purified brain
-dystroglycan (Fig. 5). Yamada et al.(29) also observed heparin inhibition of merosin binding
to 120-kDa peripheral nerve
-dystroglycan, which is similar in
size to brain
-dystroglycan (9, 10, 28) . Thus, it would appear that both
differential post-translational modification of
-dystroglycan and
structural variations between the identified extracellular ligands for
-dystroglycan may be involved in our hypothesized role for heparan
sulfate containing proteoglycans in modulating
-dystroglycan/extracellular matrix interactions.