Disruption of perlecan binding and matrix assembly by
post-translational or genetic disruption of dystroglycan function
Motoi Kanagawaa,b,c, Daniel E. Michelea,b,c,1, Jakob S. Satza,b,c, Rita Barresia,b,c,
Hajime Kusanoa,b,c, Takako Sasakif, Rupert Timplf, Michael D. Henryd,e, Kevin P. Campbella,b,c,*
aDepartment of Physiology and Biophysics, Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine,
The University of Iowa, 400 Eckstein Medical Building, Iowa City, IA 52242, USA
bDepartment of Neurology, Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa,
400 Eckstein Medical Building, Iowa City, IA 52242, USA
cDepartment Internal Medicine, Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa,
400 Eckstein Medical Building, Iowa City, IA 52242, USA
dDepartment of Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA
eDepartment of Pathology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA
fMax-Planck-Institut fur Biochemie, Martinsried D-82152, Germany
Received 13 June 2005; revised 19 July 2005; accepted 25 July 2005
Available online 11 August 2005
Edited by Micheal R. Bubb
requires LARGE-dependent glycosylation for laminin binding.
Although the interaction of dystroglycan with laminin has been
well characterized, less is known about the role of dystroglycan
glycosylation in the binding and assembly of perlecan. We report
reduced perlecan-binding activity and mislocalization of perlecan
in the LARGE-deficient Largemydmouse. Cell-surface ligand
clustering assays show that laminin polymerization promotes
perlecan assembly. Solid-phase binding assays provide evidence
for the first time of a trimolecular complex formation of dystro-
glycan, laminin and perlecan. These data suggest functional dis-
ruption of the trimolecular complex in glycosylation-deficient
? ? 2005 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Dystroglycan is a cell-surface matrix receptor that
Keywords: Dystroglycan; Laminin; Perlecan; Basement
membrane; Congenital muscular dystrophy; Largemydmouse
Dystroglycan (DG) is a transmembrane protein that links
the extracellular matrix (ECM) to the cellular cytoskeleton,
and it has multiple roles in various tissues . DG consists
of the extracellular alpha subunit (a-DG), and the transmem-
brane beta subunit (b-DG), which are encoded by the same
mRNA and cleaved in post-translational processing .
b-DG binds to intracellular dystrophin or utrophin, which
then binds to actin filaments and extracellular a-DG. a-DG
binds to several ECM proteins that contain laminin globular
(LG) domains such as laminins, agrin, and perlecan . Using
recombinant LG domains, laminins and perlecan have shown
to compete for binding to a-DG .
Recent studies demonstrate that the O-glycosylation essen-
tial for ligand-binding activity of a-DG takes place on the
mucin-like domain . Detailed analyses indicate that the N-
terminal domain of a-DG is necessary for molecular recogni-
tion by a glycosyltransferase, LARGE, and that the DG-
LARGE interaction is critical for the functional expression
of DG . Mutations in the LARGE gene have been found
in human congenital muscular dystrophy type 1D, as well as
in the Largemydmouse [6,7]. Furthermore, recent studies sug-
gest that the DG post-translational glycosylation pathway is
a convergent target for several human muscular dystrophies,
classified as ‘‘dystroglycanopathies’’ . Hypoglycosylation
of a-DG in dystroglycanopathies and Largemydmice has been
observed in conjunction with a reduction of laminin-binding
Here, we investigate roles of DG in assembly of perlecan on
the cell surface. Reduced perlecan-binding activity of DG and
Largemydmice. By controlling ligand concentration and molec-
ular interaction, we provide evidence for the first time of a tri-
molecular complex of DG, laminin, and perlecan. These data
demonstrate the mechanism of the trimolecular complex for-
mation and suggest its disruption in the pathogenesis of glyco-
sylation-deficient muscular dystrophy.
2. Materials and methods
2.1. Animals, antibodies, and proteins
Wild type (C57BL/6) and Largemydmice were bred at The University
of Iowa from stock originally obtained from Jackson Laboratories
(Bar Harbor, ME). All animal studies were authorized by the Animal
Care Use and Review Committee at The University of Iowa.
Monoclonal antibody IIH6 against a-DG and rabbit polyclonal
antibodies against perlecan (anti-PGI and anti-PGV) were described
previously [8–10]. Anti-laminin and perlecan antibodies were obtained
from Sigma and Chemicon, respectively.
Perlecan fragments domain I (PGI) and domain V (PGV) were pre-
pared as previously described [9,10]. Laminin-1 and heparan sulfate
proteoglycan (HSPG), derived from Engelbreth–Holm–Swarm (EHS)
mouse sarcoma, were obtained from Biomedical Technologies Inc.
*Corresponding author. Fax: +1 319 335 6957.
E-mail address: firstname.lastname@example.org (K.P. Campbell).
1Department of Molecular and Integrative Physiology, University of
Michigan, Ann Arbor, MI 48109-0622, USA.
0014-5793/$30.00 ? 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 579 (2005) 4792–4796 FEBS 29857
and Sigma, respectively. Purification of a-DG and enrichment of DG
with wheat germ agglutinin (WGA)-beads were also described previ-
ously [5,8]. p-Aminoethylbenzenesulfonyl fluoride (AEBSF)-treated
laminin-1 was prepared using the method described by Colognato
et al. .
2.2. Solid-phase binding assay
The solid-phase binding assay was described previously . To mea-
sure IIH6-sensitive binding, IIH6 was used at 0.06 mg/ml. In cases
where the DG-laminin complexes were tested, laminin was preincu-
bated with immobilized DG in 3% BSA-LBB (10 mM triethanolamine,
pH 7.6, 140 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2) for 16 h prior
to the addition of tested ligand. Ligand binding was detected by incu-
bating with primary antibodies followed by HRP-conjugated second-
ary antibodies. All data were triplicate means.
Immunofluorescence analysis  and laminin-clustering assay 
were described previously.
3.1. Reduction of perlecan-binding activity of DG in Largemyd
To examine perlecan-binding activity of DG in Largemyd
mice, DG preparations were enriched with WGA-chromatog-
raphy from skeletal muscle. Western blotting with antibodies
to the a-DG core protein confirmed that nearly all of the
DG in the muscle sample bound to WGA-beads (data not
shown). Solid-phase PGV binding assays showed a reduction
of more than 80% of PGV binding in Largemydmice (Fig. 1).
This indicates that perlecan-binding activity requires the spe-
cific carbohydrate modification of a-DG. Therefore, we
hypothesized that reduced ligand-binding activity of DG
may cause displaced localization of ligand proteins.
3.2. Mislocalization of perlecan/laminin in Largemydmice
We have reported previously that the Largemydmouse has a
disruption of glia limitans, the surface basement membrane in
the brain, which leads to abnormal neuronal migration .
Here, we show that perlecan does not localize in regions where
the glia limitans is disrupted (Fig. 2). In addition, the Largemyd
mouse presents with an abnormal appearance of punctuate
accumulations of laminin/perlecan throughout the cerebral
cortex in locations where DG is not normally localized,
whereas laminin/perlecan staining on microvessels appears
normal (double arrows) (Fig. 3). We also observed similar
punctuate accumulations of laminin and perlecan in brain-
specific DG-null mice (data not shown). This suggests that
perlecan interacts with laminin independently of DG, however
DG may be required for proper location of perlecan.
3.3. Cell surface assembly of perlecan with DG-facilitated
In order to understand the role of DG and laminin in mem-
brane assembly of perlecan, cell-surface ligand clustering as-
says were performed. We observed defects of the cell surface
laminin-clustering on Largemydmyoblast and fibroblast, how-
ever perlecan was not detected in these cell culture system
(R.B, M.K, and K.P.C unpublished data). Since LARGE-defi-
ciency results in a DG functional-null phenotype, we have used
genetically engineered embryonic stem (ES) cells as a model
system. We previously reported that that DG was required
for the formation of laminin clusters on the surface of individ-
ual ES cells . The clusters have been classified on the basis
of three distinct morphologies: dots, lines, and plaques .
Fig. 4A shows colocalization of clusters of DG, laminin, and
Fig. 1. Reduction of perlecan-binding activity of DG in Largemydmice.
WGA-bound materials from Largemyd(myd) or littermate control
(WT) skeletal muscle extracts were immobilized and then incubated
with various concentrations of PGV. Binding was detected with anti-
PGV antibody. The maximal binding to the control preparations was
set as 100%.
Fig. 2. Immunolocalization of laminin and perlecan in Largemyd
cerebellum. Cryoselections from Largemyd(myd) and littermate control
(WT) mice were stained with antibodies against laminin and perlecan.
Staining in blue denotes DAPI. Dotted line indicates location of
disrupted glia limitans between of cerebellar lobules. Arrows indicate
clusters of abnormally migrated granule cells.
Fig. 3. Immunolocalization of laminin and perlecan in Largemydcerebral
cortex. Cryosections from Largemyd(myd) and littermate control (WT)
mice were stained with antibodies against laminin and perlecan. The
double arrow indicates normal staining of laminin and perlecan at
microvessels within the cerebral cortex. The single arrow indicates
areas of abnormal punctuate accumulations of laminin and perlecan in
M. Kanagawa et al. / FEBS Letters 579 (2005) 4792–4796
perlecan on the surface of DG+/?ES cells which were observed
16 h after the addition of exogenous laminin-1. Treatment of
laminin-1 with AEBSF has been reported to decrease the
self-association capability of laminin . When AEBSF-
treated laminin-1 was used, dot-like clusters of laminin-1 were
most commonly observed, and such clusters were colocalized
with DG and perlecan (Fig. 4B). These findings indicate that
both DG and laminin self-association facilitates/is required
for assembly of perlecan on the cell surface.
3.4. A trimolecular complex of DG/laminin/perlecan
Although perlecan and laminin have been reported to com-
pete with each other for binding to a-DG, colocalization of
laminin and perlecan with DG led us to hypothesize that perl-
ecan assembles on a laminin oligomer whose terminal is an-
chored to a-DG. To examine this, we modified conventional
solid-phase binding assays with monoclonal antibody IIH6,
which recognizes functionally glycosylated a-DG. HSPG prep-
arations were used as ligands for this assay. We confirmed the
presence of perlecan in HSPG preparations by ELISA and
Western blotting using antibodies to PGI and PGV (data not
shown). When the HSPG preparations were incubated on
the a-DG-immobilized wells, we observed perlecan-binding
to a-DG (Fig. 5A, open squares). The presence of IIH6 inhib-
ited the perlecan-binding to a-DG (closed squares), as is the
Fig. 4. Cluster formation of laminin, DG, and perlecan on the surface of
ES cells. A laminin-clustering assay was performed on DG+/?ES cells.
DG+/?ES cells were treated with 7.5 nM laminin-1 (A) or AEBSF-
treated laminin-1 (B) and then incubated for 16 h. The cells were
subsequently immunolabeled using anti-laminin, anti-a-DG (IIH6) or
anti-perlecan antibodies, and examined by confocal microscopy. In all
panels, the antibody used for staining is shown in the upper left corner
of the panel. Each panel shows a representative small colony of ES
cells. Single, double, and triple arrowheads represent dot, linear, and
plaque clusters, respectively. Bar, 5 lm.
Fig. 5. Trimolecular complex of a-DG, laminin, and perlecan. (A)
Secondary binding of perlecan to a-DG-laminin complex. After the
preformation of a-DG-laminin complex, various concentrations of the
HSPG preparations were added with (closed circles) or without (open
circles) IIH6. The perlecan binding to immobilized a-DG without
preincubation of laminin was also measured with (closed squares) or
without (open squares) IIH6. Drawings illustrate that in the presence
of IIH6, binding of perlecan to a-DG is inhibited, whereas perlecan
binds to preformed a-DG-laminin complex through laminins on
immobilized a-DG. (B) Laminin-1-concentration dependency of the
secondary perlecan binding to a-DG-laminin complex. Various con-
centrations of laminin-1 were incubated on the a-DG-immobilized
wells and then the HSPG preparations (4 lg/ml) were added with
(closed circles) or without (open circles) IIH6. (C) Secondary binding
of PGI to laminin-1-a-DG complex. a-DG-immobilized wells were
preincubated with (right bar) or without (left bar) laminin-1 (7.5 nM),
and then binding of recombinant PGI (9 lg/ml) was analyzed.
M. Kanagawa et al. / FEBS Letters 579 (2005) 4792–4796
case for laminin-binding (data not shown). This IIH6-sensitive
binding can be defined as a direct binding of perlecan or lam-
inin to a-DG. For the formation of the trimolecular complex
of DG/laminin/perlecan, laminin-1 was preincubated on the
a-DG-immobilized wells, and then HSPG preparations were
added with or without IIH6. IIH6-insensitive perlecan binding
was detected after preincubation of laminin (open and closed
circles). Because IIH6 blocks perlecan-binding on a-DG mole-
cules that are unoccupied with laminin, the IIH6-insensitive
binding represents perlecan-binding to preformed DG-laminin
complex via laminin.
Next, we examined laminin concentrations during the pre-
formation of the a-DG-laminin complex (Fig. 5B). The
IIH6-insensitive binding of perlecan to the a-DG-laminin
complex was dependent on laminin concentrations during
preincubation (closed circles). In the absence of IIH6, bind-
ing of perlecan was inhibited by low concentrations of pre-
incubated laminin (<2.5 nM). At a higher concentration
range, the inhibitory effect was eliminated, and perlecan
binding was increased. These data indicate that laminin
and perlecan are competitive for binding to a-DG, but at
higher concentrations of laminin, perlecan is capable of
assembly on a laminin oligomer whose terminal is bound
to a-DG. PGI is known to interact with laminins but not
with a-DG [14,15]. Although we observed little binding of
PGI after incubation with immobilized a-DG, clear increases
of PGI-binding to the a-DG-laminin complex were detected
(Fig. 5C). These data demonstrate that the laminin-perlecan
interaction mediates perlecan assembly on the a-DG-laminin
A reduction in perlecan-binding activity in Largemydand the
inhibitory effect of IIH6 on the perlecan-DG interaction
suggest that similar to laminin , perlecan binding to DG is
regulated by glycosylation status of a-DG. Abnormal accumu-
lations of perlecan in the cerebral cortex appear to contain
laminin. Considering the antigen of laminin antibody used in
this study, the abnormal punctuate structures most likely con-
tain at least a1, b1, and/or c1-chain. Although perlecan and
laminin appeared to be normally localized in Largemydskeletal
muscle, electron microscopy studies showed microdisruptions
of the basement membrane . Thus, DG may be required
for proper localization and assembly of its ligand proteins into
an intact basement membrane with tissue-dependent manner.
We demonstrated that laminin self-association facilitates
assembly of perlecan on the cell surface and show evidence
for the first time of a binary interaction of perlecan and lami-
nin on their receptor DG. Consistent with previous reports ,
a competition between perlecan and laminin for binding to a-
DG was detected. Interestingly, the inhibitory effect of laminin
on the perlecan–DG interaction was reduced at higher concen-
trations of laminin during preincubation due to the secondary
binding of perlecan to a DG-anchored laminin polymer. That
is, perlecan binding becomes indirect, rather than direct, in the
presence of laminin. These data support a widely-accepted
model, the ‘‘receptor-facilitated laminin network model,’’
where both initial laminin assembly on the cell surface and
subsequent laminin self-association are necessary for the for-
mation of the ECM network .
Laminin-1 used in the in vitro binding assay is reported to
show high-affinity to DG [4,5] as well as laminin-2 which is ex-
pressed in adult skeletal muscle and brain [17,18]. Since do-
mains responsible for binding to heparin and perlecan/
nidogen complex are present in both laminin-1 and -2 , the
mechanism of DG-dependent laminin-perlecan network forma-
tion is likely similar between laminin-1 and -2. Because of
high-affinity to DG, perlecan could be a linker between DG
and laminins that show low or no affinity to DG. This is sup-
plex links laminin-6 (a3/b1/c1) on alveolar epithelial cells .
The present data indicate that the local concentrations of
competing ligands in vivo, and relative binding affinities
among these ligands and their receptors are likely critical to
the ordered assembly of matrix, the nature of the molecular
interaction between the matrix and membrane proteins, and
the molecular proximity of the laminin/perlecan lattice to the
cell surface. Clearly, it is important to elucidate an understand-
ing of the temporal and spatial distribution of local concentra-
tions of DG ligand proteins. In addition, non-DG type laminin
acceptor/receptors, such as sulfatide, have been suggested to
play important roles in ECM organization . Overall, this
study demonstrates a mechanism for the formation of a DG/
laminin/perlecan complex on the cell surface, and suggests that
its disruption might be associated with pathogenic events in
congenital muscular dystrophies.
Acknowledgements: We thank Drs. Reinhard Fassler, Steven Moore,
and Aaron Beedle, Ms. Carmen Nidey, and all members of the Camp-
bell laboratory for their critical reading of the manuscript and fruitful
discussion. We thank Jason Flanagan, Lindy McDonough, and Sarah
Anderson for expert technical assistance. We also thank Chuck Lovig
and The University of Iowa Hybridoma Facility. There are no poten-
tial financial conflicts of interest for any of the authors. This work was
supported in part by the Muscular Dystrophy Association (K.P.C.).
K.P.C. is an investigator of the Howard Hughes Medical Institute.
 Cohn, R.D. (2005) Dystroglycan: important player in skeletal
muscle and beyond. Neuromuscul. Disord. 15, 207–217.
 Ibraghimov-Beskrovnaya, O., Ervasti, J.M., Leveille, C.J.,
Slaughter, C.A., Sernett, S.W. and Campbell, K.P. (1992) Primary
structure of dystrophin-associated glycoproteins linking dystro-
phin to the extracellular matrix. Nature 355, 696–702.
 Michele, D.E. and Campbell, K.P. (2003) Dystrophin–glycopro-
tein complex: posttranslational processing and dystroglycan
function. J. Biol. Chem. 278, 15457–15560.
 Talts, J.F., Andac, Z., Gohring, W., Brancaccio, A. and Timpl, R.
(1999) Binding of the G domains of laminin alpha1 and alpha2
chains and perlecan to heparin, sulfatides, alphadystroglycan and
several extracellular matrix proteins. EMBO J. 18, 863–870.
 Kanagawa, M., Saito, F., Kunz, S., Yoshida-Moriguchi, T.,
Barresi, R., Kobayashi, Y.M., Muschler, J., Dumanski, J.P.,
Michele, D.E., Oldstone, M.B. and Campbell, K.P. (2004)
Molecular recognition by LARGE is essential for expression of
functional dystroglycan. Cell 117, 953–964.
 Longman, C., Brockington, M., Torelli, S., Jimenez-Mallebrera,
C., Kennedy, C., Khalil, N., Feng, L., Saran, R.K., Voit, T.,
Merlini, L., Sewry, C.A., Brown, S.C. and Muntoni, F. (2003)
Mutations in the human LARGE gene cause MDC1D, a novel
form of congenital muscular dystrophy with severe mental
retardation and abnormal glycosylation of alpha-dystroglycan.
Hum. Mol. Genet. 12, 2853–2861.
 Grewal, P.K., Holzfeind, P.J., Bittner, R.E. and Hewitt, J.E.
(2001) Mutant glycosyltransferase and altered glycosylation of
alpha-dystroglycan in the myodystrophy mouse. Nat. Genet. 28,
M. Kanagawa et al. / FEBS Letters 579 (2005) 4792–4796
 Michele, D.E., Barresi, R., Kanagawa, M., Saito, F., Cohn, R.D., Download full-text
Satz, J.S., Dollar, J., Nishino, I., Kelley, R.I., Somer, H., Straub,
V., Mathews, K.D., Moore, S.A. and Campbell, K.P. (2002) Post-
translational disruption of dystroglycan-ligand interactions in
congenital muscular dystrophies. Nature 418, 417–422.
 Costell, M., Mann, K., Yamada, Y. and Timpl, R. (1997)
Characterization of recombinant perlecan domain I and its
substitution by glycosaminoglycans and oligosaccharides. Eur.
J. Biochem. 243, 115–121.
 Brown, J.C., Sasaki, T., Gohring, W., Yamada, Y. and Timpl, R.
(1997) The C-terminal domain V of perlecan promotes beta1
integrin-mediated cell adhesion, binds heparin, nidogen and
fibulin-2 and can be modified by glycosaminoglycans. Eur. J.
Biochem. 250, 39–46.
 Colognato, H., Winkelmann, D.A. and Yurchenco, P.D. (1999)
Laminin polymerization induces a receptor-cytoskeleton network.
J. Cell Biol. 145, 619–631.
 Henry, M.D. and Campbell, K.P. (1998) A role for dystroglycan
in basement membrane assembly. Cell 95, 859–870.
 Henry, M.D, Satz, J.S., Brakebusch, C., Costell, M., Gustsfsson,
E., Fassler, R. and Campbell, K.P. (2001) Distinct roles for
dystroglycan, beta1 integrin and perlecan in cell surface laminin
organization. J. Cell Sci. 114, 1137–1144.
 Sasaki, T., Costell, M., Mann, K. and Timpl, R. (1998) Inhibition
of glycosaminoglycan modification of perlecan domain I by site-
directed mutagenesis changes protease sensitivity and laminin-1
binding activity. FEBS Lett. 435, 169–172.
 Ettner, N., Gohring, W., Sasaki, T., Mann, K. and Timpl, R.
(1998) The N-terminal globular domain of the laminin alpha1
chain binds to alpha1beta1 and alpha2beta1 integrins and to the
heparan sulfate-containing domains of perlecan. FEBS Lett. 430,
 Holzfeind, P.J., Grewal, P.K., Reitsamer, H.A., Kechvar, J.,
Lassmann, H., Hoeger, H., Hewitt, J.E. and Bittner, R.E. (2002)
Skeletal, cardiac and tongue muscle pathology, defective retinal
transmission, and neuronal migration defects in the Large(myd)
mouse defines a natural model for glycosylation-deficient muscle-
eye-brain disorders. Hum. Mol. Genet. 11, 2673–2687.
 Colognato, H. and Yurchenco, P.D. (2000) Form and function:
the laminin family of heterotrimers. Dev. Dyn. 218, 213–234.
 Hallmann, R., Horn, N., Selg, M., Wendler, O., Pausch, F. and
Sorokin, L.M. (2005) Expression and function of laminins in the
embryonic and mature vasculature. Physiol. Rev. 85, 979–1000.
 Jones, J.C., Lane, K., Hopkinson, S.B., Lecuona, E., Geiger,
R.C., Dean, D.A., Correa- Meyer, E., Gonzales, M., Campbell,
K., Sznajder, J.I. and Budinger, S. (2005) Laminin-6 assembles
into multimolecular fibrillar complexes with perlecan and partic-
ipates in mechanical-signal transduction via a dystroglycan-
dependent, integrin-independent mechanism. J. Cell Sci. 118,
 Li, S., Liquari, P., McKee, K.K., Harrison, D., Patel, R., Lee, S.
and Yurchenco, P.D. (2005) Laminin-sulfatide binding initiates
basement membrane assembly and enables receptor signaling in
Schwann cells and fibroblasts. J. Cell Biol. 169, 179–189.
M. Kanagawa et al. / FEBS Letters 579 (2005) 4792–4796