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(Received for publication, October 3, 1994; and in revised form, November 28, 1994) From the
In the present study we have identified the extracellular matrix
protein agrin as a major heparan sulfate proteoglycan (HSPG) in
embryonic chick brain. Using monoclonal antibodies and a polyclonal
antiserum to the core protein of a previously identified HSPG from
embryonic chick brain, our expression screened a random-primed E9 chick
brain cDNA library. Twelve cDNAs were isolated that were shown to be
identical to the chick extracellular matrix protein agrin. Western blot
analysis and immunocytochemistry confirmed that agrin is a HSPG that is
identical with the HSPG from embryonic chick brain. A polyclonal
antiserum to recombinant agrin protein recognized agrin as a diffuse
band of over 400 kDa in extracts from brain and vitreous humor. The
agrin immunoreactivity on the blot was shifted to a defined band of
approximately 250 kDa after treatment of the samples with heparitinase
or nitrous acid, and this banding pattern was indistinguishable from
immunoreactivity obtained with antibodies to the brain HSPG. We also
show that agrin binds tightly to anion exchange beads, indicating that
the molecule is highly negatively charged, which is a hallmark of all
proteoglycans. Furthermore, the agrin antiserum recognizes the affinity
purified HSPG from chick brain and vitreous humor. Immunocytochemistry
demonstrated that agrin is expressed in developing brain, and is
especially abundant in developing axonal tracts, in a distribution
identical to the staining of the brain HSPG with monoclonal antibodies.
We also show that the anti-HSPG antibodies stain the synaptic site of
the neuromuscular junction, in agreement with agrin expression. Thus,
our studies demonstrate that chick agrin is a HSPG that is prominent in
the embryonic chick brain. Since previous studies from our laboratories
have shown that this proteoglycan interacts with neural cell adhesion
molecule, our studies raise the interesting possibility that neural
cell adhesion molecule and agrin are interactive partners that may
regulate a variety of cell adhesion processes during neural
development, including synaptogenesis. Proteoglycans are a diverse class of macromolecules whose
defining feature is the addition of one or more glycosaminoglycan
(GAG( Recent studies have provided
insight into the possible functions of proteoglycans in nervous tissue,
and have begun to shed light on the molecular properties of neural
proteoglycans. These studies indicate a role for nervous tissue heparan
sulfate proteoglycans (HSPGs) in the stimulation of neurite outgrowth
during neural development(14, 15, 16) , which
is likely mediated by the interaction of HSPGs with various cell
adhesion proteins in nervous tissue. In terms of function, the binding
of HSPG to cell adhesion molecules is thought to augment the
adhesiveness of the cell adhesion proteins, and the role of HSPG in the
function of the neural cell adhesion molecule (NCAM) has been well
documented(17, 18, 19) . Previous studies
have demonstrated that HSPG binding to NCAM is required for
NCAM-mediated cell adhesion(17, 18) , and recent
studies by Akeson and co-workers (19) in which cell surface
HSPG was eliminated by heparitinase digestion confirm these
observations. It has also been suggested that heparan sulfate binding
to NCAM is required for NCAM-mediated homophilic
binding(18, 20) , thus serving as a mechanism for
modulating the adhesiveness of NCAM during development. Accordingly,
the heparan sulfate binding amino acid sequence in NCAM has been
identified(21) , and it has been shown that alteration of the
heparan sulfate binding domain of NCAM by site-directed mutagenesis
abolishes NCAM's adhesive function, thereby providing strong
evidence that HSPG plays a critical role in NCAM function(22) . We have recently initiated studies aimed at identifying and
characterizing HSPGs in chick brain, in order to obtain additional
insight into how proteoglycans contribute to the development of the
nervous system. In particular, we were interested in identifying the
chick brain HSPG that interacts with, and modulates, NCAM function. Our
studies have shown that a major chick brain HSPG, which contains a
250-kDa core protein, is expressed during early periods of chick brain
development and is capable of interacting with
NCAM(23, 24) . This HSPG has been immunopurified using
a mAb named 6D2, and the purified HSPG has been shown to promote neural
cell attachment when adsorbed onto a nitrocellulose
substratum(24) . Thus, these data strongly suggest a role for
the 6D2 HSPG in the modulation of neural cell adhesion. In the present
study we describe the molecular cloning of the HSPG core protein, using
antibody expression screening of an E9 chick brain cDNA library.
Interestingly, following the isolation of 12 cDNAs using two different
antibody preparations, we found that the putative HSPG cDNAs were
identical in sequence to the previously published chick agrin cDNA (25) . Previous studies have shown that the 220-kDa agrin
protein plays a key role in the aggregation of AChRs during
synaptogenesis in the neuromuscular junction(26) . Recent
studies have focused on the molecular characterization of agrin, and
have demonstrated that agrin is a 220-kDa extracellular matrix protein
comprised of multiple polypeptide
domains(25, 27, 28) . For example, agrin has
been shown to contain multiple protease inhibitor and laminin G
homology domains (28) , raising the possibility that these
domains contribute to its function. Agrin expression during development
has also been shown to be particularly prominent in nervous tissue and
especially brain, although its function in this tissue has yet to be
elucidated(25, 27) . It has been suggested, however,
that brain agrin might have similar and/or additional functions in
nervous tissue(29, 30) . As a means of obtaining
greater insight into the biological actions of agrin, putative
receptors for agrin have been the subject of intense investigation.
Recently,
Expression of
agrin in the vitreous body was examined according to published
protocols(23) , either prior to or following heparitinase
treatment. Agrin can be enriched from brain homogenates by binding to Q
Sepharose, as described by Hascall et al.(42) .
Homogenates from 100 E10 chick brains in CMF (see above) were
centrifuged at 15,000 The
expression of agrin in the developing chick visual system and
neuromuscular junction was conducted according to published protocols (23) . Briefly, chick heads (E10 or adult) were fixed with 4%
paraformaldehyde in phosphate-buffered saline (pH 7.4) for 1 h,
followed by cryopreservation of the specimen in 25% sucrose in CMF for
4 h. Cryostat sections through the E10 chick head or adult ocular
muscle were incubated with primary antibody (undiluted 6D2 hybridoma
supernatant or 1:500 diluted polyclonal anti-agrin fusion protein
antiserum) for 1 h, rinsed twice with CMF, and incubated with
Cy3-labeled goat anti-mouse or goat anti-rabbit secondary antibodies
diluted 1:200 for 1 h. After two additional rinses in CMF the sections
were coverslipped and examined under an epifluorescent microscope.
Figure 1:
Schematic representation of 6D2 cDNAs
isolated following screening of an E9 chick brain cDNA library with the
6D2 and 3A12 mAbs to the HSPG or a polyclonal antiserum to the HSPG
core protein. Clones pPG1-10 were isolated following screening
with the 6D2 and 3A12 mAbs, and clones pPG12, 15, 20, and 22 were
isolated using a polyclonal antiserum to the HSPG core protein. Initial
nucleotide sequence was obtained for each clone at the 5` and 3` ends,
and is indicated for each clone, and then compared to the published
chick agrin sequence. All sequences obtained for the clones were found
to be identical to the published chick agrin cDNA sequence. Consensus
sequences for 6 GAG attachment sites are indicated on the agrin map,
and are localized throughout the agrin protein, including in the
functional C-terminal domain.
Although our isolation of multiple overlapping
cDNAs that are identical to agrin is indicative of agrin encoding a
HSPG, and although Southern blots have revealed only a single gene for
agrin in chick(25) , we could not rule out the possibility that
major differences in the mRNA coding for agrin leads to the expression
of agrin as a HSPG in brain. We therefore carried out Northern analysis
of agrin mRNA expression in embryonic chick tissues using one of the
isolated proteoglycan cDNAs, pPG5. As shown in Fig. 2A,
a 10-kb mRNA was identified that was prominent in brain when compared
to heart, and this mRNA exhibited a pronounced down-regulation from E9
to E18 of brain development (Fig. 2A), consistent with
expression of the 6D2 HSPG core protein(24) . In addition, we
could identify a second, approximately 8-kb mRNA expressed in chick
brain glia, which we have shown previously to consist primarily of
astrocytes(39) . In previous studies, we have demonstrated that
the 6D2 HSPG is expressed by glia in chick brain(24) . These
data therefore show that the putative HSPG cDNAs identify a mRNA that
parallels the HSPG protein in expression. However, these data also
suggest that the HSPG mRNA is larger in molecular size than the
previously reported 8-8.5 kb agrin message in chick(25) ,
although this discrepancy is likely to be attributable to experimental
variation between different laboratories, especially in light of a
recent study by Ma et al.(44) showing a chick brain
agrin mRNA that migrates at approximately 9.5 kb.
Figure 2:
Northern analysis of agrin mRNA
expression. A, analysis of agrin mRNA expression using clone
pPG5. Poly(A)
Because our
initial cloning studies using mAbs suggested that agrin encodes a HSPG
in brain, and our Northern analyses are consistent with agrin being
expressed in brain as a single molecule and hence a proteoglycan, we
extended our analyses to confirm that agrin encodes a brain HSPG. Our
approach to confirming that agrin encodes a brain HSPG was to carry out
a second screening of our random-primed E9 brain cDNA library using a
polyclonal antiserum that was prepared to gel purified HSPG core
protein. As shown in Fig. 3A, when the 6D2 HSPG is
immunopurified using the 6D2 and 3A12 mAbs and analyzed by silver
staining, the HSPG and a minor high molecular mass 220-kDa contaminant
are isolated. It is important to emphasize that when analyzed on a
single percentage gel, rather than a gradient gel, one cannot detect
significant amounts of agrin in the absence of treatments to eliminate
HS from the HSPG (Fig. 3). Thus, in previous analyses of agrin,
which utilized single percentage gels and did not detect intact
agrin(27) , one can conclude that this occurred due to the
inability of the HSPG to enter the running gel. However, as shown in Fig. 4and Fig. 6, on gradient gels the HSPG can be
resolved as a heterogeneous smear with a molecular mass in excess of
400 kDa, in the absence of any treatments to remove HS from the
molecule.
Figure 3:
Immunopurification of 6D2 HSPG from chick
brain. A, silver stain analysis of immunopurified 6D2 HSPG
protein electrophoresed on a 6% SDS-PAGE gel. Prior to treatment (U), the HSPG migrates at the stacking and running gel
interface (arrowhead), and small amounts of a 220-kDa protein
contaminant (small asterisk) are detectable. Following nitrous
acid treatment (T), the HSPG core protein is observed (large asterisk). B, immunoblot analysis of
immunopurified HSPG protein, prior to or following nitrous acid
treatment. It can be seen that the 220-kDa contaminant is also detected
by the 6D2 mAb, and that the HSPG core protein is the major protein
component. The intact HSPG in the absence of treatment is not readily
discernible at the running/stacking gel interface (arrowhead),
and results from inefficient transfer from the gel in this
region.
Figure 4:
Specificity of a polyclonal antiserum to
the 6D2 HSPG core protein. A rabbit antiserum generated against the
immunopurified 6D2 HSPG core protein was diluted 1:250 and reacted with
total E9 chick brain protein (100 µg) left untreated (lane
1), or treated with nitrous acid (lane 2), and
immunopurified 6D2 HSPG (1 µg) left untreated (lane 3).
Protein samples were separated on 4-15% gradient gels and
transferred to nitrocellulose prior to immunoblotting with the
anti-core protein antiserum.
Figure 6:
Western blots showing that anti-agrin
antibodies recognize a heparan sulfate proteoglycan from chick brain.
The blots were stained with a polyclonal antibody to the recombinant
agrin protein C-terminal fragment named CBA-1 (
As shown in Fig. 3, by both silver staining and
immunoblotting the HSPG and 220-kDa contaminant are detectable, raising
the possibility that these two proteins may be immunologically related.
We also found that by separating nitrous acid-treated HSPG core protein
on a 5% polyacrylamide gel, it was possible to resolve these two
proteins to the point of permitting excision of only the HSPG core
protein from the gel. The gel-purified core protein was then
electroeluted and injected into rabbits to generate a polyclonal
antiserum. This antiserum is specific for agrin, as determined by
immunoblotting of total chick brain protein with or without nitrous
acid treatment to eliminate HS chains (Fig. 4). This antiserum
was used to screen 250,000 recombinants from the random-primed E9 chick
brain cDNA library, and as shown in Fig. 1, 4 cDNAs were
isolated that were also found to be identical to chick agrin. One of
these clones that was identified with the anti-core protein antiserum
was also employed in Northern analyses, and reacts with the same 10-kb
agrin mRNA in embryonic chick brain (Fig. 2B). Although
the exposure of the Northern blot with the pPG12 clone was of shorter
duration than the pPG5 blot, it is evident that both cDNAs recognize a
mRNA that is expressed at higher levels in E9 chick brain than in E18
chick brain. The blot that was probed with the pPG12 clone was
previously probed with a 500-base pair mouse
Figure 5:
Antibodies to the 6D2 HSPG recognize an
agrin fusion protein. Clone pPG5 was used to generate a fusion protein
as described under ``Experimental Procedures,'' and either
10-µg aliquots of protein from the soluble fraction of washed
inclusion bodies of uninduced (lane 1) or induced (lane
2) E. coli, or 2.5 µg of purified inclusion body
protein from induced cells, were separated on a 7.5% polyacrylamide
gel, and immunoblotted with the polyclonal antiserum to the HSPG core
protein (lanes 1 and 2) or the 6D2 mAb (lane
3).
To confirm that the
agrin HSPG is encoded by the agrin gene, a rabbit polyclonal antiserum
to an agrin protein expressed by stably transfected 293 HEK cells was
generated. The agrin used for antibody production was derived from the
well described CBA-1 C-terminal fragment of
agrin(25, 45) , which has been shown previously to
contain the AChR clustering activity of agrin(45) . This
anti-agrin antiserum was used to detect agrin in brain and vitreous
body by Western blotting. The antiserum detected agrin as a broad band
of over 400 kDa that shifted to a narrow band of 250 kDa after
treatment with heparitinase (Fig. 6, lanes 1-6).
Our experiments also show that agrin binds tightly to Q-Sepharose and
elutes from this anion exchange matrix only at ionic strength above 0.5 M NaCl (Fig. 6, compare lanes 1 and 2). Anion exchange chromatography is therefore a convenient
means of enriching for agrin from crude brain extracts. Using
Q-Sepharose chromatography, agrin can also be enriched from extracts of
chick embryo bodies excluding the brain. ( The staining
pattern of the recombinant agrin protein antiserum was
indistinguishable from the 6D2 mAb (Fig. 6, lanes
7-10), thus confirming that the agrin gene codes for a HSPG
in developing chick brain. We have also generated a polyclonal
antiserum to the pPG5 agrin fusion protein, with this antiserum also
showing reactivity with the same HSPG in E9 brain tissue and with the
purified intact HSPG (Fig. 6, lanes 11-13).
Again, the HSPG recognized by this antiserum appeared as a 400-kDa
smear that is shifted to 250 kDa following nitrous acid treatment.
Figure 7:
Fluorescent micrographs showing the
distribution of brain-derived HSPG recognized by the 6D2 mAb and agrin.
Adjacent sections through retina (R), optic nerve (ON), and optic chiasm (CH) of an E10 chick head were
stained with the 6D2 mAb (a) and a polyclonal antiserum to the
CBA-1 agrin protein fragment (b). The staining pattern of the
optic nerve and retina, with the optic fiber layer (arrow) and
the inner plexiform layer (arrowhead) exhibiting staining, are
identical. D, diencephalon. Bar: 250
µm.
Finally, the identity
between the agrin HSPG and agrin was confirmed by examining expression
of the 6D2 HSPG in adult muscle, using the 6D2 mAb. A defining feature
of agrin is its expression at the synaptic sites of the neuromuscular
junction, and its co-localization with ACh receptors(37) .
Using the 6D2 mAb to the core protein of the HSPG, we show that the
HSPG is localized to the neuromuscular junction synaptic site, as
evidenced by co-localization with
Figure 8:
The
6D2 HSPG is localized to the synaptic site of the developing
neuromuscular junction. Adult chick eye muscle (musculus obliquus
superior) was incubated with TRITC
The studies described herein were initiated in order to
obtain further information about a prominent HSPG from chick brain
which exhibits a developmentally-regulated pattern of
expression(23, 24) . Surprisingly, our cloning studies
have shown that the 6D2 HSPG core protein is identical to agrin, and
hence that agrin is a HSPG. This conclusion is supported by several
experimental observations, which include the demonstration that
antibodies to the HSPG recognize both agrin and the HSPG, that the
various HSPG antibodies only identified agrin cDNAs following
expression screening, that antibodies to agrin or an agrin fusion
protein exhibit imunoblotting patterns identical to the HSPG
antibodies, and that the 6D2 mAb to the HSPG stains the synaptic site
of the chick neuromuscular junction. In addition, we show that a
polyclonal antiserum to agrin reacts with a high molecular weight
heterogeneous protein smear by immunoblotting, which is typical for
proteoglycans. The agrin smear is shifted to a 250-kDa protein after
heparitinase treatment, in agreement with earlier studies using the 6D2
mAb to the 6D2 HSPG(23) . Agrin also binds tightly to anion
exchange matrices, showing that the molecule is highly negatively
charged, a hallmark of all proteoglycans. The core protein appears to
contain additional carbohydrate, as determined by
[ It should be noted that it was previously believed that agrin cannot
be isolated as an intact molecule, and thus proteolytic fragments of
the protein were utilized in earlier characterizations of
agrin(47) . Also, previously published Western blots using
anti-agrin antisera show only agrin fragments of about 100 kDa in
chick(48) , but never show the intact agrin protein. Likewise,
in rat agrin has been identified by immunoblotting as a protein
slightly smaller than 200 kDa(27) . Like these previous
studies, when we use single percentage gels (i.e. 6%) to
analyze agrin expression in chick brain, we only detect agrin following
elimination of HS chains from the molecule (see Fig. 3).
However, when using gradient gels to analyze agrin in brain, our data
clearly show that agrin can be isolated either from brain or vitreous
body, and is readily detectable by Western blotting in crude extracts
of brain, optic nerve, or vitreous body. In our blots, the most
abundant agrin signal is that of the intact proteoglycan, and an
immunologically related 220-kDa contaminant and smaller fragments are
only minor contaminants. An explanation for the failure to previously
detect and isolate agrin may therefore lie in the fact that the intact
proteoglycan, with a molecular mass in excess of 400 kDa, is too large
to enter even low percentage gels and may therefore have never been
detected in these earlier studies. Our most recent experiments also
show, by immunoblotting with the antiserum to the chick agrin core
protein, that agrin in mouse and cow brain exists as a proteoglycan of
over 400 kDa, with a 250-kDa core protein. ( Our
demonstration that agrin is a HSPG that is expressed primarily during
early periods of brain development is of interest, since the most
prominent expression of agrin is in brain (25; our studies here), but
the function of agrin in brain has remained elusive. Recent studies
have suggested that brain agrin may be involved in synaptogenesis in
the developing central nervous system, since it is expressed by various
neuronal cell types in brain(29, 30, 44) .
Brain agrin has also been proposed to participate in cell adhesion
processes since it is secreted by axons along their pathway of growth (49) and contains laminin-like domains that could possibly
function in cell adhesion(25, 27) . Our studies
reported here therefore provide strong support for a proposed role of
brain agrin in cell adhesion. We have shown previously that NCAM
function is modulated by HSPGs (18, 21) and that the
HSPG (agrin) is capable of binding to the heparin-binding domain of
NCAM(24) . Our recent studies have also demonstrated that
purified agrin HSPG can regulate adhesion to a functional domain of
NCAM, and most importantly, that the HSPG can serve as a substrate for
neural cell attachment(24) . With the demonstration here that
the agrin gene encodes this HSPG, it is clear that agrin function in
brain may range from promoting synaptogenesis to regulating cell
adhesion mediated by heparin-binding proteins in the central nervous
system that include NCAM, thrombospondin, laminin, and
myelin-associated glycoprotein. In addition, adhesion proteins such as
NCAM co-localize with agrin to the synaptic sites of the neuromuscular
junction(50, 51) , raising the possibility that NCAM
and agrin are interactive partners that regulate synaptogenesis during
nervous system development. Our demonstration that agrin is a HSPG
is of particular interest in view of previous studies which have shown
that heparin inhibits the ability of agrin to cluster ACh
receptors(34, 35, 36) . These studies have
shown that agrin-mediated induction of AChR clustering at the
neuromuscular junction is inhibited by soluble heparin or heparan
sulfate, with the proposal that agrin interacts with a cell surface
HSPG at the neuromuscular junction(35) . Muscle cell lines
deficient in heparan sulfate biosynthesis were also unable to form AChR
aggregates in response to exogenous agrin, again suggesting that a HSPG
is critical to agrin function(36) . Although these findings
have been interpreted as indicating that agrin may interact with a
HSPG, with this interaction critical to the AChR clustering activity of
agrin, our data presented here could also be consistent with heparin
inhibiting agrin function because of agrin's heparan sulfate
chains being required for its function. However, since GAG-deficient
muscle cells do not cluster AChRs in response to exogenous agrin, these
data suggest that the muscle cell is providing the HSPG critical to
agrin function. Although it remains possible the proteoglycan form of
agrin in muscle is needed for agrin-induced AChR aggregation, it is
also possible that a muscle HSPG distinct from agrin is necessary for
agrin activity. However, because we have recently shown that agrin can
promote cell-substratum adhesion, with this function dependent on its
heparan sulfate(24) , it is apparent that future studies will
be necessary to elucidate the possible role of agrin's heparan
sulfate chains in AChR aggregation and synaptogenesis. Recent
studies involving agrin have emphasized identifying the receptor for
this extracellular protein, since this would further one's
understanding of mechanisms underlying the function of agrin.
Preliminary studies showed that the agrin receptor is localized to the Torpedo electric organ postsynaptic membrane, consistent with
agrin's role in synaptogenesis(52) . Recent studies have
implicated
Volume 270,
Number 7,
Issue of February 17, 1995 pp. 3392-3399
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
e:46, D-60528
Frankfurt, Germany
)) sugar chains. The various classes of proteoglycans
are classified based on the type of GAG chain they
carry(1, 2) , and in many cases the identified
functions of proteoglycans are mediated by their GAG
chains(3, 4, 5) . The functions of
proteoglycans include a role in a number of critical developmental
processes, which include the regulation of cell adhesion and
recognition(6, 7, 8, 9) , the
control of cell growth and differentiation via the binding of growth
factors(10, 11, 12) , and the regulation of
gene transcription(13) .
-dystroglycan has been identified as a putative agrin
receptor(31, 32, 33) , although it remains
unclear if agrin--dystroglycan interactions in brain are necessary
for agrin function. Agrin-induced aggregation of AChRs in muscle is
also inhibited by heparin and related
polyanions(34, 35, 36) , raising the
possibility that HSPG plays a role in agrin function. Thus, our studies
reported here, describing the identification of an NCAM-binding HSPG (24) as agrin, raise the interesting possibility that agrin
plays an important role in the regulation of cell interactions during
nervous system development.
Antibodies
Antibodies used in this study that
recognize the 6D2 HSPG included the 6D2 and 3A12 mAbs, produced as
described previously(23) , and an antiserum to the HSPG core
protein. This antiserum was prepared by electroelution of the
immunopurified core protein from 5% preparative SDS-PAGE gels, followed
by injection into rabbits. Antibodies used in this study that recognize
agrin included the 5B1 mAb(37) , and antisera prepared, as
described below, to the C-terminal region of agrin and a fusion protein
encoded by the pPG5 cDNA, which was isolated following screening of an
E9 chick brain cDNA library using the 6D2 and 3A12 mAbs.Molecular Cloning of 6D2 HSPG
Monoclonal
antibodies to the HSPG protein core, named 6D2 and 3A12, were purified
from serum-free culture supernatants as described
previously(38) . mAbs (5 µg/ml) were diluted with blotto
and used to screen 750,000 recombinants from a random-primed E9 chick
brain 
Zap cDNA library (Stratagene), according to published
protocols(39) . Ten clones were identified and subsequently
plaque purified, and initial sequence information was obtained by the
dideoxy chain termination method(40) .Northern Analysis of Agrin mRNA
Expression
Expression of agrin mRNA during chick development was
examined by separating 1 µg of poly(A)
RNA on 1%
agarose gels containing 2.2 M formaldehyde, as described
previously(39) . Following transfer to nylon membrane (Nytran,
Schleicher and Schuell), the membrane was prehybridized 2 h in 5
SSPE, 50% formamide, 1 Denhardt's, 2% SDS, and
100 µg/ml sperm DNA. Agrin cDNA inserts were then isolated by
excision with EcoRI, and were labeled using random primers and
[-
P]dATP. Membranes were then hybridized 20
h with radiolabeled probe, and washed to high stringency.Production of Agrin Fusion Protein
To produce a
fusion protein to agrin, clone pPG5 was excised from pBluescript using EcoRI and was inserted into pET-23b (Novagen). The correct
orientation of the ligated insert was confirmed by restriction analysis
of mini-prep DNA, and DNA was then used to transform Escherichia
coli strain DE3 pLysS. To induce fusion protein expression, a
single colony was grown overnight in 10 ml of LB containing ampicillin
and chloramphenicol, and the overnight culture was then diluted to 500
ml and grown for 3 h at 37 °C. Induction of fusion protein
expression was for 5 h following the addition of
isopropyl-1-thio--D-galactopyranoside to a final
concentration of 0.5 mM. Fusion protein was then analyzed from
soluble and insoluble fractions, with the majority of protein being
contained in inclusion bodies. Inclusion bodies were washed with
phosphate-buffered saline containing 8 M urea, with agrin
fusion protein partitioning between the 8 M urea supernatant
and the insoluble inclusion bodies. The washed inclusion bodies were
solubilized by boiling for 10 min in SDS-PAGE sample buffer, and by
Coomassie Blue staining on SDS-PAGE gels were shown to contain only the
agrin fusion protein and a lower molecular weight protein. The agrin
fusion protein also represented the major component of the solubilized,
purified inclusion bodies, and was purified from this fraction by gel
electrophoresis on 7.5% polyacrylamide gels, followed by excision and
electroelution.Agrin Polyclonal Antibody Production
Polyclonal
antibodies to an agrin fusion protein (clone pPG5) were prepared by
purification of the pPG5 fusion protein by preparative gel
electrophoresis followed by electroelution, and injection into New
Zealand White rabbits at 3-week intervals. A polyclonal antiserum to
the C-terminal region of agrin, designated CBA-1(25) , was
prepared by stably transfecting 293 HEK cells with the agrin CBA-1
cDNA(25) , and affinity purifying the agrin protein from
conditioned media of cultures using the anti-agrin 5B1 MAb.Immunoblotting and Immunohistochemical Analysis of Agrin
Expression
The expression of agrin was analyzed by
immunoblotting using the 6D2 mAb according to published
protocols(23, 24) . For analysis of brain agrin, E9
chick brain protein was isolated by homogenizing tissue in ice-cold
calcium-magnesium free Hank's buffer (CMF), followed by
centrifugation at 48,000 g for 1 h. Aliquots of
protein (70 µg) were either left untreated or treated with nitrous
acid as described previously(24, 41) , and were then
separated on 6% polyacrylamide gels and transferred to nitrocellulose.
To analyze purified brain agrin, the HSPG was immunopurified from E9
brain tissue as described previously(24) . g for 20 min. Sodium chloride,
Triton X-100, and DNase were added to the supernatant to a final
concentration of 0.3 M, 0.5%, and 5 µg/ml, respectively,
and the solution was incubated with Q-Sepharose (Pharmacia, Piscataway,
NJ; 4 ml/200-ml homogenate) in batch. After shaking for 1 h, the beads
were allowed to settle and the unbound supernatant was decanted. After
two rinses with TBS/Tween, the beads were washed 3 times with 150 ml of
0.5 M NaCl, 4 M urea in TBS/Tween. After a short
rinse with distilled water, the beads were eluted with 4 bed volumes
(20 ml) of 1.5 M NaCl. The eluate was lyophilized, taken up
with water, and dialyzed with CMF. Agrin and vitreous body protein were
visualized by electrophoresis on 3.6-14% polyacrylamide gels,
transfer to nitrocellulose, and immunoblotting using 6D2 mAb.
Molecular Cloning of the 6D2 HSPG Core
Protein
Our previous studies have focused on the
characterization of a HSPG, and its core protein, that is involved in
the regulation of the function of NCAM (24) . Using mAbs
generated to a novel chick nervous system HSPG(23) , we have
shown that this HSPG is capable of modulating cell adhesion, binds to
NCAM, and is primarily expressed during nervous system
development(24) . To further our understanding of the molecular
and possible functional properties of this HSPG, we have initiated in
the present study the molecular cloning of the core protein of this
HSPG. Our approach was to use a mixture of two mAbs (named 6D2 and
3A12) against the core protein to screen an E9 chick brain cDNA library
prepared using random hexamer primers. Following the screening of
approximately 750,000 recombinants we isolated 10 cDNAs that were shown
by preliminary sequence analysis to be related and overlapping (Fig. 1). Interestingly, these cDNAs are identical in sequence
to the previously published chick agrin cDNA sequence(25) , a
220-kDa basement membrane protein that is a critical regulator of the
development of the neuromuscular junction(26) . Because agrin
mRNA expression is particularly abundant in
brain(25, 27) , we considered the possibility that
agrin in brain is expressed as a HSPG that would be capable of
regulating the function of heparin-binding adhesion proteins such as
NCAM. Consistent with this possibility, ProSite analysis of the
published agrin sequence in chick (25) and our cDNAs reveals
the presence of 6 glycosaminoglycan attachment site consensus
sequences, SGXG(9) , in agrin (Table 1). In
addition, these regions of agrin contain additional SG sequences, that
are preceded or followed by acidic amino acids, that may also be
capable of serving as GAG attachment sites (Table 1). Some of the
GAG attachment sites are contained within the functional, C-terminal
region of agrin (Fig. 1), although it remains to be determined
if heparan sulfate on agrin is necessary for agrin's AChR
clustering activity.
RNA (1 µg) from E9 and E18 chick
brain, E9 heart, and chick brain glia cultured from E10 chick brains
for 1 week, was separated on a 1% agarose gel and blotted with pPG5.
Note that two bands can be discerned with the agrin probe in the glial
mRNA. B, analysis of agrin mRNA expression in E9 and E18 chick
brain using clone pPG12. This blot was originally probed with a mouse
-actin DNA probe (bottom panel), and was then stripped
and probed with the pPG12 cDNA. The analysis with the pPG12 cDNA
represented the third time the blot had been probed with different
cDNAs. The data from this analysis, however, show that the
down-regulation of agrin mRNA with brain development is specific, since
actin mRNA levels show no change.
-agrin),
the 6D2 mAb (6D2), and an antiserum to the pPG5 agrin fusion
protein (-pPG5). Lane 1, crude brain homogenate
from E10 chick embryos. Lane 2, proteins eluted with 1.5 M NaCl from Q-Sepharose. The Q-Sepharose had been loaded with E9
chick brain cytosol and was extensively washed with 0.5 M NaCl
and 4 M urea prior to elution. Lanes 3 and 7, vitreous humor protein from E10 embryos. Lanes 4 and 8, vitreous humor protein digested with heparitinase. Lanes 5 and 9, HSPG from E9 chick brain
immunopurified twice using the 6D2 and 3A12 mAbs. Lanes 6 and 10, immunopurified HSPG from E9 chick brain digested with
heparitinase. Lane 11, total protein from E9 chick brain left
untreated. Lane 12, total E9 chick brain protein digested with
nitrous acid. Lane 13, immunopurified HSPG from E9 chick brain
left untreated. The immunoblots shown in lanes 1-10 were
analyzed using 3.6-14% gradient polyacrylamide gels, with
molecular weight standards shown at the left. The immunoblot shown in lanes 11-13 is from a 4-15% gradient gel, with
molecular weight standards shown to the right. Note that the
agrin from brain and vitreous humor appears as a smear with a molecular
mass exceeding 400 kDa. This smear is reduced after heparitinase or
nitrous acid treatment to a narrow band of 250 kDa. The agrin can also
be enriched by binding to an anion exchange column (lane 2).
The antisera to agrin recognize the HSPG and its core protein
immunopurified from brain using the 6D2 and 3A12 mAbs, thus showing
that the two antigens are identical. Note also that the anti-agrin
antisera exhibit an identical reactivity with chick brain protein and
the 6D2 HSPG, when compared to the anti-HSPG core protein antiserum
(see Fig. 4).
-actin DNA fragment,
prior to being stripped and probed with the pPG12 cDNA. As shown in Fig. 2B, these data demonstrate that only the agrin
mRNA levels are reduced from E9 to E18 of chick brain development. Most
important, these cloning data provide strong evidence that the agrin
cDNA encodes a HSPG in chick brain, since only agrin cDNAs were
identified following screening of the expression cDNA library with
various anti-HSPG antibodies.Production of Agrin Fusion Protein
To confirm that
the agrin clones isolated with our antibodies are authentic, and hence
that the HSPG is identical to agrin, we decided to express agrin fusion
protein using our isolated cDNAs, and then generate an antiserum to
this fusion protein. In these experiments we used the pPG5 cDNA, which
is an approximately 1.4-kb cDNA consisting entirely of open reading
frame sequence. This cDNA was excised from pBluescript and inserted
into the pET-23b expression vector, and large scale production of
fusion protein was achieved following induction of E. coli DE3
pLysS cells with isopropyl-1-thio--D-galactopyranoside. (
)When fusion protein from the soluble fraction of washed
inclusion bodies was immunoblotted with an antiserum to the HSPG core
protein, intense immunoreactivity was observed (Fig. 5, lanes 1 and 2). Although low levels of
immunoreactivity were detected in uninduced E. coli, it is
also apparent that pronounced synthesis of agrin fusion protein
occurred following induction of the cells with
isopropyl-1-thio--D-galactopyranoside (Fig. 5, lanes 1 and 2). Using the 6D2 mAb and protein from
purified inclusion bodies isolated from induced E. coli,
intense immunoreactivity with the agrin fusion protein could also be
detected (Fig. 5, lane 3). Thus, these data show that
both monoclonal and polyclonal antibodies to a brain HSPG recognize an
agrin fusion protein, and hence recognize agrin.
)Agrin Is Expressed in Developing Axonal Tracts and the
Neuromuscular Junction
As a final means of obtaining evidence
that the agrin HSPG and agrin are identical molecules, we examined the
expression of the HSPG and agrin in the developing chick visual system,
and the HSPG in adult muscle. Our analysis of agrin in developing chick
brain was of particular interest, since previous information regarding
the expression of agrin during chick brain development is not
available. We have previously shown that the 6D2 HSPG is expressed
abundantly in chick optic nerve and tectum(23) , and therefore
compared the HSPG and agrin expression in the chick visual system. As
shown in Fig. 7a, the 6D2 mAb intensely stains E10
chick retina, optic nerve, and optic chiasm, and the polyclonal
antiserum to the CBA-1 agrin fragment exhibits an identical
immunostaining pattern. Thus, agrin expression in the developing chick
visual system is indistinguishable from the 6D2 HSPG. Most importantly,
these data indicate that agrin is abundantly expressed in at least one
developing nerve tract, the optic pathway.
-bungarotoxin binding sites,
with lower levels of expression at extra-synaptic sites (Fig. 8). A similar distribution of the 6D2 HSPG has been
observed in embryonic pectoralis muscle. (
)This expression
pattern in muscle is identical to that of agrin(37) , providing
additional support for the conclusion that agrin is a nervous system
HSPG.
-bungarotoxin (A) or
6D2 mAb and fluorescein-conjugated anti-mouse secondary antibody (B), and visualized by epifluorescence microscopy. Expression
of the HSPG can be observed at the synaptic site of the neuromuscular
junction, as well as at extrasynaptic sites. The asterisk indicates a blood vessel. Bar: 10
µm.
H]glucosamine labeling(24) , but does
not contain other classes of GAG chains(23, 24) .
These data therefore show that agrin is the core protein of a large
HSPG. Lastly, studies carried out which have examined bovine kidney
basement membrane HSPGs suggested that tryptic fragments of a kidney
HSPG core protein exhibited high homology to rat agrin(46) ,
providing additional support for our conclusion that agrin is a HSPG. 
)-dystroglycan as a putative agrin
receptor(31, 32, 33) , with this protein
being distributed over the entire muscle surface, including the
postsynaptic membrane. Our studies provide evidence that NCAM may also
function as an agrin-binding protein, since immunopurified agrin is
capable of binding to the heparin-binding domain of NCAM (24) and antibodies to NCAM partially inhibit cell adhesion to
an agrin substratum(24) . Thus, since NCAM and agrin
co-localize at the synaptic site of the neuromuscular
junction(50) , it is of interest to consider the possibility
that these two proteins may participate as binding partners in the
regulation of synaptogenesis. One can also speculate that additional
agrin-binding proteins may also exist in view of the presence of
heparan sulfate on the protein, and therefore a multitude of
heparin-binding proteins can serve as putative agrin-binding proteins.
With this capability of serving as a multifunctional protein, agrin may
have the potential to regulate the function of a diverse population of
nervous system proteins, and thus future studies will have the
opportunity to explore the function of agrin in a variety of
developmental processes.
)))))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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