©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Agrin Is a Heparan Sulfate Proteoglycan (*)

(Received for publication, October 3, 1994; and in revised form, November 28, 1994)

Guoshan Tsen (1) Willi Halfter (2) Stephan Kröger (3) Gregory J. Cole (1)(§)

From the  (1)Neurobiotechnology Center and Department of Cell Biology, Neurobiology and Anatomy, The Ohio State University, Columbus, Ohio 43210, the (2)Department of Neurobiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, and the (3)Max-Planck-Institute for Brain Research, Department of Neuroanatomy, Deutschordenstrabetae:46, D-60528 Frankfurt, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Proteoglycans are a diverse class of macromolecules whose defining feature is the addition of one or more glycosaminoglycan (GAG(^1)) 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) .

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, alpha-dystroglycan has been identified as a putative agrin receptor(31, 32, 33) , although it remains unclear if agrin-alpha-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.


EXPERIMENTAL PROCEDURES

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 times SSPE, 50% formamide, 1 times 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 [alpha-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-beta-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 times 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) .

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 times 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.

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.


RESULTS

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.


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) 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 beta-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.



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 (alpha-agrin), the 6D2 mAb (6D2), and an antiserum to the pPG5 agrin fusion protein (alpha-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).



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 beta-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-beta-D-galactopyranoside. (^2)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-beta-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.


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. (^3)

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.

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.


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 alpha-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. (^4)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.


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 alpha-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.




DISCUSSION

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 [^3H]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.

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. (^5)

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 alpha-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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant NS29934 (to G. J. C.) and National Science Foundation Grant BNS-9021474 (to W. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Neurobiotechnology Center, The Ohio State University, 184 Rightmire Hall, 1060 Carmack Rd., Columbus, OH 43210. Tel.: 614-292-1205; Fax: 614-292-5379.

(^1)
The abbreviations used are: GAG, glycosaminoglycan; HSPG, heparan sulfate proteoglycan; NCAM, neural cell adhesion molecule; mAb, monoclonal antibody; AChR, acetylcholine receptor; PAGE, polyacrylamide gel electrophoresis; CMF, calcium magnesium free; kb, kilobase(s).

(^2)
G. J. Cole and G. Tsen, unpublished data.

(^3)
W. Halfter, unpublished data.

(^4)
S. Kröger, unpublished data.

(^5)
W. Halfter, unpublished data.


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