WO1995013298A1 - Recepteur d'agrine - Google Patents

Recepteur d'agrine Download PDF

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Publication number
WO1995013298A1
WO1995013298A1 PCT/US1994/005387 US9405387W WO9513298A1 WO 1995013298 A1 WO1995013298 A1 WO 1995013298A1 US 9405387 W US9405387 W US 9405387W WO 9513298 A1 WO9513298 A1 WO 9513298A1
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agrin
protein
binding
seq
receptor
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PCT/US1994/005387
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English (en)
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Justin Fallon
Mark A. Bowe
Beth A. Mckechnie
Mary A. Nastuk
John D. Leszyk
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Worcester Foundation For Experimental Biology
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Priority to AU72006/94A priority Critical patent/AU7200694A/en
Publication of WO1995013298A1 publication Critical patent/WO1995013298A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • This invention relates to certain agrin binding receptors which are capable of binding to agrin in a calcium dependent fashion.
  • This invention also relates to substantially pure oligopeptides from these isolated agrin receptors which can be characterized by the molecular weight and association with agrin binding.
  • the invention also relates to the use of the isolated protein in assays and assay kits for the detection of compounds which can bind agonistically or antagonistically to the agrin receptor.
  • the invention also relates to the use of the proteins of the invention for the screening of inhibitors or facilitators of binding of agrin to the agrin receptor.
  • the invention also relates to the generation of polyclonal and monoclonal antibodies which will bind to the agrin receptor and the use of these antibodies as inhibitors, immunopurification agents, and labeling agents.
  • the invention relates to the use of the agrin receptor portion as a means for affinity purifying agrin and agrin related proteins from in vitro and in vivo sources.
  • the invention also relates to the use of agrin receptor protein as a specific inhibitor of agrin binding in vitro and in vivo.
  • the invention relates to the use of agrin receptor and the identification of binding domains on agrin protein.
  • the invention also relates to specific amino acid sequences that are part of agrin receptor subunits.
  • Agrin, agrin related proteins and agrin receptors play a vital role in directing the formation and specialization of pre- and post-synaptic neuromuscular junctions
  • Agrin and agrin related proteins have been characterized as being involved in the clustering of AChRs (acetylcholine receptors), acetylcholinesterase (AChE), and other post synaptic components on the surface of cultured myotubes (Fallon et al., 1985, Nature 315: 571-574).
  • Agrin has been found associated with both normal and damaged synaptic basal lamina, which is capable of directing the specialization of both pre- and post-synaptic neuromuscular junctions
  • Agrin is best characterized by the induction of AChR clustering on the myotubule surface, however evidence indicates that this is not the result of agrin mediated cross linking of the AChR on the cell surface, but rather via intracellular signal transduction events via a unique receptor (Nastuk et al., 1991, Neuron 7: 807- 818).
  • Agrin-induced AChR aggregates arise from the lateral migration of preexisting receptors, and extracellular calcium is required (Godfrey et al., 1984, J. Cell Bio. 99: 615-627; Henderson et al., 1984, J. Neurosci. 4: 3140-3150).
  • Agrin induced clustering can be inhibited by phorbol esters, metabolic energy inhibitors, or infection with Rous sarcoma virus (Wallace, 1988, /. Cell Biol. 107: 267-278; Anthony et al, 1988, /. Cell Biol. 106: 1713-1721 ).
  • Agrin is a single chain, multidomain extracellular matrix protein of about 200 kD (reviewed by Nastuk and Fallon, 1993, Trends in Neurosci. 16: 72-76). Stable, bioactive proteolytic breakdown products of the molecule purified from Torpedo electric organ are 150 kD and 95 kD.
  • the amino-terminal half of the agrin protein consists of nine-tandem repeats, each repeat homologous to Kazel-type protease- inhibitor domains. There is also approximately 39% homology with the Bl, B2 and S- laminin chains, which may implicate this area in binding to the basal lamina component entactin (nidogen).
  • the carboxy-terminal half of the protein has homology to the cysteine-rich motifs of epidermal growth factor (EGF).
  • the carboxy half roughly corresponds with the 95 kD portion, and can mediate all of agrin 's known functions.
  • the 95 kD form of agrin is able to induce AChR clustering
  • a truncated form of the protein called the 70 kD form will not induce clustering of AChR.
  • a putative agrin receptor was first demonstrated to exist, by the present inventors, on chicken myotube plasma membrane showing agrin binding in the presence and absence of AChRs. The distribution of the agrin receptors was not dependent on the presence of AChR (Nastuk et al., 1991, Neuron 7: 807-818).
  • Dissociated mouse embryonic (ED 9-10) spinal cord cells displayed a subpopulation of neurons which also demonstrated agrin binding (Nastuk and Fallon, 1991, Soc. Neurosci. Absti-. 17: 219).
  • Agrin may play a role in the development of pathological conditions which exhibit motomeuron degeneration.
  • Agrin appears crucial to the proper formation of the neuromuscular junction, which occurs when a nerve terminal contacts a muscle fiber, and to the reestablishment of injured synaptic connections .
  • the proper formation of such an intricate structure during development requires the localized cell surface specialization and accumulation of nicotinic acetylcholine receptors (nAChRs) and acetylcholinesterase (AChE) at the synapse beneath the nerve terminal (Patthy and Nikolics, 1993, Trends in Neurosci. 16: 76-81).
  • nAChRs nicotinic acetylcholine receptors
  • AChE acetylcholinesterase
  • agrin When damage occurs, reinnervation of the muscle is topographically specific, preferentially reforming connections at original synaptic sites (McMahon and Slater, 1984, J. Cell Bio., 98: 1453-73).
  • the biological activity of agrin is not limited to AChR aggregation on myotubues. It has been shown that a dozen molecules undergo redistribution in response to agrin (Nastuk and Fallon, 1993, Trends in Neurosci. 16: 72-76). High concentrations of neuro transmitter receptors characterize both neuromuscular junctions as well as neuron-neuron synapses in the brain and peripherial nervous system.
  • Agrin protein and mRNA is expressed by cells of the brain, and can be detected by western and northern blotting.
  • Agrin mRNA is found not only in motor neurons, but also in Purkinje cells and retinal ganglion cells that project exclusively within the central nervous system (Smith et al., 1992 , Molec. Cell. Neurosci. 3: 406-417).
  • the purified agrin receptor would be invaluable for the demonstration of the presence of compounds which could bind agonistically, or antagonistically with the receptor.
  • the purified receptor protein allows for the generation of antibodies which will bind to the protein and thereby allow for the labeling, identification, and purification of agrin receptor proteins from a variety of tissues from any creatures with a nervous system.
  • the agrin receptor protein can be used as a means for screening tissues for the presence or absence of agrin, as an indicator of pathological conditions, or as a means for manipulation of tissue interactions dependent on agrin.
  • the agrin receptor protein allows for the identification of substances which will bind to the agrin receptor.
  • the agrin receptor protein allows for the identification of substances which will enhance or inhibit the binding of agrin or agrin-related proteins with the agrin receptor.
  • This invention provides for a substantially purified oligopeptide which is characterized by the ability to bind agrin, having a molecular weight of about 190 kD as measured by SDS polyacrylamide gel electrophoresis.
  • the substantially purified protein is characterized by the ability to bind agrin, and has one polypeptide of a molecular weight of about 190 kD and a second polypeptide of a molecular weight of about 50 kD, as measured by SDS polyacrylamide gel electrophoresis.
  • the invention also encompasses substantially purified oligopeptides or fragments thereof, of the protein identified as the agrin receptor which is characterized by the ability to inhibit the binding of agrin or agrin related proteins to the agrin receptor.
  • the invention encompasses the use of substantially purified oligopeptides or fragments thereof, of the protein identified as the agrin receptor, as inhibitors of the clustering activity associated with agrin binding at pre- and postsynaptic membranes. This clustering including those structures listed in table I.
  • the invention encompasses the use of substantially purified oligopeptides or fragments thereof, of the protein identified as the agrin receptor, as activators of the clustering activity associated with agrin binding at pre- and postsynaptic membranes.
  • This clustering including those structures listed in table I.
  • the protein and fragments thereof are glycosylated.
  • the agrin receptor of the invention may be isolated and purified from many sources, among these would be nervous tissues of any vertebrate or invertebrate animal.
  • the agrin receptor is isolated from Torpedo electric organ.
  • the receptor protein will allow for the determination of amino acid residue sequence information for the agrin receptor protein. This sequence information will allow for the determination of nucleic acid sequences which encode and translate the agrin receptor.
  • one embodiment of the agrin receptor of the instant invention is produced using recombinant DNA technology where by an expression vector is used to express, translate, and produce the peptides of the instant invention. This expression can take place in bacterial, yeast, mammalian, plant, fungal, or insect cells or variations thereof.
  • the expression vector may be used for the introduction into embryonic cells for the production of transgenic animals, plants or insects, or for gene therapy.
  • the instant invention provides amino acid sequences which are part of the agrin receptor subunits as listed in Table III.
  • the instant invention provides for the construction of natural or syntheic peptides of the disclosed sequences, as well as for recombinant DNA vectors for the expression of peptides from corresponding DNA sequences, or the generation of antisense RNA from corresponding inverse DNA sequences.
  • the present invention also provides for methods of screening for compounds which can inhibit binding of agrin to the agrin receptor, comprising the steps of combining an amount of protein of receptor with an amount of compound to be tested, followed by the administration of an amount of agrin, where one of the components of the assay is labeled so that a quantitative determination of binding can be made.
  • the present invention also provides methods of screening compounds for binding with the agrin receptor comprising the steps of combining an amount of receptor with an amount of compound to be tested, where one of the components is labeled so that a qualitative measurement of binding can be made. Such screening can be accomplished so as to identify potential neuro-toxins, neuro-trophic compounds, agonists, antagonist, or enzymes which may be a part of the processes of agrin binding and the effects of such binding to the receptor.
  • the present invention also encompasses the production of antibodies which are immunoreactive with the agrin receptor protein.
  • the antibodies are monoclonal antibodies.
  • a particular embodiment is the monoclonal antibody, mAb-AgRl, which is produced by the cell line AgRl.
  • the present invention thus encompass the use of agrin protein to produce antibodies which will immunoreact with the agrin protein and inhibit the specific binding of the monoclonal antibody mAb-AgRl with the agrin receptor.
  • the present invention thus encompasses cell lines which can express antibodies that are able to bind to the agrin receptor.
  • a specific embodiment of this is the cell line AgRl which produces the monoclonal antibody mAb-AgRl.
  • the antibodies of the present invention can bind to the various epitopes of the agrin receptor, these epitopes encompassing protein quaternary, tertiary and secondary structure epitopes which are based upon the primary structure of the agrin receptor protein.
  • the antibodies of the present invention can recognize carbohydrate epitopes associated with the agrin receptor protein and the functional derivatives of the carbohydrate moieties generated by natural or induced modification.
  • the present invention also provides for methods for stimulating or inhibiting the activity of the agrin receptor consisting of the administration of an effective amount of the monoclonal antibody. Also provided is a method for stimulating or inhibiting the aggregation of AChR (acetylcholine receptors) on neural tissues comprising the administration of an effective amount of mAb-AgRl.
  • AChR acetylcholine receptors
  • the antibodies of the instant invention can be used for immunopurification, immunolabling, immunodiagnostic and immunoassay procedures for the detection and isolation of agrin receptor proteins. These methods can be embodied in kits consisting of aliquoted antibody solution and control solution for the quantification of agrin receptor proteins, or agrin content of various tissues.
  • the present invention also teaches the use of a 70 kD agrin protein which is capable of binding to agrin receptors without stimulating AChR aggregation. This being a useful means of binding agrin to target membranes with agrin receptors, without subsequent AChR clustering.
  • Figure 1 illustrates the Ligand Overlay Assay of Torpedo electric organ postsynaptic membranes.
  • Far left lane lane 1
  • Silver stain of immunoaffinity (mAb-AgRl) purified agrin receptor polypeptides Note the 190 kD and 50 kD proteins.
  • Lane 4 same sample as in lane 3, overlay in the absence of calcium (lane 4), overlay in the absence of agrin (lane 4).
  • Figure 2 illustrates SDS-PAGE analysis of the purified 190 kD and 50 kD polypeptides.
  • Figure 3 (a) is a diagram showing the elution profile of TEO agrin binding protein with inset showing analysis of specific fractions by SDS-PAGE and silver-staining.
  • (b) is a graph showing the agrin binding activity is retained even when AChR is depleted.
  • Figure 4 is a visualized blot of intact and alkaline stripped membrane proteins isolated from TEO and a correlation with agrin binding as measured using 125 I-labeled anti- agrin monoclonal antibodies.
  • Figure 5 is a graph illustrating the calcium sensitive, saturable binding of agrin to synaptic membrane receptors as measured by RIA.
  • Figure 6 (a) is a graph illustrating the calcium sensitive agrin binding by membrane receptors as measured by RIA as intact or stripped membranes.
  • 6 (b) is a graph demonstrating that Agrin binds to rat brain synaptosomes, and is calcium sensitive, as shown by RIA.
  • Figure 7 is a diagram of the agrin protein and the relationship between the 90 kD and
  • Figure 8 is a western blot demonstrating the binding of the 90 kD and 70 kD fragments to agrin receptors on isolated membranes.
  • Figure 9 is a diagram illustrating the relationship between the Agrin Receptor Subunit sequences and the human dystroglycan precursor protein. Detailed Description of the Invention
  • TEO Torpedo californica electric organ
  • OG octyl- ⁇ -D-glucopyranoside
  • the homogenate was centrifuged twice for 10 min at 6,000 x g and the resulting supernatant then centrifuged for 1 hr at 100,000 x g.
  • the pellet was resuspended in buffer B (10 mM NaH2PO4, 1 mM EDTA, 1 mM EGTA, 100 ug/ml PMSF, 0.02% sodium azide, pH 7.4), homogenized, layered onto a continuous 25%-40% sucrose gradient, and centrifuged for 6 hr at 100,000 x g.
  • Acetylcholine receptor (AChR)-rich fractions determined by SDS-PAGE (sodium dodecyl sulfate-poly acrilamide gel electrophoresis) , were then pooled.
  • the four AChR subunits were identified by comigration with purified AChR generously provided by A. Karlin. Protein concentrations, determined according to Bradford (1976, Anal. Biochem. 72: 248-254), ranged from 0.4 to 0.6 mg/ml for the AChR-rich pools.
  • Membranes from Torpedo liver were prepared exactly as described above.
  • FIG. 1 illustrates the Ligand Overlay Assay of Torpedo electric organ postsynaptic membranes.
  • the far left lane (lane 1) shows the silver-stain of immunoaffinity purified agrin receptor polypeptides from the mAb-AgRl immunoaffinity column. This purified product resolved into two bands of about 190 kD and 50 kD (arrows).
  • Autoradiography of the agrin overlay assay shows that agrin bound ( 125 I labeled-anti-agrin mAb detected) to the single 190 kD band (lane 2).
  • Agrin bound to a single band in the presence of Ca 44" (lane 3), but not in the absence of Ca "1-1" (lane 4).
  • a second control lane received no agrin (lane 5).
  • Molecular weight standards are from the top, 200, 116, 97, 68, 45, and 29 kD and are indicated by the plain lines.
  • the protein content of unpurified postsynaptic membranes is illustrated in Figure 2 (lane 1, silver stain) and contains multiple proteins.
  • Solubilization of postsynaptic membranes was accomplished, optimally, by incubating untreated or alkaline stripped membranes in 25 mM octyl- ⁇ -D- glucopyranoside in 50 mM NaCl, 0.1 mM PMSF in sucrose-free buffer B, pH 7.4 for 18 hours at 4°C. Detergent- treated membranes were centrifuged at 100,000 x g f or 3 hr and the supernatant collected for further analysis.
  • the conditions for solubilization of the agrin receptor is distinct from that for AChR, which was found to be 34 mM octyl- ⁇ -D-glucopyranoside, 0 mM NaCl as determined by SDS-PAGE and 1 5 I- ⁇ - bungarotoxin binding.
  • Example 2 Determination of Agrin Receptor by SDS-PAGE/Ligand Overlay Assay and by Non-denaturing Conditions using Ligand Affinity Chromato raphy Agrin was purified over 3,000 fold from Torpedo electric organ as described previously (Nastuk et al., 1991, Neuron 7: 807-818).
  • Agrin bioreactivity is expressed in Units (U)/ml, which refers to the amount of agrin required to induce half-maximal AChR clustering on myotubes cultured in 1 ml of media (Godfrey et al., 1984, J. Cell Biol. 99: 615-627).
  • U Units
  • One unit is estimated to correspond with 10" 12 to 10 ⁇ 13 M agrin (Nitkin et al., 1987, J. Cell Biol. 105: 2471-2478).
  • agrin activity of purified agrin was tested as follows. Membranes were pelleted and resuspended in HEPES buffered MEM (pH 7.2; MEM-H) with 10% horse serum/1% BSA and incubated for 30 min at 4°C with 3 U of purified agrin in 20 ul of membranes with gentle agitation. Incubations (in duplicate) were carried out in the presence or absence of 2 mM EGTA. Maximal agrin activity under these conditions was determined by incubating agrin without membranes. After incubation, pelleted membranes and 15 ul of each supernatant was used for overnight stimulation of chick myotube cultures.
  • Myotubes were labeled with rhodamine- ⁇ -bungarotoxin and AChR clustering activity determined.
  • the clustering ability of agrin effects many synapse components, these are summarized in table I, phase indicated in relation to agrin binding (from Nastuk and Fallon, 1993, Trends in Neurosci. 16: 72-76).
  • Acetylcholineseterase (globular) early
  • Acetylcholinesterase (A 12 asymmetric) ?
  • agrin receptors The presence of agrin receptors was demonstrated as follows. Alkaline- stripped postsynaptic membranes were resuspended in 150 U of agrin for 1 hr at 4°C in 50 mM NaCl, 5% glycerol, 10 mM NaPO4, pH 7.4 with 1 mM calcium or 1 mM EGTA. The membranes were solubilized in SDS sample buffer with no reducing agents, separated on 7.5% SDS-PAGE gels. The gels were then blotted onto nitrocellulose. The bound agrin was demonstrated by the binding of mAb 11D2, an anti-agrin monoclonal antibody (Nitkin et al., 1987, J. Cell Bio. 105: 2471-2478).
  • the mAb 11D2 was visualized by incubating the blots with 11D2 and biotinylated horse anti-mouse IgG, and avidin-biotin-HRP (horse radish peroxidase) (Vector). Bound antibody was detected by chemiluminescence (ECL, Amersham), and indicated the presence of agrin bound to protein blotted to the nitrocellulose.
  • solubilized proteins separated by SDS-PAGE were either stained with silver or blotted to nitrocellulose.
  • the separated blotted proteins were then blocked with 5% non-fat dry milk.
  • the indicated areas were then treated with purified TEO agrin in the presence of 1 mM Ca ++ or 1 mM EDTA.
  • the blots were then incubated with 125 I-anti-agrin mAb 6D4 (Fallon et al., 1985, Nature 315: 571-4), washed, and dried; agrin-binding bands were then revealed by autoradiography.
  • Agrin binding fractions of gel-filtered membrane proteins were iodinated (lodogen method, Pierce). Labeled proteins were pre-incubated with agrin for 1 hr in the presence of 1 mM C ⁇ ++ or EGTA to form ligand-receptor complexes, and were then applied overnight to anti-agrin immunoaffinity columns (mAb-5Bl and mAb-6D4 cross- linked to Protein A agarose beads by the method of Schneider, C. et al., 1982, J. Biol. Chem. 257: 10766-10769) , washed, and eluted with 10 mM EGTA.
  • Figure 2 illustrates SDS-PAGE analysis of the purified receptor yielding 190 kD and 50 kD polypeptides (arrows).
  • Lane 1 is a silver-stain of initial biochemical purification of TEO membrane proteins showing many proteins present.
  • Lane 2 are the postsynaptic membranes after purification by immunoaffinity column (same as lane 1 of Figure 1).
  • Lanes 3 and 4 shows the elutes from agrin affinity chromatography. The peak fractions from the gel-filtration column ( Figure 3) were iodinated and applied to agrin affinity columns in the presence of calcium (lane 3) or the absence of calcium (lane 4).
  • Lane 3 is an autoradiogram of the mAb-AgRl immunoprecipitate of the material eluted by EGTA from the agrin ligand- affinity column with calcium present.
  • the column bound 125 I-labeled 190 kD and 50 kD polypeptides (arrows) in the presence but not in the absence of calcium.
  • Molecular weight standards are the same as Figure 1. Lines to the right indicate (from the top) Na-ATPase and AChR ⁇ , gamma, ⁇ and ⁇ subunits. Additional bands of 48, 55 and 65 kD also bound in a Ca ++ independent manner.
  • Solubilized membrane proteins isolated as above were made 0.5 M in NaCl and also applied to immunoaffinity columns which were prepared with mAb-AgRl covalently linked to the column matrix (Schneiden et al., 1982, J. Biol. Chem. 257: 10766-10769). The columns were washed in 1.0 M NaCl. The 190 kD and 50 kD polypeptides coeluted from this column. This is shown in Figure 2. The agrin binding activity co ⁇ esponded with the elution of these polypeptides, and this is shown in Figure 3.
  • the calcium sensitive eluates from the agrin ligand affinity columns (from example 2) containing the 190 kD and 50 kD polypeptides were then applied to mAb- AgRl immunoaffinity columns.
  • the final eluate from these immunoaffinity columns was found to contain the same 190 kD and 50 kD polypeptides, and to retain agrin binding activity as demonstrated by the ligand overlay assay.
  • agrin bound to the 190 kD purified polypeptide As shown with the ligand overlay assays of the crude membrane preparations, agrin bound to the 190 kD purified polypeptide, and this is shown in Figure 2.
  • Postsynaptic membrane from Torpedo californica electric organ were prepared as described previously. Membrane proteins were alkaline-treated as described below, to remove peripheral membrane proteins, solubilized in 25 mM OG, and fractionated on Superose 6. Agrin binding was quantitated by a solid phase, ligand-binding RIA (described below), under sub-saturating conditions.
  • FIG. 3 (a) is a diagram showing the elution profile of TEO agrin binding protein.
  • the 190 kD and 50 kD polypeptides migrate as a complex during gel filtration chromatography.
  • Agrin binding activity and OD28O of membrane proteins fractionated by gel-filtration. Shown as an inset is SDS-PAGE and silver-stain of mAb-AgRl immunoprecipitates of the indicated fractions. Note there is only a single peak of agrin binding activity in these membranes.
  • a 190 kD and 50 kD protein co- eluted with this peak of agrin binding. Peak fractions of AChR and Na, K-ATPase are indicated by the arrowheads at fraction #29, and #33, respectively.
  • Column fractions were applied to a mAb-AgRl immunoaffinity column (mAb crosslinked to Protein-A agarose beads), washed, and eluted with 0.1 M diethylamine (pH 11.
  • Figure 3 (a) shows the agrin binding activity as eluting in a single peak with the 190 kD/50 kD complex. This peak eluted 1 to 2 fractions earlier (larger stokes radius) than the elution of the AChR complex, which has a native Mr of 250 kD (data not shown). Results are the means ⁇ S.E.M. of 4 experiments. AChR concentration was determined by 125 I- ⁇ -bungarotoxin binding and confirmed by SDS-PAGE; Na/K- ATPase was revealed by SDS-Page and silver staining. Insert immunoaffinity eluates were separated by SDS-PAGE and silver stained.
  • the AChR and the agrin receptor could be separated from the AChR under mild detergent, salt, and pH, conditions which favor the preservation of macromolecular complexes. This data suggests that the AChRs and the agrin receptors are not tightly associated with one another in the membrane.
  • Figure 3 (b) is a bar graph showing the results of the use of ⁇ -bungarotoxin affinity columns, and the flow-through levels of AChR and agrin receptors. Solubilized membranes were incubated with buffer only (start), or with 10 "8 M biotinylated- ⁇ -bungarotoxin (AChR depleted), or without ⁇ -bungarotoxin (control), followed by strptavidin-agarose beads. More than 95% of the AChR was removed as measured by SDS-PAGE and scanning densitometry of the AChR ⁇ -subunit. Agrin receptor binding of agrin was measured by RIA. The agrin receptor does not co- precipitate with AChRs.
  • Both the 190 kD and the 50 kD polypeptides are integral membrane proteins since they are resistant to alkaline extraction ( Figure 2), and have carbohydrate modifications demonstrating sensitivity to glycosidases.
  • Alkaline treatment (pH 11) of Torpedo membranes is known to extract peripherial membrane proteins such as 43 kD, 58 kD, and dystrophin-like polypeptides (Neubig et al, 1979, JV. ⁇ .S. USA 76: 690-694; Yeadon, et al., 1991, J. Cell Biol. 115: 1069-1076).
  • these stripped membranes retain integral membrane proteins with hydrophobic domains, such as AChR.
  • FIG. 4 is a visualized blot of intact and alkaline stripped membrane proteins isolated from TEO and a correlation with agrin binding as measured using Horseradish peroxidase (HRP)-labeled anti-agrin monoclonal antibodies.
  • HRP Horseradish peroxidase
  • Figure 4 demonstrates alkaline stripping extracts peripherial membrane proteins as shown in lanes 1 vs. 2. The stripped membranes retaining integral membrane proteins after treatment at pH 11, lane 2. Intact TEO membrane is shown in lane 1 , all proteins visualized by coomassie blue staining. The well characterized 43 kD and 300 kD dystrophin-like cytoplasmic peripherial membrane proteins are removed by this treatment (arrows). The position of the four AChR subunits are indicated, ⁇ , ⁇ , ⁇ , ⁇ .
  • Western blot probed with WGA of proteins extracted at pHl 1 (lane 3) and extracted at pH 12 (lane 4) show that the same set of peripherial membrane glycoproteins are extracted by both treatments.
  • FIG. 5 is a bar graph illustrating the agrin binding to intact and pH 1 1 stripped membranes, in a calcium sensitive manner. Equal volumes of intact or alkaline stripped membranes were assayed by agrin binding RIA (as described in example 6). The results show that agrin binding is quantitatively recovered following membrane alkaline stripping, with complete retention of calcium sensitivity.
  • Table II shows the results of lectin affinity chromatography depletion of the agrin receptor.
  • Various lectins were used and the residual actin binding activity measured. Alkaline stripped, detergent solubilized postsynaptic membranes were incubated with the indicated lectin-substituted agarose beads. After incubation, the level of agrin binding activity in the supernatants was determined by RIA. This demonstrates that the agrin receptor is most likely glycoprotein, and sensitive to the carbohydrate structure.
  • Monoclonal antibody to the agrin receptor was generated using standard fusion tehchniques, with the following modification.
  • Mice were immunized by injection of alkaline- stripped postsynaptic membranes from TEO in RIBI adjuvant (Ribi, 1984, J. Biol. Resp. Mod. 3: 1-9) subcutaneously at the ankle. After 6 injections at 5-7 day intervals the mice were sacrificed and the popliteal an inguinal lymph nodes harvested and fused with the Agl08 myeloma cell line. Hybridoma supernatants were screened for antibodies that immunoprecipitated the agrin receptor.
  • agrin was mixed with 15 ul of solubilized postsynaptic membranes to form agrin-agrin receptor complexes.
  • the hybridoma supernatants were then added, incubated overnight, and then applied to sepharose bound anti-mouse Ig.
  • Agrin/agrin-receptor/anti-agrin receptor complex was thus precipitated associated with the sepharose beads.
  • the agrin remaining in the supernatant was measured by a two site RIA where anti-agrin mAb-6D4 was immobilized on microwells, agrin (supernatant) added, and the captured agrin quantitated by incubation with iodinated anti-agrin mAb-5Bl.
  • the cell line producing mAb-AgRl has been deposited with the American Type Culture Collection (ATCC, Rockville, M.D.) under the Budapest Treaty and identified as mouse myeloma/B-cell hybridoma, anti-AgR-MAb-3B3, with the assigned designation number HB 11530, on January 21, 1994.
  • a RIA radio immunoassay
  • the initial blocking step, the agrin incubation, and the subsequent wash in MEM-H were performed with or without 2 mM EGTA.
  • the peptides GRGDSP (100 uM; Gly-Arg-Gly-Asp-Ser-Pro; Telios) and LRE (300 uM; Leu-Arg-Glu; from J. Sanes) were added during the agrin binding step, although neither peptide inhibited binding.
  • the binding of exogenous agrin to the membranes is measured as a function of bound radiolabelled anti-agrin antibody.
  • FIG. 5 -3- shows that the calcium-dependent agrin binding to TEO membranes is saturable. In the absence of calcium, binding is reduced by more than 5- fold, and rises linearly with increasing ligand concentration. No agrin binding was observed on Torpedo liver membranes, with or without the presence of calcium. Half- maximal agrin binding is observed at an agrin concentration of about 250 U/ml, this co ⁇ esponding to ⁇ 10 ⁇ 10 M agrin. This low ligand concentration suggests a high affinity binding to the agrin receptor.
  • Figure 5 is a graph showing the binding of agrin to membrane receptors as measured by RIA (described in example 6) and demonstrates saturation. In the absence of calcium, agrin binding is not saturable.
  • Figure 6 (a) is a graph which demonstrates the calcium sensitive binding of agrin to the membrane receptors as measured by RIA. Agrin binding to postsynaptic membranes was measured under varying calcium concentrations. Optimal agrin binding is observed at 1-2 mM calcium, which agrees with the calcium optima for agrin's AChR clustering activity.
  • Figure 6 (b) is a graph which demonstrates that agrin binds to adult rat brain synaptosomes in a calcium dependent manner.
  • Rat brain synaptosomes were prepared according to Watkins et al., (1984, J. Physiol..London 153: 35P). Synaptosomes were immobilized on poly-lysine coated microwells and agrin binding was determined as above. These data indicate that agrin receptors are expressed in the rat brain.
  • Example 7 Agrin Binding Domain of 95 kD and 70 kD Polypeptides Purification of agrin protein yielded two major polypeptide fragments referred to as 95 and 70 kP, based on the apparent Mr under reducing conditions. These fragments have identical amino termini, with the 70 kP polypeptide being truncated at its carboxyl end. We find that both the 95 kP and the 70 kP fragments bind to the TEO membranes as measured by RIA. This result shows that at least one of the receptor binding domains of agrin is located in the region delineated by the 70 kP fragment.
  • FIG. 7 is a schematic diagram of the structure of agrin. The amino terminal
  • Torpedo agrin (shaded portion) has not been sequenced; a consensus domain orgainization for this region based on rat and chick sequence is shown for illustrative purposes.
  • FIG. 8 shows the identification of agrin polypeptides which bind to postsynaptic membranes. Shown is a western blot probed with anti-agrin antibody 11D2 (lanes 1-4) or an i ⁇ elevant IgG (lanes 5-8). Lanes 1, 5: starting agrin preparations of 150 U. Lanes 2-8: pellets of alkaline stripped postsynaptic membranes (25 ug) after incubation with: no agrin (lane 2, 6); or agrin (150 U) in the presence of calcium (lane 3, 7), or the absence of calcium (lane 4, 8). Both the 95 kD and the 70 kD agrin polypeptide fragments bind postsynaptic membranes in a calcium dependent manner. It is also noteable that the starting membrane preparations showed a lack of detectable agrin.
  • Immunoaffinity-purified agrin receptor subunits (approximately 20 ug of total protein) were electrophoresed on a discontinuous SDS-PAGE separating gel (upper half 5%-7%, lower half 11% acrylamide). The polypeptides were electroblotted for 3 hr. at 2 A onto Immobilon PVDF membranes (Millipore) in 10% methanol, 25 mM Tris, 192 mM glycine (pH 8.3).
  • the 190 kDa- and 50 kDa-bearing bands were excised, minced, and subjected to in situ enzymatic digestion with 0.5 ug of endoproteinase Lys-C (190 kDa polypeptide) or 0.25 ug of trypsin (50 kDa polypeptide), essentially as described (Fernandez, et al., 1994, Anal. Biochem. 218:112-117).
  • the released peptides were separated with a Hewlett Packard 1090M HPLC system equipped with a UV diode a ⁇ ay detector, using a Brownlee Aquapore C8 microbore column (1 x 250 mm) with 0.1% TFA as solvent A and 0.08% TFA in acetonitrile-water (70:30 v/v) as solvent B.
  • a linear gradient was developed from 0 to 55% in solvent B over a period of 90 min at a flow rate of 50 ul/min.
  • the peptide peaks were collected manually in 1.5 ml microfuge tubes.
  • the native agrin receptor is a heteromeric complex of two membrane glycoproteins (190 kDa and 50 kDa) that are related to the dystrophin-associated glycoproteins ⁇ - and ⁇ -dystroglycan respectively.
  • the sequences obtained show that the peptides are related to the dystroglycans.
  • a sequence alignment with the human dystroglycan precursor sequence [SEQ ID NO. 7] is shown in Figure 9.
  • Gly Val Pro lie lie Phe Ala Asp Glu Leu Asp Asp Ser Lys Pro Pro 1 5 10 15
  • Glu Val Lys lie Pro Ser Asp Thr Phe Tyr Asp His Glu Asp Thr Thr

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Abstract

La présente invention se rapporte à des récepteurs d'agrine et à des peptides associés à ces récepteurs d'agrine. Cette invention se rapporte également à la production d'anticorps contre le récepteur d'agrine, ainsi qu'à l'utilisation du récepteur d'agrine dans des méthodes de diagnostic, de test et de marquage. La présente invention décrit des séquences d'acides aminés spécifiques s'appliquant à des sous-unités du récepteur d'agrine.
PCT/US1994/005387 1993-11-10 1994-05-16 Recepteur d'agrine WO1995013298A1 (fr)

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Publication number Priority date Publication date Assignee Title
US7335637B2 (en) 1999-11-18 2008-02-26 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US7759314B2 (en) 2001-08-15 2010-07-20 Brown University Treatment of muscular dystrophies and related disorders
US8691766B2 (en) 1999-11-18 2014-04-08 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US9958458B2 (en) 2010-12-27 2018-05-01 Brown University Therapeutic and diagnostic methods involving biglycan and utrophin
US9969785B2 (en) 2010-05-17 2018-05-15 Brown University Biglycan mutants and related therapeutics and methods of use

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BOWE MA. ET AL.;: "Prodcution of monoclonal antibodies to the agrin receptor", SOCIETY FOR NEUROSCIENCE ABSTRACT, vol. 19, no. 1-3, 7 November 1993 (1993-11-07), pages 1272 *
FALLON J R ET AL.;: "Characterization of a putative agrin receptor from torpedo electric organ", SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 17, 1991, pages 179 *
FALOON, J.R. ET AL;: "The putative agrin receptor", THE JOURNAL OF CELLULAR BIOLOGY, vol. 0, no. 16E, 1992, NEW YORK, USA, pages 222 *
MA J; FALLON JR;: "Biochemical characterization of putative agrin receptor from torpedo electric organ", SOCIETY FOR NEUROSCIENCE ABSTRACT, vol. 18, 1992, pages 414 *
MA J-Y; NASTUK M A; FALLON JR: "Characterization of a putative agrin receptor from torpedo electric organ", JOURNAL OF CELL BIOLOGY, vol. 115, no. 3, 1991, pages 100A *
NASUK, M.A. ET AL.;: "The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholone receptor clustering", NEURON, vol. 7, no. 5, November 1991 (1991-11-01), pages 807 - 818 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7335637B2 (en) 1999-11-18 2008-02-26 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US7816322B2 (en) 1999-11-18 2010-10-19 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US8658596B2 (en) 1999-11-18 2014-02-25 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US8691766B2 (en) 1999-11-18 2014-04-08 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US7759314B2 (en) 2001-08-15 2010-07-20 Brown University Treatment of muscular dystrophies and related disorders
US8822418B2 (en) 2001-08-15 2014-09-02 Brown University Treatment of muscular dystrophies and related disorders
US9969785B2 (en) 2010-05-17 2018-05-15 Brown University Biglycan mutants and related therapeutics and methods of use
US9958458B2 (en) 2010-12-27 2018-05-01 Brown University Therapeutic and diagnostic methods involving biglycan and utrophin

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