Expression, identification and biological effects of the novel recombination protein, PACAP38-NtA, with high bioactivity.

Pituitary adenylate cyclase­activating polypeptide (PACAP) is a type of neuropeptide with multiple biological functions. However, it has a short half­life period in the body, ~3 to 5 min, restricting its further development as a drug that can promote the recovery of nerve injury. In vitro and in vivo experiments have shown that PACAP can repair the epithelial cell on the surface of the injured cornea, as PACAP can act on the trigeminal nerve cell to secrete other active neurotransmitters, which can promote corneal epithelial cell proliferation and differentiation. In the present study, PACAP is connected to the N­terminal agrin domain (NtA) with a genetic engineering method, which allows the function of repairing the injured nerve. Notably, the recombinant polypeptide can interact with laminin, improving the biological effect of PACAP in repairing the injured nerve. In the study, the recombinant protein was constructed by combining PACAP38 and NtA by genetic engineering, and it is expressed in the pronucleus escherichia coli. The recombinant protein, PACAP38­NtA, is obtained with a two­step purification method, including anion­exchange chromatography and Ni­affinity chromatography, with the purity reaching >90%. The in vitro experiment has shown that this recombinant protein not only has the neurotrophy and neural restoration function of PACAP, but also has the function of an anchoring protein as laminin interacts with NtA. According to the in vitro anti­apoptosis, PC12 axon growth and ELISA experiments, this protein has the biological activity of a recombinant protein. PACAP38­NtA also has an anchoring function as NtA and laminin interact with good biological activity.

T-shaped arrangement of the recombinant agrin G3-IgG Fc protein.

Agrin is a large heparin sulphate proteoglycan with multiple domains, which is located in the extracellular matrix. The C-terminal G3 domain of agrin is functionally one of the most important domains. It harbors an α-dystroglycan binding site and carries out acetylcholine receptor clustering activities. In the present study, we have fused the G3 domain of agrin to an IgG Fc domain to produce a G3-Fc fusion protein that we intend to use as a tool to investigate new binding partners of agrin. As a first step of the study, we have characterized the recombinant fusion protein using a multidisciplinary approach using dynamic light scattering, analytical ultracentrifugation and small angle X-ray scattering (SAXS). Interestingly, our SAXS analysis using the high-resolution structures of G3 and Fc domain as models indicates that the G3-Fc protein forms a T-shaped molecule with the G3 domains extruding perpendicularly from the Fc scaffold. To validate our models, we have used the program HYDROPRO to calculate the hydrodynamic properties of the solution models. The calculated values are in excellent agreement with those determined experimentally.

Agrin binds BMP2, BMP4 and TGFbeta1.

The C-terminal 95 kDa fragment of some isoforms of vertebrate agrins is sufficient to induce clustering of acetylcholine receptors but despite two decades of intense agrin research very little is known about the function of the other isoforms and the function of the larger, N-terminal part of agrins that is common to all isoforms. Since the N-terminal part of agrins contains several follistatin-domains, a domain type that is frequently implicated in binding TGFbetas, we have explored the interaction of the N-terminal part of rat agrin (Agrin-Nterm) with members of the TGFbeta family using surface plasmon resonance spectroscopy and reporter assays. Here we show that agrin binds BMP2, BMP4 and TGFbeta1 with relatively high affinity, the K(D) values of the interactions calculated from SPR experiments fall in the 10(-8) M-10(-7) M range. In reporter assays Agrin-Nterm inhibited the activities of BMP2 and BMP4, half maximal inhibition being achieved at approximately 5x10(-7) M. Paradoxically, in the case of TGFbeta1 Agrin N-term caused a slight increase in activity in reporter assays. Our finding that agrin binds members of the TGFbeta family may have important implications for the role of these growth factors in the regulation of synaptogenesis as well as for the role of agrin isoforms that are unable to induce clustering of acetylcholine receptors. We suggest that binding of these TGFbeta family members to agrin may have a dual function: agrin may serve as a reservoir for these growth factors and may also inhibit their growth promoting activity. Based on analysis of the evolutionary history of agrin we suggest that agrin's growth factor binding function is more ancient than its involvement in acetylcholine receptor clustering.

Identification and function of agrin expressed in rat brain microvessels.

Agrin isoforms in the brain microvessels were investigated. Brain microvessels prepared from rat cerebral cortex expressed the short amino terminal (SN) isoform, which contains a transmembrane sequence of SN agrin, and the long amino terminal (LN) isoform, which contains a signal sequence for secretion of LN agrin, were detected in the microvessels. The expression of LN form mRNA in comparison with that of SN form was higher in the brain microvessels than in the cerebral cortex, indicating preferential expression of LN form agrin in the microvessels. In addition, the amino-terminal sequence of agrin expressed in the rat brain microvessels was determined by rapid amplification of 5' cDNA end (5'-RACE). The sequence contained a cluster of hydrophobic amino acids, which may form a signal sequence for secretion. Transfection of expression vector coding amino-terminal sequence of LN agrin fused with green fluorescence protein (GFP) into human embryonic kidney (HEK293) cells caused expression of the protein in the cell as well as in the medium, whereas SN agrin fused with GFP expressed at the cell surface. These results show that the major form of agrin expressed in the brain microvessels is of basal lamina-associated LN agrin and suggest that LN agrin may play a role in molecular organization at the interface between the parenchyma cells and microvessels.

Identification of agrinSN isoform and muscle-specific receptor tyrosine kinase (MuSK) [corrected] in sperm.

We previously demonstrated several nicotinic acetylcholine receptor (nAChR) subunits and associated proteins in human sperm. Here, we identified in sperm for the first time two additional nAChR-associated molecules: (1) agrin(SN)Z(+) in human sperm localized in the posterior post-acrosomal, neck, and flagellar mid-piece regions; (2) a low-molecular weight isoform of muscle-specific receptor tyrosine kinase in human and mouse sperm localized in the flagellar mid-piece of human sperm.

Identification and distribution of heparan sulfate proteoglycans in the white muscle of Atlantic cod (Gadus morhua) and spotted wolffish (Anarhichas minor).

Heparan sulfate proteoglycans (HSPGs) were identified in pre-rigor muscle of two species of cold water fish, Atlantic cod (Gadus morhua) and spotted wolffish (Anarhichas minor) by biochemical and immunological methods. The distribution was described by immunohistology. Special emphasis was directed to the extracellular matrix (ECM) HSPGs perlecan and agrin. In vivo 35S-sulfate labeling combined with ultracentrifugation in CsCl2, DEAE chromatography and scintillation counting of the eluates, revealed that the content of 35S-labeled PGs was much higher in wolffish than in cod. A considerable proportion of the 35S-sulfated PGs in both species was HSPG, as judged by nitrous acid degradation. HSPG represented, however, a higher proportion of the 35S-sulfated PGs in cod compared to wolffish. Dot blot and electrophoresis/western blot using two different HS-mAbs, 10E4 and HepSS-1 indicated structural differences in the HS-chains of the PGs present. This observation was strengthened by immunohistochemistry, showing that both mAbs detected epitopes in the pericellular area, but the staining patterns were not superimposable. Two different agrin isoforms were identified in both species. Furthermore, in the white muscle of both cod and wolffish, perlecan mAb (A7L6) showed positive staining restricted to the transition between myocommata and myofibers.

Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo.

Aggregation of acetylcholine receptors (AChRs) in muscle fibers by nerve-derived agrin plays a key role in the formation of neuromuscular junctions. So far, the effects of agrin on muscle fibers have been studied in culture systems, transgenic animals, and in animals injected with agrin--cDNA constructs. We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results. Both neural and muscle agrin bind uniformly to the surface of innervated and denervated muscle fibers along their entire length. Neural agrin causes a dose-dependent appearance of AChR aggregates, which persist > or = 7 wk after a single application. Muscle agrin does not cluster AChRs and at 10 times the concentration of neural agrin does not reduce binding or AChR-aggregating activity of neural agrin. Electrical muscle activity affects the stability of agrin binding and the number, size, and spatial distribution of the neural agrin--induced AChR aggregates. Injected agrin is recovered from the muscles together with laminin and both proteins coimmunoprecipitate, indicating that agrin binds to laminin in vivo. Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo.

Agrin isoforms with distinct amino termini: differential expression, localization, and function.

The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons. Little is known about its roles at sites other than the neuromuscular junction, or about how its expression and subcellular localization are regulated in any tissue. Here we demonstrate that the murine agrin gene generates two proteins with different NH(2) termini, and present evidence that these isoforms differ in subcellular localization, tissue distribution, and function. The two isoforms share approximately 1,900 amino acids (aa) of common sequence following unique NH(2) termini of 49 or 150 aa; we therefore call them short NH(2)-terminal (SN) and long NH(2)-terminal (LN) isoforms. In the mouse genome, LN-specific exons are upstream of an SN-specific exon, which is in turn upstream of common exons. LN-agrin is expressed in both neural and nonneural tissues. In spinal cord it is expressed in discrete subsets of cells, including motoneurons. In contrast, SN-agrin is selectively expressed in the nervous system but is widely distributed in many neuronal cell types. Both isoforms are externalized from cells but LN-agrin assembles into basal laminae whereas SN-agrin remains cell associated. Differential expression of the two isoforms appears to be transcriptionally regulated, whereas the unique SN and LN sequences direct their distinct subcellular localizations. Insertion of a "gene trap" construct into the mouse genome between the LN and SN exons abolished expression of LN-agrin with no detectable effect on expression levels of SN-agrin or on SN-agrin bioactivity in vitro. Agrin protein was absent from all basal laminae in mice lacking LN-agrin transcripts. The formation of the neuromuscular junctions was as drastically impaired in these mutants as in mice lacking all forms of agrin. Thus, basal lamina-associated LN-agrin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.

Alternative splicing of agrin regulates its binding to heparin alpha-dystroglycan, and the cell surface.

Agrin is a basal lamina molecule that directs key events in postsynaptic differentiation, most notably the aggregation of acetylcholine receptors (AChRs) on the muscle cell surface. Agrin's AChR clustering activity is regulated by alternative mRNA splicing. Agrin splice forms having inserts at two sites (y and z) in the C-terminal region are highly active, but isoforms lacking these inserts are weakly active. The biochemical consequences of this alternative splicing are unknown. Here, the binding of four recombinant agrin isoforms to heparin, to alpha-dystroglycan (a component of an agrin receptor), and to myoblasts was tested. The presence of a four-amino acid insert at the y site is necessary and sufficient to confer heparin binding ability to agrin. Moreover, the binding of agrin to alpha-dystroglycan is inhibited by heparin when this insert is present. Agrin binding to the cell surface showed analogous properties: heparin inhibits the binding of only those agrin isoforms containing this four-amino acid insert. The results show that alternative splicing of agrin regulates its binding to heparin and suggest that agrin's interaction with alpha-dystroglycan may be modulated by cell surface glycosaminoglycans in an isoform-dependent manner.

NCAM-mediated adhesion of transfected cells to agrin.

The vertebrate neural cell adhesion molecule NCAM mediates heterophilic adhesion to heparan sulfate proteoglycans in embryonic chick brain membranes. In this study, mouse L cells transfected with chicken NCAM were used to identify two of these ligands as agrin and the target of the 6C4 monoclonal antibody. A third heparan sulfate proteoglycan, perlecan, appeared not to support NCAM-mediated adhesion. Enzymatic degradation of chondroitin sulfates decreased adhesion in agrin-containing membrane fractions but increased adhesion if the agrin had previously been removed by immunoprecipitation, suggesting that interactions between heparan sulfate and chondroitin sulfate proteoglycans have important influences on adhesion. Our experiments support the view that NCAM can interact with multiple, but not with all, heparan sulfate and chondroitin sulfate proteoglycans in chick brain membranes in both positive and negative ways to influence cell adhesion.

Agrin binding to alpha-dystroglycan. Domains of agrin necessary to induce acetylcholine receptor clustering are overlapping but not identical to the alpha-dystroglycan-binding region.

The synaptic basal membrane protein agrin initiates the aggregation of acetylcholine receptors at the postsynaptic membrane of the developing neuromuscular junction. Recently, alpha-dystroglycan was found to be a major agrin-binding protein on the muscle cell surface and was therefore considered a candidate agrin receptor. Employing different truncation fragments of agrin, we determined regions of the protein involved in binding to alpha-dystroglycan and to heparin, an inhibitor of alpha-dystroglycan binding. Deletion of a 15-kDa fragment from the C terminus of agrin had no effect on its binding to alpha-dystroglycan from rabbit muscle membranes, even though this deletion completely abolishes its acetylcholine receptor aggregating activity. Conversely, deletion of a central region does not affect agrin's clustering activity, but reduced its affinity for alpha-dystroglycan. Combination of these two deletions resulted in a fragment of approximately 35 kDa that weakly bound to alpha-dystroglycan, but displayed no clustering activity. All of these fragments bound to heparin with high affinity. Thus, alpha-dystroglycan does not show the binding specificity expected for an agrin receptor. Our data suggest the existence of an additional component on the muscle cell surface that generates the observed ligand specificity.

Distribution of alpha-dystroglycan during embryonic nerve-muscle synaptogenesis.

The distribution of alpha-dystroglycan (alpha DG) relative to acetylcholine receptors (AChRs) and neural agrin was examined by immunofluorescent staining with mAb IIH6 in cultures of nerve and muscle cells derived from Xenopus embryos. In Western blots probed with mAb IIH6, alpha DG was evident in membrane extracts of Xenopus muscle but not brain. alpha DG immunofluorescence was present at virtually all synaptic clusters of AChRs and neural agrin. Even microclusters of AChRs and agrin at synapses no older than 1-2 h (the earliest examined) had alpha DG associated with them. alpha DG was also colocalized at the submicrometer level with AChRs at nonsynaptic clusters that have little or no agrin. The number of large (> 4 microns) nonsynaptic clusters of alpha DG, like the number of large nonsynaptic clusters of AChRs, was much lower on innervated than on noninnervated cells. When mAb IIH6 was included in the culture medium, the large nonsynaptic clusters appeared fragmented and less compact, but the accumulation of agrin and AChRs along nerve-muscle contacts was not prevented. It is concluded that during nerve-muscle synaptogenesis, alpha DG undergoes the same nerve-induced changes in distribution as AChRs. We propose a diffusion trap model in which the alpha DG-transmembrane complex participates in the anchoring and recruitment of AChRs and alpha DG during the formation of synaptic as well as nonsynaptic AChR clusters.

Agrin is a heparan sulfate proteoglycan.

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.