Figure 1.
Domain architectures of agrins.
Different domain-types of agrins are: N-terminal agrin domain, NtA (NtA; light purple); Follistatin-domains, (FS; blue), laminin-EGF domains (LE; light brown), SEA-domain (SEA; purple); EGF-domains (E; orange), laminin G domains (LG; light red). Vertebrate agrins (represented here by Rattus norvegicus agrin) have two alternative N-termini. The secreted form has a signal sequence (SP; orange) and a laminin-binding N-terminal agrin domain, whereas the membrane-bound form has a short intracellular region and a transmembrane helix (vertical black bar). Alternative splicing in the C-terminal part of vertebrate agrins at B/z site gives rise to agrin isoforms that differ in their ability to cluster acetylcholin receptors. The two arrows indicate the neurotrypsin cleavage sites (α and β sites) of vertebrate agrins. Note that the neurotrypsin cleavage sites are not conserved in invertebrate agrins. The solid red line indicates the domain structure of recombinant Agrin_Nterm used in the present study. The lower part of the figure shows the structures of agrins of invertebrate species representing Urochordates (Ciona intestinalis), Echonoderms (Strongylocentrotus purpuratus), Arthropods (Apis mellifera), Nematodes (Caenorhabditis elegans) and Placozoa (Trichoplax adhaerens); for details see TableS1. Note that agrin of the Arthropod Tribolium castaneum has the same domain architecture as agrin of Apis mellifera.
Figure 2.
Isolation of Agrin-Nterm by affinity chromatography.
Dialyzed culture fluid of Drosophila melanogaster S2 cells expressing recombinant Agrin_Nterm was applied onto a Ni affinity column, the column was washed with 10 column volumes of 20 mM Tris-HCl buffer, pH 7.9 containing 500 mM NaCl and 5 mM imidazole, then with 5 column volumes of 20 mM Tris-HCl buffer, pH 7.9 containing 500 mM NaCl and 30 mM imidazole (first arrow) and the bound protein was eluted with 20 mM Tris-HCl buffer, pH 7.9 containing 300 mM imidazole (second arrow). The right panel shows the SDS-PAGE of the affinity chromatography, St indicates the pattern of the Low Molecular Weight Calibration Kit (Amersham Pharmacia Biotech, Uppsala, Sweden; Mr values: 14,400, 20,100, 30,000, 45,000, 66,000, and 97,000). The horizontal bars indicate the pooled fractions containing Agrin-Nterm.
Figure 3.
Purification of Agrin-Nterm by gel chromatography.
Recombinant protein isolated by Ni affinity chromatography (seeFig. 2.) was chromatographed on a AKTA purifier 10 System using a Superdex gel filtration column (HiLoad 10/300 Superdex 200 GL) and fractions containing pure Agrin-Nterm (horizontal bar) were pooled. Lane a of the insert shows SDS-PAGE of Agrin-Nterm of the pooled factions of AKTA chromatography, lane b indicates the pattern of the Low Molecular Weight Calibration Kit (Amersham Pharmacia Biotech, Uppsala, Sweden; Mr values: 14,400, 20,100, 30,000, 45,000, 66,000, and 97,000).
Figure 4.
Characterization of the interaction of BMP2, BMP4 and TGFβ1 with Agrin-Nterm by SPR assays.
Sensorgrams of the interactions of (A) Agrin-Nterm (660, 1320, 1980, 2640 and 3300 nM) with BMP2; (B) Agrin-Nterm (660, 1320, 2640, 3960 and 5280 nM) with BMP4; (C) Agrin-Nterm (360, 900, 1800, 2160 and 2700 nM) with TGFβ1. Various concentrations of Agrin-Nterm in 20 mM HEPES buffer, pH 7.5, containing 150 mM NaCl, 5 mM EDTA, 0.005% Tween 20 were injected over TGFβ1 or BMPs immobilized on CM5 sensorchips. For each type of experiment, one set of representative data of three parallels is shown. For the sake of clarity the concentrations of Agrin-Nterm are not indicated in the panels; in each case SPR response increased parallel with the increase of Agrin-Nterm concentration.
Table 1.
Kinetic parameters of the interaction of Agrin-Nterm with different members of the TGFβ family.
Figure 5.
Effect of Agrin-Nterm on the binding of BMP2, BMP4 and TGFβ1 to the extracellular domain of their cognate receptors, monitored by surface plasmon resonance.
A) Sensorgrams of the interactions of immobilized ECD of BMPRIA with 40 nm BMP2 preincubated with 0 nM, 12 nM, 29 nM, 58 nM, 88 nM, 146 nM and 293 nM of Agrin-Nterm. B) Sensorgrams of the interactions of immobilized ECD of BMPRIA with 40 nm BMP4 preincubated with 0 nM, 220 nM, 440 nM, 660 nM, 880 nM, 1100 nM and 1540 nM of Agrin-Nterm. C) Sensorgrams of the interactions of immobilized ECD of TGF-βsRII with 4 nm TGFβ1 preincubated with 0 nM, 912 nM, 1824 nM, 2736 nM and 4560 nM of Agrin-Nterm. Growth factors were preincubated with Agrin-Nterm in 20 mM HEPES buffer, pH 7.5 containing 150 mM NaCl, 5 mM EDTA, 0.005% Tween20 for 30 min at room temperature and were injected over CM5 sensorchips containing immobilized ECD of receptors. For the sake of clarity the concentrations of Agrin-Nterm are not indicated in the panels; in each case SPR response decreased parallel with the increase of Agrin-Nterm concentration. Panels on the right indicate the concentration dependence of the inhibitory effect of Agrin-Nterm as monitored by changes in observed association rate, kobs.
Figure 6.
Effect of Agrin-Nterm on growth factor activities.
Panel A) HepG2-BRA cells were incubated for 17 hours with 250 pM BMP2 preincubated with different concentrations of Agrin-Nterm. Panel B) HepG2-BRA cells were incubated for 17 hours with 250 pM BMP4 preincubated with different concentrations of Agrin-Nterm. Panel C) Mink lung epithelial cells MLEC-clone32 were incubated for 17 hours with 16 pM TGFβ1 preincubated with different concentrations of Agrin-Nterm; The luciferase activities were normalized to the protein content of the wells and background values obtained from control cells were subtracted. The figure shows the mean values of three parallel experiments.