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• W2020083886 abstract "Netrins were first identified as neural guidance molecules, acting through receptors that are members of the DCC and UNC-5 family. All netrins share structural homology to the laminin N-terminal domains and the laminin epidermal growth factor-like domains of laminin short arms. Laminins use these domains to self-assemble into complex networks. Here we demonstrate that netrin-4 is a component of basement membranes and is integrated into the laminin polymer via interactions with the lamininγ1 andγ3 short arms. The binding is mediated through the laminin N-terminal domain of netrin-4. In contrast to netrin-4, other members of the netrin family do not bind to these laminin short arms. Moreover, a truncated form of netrin-4 completely inhibits laminin-111 self-assembly in vitro, and full-length netrin-4 can partially disrupt laminin self-interactions. When added to explant cultures, netrin-4 retards salivary gland branching morphogenesis. Netrins were first identified as neural guidance molecules, acting through receptors that are members of the DCC and UNC-5 family. All netrins share structural homology to the laminin N-terminal domains and the laminin epidermal growth factor-like domains of laminin short arms. Laminins use these domains to self-assemble into complex networks. Here we demonstrate that netrin-4 is a component of basement membranes and is integrated into the laminin polymer via interactions with the lamininγ1 andγ3 short arms. The binding is mediated through the laminin N-terminal domain of netrin-4. In contrast to netrin-4, other members of the netrin family do not bind to these laminin short arms. Moreover, a truncated form of netrin-4 completely inhibits laminin-111 self-assembly in vitro, and full-length netrin-4 can partially disrupt laminin self-interactions. When added to explant cultures, netrin-4 retards salivary gland branching morphogenesis. Netrins were first isolated as long range guidance cues, acting in early embryogenesis by regulating the migration of neurons and the axonal growth cone (1Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1158) Google Scholar). These proteins showed either chemoattractive or chemorepulsive effects upon distinct sets of cells, hence cells expressing netrin-1 can mimic the ability of the floor plate to repel the growth cones of trochlear motor neurons in vitro, while attracting the axons of spinal commissural neurons (2Serafini T. Colamarino S.A. Leonardo E.D. Wang H. Beddington R. Skarnes W.C. Tessier-Lavigne M. Cell. 1996; 87: 1001-1014Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar, 3Kennedy T.E. Serafini T. de la Torre de la Torre.J. Tessier-Lavigne M. Cell. 1994; 78: 425-435Abstract Full Text PDF PubMed Scopus (1128) Google Scholar, 4Colamarino S.A. Tessier-Lavigne M. Cell. 1995; 81: 621-629Abstract Full Text PDF PubMed Scopus (510) Google Scholar). Consequently, netrin-1 is considered a bifunctional guidance cue. This activity has been shown to relate to expression levels of specific receptors from either the DCC or UNC-5 families (5Keino-Masu K. Masu M. Hinck L. Leonardo E.D. Chan S.S. Culotti J.G. Tessier-Lavigne M. Cell. 1996; 87: 175-185Abstract Full Text Full Text PDF PubMed Scopus (874) Google Scholar, 6Hedgecock E.M. Culotti J.G. Hall D.H. Neuron. 1990; 4: 61-85Abstract Full Text PDF PubMed Scopus (727) Google Scholar, 7Leung-Hagesteijn C. Spence A.M. Stern B.D. Zhou Y. Su M.W. Hedgecock E.M. Culotti J.G. Cell. 1992; 71: 289-299Abstract Full Text PDF PubMed Scopus (343) Google Scholar, 8Chan S.S. Zheng H. Su M.W. Wilk R. Killeen M.T. Hedgecock E.M. Culotti J.G. Cell. 1996; 87: 187-195Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 9Leonardo E.D. Hinck L. Masu M. Keino-Masu K. Ackerman S.L. Tessier-Lavigne M. Nature. 1997; 386: 833-838Crossref PubMed Scopus (426) Google Scholar). Five members of the netrin gene family have been identified in mammals: netrin-1 (2Serafini T. Colamarino S.A. Leonardo E.D. Wang H. Beddington R. Skarnes W.C. Tessier-Lavigne M. Cell. 1996; 87: 1001-1014Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar, 10Meyerhardt J.A. Caca K. Eckstrand B.C. Hu G. Lengauer C. Banavali S. Look A.T. Fearon E.R. Cell Growth & Differ. 1999; 10: 35-42PubMed Google Scholar), netrin-3 (11Van Raay T.J. Foskett S.M. Connors T.D. Klinger K.W. Landes G.M. Burn T.C. Genomics. 1997; 41: 279-282Crossref PubMed Scopus (47) Google Scholar, 12Wang H. Copeland N.G. Gilbert D.J. Jenkins N.A. Tessier-Lavigne M. J. Neurosci. 1999; 19: 4938-4947Crossref PubMed Google Scholar), netrin-4 (13Koch M. Murrell J.R. Hunter D.D. Olson P.F. Jin W. Keene D.R. Brunken W.J. Burgeson R.E. J. Cell Biol. 2000; 151: 221-234Crossref PubMed Scopus (137) Google Scholar), netrin-G1, and netrin-G2 (14Nakashiba T. Ikeda T. Nishimura S. Tashiro K. Honjo T. Culotti J.G. Itohara S. J. Neurosci. 2000; 20: 6540-6550Crossref PubMed Google Scholar, 15Nakashiba T. Nishimura S. Ikeda T. Itohara S. Mech. Dev. 2002; 111: 47-60Crossref PubMed Scopus (110) Google Scholar, 16Yin Y. Miner J.H. Sanes J.R. Mol. Cell. Neurosci. 2002; 19: 344-358Crossref PubMed Scopus (73) Google Scholar). All netrin genes encode secreted proteins, but in contrast to netrin-1, -3, and -4, netrin-G1 and -G2 are membrane-bound via glycosylphosphatidylinositol anchors. All netrins share homology to the N-terminal parts of the laminin short arms (Fig. 2), being formed of a laminin N-terminal (LN) 2The abbreviations used are: LN, laminin N-terminal domains; LE, laminin epidermal growth factor-like domains; Δ, truncated; EGF, epidermal growth factor; EHS, Engelbreth-Holm-Swarm; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; BS3, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester); P, postnatal day; E, embryonic day; RU, response unit. 2The abbreviations used are: LN, laminin N-terminal domains; LE, laminin epidermal growth factor-like domains; Δ, truncated; EGF, epidermal growth factor; EHS, Engelbreth-Holm-Swarm; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; BS3, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester); P, postnatal day; E, embryonic day; RU, response unit. module followed by about three LEa (laminin-EGF) modules and a C-terminal NTR (netrin) domain also designated as C domain. Recently netrins and their receptors have been shown to be significant in developmental and physiological events outside the nervous system, including their acting as angiogenic factors (17Park K.W. Crouse D. Lee M. Karnik S.K. Sorensen L.K. Murphy K.J. Kuo C.J. Li D.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16210-16215Crossref PubMed Scopus (267) Google Scholar, 18Lu X. Le Noble F. Yuan L. Jiang Q. De Lafarge B. Sugiyama D. Breant C. Claes F. De Smet F. Thomas J.L. Autiero M. Carmeliet P. Tessier-Lavigne M. Eichmann A. Nature. 2004; 432: 179-186Crossref PubMed Scopus (431) Google Scholar, 19Nguyen A. Cai H. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6530-6535Crossref PubMed Scopus (139) Google Scholar) and being involved in epithelial branching morphogenesis of the lung, intestine, pancreas, and mammary gland (20Liu Y. Stein E. Oliver T. Li Y. Brunken W.J. Koch M. Tessier-Lavigne M. Hogan B.L. Curr. Biol. 2004; 14: 897-905Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 21Dalvin S. Anselmo M.A. Prodhan P. Komatsuzaki K. Schnitzer J.J. Kinane T.B. Gene Expr. Patterns. 2003; 3: 279-283Crossref PubMed Scopus (34) Google Scholar, 22De Breuck S. Lardon J. Rooman I. Bouwens L. Diabetologia. 2003; 46: 926-933Crossref PubMed Scopus (54) Google Scholar, 23Srinivasan K. Strickland P. Valdes A. Shin G.C. Hinck L. Dev. Cell. 2003; 4: 371-382Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 24Jiang Y. Liu M.T. Gershon M.D. Dev. Biol. 2003; 258: 364-384Crossref PubMed Scopus (152) Google Scholar). Furthermore, netrin-1 has been suggested as a ligand for the laminin receptors integrins α6β4 and α3β1 and hence has been described as acting as an adhesive cue (25Yebra M. Montgomery A.M. Diaferia G.R. Kaido T. Silletti S. Perez B. Just M.L. Hildbrand S. Hurford R. Florkiewicz E. Tessier-Lavigne M. Cirulli V. Dev. Cell. 2003; 5: 695-707Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). We have previously shown that netrin-4 is also a component of basement membranes in a variety of tissues (13Koch M. Murrell J.R. Hunter D.D. Olson P.F. Jin W. Keene D.R. Brunken W.J. Burgeson R.E. J. Cell Biol. 2000; 151: 221-234Crossref PubMed Scopus (137) Google Scholar). Basement membranes are specialized extracellular matrices underlying all endothelia and epithelia and surrounding many forms of mesenchymal cells (26Yurchenco P.D. Wadsworth W.G. Curr. Opin. Cell Biol. 2004; 16: 572-579Crossref PubMed Scopus (88) Google Scholar). They have both physical and signaling functions that alter with both tissue type and stage of development. In addition to forming surfaces to which cells attach, they also transmit force between cells and the surrounding extracellular matrix. Basement membrane components may also regulate many aspects of intracellular activity by signaling via the integrin, dystroglycan, and syndecan receptor families. Furthermore, basement membranes can act as reservoirs for cytokines and growth factors, e.g. members of the fibroblast growth factor family. Finally, signaling by other cytokines may be complemented by basement membrane-induced signals, as seen with neurotrophins. Hence basement membrane-induced signaling plays an important role in cell survival and differentiation as well as in cell migration and as an axonal guidance cue. Laminins are the major noncollagenous proteins of basement membranes and are crucial in its formation. Laminin-111, the prototype laminin, is a cross-like shaped molecule formed as a multidomain heterotrimer assembled of one α1, one β1, and one γ1 chain (27Chung A.E. Jaffe R. Freeman I.L. Vergnes J.P. Braginski J.E. Carlin B. Cell. 1979; 16: 277-287Abstract Full Text PDF PubMed Scopus (240) Google Scholar, 28Timpl R. Rohde H. Robey P.G. Rennard S.I. Foidart J.M. Martin G.R. J. Biol. Chem. 1979; 254: 9933-9937Abstract Full Text PDF PubMed Google Scholar, 29Aumailley M. Bruckner-Tuderman L. Carter W.G. Deutzmann R. Edgar D. Ekblom P. Engel J. Engvall E. Hohenester E. Jones J.C. Kleinman H.K. Marinkovich M.P. Martin G.R. Mayer U. Meneguzzi G. Miner J.H. Miyazaki K. Patarroyo M. Paulsson M. Quaranta V. Sanes J.R. Sasaki T. Sekiguchi K. Sorokin L.M. Talts J.F. Tryggvason K. Uitto J. Virtanen I. von der Mark K. Wewer U.M. Yamada Y. Yurchenco P.D. Matrix Biol. 2005; 24: 326-332Crossref PubMed Scopus (663) Google Scholar). The laminin trimer has one long and three short arms, the latter being formed from the three free N-terminal ends of the α1, β1, and γ1 chains (30Beck K. Dixon T.W. Engel J. Parry D.A. J. Mol. Biol. 1993; 231: 311-323Crossref PubMed Scopus (83) Google Scholar, 31Beck K. Hunter I. Engel J. FASEB J. 1990; 4: 148-160Crossref PubMed Scopus (659) Google Scholar). These parts of the β1 and γ1 chains each contain two globular domains, designated LN and L4/LF. The globules are interspersed by multiple LE modules, forming rods in domains LEa and LEb. Certain laminin chains have been described as maintaining the domain structure of those in the original laminin-111 (e.g. α2 and β2), whereas others have N-terminal truncations, lacking an LN domain. So far 15 laminin isoforms have been shown to occur (29Aumailley M. Bruckner-Tuderman L. Carter W.G. Deutzmann R. Edgar D. Ekblom P. Engel J. Engvall E. Hohenester E. Jones J.C. Kleinman H.K. Marinkovich M.P. Martin G.R. Mayer U. Meneguzzi G. Miner J.H. Miyazaki K. Patarroyo M. Paulsson M. Quaranta V. Sanes J.R. Sasaki T. Sekiguchi K. Sorokin L.M. Talts J.F. Tryggvason K. Uitto J. Virtanen I. von der Mark K. Wewer U.M. Yamada Y. Yurchenco P.D. Matrix Biol. 2005; 24: 326-332Crossref PubMed Scopus (663) Google Scholar). Laminins self-assemble into a network through Ca2+-dependent interactions between their N-terminal parts (32Yurchenco P.D. Tsilibary E.C. Charonis A.S. Furthmayr H. J. Biol. Chem. 1985; 260: 7636-7644Abstract Full Text PDF PubMed Google Scholar), and polymerization of laminin-111 may be inhibited by proteolytic laminin fragments that contain LN domains (33Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). Netrin-4 is the most recently described soluble netrin family member, and its biological significance is still poorly understood. We have shown that netrin-4 is widely expressed with the protein being concentrated in certain basement membranes and having a spatial expression that broadens during later development. Here we demonstrate the integration of netrin-4 into the basement membrane via the binding to the N-terminal region of the laminin γ1 chain. Furthermore, we show the significance of netrin-4 in basement membrane assembly and its effect upon branching morphogenesis. Recombinant Expression and Purification of Murine Netrin and Laminin-derived Proteins—The following full-length or truncated netrin forms were amplified by PCR and subcloned into an episomal expression vector: mouse netrin-1 (U65418), nucleotides 1–1368 with three additional nucleotides after nucleotide 69, which leads to an insertion of an additional glycine and a single amino acid substitution valine to leucine at nucleotides 259–261 (present in all the sequenced clones, all of which were independent PCR products); mouse netrin-4 (AF281278), nucleotides 311–2166; mouse Δnetrin-4 (AF281278), nucleotides 311–1672; and mouse netrin-G1e (AB038663), nucleotides 52–1305. The PCR products were subcloned (rapid DNA ligation kit, Roche Diagnostics) into a modified pCEP-Pu (13Koch M. Murrell J.R. Hunter D.D. Olson P.F. Jin W. Keene D.R. Brunken W.J. Burgeson R.E. J. Cell Biol. 2000; 151: 221-234Crossref PubMed Scopus (137) Google Scholar) expression vector (an 8-histidine tag and a thrombin cleavage site were introduced either at the N-terminal or the C-terminal end of the protein sequence). On the basis of the Δnetrin-4 and Δnetrin-G1e constructs, the following Δnetrin-4 deletion and Δnetrin-4-netrin-G1 hybrid constructs were cloned: Δnetrin-4 (LN + EGF 1–2), nucleotides 311–1432; Δnetrin-4 (LN + EGF 1 + 3), nucleotides 311–1242 and 1432–1672; netrin-4 (EGF 1–3.5), nucleotides 1031–1672; Δnetrin-4 (LN) + netrin-G1 (EGF 1–3), nucleotides 311–1036 (netrin-4) and 889–1305 (netrin-G1e); netrin-G1 (LN) + netrin-4 (EGF 1–3.5), nucleotides 52–891 (netrin-G1e) and 1040–1672 (netrin-4). In addition, a fragment of the laminin γ1 chain (J02930), nucleotides 292–1668, containing the N-terminal LN domain and four LEa repeats and a fragment of the laminin γ3 chain (NM 011836), nucleotides 148–3133, containing domains LN, LEa, L4, and LEb, were amplified by PCR and subcloned into the modified pCEP-Pu expression vector. The expression vectors were transfected into 293-EBNA cells with FuGENE 6 transfection reagent (Roche Diagnostics), and selected clones with the highest protein expression were expanded for large scale production. The purification of the secreted proteins was performed as described previously (13Koch M. Murrell J.R. Hunter D.D. Olson P.F. Jin W. Keene D.R. Brunken W.J. Burgeson R.E. J. Cell Biol. 2000; 151: 221-234Crossref PubMed Scopus (137) Google Scholar). Antibody Production—For antibody KR1, a rabbit was immunized with purified recombinant Δnetrin-4. The antiserum was affinity-purified by applying it to a Sepharose column to which mouse Δnetrin-4 protein had been coupled. Bound antibodies were eluted with triethylamine, pH 11.5, immediately neutralized, and dialyzed against PBS. For antibody KR 24, a rabbit was immunized with purified recombinant netrin-1, and the antiserum was affinity-purified as described for antibody KR1. Immunofluorescence Microscopy—Newborn (P2) and adult mouse tissues were embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Europe). 7-μm-thick sections were cut with a Leica cryostat and stored at –20 °C. For use, slides were returned to room temperature, fixed, washed in PBS, and blocked with 0.2% Tween 20 in PBS for 1 h at room temperature. Slides were fixed in 4% paraformaldehyde for 5 min, washed, blocked with 5% goat serum, and incubated with antibodies against netrin-4 (KR1, see under “Antibody Production”) and the laminin γ1 chain (rat anti-laminin β2 chain monoclonal antibody, Chemicon). After incubation with secondary antibodies (Cy3-labeled goat anti-rabbit IgG, Jackson ImmunoResearch; or Alexa 488-labeled goat anti-rat IgG, Molecular Probes), mounted sections were observed under a laser scanning confocal microscope (Leica) scanning the sections 16 times. ELISA Style Ligand Binding Assay—Unless otherwise specified, all solutions used contained 2 mm CaCl2. For testing the divalent cation dependence of interactions, no CaCl2, 2 mm CaCl2, or 20 mm EDTA was added to the solutions. Purified proteins were diluted in TBS, pH 7.4, and coated at 10 μg/ml (500 ng/well) overnight at room temperature onto 96-well plates (Nunc Maxisorb). After washing with TBS containing 0.05% Tween 20, plates were blocked for 2 h at room temperature with TBS containing either 5% milk powder or 1% bovine serum albumin. Ligands were diluted to concentrations between 0.001 and 50 nm and incubated in the wells for 1 h at room temperature. After extensive washing with TBS containing 0.05% Tween 20, bound ligands were detected with specific polyclonal rabbit antibodies directed against netrin-1 and netrin-4, respectively, followed by horseradish peroxidase-conjugated swine anti-rabbit immunoglobulins (DAKO Cytomation) and tetramethylbenzidine as substrate. Absorption was measured at 450 nm after stopping the reaction with 10% sulfuric acid. Surface Plasmon Resonance Binding Assays—Assays were performed using a Biacore 2000 (BIAcore AB). Coupling of proteins to the CM5 chip was performed in 25 mm sodium acetate, pH 5.0, at a flow rate of 5 μl/min. A 7-min pulse of 0.05 mm N-hydroxysuccinimide, 0.2 m N-ethyl-N′-dimethylaminopropyl carbodiimide was used to activate the surface. The protein was injected until the desired amount was coupled (500–1000 RU), and excess reactive groups were deactivated by a 7-min pulse of 1 m ethanolamine HCl, pH 8.5. Measurements were carried out in HBS (20 mm Hepes, 150 mm NaCl, pH 7.4) containing 2 mm CaCl2 and 0.005% P20 at a flow of 25 μl/min. The injection of 100 μl of protein solution (0.05–1 μm) was followed by a 400-s dissociation. Each analysis was carried out at six different concentrations. The data were analyzed with BIAe-valuation software 3.2 according to the Langmuir model for 1 to 1 binding. All binding curves were fitted with an accuracy of χ2 <1% of Rmax (maximal RU). The mean ka, kd, and KD values for the six concentrations are given in Table 2.TABLE 2Surface plasmon resonance analysis of netrin-4 binding to laminin γ1AnalytekakdKDm-1 s-1s-1mNetrin-4 (LN + EGF 1-3.5)Laminin γ1 (LN + EGF 1-4)5.70 × 1041.24 × 10-33.71 × 10-8Laminin γ1 (LN + EGF 1-4)Netrin-4 (LN + EGF 1-3.5)1.83 × 1051.29 × 10-31.69 × 10-8Laminin γ1 (LN + EGF 1-4)Netrin-G1 (LN + EGF 1-3)NBaNB indicates no binding.NBNBLaminin γ1 (LN + EGF 1-4)Netrin-4 (LN + EGF 1/3)3.88 × 1041.32 × 10-35.66 × 10-8Laminin γ1 (LN + EGF 1-4)Netrin-4 (LN + EGF 1-2)2.60 × 1059.94 × 10-41.39 × 10-8Laminin γ1 (LN + EGF 1-4)Netrin-4 (LN) + netrin-G1 (EGF 1-3)4.92 × 1046.24 × 10-35.82 × 10-7Laminin γ1 (LN + EGF 1-4)Netrin-4 (EGF 1-3.5)NBNBNBLaminin γ1 (LN + EGF 1-4)Netrin-G1 (LN) + netrin-4 (EGF 1-3.5)NBNBNBa NB indicates no binding. Open table in a new tab Cross-linking Assays—Cross-linking assays were carried out using the lysine side chain-reactive cross-linker bis[sulfosuccinimidyl]suberate (Pierce). The reaction was carried out at a protein concentration of 2.8 μm in a final volume of 50 μlof PBS, pH 7.4. The cross-linker was used at concentrations from 0.5 to 2 mm. The reaction was allowed to continue for 1 h on ice and was stopped by the addition of 10 μl of 1 m Tris-HCl, pH 8.0. Organ Culture—Submandibular glands from embryonic day 13.5 BL/6 mice were placed on a Nucleopore filter (Whatman) and cultured at the air/medium interface. Cultures were carried out in serum-free Dulbecco’s modified Eagle’s medium/F-12 supplemented with 2 mm l-glutamine, 50 μg/ml transferrin, and penicillin/streptomycin (Invitrogen). For testing the effects of full-length netrin-4 protein and Δnetrin-4, lacking the netrin C domain and Δlaminin γ1, these were added to the culture medium at a final concentration of 50 μg/ml at the onset of organ culture. For negative controls the same amount of PBS was added. Medium was changed every day. After 3 days the explants were photographed under a microscope and embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Europe). Sections were cut and stained as described above. Aggregation of Laminin-111-Nidogen Complexes—Laminin-111 (laminin-111-nidogen complexes) was extracted from mouse EHS tumor tissue (kindly provided by Juörgen Engel and Therese Schuthess, EDTA extraction method of Paulsson et al. (34Paulsson M. Aumailley M. Deutzmann R. Timpl R. Beck K. Engel J. Eur. J. Biochem. 1987; 166: 11-19Crossref PubMed Scopus (330) Google Scholar)), and Δnetrin-4 in 0.02 m Tris-HCl, 0.15 m NaCl, 0.05 mm EDTA, pH 7.4, was centrifuged for 15 min at 10,000 × g at room temperature. Aliquots of laminin-111 (0.67 mg/ml, Fig. 6A, and 0.5 mg/ml, Fig. 6B) were pre-equilibrated at 37 °C for 30 min in the cuvettes of a thermostatted Beckman DU 640 spectrophotometer. To one sample 5 mm EDTA was added, and to the other samples prewarmed Δnetrin-4 (0.21 mg/ml, Fig. 6A, and 0.14 mg/ml, Fig. 6B) or full-length netrin-4 (0.19 mg/ml, Fig. 6B) was added. After supplementing all samples with 1 mm CaCl2, turbidity development was monitored as absorbance at 360 nm. After 300 min EDTA (5 mm) was added to such samples that had not been kept in the presence of EDTA for the duration of the experiment. In a second independent polymerization assay, laminin-111 (0.6 mg/ml, laminin-111-nidogen complex) in 0.02 m Tris-HCl, 0.15 m NaCl, 0.05 mm EDTA, pH 7.4, was incubated at 37 °C for 3 h after the initiation of polymerization by addition of 1 mm CaCl2 (35Paulsson M. J. Biol. Chem. 1988; 263: 5425-5430Abstract Full Text PDF PubMed Google Scholar, 36Cheng Y.S. Champliaud M.F. Burgeson R.E. Marinkovich M.P. Yurchenco P.D. J. Biol. Chem. 1997; 272: 31525-31532Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Aggregation was assayed after centrifugation at 13,000 × g for 10 min by SDS-PAGE under reducing conditions on 8% polyacrylamide gels. When desired 10 mm EDTA or serial dilutions of Δnetrin-4 or Δnetrin-1, giving a 1:1 to 1:125 molar ratio between netrin and laminin, were added prior to incubation. Netrin-4 Expression in Newborn and Adult Mice—We described previously the expression pattern of netrin-4 in the adult mouse and showed that it is present in basement membranes in a variety of tissues (13Koch M. Murrell J.R. Hunter D.D. Olson P.F. Jin W. Keene D.R. Brunken W.J. Burgeson R.E. J. Cell Biol. 2000; 151: 221-234Crossref PubMed Scopus (137) Google Scholar). To extend these observations, the netrin-4 expression in kidney was studied during development, initially by indirect immunofluorescence microscopy with a netrin-4-specific polyclonal antibody on kidney sections from day 14.5 (E14.5) and day 16.5 (E16.5) mouse embryos, newborn mice (P2), and 3–4-month-old adult mice (for antibody specificity see supplemental Fig. 1). Surprisingly, expression was not observed until birth (Fig. 1B), with no immunostaining of the embryonic kidney (not shown). Postnatally expression increases, so that while at P2 netrin-4 staining was mainly localized to the glomerular basement membrane and the vascular basement membranes (Fig. 1, B and C), and in the adult kidney the tubular basement membrane and that of the tissue capsule also stained for netrin-4 (Fig. 1, E and F). A strong co-localization of netrin-4 and the laminin γ1 subunit was observed, although netrin-4 was also observed at non-basement membrane sites (Fig. 1, C and F), for instance in the mesangium of the glomerulus. This suggests that netrin-4 expression occurs as epithelial structures mature, e.g. the kidney tubules. Netrin-4 Interactions with Laminin LN Domains—The basement membrane localization of netrin-4 suggests its involvement in protein-protein interactions. Its marked homology to the LN domains of laminin, which participate on laminin self-interactions, raised the possibility that netrin-4, as well as other members of the netrin family, could be a laminin-binding protein. Hence, recombinant proteins were produced that corresponded to the N-terminal part of laminin chains or were full-length or modified forms of netrin-1 or netrin-4 (Fig. 2). Initially, surface plasmon resonance-based assays were carried out to identify any possible binding between short arms of laminins and netrin-1 and -4. This showed a strong binding of the Δlaminin γ1 chain-(LN + LEa) and γ3 chain (LN + LEa + L4 + LEb)-derived proteins to Δnetrin-4 (LN + EGF 1–3.5; Fig. 3) but not to Δnetrin-1 (LN + EGF 1–3; data not shown); the calculated KD was 2.35 × 10–8 m for the γ1 chain fragment (Fig. 3A) and 1.96 × 10–8 m for the γ3 chain short arm (Fig. 3B). In addition binding to the Δlaminin β1 chain (LN + LEa) could be observed (not shown). The following experiments focused on the Δlaminin γ1 chain, since this subunit is common to most laminin heterotrimers (29Aumailley M. Bruckner-Tuderman L. Carter W.G. Deutzmann R. Edgar D. Ekblom P. Engel J. Engvall E. Hohenester E. Jones J.C. Kleinman H.K. Marinkovich M.P. Martin G.R. Mayer U. Meneguzzi G. Miner J.H. Miyazaki K. Patarroyo M. Paulsson M. Quaranta V. Sanes J.R. Sasaki T. Sekiguchi K. Sorokin L.M. Talts J.F. Tryggvason K. Uitto J. Virtanen I. von der Mark K. Wewer U.M. Yamada Y. Yurchenco P.D. Matrix Biol. 2005; 24: 326-332Crossref PubMed Scopus (663) Google Scholar). We also tested the laminin γ1 chain-netrin-4 interaction in an ELISA style ligand-binding assay. After coating the Δlaminin γ1 protein (LN + LEa) onto wells, increasing concentrations of Δnetrin-4 (LN + EGF 1–3.5) were added, and binding with an apparent KD of 1.38 × 10–11 m was detected (not shown). In addition, we investigated whether Δnetrin-4 also binds to native laminin-111 isolated from mouse EHS tumor. ELISA style assays were carried out with laminin-111 as the immobilized ligand, and increasing concentrations of Δnetrin-1 and Δnetrin-4 were added to the solution (Fig. 4). ΔNetrin-1 failed to bind laminin-111, whereas Δnetrin-4 bound to native laminin-111 with an apparent KD of 3.70 × 10–11 m, similar to the one observed in the experiment with recombinant Δlaminin γ1 (LN + LEa) chains in the same kind of assay. These results suggest that the netrin-4 binding activity for laminin-111 resides in the LN and LE domains of the laminin γ1 chain. The laminin γ1 chain-netrin-4 interaction was also studied using the water-soluble covalent cross-linking agent BS3. As shown previously and in this cross-linking experiment, the N-terminal Δlaminin γ1 chain fragments do not self-interact (Fig. 5A) (37Odenthal U. Haehn S. Tunggal P. Merkl B. Schomburg D. Frie C. Paulsson M. Smyth N. J. Biol. Chem. 2004; 279: 44504-44512Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). ΔNetrin-4 on the other hand shows self-interaction as well as an interaction with Δlaminin γ1 chain fragments (Fig. 5, A and B). Remarkably, Δnetrin-4 binding to an N-terminal Δlaminin γ1 chain fragment (LN + LEa) results only in the formation of dimers, whereas the Δnetrin-4 self-interaction results in multimers. As expected no interaction between netrin-1 and the Δlaminin γ1 chain was detected (Fig. 5C). The Netrin-4 Interaction with Laminin LN and LE Domains Is Independent of Divalent Cations—To analyze whether Ca2+ ions affect the netrin-4-laminin binding, ELISA style ligand binding assays were performed either in the absence of added CaCl2 or after the addition of either 2 mm CaCl2 or excess EDTA. Binding was observed under all conditions and had similar apparent KD values (1.38 × 10–11 m in the absence of added CaCl2; 1.07 × 10–11 m in the presence of 2 mm CaCl2 and 1.64 × 10–11 m in the presence of excess EDTA) (supplemental Fig. 2A). Some laminin LN domains undergo a conformational change in the presence of Ca2+ (37Odenthal U. Haehn S. Tunggal P. Merkl B. Schomburg D. Frie C. Paulsson M. Smyth N. J. Biol. Chem. 2004; 279: 44504-44512Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To determine whether the conformation of Δnetrin-4 is also influenced by Ca2+, circular dichroism spectra of Δnetrin-4 were recorded in the absence of CaCl2, in the presence of 2 mm CaCl2, and after the addition of excess EDTA (Table 1; supplemental Fig. 2B). In contrast to the laminin LN domains, no significant conformational changes in Δnetrin-4 could be observed upon addition of CaCl2 or EDTA. Apparently neither the conformation of the netrin-4 LN domain nor its interaction with the Δlaminin γ1 subunit requires the presence of divalent cations, suggesting a different mechanism of interaction than between laminins.TABLE 1Secondary structures of Δnetrin-1 and Δnetrin-4Proteinα-Helixβ-Strandβ-TurnUnorderedCONTINCDSSTRCONTINCDSSTRCONTINCDSSTRCONTINCDSSTR%%%%Secondary structure of Δnetrin-1Δnetrin-16.3640.93920.92131.833Δnetrin-1 + 2 mm CaCl210.6743.65219.31826.521Δnetrin-1 + 4 mm EDTA8.6742.23820.32228.832Secondary structure of Δnetrin-4Δnetrin-4610434020.51830.532Δnetrin-4 + 2 mm CaCl24.9546.34220.32128.631Δnetrin-4 + 4 mm EDTA7.1639.75322.92130.320 Open table in a new tab Localization of the Lami" @default.
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• W2020083886 title "Binding of Netrin-4 to Laminin Short Arms Regulates Basement Membrane Assembly" @default.
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