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• W2076667246 abstract "Recently a novel laminin γ3 chain was identified in mouse and human and shown to have the same modular structure as the laminin γ1 chain. We expressed two fragments of the γ3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3–5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The γ3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the β2 VI/V fragment. The γ3 III3–5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the γ1 III3–5 fragment. These data suggested that laminins containing the γ3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against γ3 VI/V and γ3 III3–5 showed no cross-reaction with homologous fragments from the γ1 and γ2 chains of laminin and allowed the establishment of γ chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20–100-fold lower content of the γ3 chain compared with the γ1 chain in various tissue extracts of adult mice. The expression of γ3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the γ3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the γ3 chain to basement membranes of these tissues. Recently a novel laminin γ3 chain was identified in mouse and human and shown to have the same modular structure as the laminin γ1 chain. We expressed two fragments of the γ3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3–5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The γ3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the β2 VI/V fragment. The γ3 III3–5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the γ1 III3–5 fragment. These data suggested that laminins containing the γ3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against γ3 VI/V and γ3 III3–5 showed no cross-reaction with homologous fragments from the γ1 and γ2 chains of laminin and allowed the establishment of γ chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20–100-fold lower content of the γ3 chain compared with the γ1 chain in various tissue extracts of adult mice. The expression of γ3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the γ3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the γ3 chain to basement membranes of these tissues. Basement membranes are specialized structures of the extracellular matrix with multiple functions (1Timpl R. Brown J.C. BioEssays. 1996; 18: 123-132Crossref PubMed Scopus (575) Google Scholar). As thin condensed matrices, they divide the cells of the parenchymal tissues from the interstitial matrix. Their main components are collagen type IV variants, laminins, nidogens, and perlecan (2Timpl R. Curr. Opin. Cell Biol. 1996; 8: 618-624Crossref PubMed Scopus (547) Google Scholar). Laminins constitute a family of heterotrimeric proteins (αβγ) which are mainly localized in basement membranes and are involved in cell matrix and various other protein interactions. Eleven different chains (α1–α5, β1–β3, γ1–γ3) have been identified and sequenced, and they assemble fifteen different isoforms, laminin-1–15 (3Sasaki T. Timpl R. Guidebook to the Extracellular Matrix, Anchor, and Adhesion Proteins. Sambook and Tooze, Oxford1999: 434-443Google Scholar, 4Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1024) Google Scholar, 5Libby R.T. Champliaud M-F. Claudepierre T. Xu Y. Gibbons E.P. Koch M. Burgeson R.E. Hunter D.D. Brunken W.J. J. Neurosci. 2000; 20: 6517-6528Crossref PubMed Google Scholar). These chains share a 600-residue domain II/I that oligomerizes into a rod-like coiled-coil structure forming the long arm of laminins. The N-terminal short arms consist of rod-like elements on the basis of tandem arrays of laminin-type epidermal growth factor-like (LE) modules (6Stetefeld J. Mayer U. Timpl R. Huber R. J. Mol. Biol. 1996; 257: 644-657Crossref PubMed Scopus (109) Google Scholar) and several globular domains, referred to as laminin N-terminal (LN), 1The abbreviations used are: LN, laminin N-terminal; LE, laminin-type epidermal growth factor-like; PBS, phosphate-buffered saline. 1The abbreviations used are: LN, laminin N-terminal; LE, laminin-type epidermal growth factor-like; PBS, phosphate-buffered saline. L4 modules, and domains IVb (α3B and α5 chains)/IV (β1 and β2 chains), which have not been classified to date. All α chains share a unique C-terminal G domain, which consists of five laminin G modules (7Timpl R. Tisi D. Talts J.F. Andac Z. Sasaki T. Hohenester E. Matrix Biol. 2000; 19: 309-317Crossref PubMed Scopus (254) Google Scholar). Most of these modules are also shared by several other extracellular proteins, such as the proteoglycans perlecan and agrin. A newly identified γ3 chain shares the same domain structure as the γ1 chain (8Koch M. Olsen P. Albus A. Jin W. Hunter D. Brunken W.J. Burgeson R.E. Champliaud M-F. J. Cell Biol. 1999; 145: 605-617Crossref PubMed Scopus (213) Google Scholar, 9Iivanainen A. Morita T. Tryggvason K. J. Biol. Chem. 1999; 274: 14107-14111Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The domain VI (LN module) of laminin γ3 shows the highest sequence identity with that of the γ1 chain. The LN modules of laminin chains are essential for self-polymerization into large non-covalent networks, as shown for laminin-1 and laminin-2 (10Yurchenco P.D. Cheng Y-S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar, 11Cheng 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 (202) Google Scholar). This self-assembly is considered to be crucial for the integrity of basement membranes (12Yurchenco P.D. Extracellular Matrix Assembly and Structure. Academic Press, San Diego, CA1994: 351-388Crossref Google Scholar). The γ3 chain is a component of four isoforms, laminin-12 (α2β1γ3), -13 (α3β2γ3), -14 (α4β2γ3), and -15 (α5β2γ3) (5Libby R.T. Champliaud M-F. Claudepierre T. Xu Y. Gibbons E.P. Koch M. Burgeson R.E. Hunter D.D. Brunken W.J. J. Neurosci. 2000; 20: 6517-6528Crossref PubMed Google Scholar, 8Koch M. Olsen P. Albus A. Jin W. Hunter D. Brunken W.J. Burgeson R.E. Champliaud M-F. J. Cell Biol. 1999; 145: 605-617Crossref PubMed Scopus (213) Google Scholar). There are interesting theories that these laminins also participate in self-assembly and that they co-polymerize with other isoforms. Nidogen-1 is a ubiquitously expressed basement membrane protein consisting of three globular domains (G1–G3) connected by a link and a rod-like segment. It has been shown to bind several extracellular proteins through different domains and has been proposed to act as a connecting element for basement membrane assembly (1Timpl R. Brown J.C. BioEssays. 1996; 18: 123-132Crossref PubMed Scopus (575) Google Scholar, 13Miosge N. Sasaki T. Timpl R. Matrix Biol. 2002; 21: 611-621Crossref PubMed Scopus (68) Google Scholar). The G3 domain of nidogen-1 binds to a single laminin-type epidermal growth factor-like (LE) module of the γ1 chain with high affinity (14Mayer U. Nischt R. Pöschl E. Mann K. Fukuda K. Gerl M. Yamada Y. Timpl R. EMBO J. 1993; 12: 1879-1885Crossref PubMed Scopus (244) Google Scholar, 15Pöschl E. Fox J.W. Block D. Mayer U. Timpl R. EMBO J. 1994; 13: 3741-3747Crossref PubMed Scopus (85) Google Scholar), and this binding is thought to be particularly important for basement membrane assembly. Nidogen-2 is another isoform and also binds several matrix proteins with affinities different from nidogen-1 (16Kohfeldt E. Sasaki T. Göhring W. Timpl R. J. Mol. Biol. 1998; 282: 99-109Crossref PubMed Scopus (199) Google Scholar, 17Salmivirta K. Talts J.F. Olsson M. Sasaki T. Timpl R. Ekblom P. Exp. Cell Res. 2002; 279: 188-201Crossref PubMed Scopus (87) Google Scholar), although the binding repertoire is largely overlapping. Site-directed mutagenesis demonstrates the crucial amino acids for the nidogen binding in the laminin γ1 LE module γ1 III4 (18Pöschl E. Mayer U. Stetefeld J. Baumgartner R. Holak T. Huber R. Timpl R. EMBO J. 1996; 15: 5154-5159Crossref PubMed Scopus (69) Google Scholar) and subsequently these data are confirmed by the crystal structure of the complex between nidogen and laminin fragments (19Takagi J. Yang Y. Liu J-H. Wang J.-H. Springer T.A. Nature. 2003; 424: 969-974Crossref PubMed Scopus (112) Google Scholar). The γ3 chain contains a γ1-like nidogen binding motif with only a single conservative amino acid substitution (8Koch M. Olsen P. Albus A. Jin W. Hunter D. Brunken W.J. Burgeson R.E. Champliaud M-F. J. Cell Biol. 1999; 145: 605-617Crossref PubMed Scopus (213) Google Scholar, 9Iivanainen A. Morita T. Tryggvason K. J. Biol. Chem. 1999; 274: 14107-14111Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), suggesting that γ3-containing laminins should be capable of associating with other basement membrane molecules through nidogens. The γ3 chain has been localized at the apical surface of ciliated epithelial cells of lung, oviduct, epididymis, ductus deferens, and seminiferous tubules, and it was reported that the γ3 chain is a non-basement membrane-associated laminin chain (8Koch M. Olsen P. Albus A. Jin W. Hunter D. Brunken W.J. Burgeson R.E. Champliaud M-F. J. Cell Biol. 1999; 145: 605-617Crossref PubMed Scopus (213) Google Scholar). The laminin isoforms containing the γ3 chain are widely expressed in different compartments of the retina (5Libby R.T. Champliaud M-F. Claudepierre T. Xu Y. Gibbons E.P. Koch M. Burgeson R.E. Hunter D.D. Brunken W.J. J. Neurosci. 2000; 20: 6517-6528Crossref PubMed Google Scholar). For this study, we prepared recombinant fragments of the laminin γ3 chain and compared the binding properties with those of the γ1 and γ2 chains. These fragments allowed us to raise specific antibodies that are useful for quantitative analyses and examination of the distribution of the γ3 chain in tissues. We systematically localized the laminin γ1 and γ3 chains during mouse organogenesis from day 12 to day 18 and in adult organs at the light microscopic level as well as at the ultrastructural level and found it to be a true basement membrane component. In addition, we performed immunogold double labeling and showed a co-localization of the laminin γ3 chain and nidogen-1 in basement membranes in vivo. Mouse (20Fox J.W. Mayer U. Nischt R. Aumailley M. Reinhardt D. Wiedemann H. Mann K. Timpl R. Krieg T. Engel J. Chu M-L. EMBO J. 1991; 10: 3137-3146Crossref PubMed Scopus (375) Google Scholar) and human nidogen-1 (21Mayer U. Zimmermann K. Mann K. Reinhardt D. Timpl R. Nischt R. Eur. J. Biochem. 1995; 227: 681-686Crossref PubMed Scopus (45) Google Scholar) and mouse (17Salmivirta K. Talts J.F. Olsson M. Sasaki T. Timpl R. Ekblom P. Exp. Cell Res. 2002; 279: 188-201Crossref PubMed Scopus (87) Google Scholar) and human nidogen-2 (16Kohfeldt E. Sasaki T. Göhring W. Timpl R. J. Mol. Biol. 1998; 282: 99-109Crossref PubMed Scopus (199) Google Scholar) were obtained as recombinant products. Laminin-1 was purified from the mouse Engelbreth-Holm-Swarm tumor (22Paulsson M. Aumailley M. Deutzmann R. Timpl R. Beck K. Engel J. Eur. J. Biochem. 1987; 166: 11-19Crossref PubMed Scopus (329) Google Scholar). The laminin-1 fragments E4 and P1 were purified as described previously (23Ott U. Odermatt E. Engel J. Furthmayr H. Timpl R. Eur. J. Biochem. 1982; 123: 63-72Crossref PubMed Scopus (201) Google Scholar). The recombinant fragment γ1 III3–5 (14Mayer U. Nischt R. Pöschl E. Mann K. Fukuda K. Gerl M. Yamada Y. Timpl R. EMBO J. 1993; 12: 1879-1885Crossref PubMed Scopus (244) Google Scholar) and γ2N, γ2 LE4–6, and β3 VI/V (24Sasaki T. Göhring W. Mann K. Brakebusch C. Yamada Y. Fässler R. Timpl R. J. Mol. Biol. 2001; 314: 751-763Crossref PubMed Scopus (94) Google Scholar) have already been described. Other recombinant fragments of mouse laminin included β1 VI/V, β2 VI/V, and γ1 VI/V (Fig. 1), 2N. Gersdorff, E. Kohfeldt, T. Sasaki, R. Timpl, and N. Miosge, unpublished data. which were prepared by established procedures (25Garbe J.H.O. Göhring W. Mann K. Timpl R. Sasaki T. Biochem. J. 2002; 362: 213-221Crossref PubMed Scopus (54) Google Scholar). The templates used were mouse γ3 cDNA clone γ3_3pB provided by Prog. Dr. K. Tryggvasson for γ3 III3–5 and RNA from mouse testis for γ3 VI/V fragment. The sense and antisense primer for γ3 III3–5 were 5-′GTCAGCTAGCGCCCTGTCCGTGCCCTGG-3′ and 5-′GTCACTCGAGCTAGCTCTGGCAGCCCCTCCC-3′, respectively, and for γ3 VI/V, 5-′GTCAGCTAGCAGTGCAGAGCGGGG-3′ and 5-′GTCACTCGAGCTAGCTGCTGCAGCCCACTGG-3′, respectively. They were used for amplification by PCR. These primers introduced at the 5′ end a NheI site and at the 3′ end a stop codon followed by a XhoI site. A NheI/XhoI cDNA fragment for γ3 III3–5 as well as that for γ3 VI/V was inserted into the episomal expression vector pCEP-Pu containing the BM-40 signal peptide (26Kohfeldt E. Mauer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 217-221Crossref Scopus (201) Google Scholar). These vectors were used to transfect 293-Epstein-Barr virus nuclear antigen-1 cells, and serum-free medium was collected from these cells (26Kohfeldt E. Mauer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 217-221Crossref Scopus (201) Google Scholar). Conditioned medium was passed over a DEAE-cellulose column equilibrated with 0.05 m Tris-HCl, pH 8.6, and eluted with a linear 0–0.5 m NaCl gradient. Most of fragment γ3 III3–5 did not bind to the column, whereas fragment γ3 VI/V was eluted at 0.1–0.2 m NaCl. They were further purified on a Superose 12 column (HR 16/50) equilibrated with 0.2 m ammonium acetate, pH 6.8. Solid phase assays with laminin fragments immobilized on the plastic wells of microtitre plates and soluble nidogens followed published procedures (27Aumailley M. Wiedemann H. Mann K. Timpl R. Eur. J. Biochem. 1989; 184: 241-248Crossref PubMed Scopus (169) Google Scholar). Binding was detected by antibodies specific for each nidogen isoform. The radioligand inhibition assay with 125I-labeled laminin fragment P1 and recombinant nidogen-1 (14Mayer U. Nischt R. Pöschl E. Mann K. Fukuda K. Gerl M. Yamada Y. Timpl R. EMBO J. 1993; 12: 1879-1885Crossref PubMed Scopus (244) Google Scholar) was also used. The laminin-1 polymerization inhibition assay as well as surface plasmon resonance assays (BIAcore 1000, Stevenage, Hertsfordshire, UK) were carried out as described previously (25Garbe J.H.O. Göhring W. Mann K. Timpl R. Sasaki T. Biochem. J. 2002; 362: 213-221Crossref PubMed Scopus (54) Google Scholar). Immunization of rabbits, affinity purification of antibodies, enzymelinked immunosorbent assay titration and inhibition radioimmunoassays were carried out using established protocols. Extraction of mouse tissue was performed with neutral buffer containing 10 mm EDTA followed by the same buffer containing detergents, and both buffers contained protease inhibitors (28Sasaki T. Wiedemann H. Matzner M. Chu M-L. Timpl R. J. Cell Sci. 1996; 109: 2895-2904Crossref PubMed Google Scholar). Female New Mexico Research Institute (NMRI) mice were kept on a normal day/night cycle and received Altromin commercial food and water ad libitum. The day on which, at 11:00 a.m., a vaginal plug was detected after a mating period of 3 h, was designated as day 0 of gestation. On the respective days of gestation, beginning with day 12, pregnant mice were anesthetized with ether and sacrificed by cervical dislocation. After dissection of the uterine horns, the embryos (day 12) or fetuses (days 14, 16, and 18) were removed. Three embryos or fetuses of each developmental stage were investigated. For light microscopic analysis of adult tissues, the various organs from three different three-month-old NMRI mice were taken. Fixation and Preparation of Tissues—For light microscopy, all specimens were fixed by immersion in 4% paraformaldehyde in phosphate buffer, pH 7.2, at 4 °C. They were then dehydrated in an ascending series of ethanol from 30 to 100% and embedded in paraffin (29Miosge N. Köther F. Heinemann S. Kohfeldt E. Herken R. Timpl R. Histochem. Cell Biol. 2000; 113: 115-124Crossref PubMed Scopus (35) Google Scholar). Serial sections of 5 μm were cut with a Reichert's microtome. Every fifth section was stained with hematoxylin for topological orientation within the anatomical regions examined, and staging of the embryos or fetuses was achieved by comparison with the appropriate Theiler stages. For the ultrastructural approach, the tissue pieces were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde for 15 min, dehydrated in a graded series of ethanol up to 70%, and embedded in the acrylic resin LR-Gold (London Resin Company, Reading, UK). Semithin (1-μm) and ultrathin (0.8-μm) sections were cut according to procedures previously described in detail (29Miosge N. Köther F. Heinemann S. Kohfeldt E. Herken R. Timpl R. Histochem. Cell Biol. 2000; 113: 115-124Crossref PubMed Scopus (35) Google Scholar). Light Microscopic Immunohistochemistry—For the light microscopic immunohistochemistry, sections were deparaffinized, rehydrated, and rinsed for 10 min in PBS. Endogenous peroxidase was blocked by incubation in 3% H2O2 in methanol for 45 min in the dark. Each of the reaction steps was followed by rinsing for 10 min in PBS. The sections were pretreated for 5 min with 10 μg/ml protease XXIV (Sigma). The anti-laminin γ1 antibody, the anti-laminin γ3 III3–5 antibody, and the laminin γ3 VI/V antibody were used at a dilution of 1:100 for 1 h at room temperature. The anti-nidogen-1 antibody was also used at a dilution of 1:100 for 1 h at room temperature. The peroxidase-anti-peroxidase method followed the previously described procedures (29Miosge N. Köther F. Heinemann S. Kohfeldt E. Herken R. Timpl R. Histochem. Cell Biol. 2000; 113: 115-124Crossref PubMed Scopus (35) Google Scholar). As negative controls, normal rabbit IgGs and the corresponding preimmune sera were used instead of the primary antibodies, at similar concentrations. No immunostaining was observed. Immunogold Histochemistry—For single labeling using immunogold histochemistry, the tissue sections were incubated for 1 h at 4 °C with the antibodies against laminin γ3 VI/V and laminin γ3 III3–5. The sections were rinsed in PBS and incubated with the 16-nm gold-coupled goat anti-rabbit IgG diluted 1:20 in PBS for 16 h at 4 °C. Thereafter, the sections were rinsed with water and stained with uranyl acetate (10 min) and lead citrate (8 min). The sections were examined with a LEO 906E electron microscope. For double labeling, all sections were incubated for 5 min at room temperature with 1% bovine serum albumin in PBS and then rinsed in PBS. Thereafter, anti-laminin γ3 antibody diluted 1:100 in PBS was applied for 1 h at 4 °C. After a rinse with PBS, the gold-coated (16-nm) goat anti-rabbit antibody diluted 1:200 was applied for 20 min at room temperature. The sections were thoroughly rinsed with PBS. The gold-coated (8-nm) anti-nidogen-1 antibody diluted 1:100 in PBS was then incubated. Colloidal gold particles were prepared and coupled to the antibodies according to our standard protocols (13Miosge N. Sasaki T. Timpl R. Matrix Biol. 2002; 21: 611-621Crossref PubMed Scopus (68) Google Scholar). All sections were finally rinsed with water and stained with uranyl acetate (10 min) and lead citrate (8 min). As the control for the double labeling experiments, we also applied a monoclonal anti-nidogen-1 antibody (JF4). This approach avoids possible cross-links between the two rabbit IgGs used in the double labeling described above and yielded identical results. Expression and Purification of Recombinant Proteins—Previous recombinant studies with the LN module of the α1 (30Ettner N. Göhring W. Sasaki T. Mann K. Timpl R. FEBS Lett. 1998; 430: 217-221Crossref PubMed Scopus (82) Google Scholar) and the β1 and γ1 chains 3T. Sasaki, unpublished data. have shown that this module does not represent an autonomous folding unit and requires the addition of several adjacent, rod-like LE modules from domain V to achieve efficient production in mammalian cells. Therefore, we prepared the LN module of the γ3 chain together with the complete domain V (four LE modules) (position 29–488), as already performed for the γ1 VI/V fragment. Domain γ3 III3–5 (three LE modules) corresponds to amino acid 766–927 of the laminin γ3 chain, a region homologous to the laminin γ1 chain exhibiting a high affinity binding site for nidogen-1. These fragments were produced in human 293-EBNA cells transfected with episomal expression vectors (26Kohfeldt E. Mauer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 217-221Crossref Scopus (201) Google Scholar). These cells secreted the recombinant fragments in sufficient amounts into serum-free culture media, which were used for purification by conventional chromatography. The purified fragments showed a single 55-kDa band for domain γ3 VI/V (Fig. 1, lane 1) and a 23-kDa band for domain γ3 III3–5 (Fig. 1, lane 5) in SDS-polyacrylamide gel electrophoresis. The purity was also confirmed by a single N-terminal ADMGS(C)YDGV sequence for γ3 VI/V, in agreement with the presence of a typical signal peptide and an APLAP(C)P(C)PGQ sequence for γ3 III3–5, the first four residues being derived from the signal peptide cleavage region of the vector. Inhibition of Laminin-1 Polymerization—A major function of LN modules is the promotion of self-assembly of laminins into non-covalent quasihexagonal networks in a Ca2+-dependent fashion, as shown for laminin-1 (10Yurchenco P.D. Cheng Y-S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar) and subsequently for α2 chain-containing laminins (11Cheng 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 (202) Google Scholar). In an earlier experiment, we developed a quantitative inhibition radioimmunoassay to study the interference of the different α chain fragments (25Garbe J.H.O. Göhring W. Mann K. Timpl R. Sasaki T. Biochem. J. 2002; 362: 213-221Crossref PubMed Scopus (54) Google Scholar). In the present study, we used this assay for the β and γ chain fragments. The assay was designed to allow the polymerization of 30–38% of laminin-1 (subsequently set to 100%), which was reduced to only a few percent in the presence of EDTA (Table I). The inhibitors were added in stoichiometric amounts (0.32 μm) or in five-fold molar excess (1.6 μm) prior to starting the polymerization. Lower activities for β3 VI/V and γ3 VI/V and distinctly low activity for β2 VI/V at both concentrations were observed (Table I). As a positive control, we used the laminin E4 fragment corresponding to β1 VI/V, which was used in previous inhibition studies (10Yurchenco P.D. Cheng Y-S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar), and found activity approximately identical to that of recombinant β1 VI/V and γ1 VI/V. Surface plasmon resonance assays were used to examine the binding to immobilized laminin-1. This demonstrated a similar binding of γ1 VI/V (Kd = 0.245 μm) and β1 VI/V (Kd = 0.275 μm), and lower affinities were observed for β3 VI/V (Kd = 1.05 μm) and for γ3 VI/V (Kd = 1.75 μm). No binding was detected for β2 VI/V up to the highest concentration used (4 μm).Table IInhibition of laminin-1 polymerization by N-terminal (VI/V) fragments of laminin β and γ chainsInhibitors% Inhibition0.32 μm1.6 μmβ1 VI/V28 ± 370 ± 7Fragment E432 ± 1464 ± 19β2 VI/V0 ± 911 ± 20β3VI/V25 ± 439 ± 23γ1 VI/V40 ± 1076 ± 3γ3 VI/V10 ± 1530 ± 21EDTA85 ± 6 Open table in a new tab Binding to Nidogens—The mouse laminin γ1 chain fragment γ1 III3–5 was shown to bind to nidogen-1 with high affinity (14Mayer U. Nischt R. Pöschl E. Mann K. Fukuda K. Gerl M. Yamada Y. Timpl R. EMBO J. 1993; 12: 1879-1885Crossref PubMed Scopus (244) Google Scholar). Most of the amino acids that interact with the nidogen G3 domain are conserved in human and mouse laminin γ3 chains (19Takagi J. Yang Y. Liu J-H. Wang J.-H. Springer T.A. Nature. 2003; 424: 969-974Crossref PubMed Scopus (112) Google Scholar), suggesting that the γ3 chain also binds to nidogens. We prepared the mouse laminin γ3 III3–5 fragment to compare the binding to nidogen isoforms with that of γ1 and γ2 chains. Solid phase assays were used to test the binding of nidogen-1 and nidogen-2 to immobilized γ1 III3–5 (Fig. 2A) and γ3 III3–5 (Fig. 2B). Both mouse nidogen isoforms bound better to γ1 III3–5, and the half-maximal binding for mouse nidogen-1, human nidogen-1, mouse nidogen-2, and human nidogen-2 were 0.2–0.3, 0.4–0.5, 1–2, and 3–4 nm, respectively. The concentrations of half-maximal binding to the γ3 III3–5 fragment were calculated, 0.8–1 nm for both types of nidogen-1 and 20–30 nm for both types of nidogen-2. No nidogen isoforms showed any binding to the γ2 LE4–6 fragment, the homologous region of laminin γ2 chain in the solid phase assay. The inhibitory activity of these γ chain fragments on the interaction between laminin fragment P1 and nidogen-1 was analyzed by radioligand inhibition assays (Fig. 3). The inhibitory activity of the γ1 III3–5 fragment was identical to the P1 fragment, and the concentration required for 50% inhibition (IC50) was found to be 0.08 nm. The IC50 of γ3 III3–5 was 20 nm and that of γ2 LE4–6 was >10 μm. Surface plasmon resonance assays were performed using III3–5 fragments as immobilized ligands. Nidogens showed high affinity binding to γ1 III3–5 but no binding was seen to γ3 III3–5 (data not shown).Fig. 3Radioligand inhibition assay of laminin-nidogen interaction by recombinant fragments from mouse laminin γ1, γ2, and γ3 chains. The test consisted of 125I-labeled laminin fragment P1 and recombinant nidogen-1. Inhibitors used were fragment P1 (○), the recombinant fragments γ1 III3–5 (▴), γ3 III3–5 (□), γ2 LE4–6 (▵), and reduced and alkylated γ3 III3–5 (▪).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunological Assays—Rabbit antisera were prepared against recombinant γ3 VI/V and γ3 III3–5 fragments. These antisera had high titers against the antigen used for immunization and were only marginally cross-reactive with the fragments obtained from γ1 and γ2 chains by enzyme-linked immunosorbent assay (data not shown) and by inhibition radioimmunoassays (Fig. 4). Several tissues from laminin γ3 knock-out mice tested for both antibodies did not show any staining (data not shown). In inhibition radioimmunoassays, the half-maximal inhibitions were achieved at 0.1 nm for γ3 VI/V (Fig. 4A) and at 0.06 nm for γ3 III3–5 (Fig. 4B). A >1000-fold excess of homologous fragments obtained from other γ chains did not show inhibition. Some adult mouse tissues were extracted with EDTA and detergents and examined by this assay together with the γ1 VI/V assay. However, mouse tissues showed inhibitions in the γ3 VI/V assay but not in the γ3 III3–5 assay. Therefore, the amount of the γ3 chain was quantified by the γ3 VI/V assay. The much lower amounts of the γ3 chain (1–6% of the γ1 chain of laminin) were found in the tissue extracts examined (Table II).Table IIContents (pmol/g tissue) of laminin γ1 and γ3 chain determined by inhibition radioimmunoassays in extracts of adult mouse tissuesTissueAssay forγ1 VI/Vγ3 VI/VSkin422.6Lung1451.8Stomach1632.0Intestine1321.3Kidney692.1 Open table in a new tab Immunolocalization of Laminin γ1 and γ3 Chains during Embryonic Development and in Adult Tissues—From day 12 to day 18, and in adult mouse tissue, staining for the laminin γ1 chain was seen in almost all epithelial basement membrane zones in all consecutive stages of development, as already known for a long time (Table III). In the skin, for example, the dermal-epidermal basement membrane zone exhibited staining, as did the basement membrane zones of hair follicles, but neither the fibroblasts nor the keratinocytes showed any staining (Fig. 5A). In all stages of kidney organogenesis, only basement membrane zones of the consecutive stages of glomeruli development (comma, S-shaped, early glomeruli) and those of the tubules were positive for the γ1 chain (Fig. 5B). In the small and large intestine, the epithelial cells were not stained, whereas the basement membrane zones underlying the developing epithelium were stained for the γ1 chain (Fig. 5C).Table IIILocalization of laminin γ1 and γ3 chains during mouse embryonic development and in adult tissuesOrgan anlage or developing organStages of development and type of γ chainE12E14E16E18Adu" @default.
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• W2076667246 date "2005-06-01" @default.
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• W2076667246 title "Laminin γ3 Chain Binds to Nidogen and Is Located in Murine Basement Membranes" @default.
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• W2076667246 doi "https://doi.org/10.1074/jbc.m501875200" @default.
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