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• W2064424062 abstract "Ten-m/Odz/teneurins are a new family of four distinct type II transmembrane molecules. Their extracellular domains are composed of an array of eight consecutive EGF modules followed by a large globular domain. Two of the eight modules contain only 5 instead of the typical 6 cysteine residues and have the capability to dimerize in a covalent, disulfide-linked fashion. The structural properties of the extracellular domains of all four mouse Ten-m proteins have been analyzed using secreted, recombinant molecules produced by mammalian HEK-293 cells. Electron microscopic analysis supported by analytical ultracentrifugation data revealed that the recombinant extracellular domains of all Ten-m proteins formed homodimers. SDS-PAGE analysis under nonreducing conditions as well as negative staining after partial denaturation of the molecules indicated that the globular COOH-terminal domains of Ten-m1 and -m4 contained subdomains with a pronounced stability against denaturing agents, especially when compared with the homologous domains of Ten-m2 and -m3. Cotransfection experiments of mammalian cells with two different extracellular domains revealed that Ten-m molecules have also the ability to form heterodimers, a property that, combined with alternative splicing events, allows the formation of a multitude of molecules with different characteristics from a limited set of genes. Ten-m/Odz/teneurins are a new family of four distinct type II transmembrane molecules. Their extracellular domains are composed of an array of eight consecutive EGF modules followed by a large globular domain. Two of the eight modules contain only 5 instead of the typical 6 cysteine residues and have the capability to dimerize in a covalent, disulfide-linked fashion. The structural properties of the extracellular domains of all four mouse Ten-m proteins have been analyzed using secreted, recombinant molecules produced by mammalian HEK-293 cells. Electron microscopic analysis supported by analytical ultracentrifugation data revealed that the recombinant extracellular domains of all Ten-m proteins formed homodimers. SDS-PAGE analysis under nonreducing conditions as well as negative staining after partial denaturation of the molecules indicated that the globular COOH-terminal domains of Ten-m1 and -m4 contained subdomains with a pronounced stability against denaturing agents, especially when compared with the homologous domains of Ten-m2 and -m3. Cotransfection experiments of mammalian cells with two different extracellular domains revealed that Ten-m molecules have also the ability to form heterodimers, a property that, combined with alternative splicing events, allows the formation of a multitude of molecules with different characteristics from a limited set of genes. epidermal growth factor-like N-ethylmaleimide guanidinium chloride nickel-charged nitrilotriacetic acid hexahistidine monoclonal antibody cytomegalovirus The Ten-m/Odz protein was first found in Drosophilawhere it was proposed to be either a secreted tenascin-like molecule (1Baumgartner S. Martin D. Hagios C. Chiquet-Ehrismann R. EMBO J. 1994; 13: 3728-3740Crossref PubMed Scopus (142) Google Scholar) or type I transmembrane receptor (2Levine A. Bashan-Ahrend A. Budai-Hadrian O. Gartenberg D. Menasherow S. Wides R. Cell. 1994; 77: 587-598Abstract Full Text PDF PubMed Scopus (122) Google Scholar). We have subsequently identified and characterized the mouse Ten-m and found that it characterizes a new family of genes composed of 4 members (Ten-m1–4). The biochemical analysis of recombinant fragments of mouse Ten-m1 and alkaline phosphatase fusion proteins revealed that Ten-m1 is expressed as a type II transmembrane molecule. Furthermore, we could demonstrate that two of the eight tandemly arranged EGF1 modules present in the extracellular domain of mouse Ten-m1 containing 5 instead of 6 cysteine residues facilitate the dimerization of two molecules in a covalent, disulfide-linked fashion. Members of the Ten-m family have in the meantime also been described in rat (3Otaki J.M. Firestein S. Dev. Biol. 1999; 212: 165-181Crossref PubMed Scopus (37) Google Scholar), chicken (4Minet A.D. Rubin B.P. Tucker R.P. Baumgartner S. Chiquet-Ehrismann R. J. Cell Sci. 1999; 112: 2019-2032Crossref PubMed Google Scholar, 5Rubin B.P. Tucker R.P. Martin D. Chiquet-Ehrismann R. Dev. Biol. 1999; 216: 195-209Crossref PubMed Scopus (78) Google Scholar, 6Tucker R.P. Martin D. Kos R. Chiquet-Ehrismann R. Mech. Dev. 2000; 98: 187-191Crossref PubMed Scopus (27) Google Scholar, 7Tucker R.P. Chiquet-Ehrismann R. Chevron M.P. Martin D. Hall R.J. Rubin B.P. Dev. Dyn. 2001; 220: 27-39Crossref PubMed Scopus (55) Google Scholar), human (8Brandau O. Schuster V. Weiss M. Hellebrand H. Fink F.M. Kreczy A. Friedrich W. Strahm B. Niemeyer C. Belohradsky B.H. Meindl A. Hum. Mol. Genet. 1999; 8: 2407-2413Crossref PubMed Scopus (94) Google Scholar, 9Minet A.D. Chiquet-Ehrismann R. Gene. 2000; 257: 87-97Crossref PubMed Scopus (52) Google Scholar), zebrafish (10Mieda M. Kikuchi Y. Hirate Y. Aoki M. Okamoto H. Mech. Dev. 1999; 87: 223-227Crossref PubMed Scopus (37) Google Scholar), and Caenorhabditis elegans (11Wilson R. Ainscough R. Anderson K. Baynes C. Berks M. Bonfield J. Burton J. Connell M. Copsey T. Cooper J. Coulson A. Craxton M. Dear S., Du, Z. Durbin R. et al.Nature. 1994; 368: 32-38Crossref PubMed Scopus (1439) Google Scholar). The expression pattern of Ten-m/Odz in flies and mammals suggests important roles during as well as after development. InDrosophila embryogenesis, Ten-m/Odz is expressed in seven stripes during the blastoderm stage (12Baumgartner S. Chiquet-Ehrismann R. Mech. Dev. 1993; 40: 165-176Crossref PubMed Scopus (85) Google Scholar) and later also in the central nervous system (1Baumgartner S. Martin D. Hagios C. Chiquet-Ehrismann R. EMBO J. 1994; 13: 3728-3740Crossref PubMed Scopus (142) Google Scholar), heart (2Levine A. Bashan-Ahrend A. Budai-Hadrian O. Gartenberg D. Menasherow S. Wides R. Cell. 1994; 77: 587-598Abstract Full Text PDF PubMed Scopus (122) Google Scholar), and eye (13Levine A. Weiss C. Wides R. Dev. Dyn. 1997; 209: 1-14Crossref PubMed Scopus (28) Google Scholar). Expression studies of Ten-m1–4 in adult mouse tissues showed a widespread expression with the highest levels in the brain (14Oohashi T. Zhou X.H. Feng K. Richter B. Morgelin M. Perez M.T., Su, W.D. Chiquet-Ehrismann R. Rauch U. Fässler R. J. Cell Biol. 1999; 145: 563-577Crossref PubMed Scopus (95) Google Scholar, 15Ben-Zur T. Feige E. Motro B. Wides R. Dev. Biol. 2000; 217: 107-120Crossref PubMed Scopus (76) Google Scholar). In chicken, both teneurin-1 (corresponding to Ten-m1) and teneurin-2 (corresponding to Ten-m2) are expressed in neurons of the developing visual system (4Minet A.D. Rubin B.P. Tucker R.P. Baumgartner S. Chiquet-Ehrismann R. J. Cell Sci. 1999; 112: 2019-2032Crossref PubMed Google Scholar). Furthermore, teneurin-2 mRNA and protein are also found in the developing limbs, somites, and craniofacial mesenchyme (7Tucker R.P. Chiquet-Ehrismann R. Chevron M.P. Martin D. Hall R.J. Rubin B.P. Dev. Dyn. 2001; 220: 27-39Crossref PubMed Scopus (55) Google Scholar). During the segmentation period of zebrafish, Ten-m3 is expressed in the somites, notochord, pharyngeal arches, and the brain, whereas the expression of Ten-m4 is restricted mainly to the brain (10Mieda M. Kikuchi Y. Hirate Y. Aoki M. Okamoto H. Mech. Dev. 1999; 87: 223-227Crossref PubMed Scopus (37) Google Scholar). Genetic studies of the fly Ten-m/Odz revealed a crucial role during segmentation and identified the Ten-m/Odz gene as the first pair rule gene that does not encode a transcription factor. Loss of Ten-m/Odz results in a typical deletion of cuticle segments, which appear in a reiterative manner along the body axis of the hatched larvae (1Baumgartner S. Martin D. Hagios C. Chiquet-Ehrismann R. EMBO J. 1994; 13: 3728-3740Crossref PubMed Scopus (142) Google Scholar). The function of Ten-m/Odz genes in vertebrates, however, is unknown. It has been reported that various forms of stress including alkylating agents or UV light can trigger the activation of mouse Ten-m/Odz 4 (16Wang X.Z. Kuroda M. Sok J. Batchvarova N. Kimmel R. Chung P. Zinszner H. Ron D. EMBO J. 1998; 17: 3619-3630Crossref PubMed Scopus (267) Google Scholar). The induction of rat neurestin (corresponding to Ten-m2/Odz 2) in external tufted cells during regeneration of olfactory sensory neurons suggests a possible function in synapse formation and morphogenesis (3Otaki J.M. Firestein S. Dev. Biol. 1999; 212: 165-181Crossref PubMed Scopus (37) Google Scholar). Ectopic expression of a splice variant of teneurin-2 in neuronal cells significantly increased the number of filopodia and the formation of enlarged growth cones (5Rubin B.P. Tucker R.P. Martin D. Chiquet-Ehrismann R. Dev. Biol. 1999; 216: 195-209Crossref PubMed Scopus (78) Google Scholar), suggesting a role in actin dynamics. All four mouse Ten-m protein chains are 2700–2800 amino acids long and lack signal peptides at the NH2 terminus, but they contain short hydrophobic stretches characteristic of transmembrane proteins. These hydrophobic domains are present about 300–400 amino acids after the translation start. Approximately 200 amino acids COOH-terminal to this transmembrane region are eight consecutive EGF-like repeats. In all Ten-m/Odz genes the second as well as the fifth EGF module contain an odd number of cysteine residues. They mediate the covalent dimerization of two Ten-m proteins. The sequence similarity of the EGF repeats between the mouse Ten-m homologues ranges from 65 to 72%, whereas other parts are less conserved. The large COOH-terminal domains distal to the EGF repeats, for example, have similarities ranging between 58 and 68% (14Oohashi T. Zhou X.H. Feng K. Richter B. Morgelin M. Perez M.T., Su, W.D. Chiquet-Ehrismann R. Rauch U. Fässler R. J. Cell Biol. 1999; 145: 563-577Crossref PubMed Scopus (95) Google Scholar). It has been shown recently for chicken teneurin-2 that the large COOH-terminal domain, constituting about 70% of the molecular mass, can be spliced alternatively (7Tucker R.P. Chiquet-Ehrismann R. Chevron M.P. Martin D. Hall R.J. Rubin B.P. Dev. Dyn. 2001; 220: 27-39Crossref PubMed Scopus (55) Google Scholar). Outside of the EGF repeats, the Ten-m/Odz family sequences bear no similarity to any other eukaryotic sequences (15Ben-Zur T. Feige E. Motro B. Wides R. Dev. Biol. 2000; 217: 107-120Crossref PubMed Scopus (76) Google Scholar). However, the COOH-terminal part harbors 26 repetitive sequence motifs termed YD repeats, which are most similar to the core of therhs elements of Escherichia coli. Related repeats in toxin A of Clostridium difficile bind specific carbohydrates (4Minet A.D. Rubin B.P. Tucker R.P. Baumgartner S. Chiquet-Ehrismann R. J. Cell Sci. 1999; 112: 2019-2032Crossref PubMed Google Scholar). In the present study, we characterized the properties of the extracellular domains of all four mouse Ten-m/Odz family members. They have essentially identical arrays of EGF repeats but show different cysteine patterns in the appending COOH-terminal domains. Ten-m2 and Ten-m4 contain an uneven number of this amino acid. We show that the recombinantly produced extracellular domains of Ten-m1–4 can form homodimers. Differences in the cysteine patterns in the globular COOH-terminal domains appear to affect the stability of the tertiary structures, whereas all four mouse Ten-m molecules share the same dimeric quaternary structure. In addition, we demonstrate that the Ten-m molecules have the ability to form heterodimers, a property allowing the formation of a multitude of molecules from a limited set of genes. The extracellular domains of Ten-m2 (starting at serine 572), Ten-m3 (starting at glutamic acid 513), Ten-m4 (starting at serine 564), and the globular COOH-terminal parts of Ten-m1 (Gten-m1; starting at glutamic acid 799) and Ten-m3 (Gten-m3; starting at glutamic acid 787) (Table I) were linked to the signal peptide of BM-40 via the sequence APLA (Ten-m3) (17Mayer 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) or APLGRGSHHHHHHGGLA (Ten-m2, Ten-m4, Gten-m1, and Gten-m3), which can be detected by the anti-RGS (H4) antibody (Qiagen). The latter property allows affinity purification on Ni-NTA-Sepharose (Qiagen) (18Janknecht R. de Martynoff G. Lou J. Hipskind R.A. Nordheim A. Stunnenberg H.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8972-8976Crossref PubMed Scopus (414) Google Scholar). The DNA was inserted into a eukaryotic expression plasmid driven by a CMV promoter (pRC/CMV, Invitrogen) and containing a puromycin resistance gene (19Kohfeldt E. Göhring W. Mayer U. Zweckstetter M. Holak T.A. Chu M.L. Timpl R. Eur. J. Biochem. 1996; 238: 333-340Crossref PubMed Scopus (30) Google Scholar). Upon transfection into human embryonic kidney cells (HEK-293 cells, American Type Culture Collection) using LipofectAMINE (Invitrogen) puromycin-resistant clones were isolated as described earlier (14Oohashi T. Zhou X.H. Feng K. Richter B. Morgelin M. Perez M.T., Su, W.D. Chiquet-Ehrismann R. Rauch U. Fässler R. J. Cell Biol. 1999; 145: 563-577Crossref PubMed Scopus (95) Google Scholar). Positive clones were identified by 5% SDS-PAGE and Coomassie Blue staining or Western blotting using mouse anti-RGS (H4) antibody (Qiagen, Stockholm, Sweden). For heterodimer analysis HEK-293 cells expressing the His-tagged extracellular domain of Ten-m2 were co-transfected with the non-His-tagged Ten-m1 or Ten-m3 extracellular domains, respectively, using the same eukaryotic expression vector described above (pRC/CMV, Invitrogen) but containing a neomycin resistance gene. Clones resistant to puromycin (1 μg/ml) as well as G418 (1.2 mg/ml) were identified by Western blot.Table IPredicted characteristics of the proteins expressed in HEK-293 cellsConstructNH2-terminal amino acidNH2-terminal sequenceCalculated molecular massPotential N-glycosylation sitesTen-m1E 526APLAE IMDDC STNCN GNGEC24713Ten-m2S 572APLGR GSHHH HHHGG LASVQ24513Ten-m3E 513APLAE SVVEC PRNCH GNGEC24613Ten-m4S 564APLGR GSHHH HHHGG LASVD24811Gten-m1E 799APLGR GSHHH HHHGG LAEML22013Gten-m3E 787APLGR GSHHH HHHGG LAETL21913The sequences derived from the respective mouse Ten-m cDNAs starts with the NH2-terminal amino acid as indicated. The NH2-terminal sequence is derived from the proteolytic processing site of the BM 40 signal peptide (17Mayer 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). The molecular mass was calculated on the basis of the NH2-terminal sequence and the Ten-M C-termini predicted from the respective cDNA sequences. The number of potential N-glycosylation sites is based on the N-X-S/T recognition motif excluding proline residues at position 2. Open table in a new tab The sequences derived from the respective mouse Ten-m cDNAs starts with the NH2-terminal amino acid as indicated. The NH2-terminal sequence is derived from the proteolytic processing site of the BM 40 signal peptide (17Mayer 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). The molecular mass was calculated on the basis of the NH2-terminal sequence and the Ten-M C-termini predicted from the respective cDNA sequences. The number of potential N-glycosylation sites is based on the N-X-S/T recognition motif excluding proline residues at position 2. The procedure has been described as the rat lymph node method (20Kishiro Y. Kagawa M. Naito I. Sado Y. Cell Struct. Funct. 1995; 20: 151-156Crossref PubMed Scopus (163) Google Scholar) for raising monoclonal antibodies (mAb). Briefly, WKY/NCRj rats (Charles River Japan, Yokohama, Japan) were immunized in the hind footpads with the emulsion of the recombinant protein and Freund's complete adjuvant. Three weeks later the rats were killed, and lymphocytes from the medial iliac lymph nodes were fused with mouse myeloma cells (SP2/0-Ag14). Supernatants from hybridoma cultures were screened by enzyme-linked immunosorbent assay using the recombinant protein as immobilized ligand. A subsequent screening was performed by indirect immunofluorescence for Ten-m1 and Ten-m3 using sections from mouse testis and mouse brain, respectively. The specificity of the mAbs TO4 and HG31, which were raised against the extracellular domains of Ten-m1 and Ten-m3, respectively, were tested by Western assays using the recombinant extracellular domains of Ten-m1, Ten-m2, and Ten-m3. Briefly, 50 ng of purified recombinant protein was separated under nonreducing condition on 6% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Hybond-P,Amersham Biosciences) in Tris/glycine buffer containing 10% methanol for 1 h with 100 V using the Bio-Rad mini-gel system. The membranes were blocked with 5% nonfat dry milk in TBST (20 mm Tris-HCl, pH7.6, 150 mm NaCl, 0.1% Tween 20), incubated with protein G-purified TO4 (1:1000) and HG 31 supernatant (1:3000), respectively, and developed with horseradish peroxidase-conjugated secondary antibody in TBST containing 5% nonfat dry milk and the ECL+ detection system (AmershamBiosciences). Serum-free conditioned medium was dialyzed (three times, 6 h each time) against 50 mm NaH2PO4, pH 8, 300 mm NaCl, 0.5 mm NEM, and 1 mmimidazole supplemented with freshly added 0.5 mmphenylmethylsulfonyl fluoride. 1 ml of the Ni-NTA slurry was added to 50 ml of conditioned medium, incubated with Ni-NTA matrix at 4 °C for at least 6 h, loaded into an empty column, and washed 15 times the column volume with wash buffer (50 mmNaH2PO4, pH 8, 300 mm NaCl, 20 mm imidazole, 0.5 mm NEM). The protein was finally released with 5 ml elution buffer (50 mmNaH2PO4, pH 8, 300 mm NaCl, 250 mm imidazole, 0.5 mm NEM). The eluate was concentrated to less than 1 ml by centrifugation through membranes with a cut-off of 10 kDa (Amicon). The concentrated protein was applied to a Superose 6 column equilibrated with 10 mm HEPES, pH 7.4, 500 mm NaCl, and 0.5 mm NEM. Fractions containing the purified molecules were dialyzed against with 10 mm HEPES, pH 7.4, 150 mm NaCl and stored at −80 °C. For heterodimer analysis of recombinant Ten-m1 and Ten-m2, 1 ml of conditioned medium from the co-transfected cells was dialyzed against 50 mmNaH2PO4, pH 8, 300 mm NaCl, 0.5 mm NEM, and 1% bovine serum albumin plus 20 mmimidazole, each for 6 h for three times at 4 °C and then incubated with Ni-NTA beads overnight. The beads were washed with the same buffer containing 20 mm imidazole and finally eluted two times with the same buffer containing 250 mm imidazole. Specific bands were detected with TO4 (1:1000) mAb and anti-RGS (H4) (1:50,000) antibody on Western blot. To analyze the heterodimers of the extracellular domains of Ten-m3 and Ten-m2, 1 ml of serum-free conditioned medium of cotransfected cells or a mixture of 0.5 ml of recombinant Ten-m3 and 0.5 ml of recombinant His-tagged Ten-m2 was applied to Ni-NTA beads. The beads were washed with 20 mm imidazole and finally eluted two times with 250 mm imidazole. Bands were detected with HG31 (1:3000) mAb and anti-RGS (H4) (1:50,000) antibody by Western blot. N-glycosidase F (Roche Molecular Biochemicals) treatment (2 units) of 12 μg of purified recombinant extracellular domain of Ten-m1 or Ten-m2 was carried out at 37 °C for 16 h in 20 mm phosphate buffer, pH 7.2, containing 0.5% octylglucoside, 1 mm phenylmethylsulfonyl fluoride, and 10 mm EDTA. Subsequently the samples were denatured by heating to 97 °C for 5 min in the presence of 1% SDS in 20 mm phosphate buffer, pH 7.2. A Beckman model XLA analytical Ultracentrifuge equipped with absorption optics was employed. Sedimentation velocity runs were performed in 12-mm double sector cells at rotor speeds of 40,000 and 52,000 rpm. Sedimentation equilibrium runs were performed at 4,400 rpm using the same cells but at a filling height of 1.5–3 mm only. Sedimentation coefficientss 20, w are corrected to standard conditions (water at 20 °C) (21Van Holde K.E. Physical Biochemistry. Prentice-Hall, Englewood Cliffs, NJ1971: 98-120Google Scholar). The molecular masses, M, were calculated from sedimentation equilibrium runs using a floating base-line computer program that adjusts the base-line absorption to obtain the best linear fit of lnA versus r 2 (A = absorbance, r = distance from the rotor axis). A partial specific volume of 0.70 cm3/g was used, which was calculated for proteins with 30% glycosylation (22Ralson G. Introduction to Analytical Ultracentrifugation. Beckman Instruments, Fullerton, CA1993Google Scholar). Frictional ratiosf/f 0 were calculated from the sedimentation coefficients and molecular masses according to Van Holde (21Van Holde K.E. Physical Biochemistry. Prentice-Hall, Englewood Cliffs, NJ1971: 98-120Google Scholar), and axial ratios of ellipsoids of revolution a/b were determined from Perrin's table (21Van Holde K.E. Physical Biochemistry. Prentice-Hall, Englewood Cliffs, NJ1971: 98-120Google Scholar). All measurements were performed in 10 mm HEPES, 150 mm NaCl at 20 °C. Glycerol spraying/rotary shadowing, negative staining, and evaluation of the data from electron micrographs were carried out as described previously (23Engel J. Furthmayr H. Methods Enzymol. 1987; 145: 3-78Crossref PubMed Scopus (136) Google Scholar). For negative staining 5-μl samples of different Ten-m preparations (typical concentrations of about 10 μg/ml in Tris-buffered saline) were adsorbed to 400-mesh carbon-coated copper grids, washed briefly with water, and stained on two drops of freshly prepared 0.75% uranyl formate. The grids were rendered hydrophilic by glow discharge at low pressure in air. For glycerol spraying/rotary shadowing, Ten-m samples were dialyzed overnight at 4 °C against 0.2 m ammonium hydrogen carbonate, pH 7.9. They were mixed with equal volumes of 80% glycerol and sprayed onto freshly cleaved mica pieces with a nebulizer designed for small volumes. They were dried in a high vacuum for 2 h and shadowed under rotation with 2 nm platinum/carbon at a 9° angle, followed by coating with a stabilizing 10-nm carbon film. Specimens were observed in a Jeol JEM 1230 electron microscope operated at 80 kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 CCD camera. Molecular masses of globular protein domains from negatively stained images were estimated as described previously (23Engel J. Furthmayr H. Methods Enzymol. 1987; 145: 3-78Crossref PubMed Scopus (136) Google Scholar). The extracellular domains of Ten-m2, -m3 and -m4, starting with the first EGF module, were recombinantly expressed in HEK-293 cells and purified from serum-free conditioned culture medium. The rotary shadowing electron microscopic image showed that the recombinant extracellular domain of all mouse Ten-m family members form similar cherry-like structures of two globular domains connected by two extended rods (Fig. 1) as previously observed for the recombinant extracellular domain of Ten-m1 (14Oohashi T. Zhou X.H. Feng K. Richter B. Morgelin M. Perez M.T., Su, W.D. Chiquet-Ehrismann R. Rauch U. Fässler R. J. Cell Biol. 1999; 145: 563-577Crossref PubMed Scopus (95) Google Scholar). In some images the connecting part between the two globular domains was extremely extended. In such cases the distances between the two globular domains was up to 30 nm (Fig. 1). Separation of all four recombinantly expressed extracellular domains of the Ten-m family proteins by 6% SDS-PAGE under reducing conditions revealed apparent molecular masses of about 225 kDa for all four Ten-m proteins (Fig. 2 A). Gel separation under nonreducing conditions showed significant differences in the migratory behavior of the four samples (Fig. 2 B) dividing the extracellular domains of the four Ten-m molecules into two subfamilies, one consisting of Ten-m1 and Ten-m4 (Fig. 2 B,lanes 1 and 2), which migrate considerably slower on a 6% SDS-PAGE than the second subfamily, consisting of Ten-m2 and Ten-m3 (Fig. 2 B, lanes 3 and 4). In our previous study (14Oohashi T. Zhou X.H. Feng K. Richter B. Morgelin M. Perez M.T., Su, W.D. Chiquet-Ehrismann R. Rauch U. Fässler R. J. Cell Biol. 1999; 145: 563-577Crossref PubMed Scopus (95) Google Scholar) Ten-m1 was proposed to be a dimer mainly on the basis of electron microscopic observations, although a thorough SDS-PAGE analysis with technically adequate markers had not been performed. Application of a 3–12% gradient SDS-PAGE allowed the extracellular domains of Ten-m1 to migrate 4.5 cm into the polyacrylamide gel and decreased the apparent differences between the migratory abilities of the extracellular domains of the Ten-m molecules (Fig. 2 C). Apparent molecular masses derived from this gel using laminin (850 kDa), nidogen (150 kDa), and myosin (200 kDa) for calibration were 550 kDa for Ten-m1 and Ten-m4 and 440 kDa for Ten-m2 and Ten-m3. For the later proteins an additional band was seen migrating with the same apparent molecular mass of 225 kDa as the reduced protein, thus probably corresponding to unlinked monomers. To test whether differential N-linked glycosylation was responsible for the observed differences, the recombinant extracellular domains of Ten-m1 and Ten-m2 were subjected to N-glycosidase F treatment. This treatment reduced the apparent molecular masses of both molecules to a similar extent on SDS-PAGE ruling outN-linked glycosylation as the cause of the different migratory behaviors (results not shown). Despite their different migratory abilities on SDS-PAGE, the extracellular domains of Ten-m proteins from the two different subfamilies had identical elution profiles when subjected to gel permeation chromatography on Superose 6 (results not shown). It is possible that molecular mass determination on SDS-PAGE under nonreducing conditions might be affected by particular tertiary or quaternary structures, which could be expected to be more different in molecules with unrelated sequences, like Ten-m 1 and laminin, than in molecules with closely related sequences, like Ten-m1 and Ten-m2. To obtain an accurate molecular mass independent of the structural peculiarities of the subjected substance, we performed ultracentrifugation experiments. The equilibrium ultracentrifugation data showed that both the recombinant extracellular domains of Ten-m1 and Ten-m2 had under nonreducing condition approximately the same average molar mass of 500–550 kDa. Experimental values are M = 515,000 ± 60,000 for Ten-m1 and 545,000 ± 60,000 for Ten-m2 (Fig. 3). The comparison of these values with the calculated molecular mass of a single polypeptide chain of the recombinantly expressed sequence of Ten-m1 (247 kDa) and Ten-m2 (245 kDa), both containing 13 potential N-glycosylation sites (Table I), indicates that the extracellular domains of both Ten-m proteins assume a similar dimeric quaternary structure. This result underlines the observations using rotary shadowing electron microscopy (Fig. 1) and indicates that, despite their differences in mobility on SDS-PAGE, all Ten-m molecules are dimeric type II transmembrane molecules covalently linked only via their EGF module arrays. Sedimentation velocity experiments with the recombinant extracellular domains of Ten-m1 and Ten-m2 gave sedimentation coefficient values ofs 20, w = 15.4 and 16.2 S, respectively. Frictional ratios calculated with these values and M = 500,000 aref/f 0 = 1.23 and 1.17, respectively. This indicates an axial ratio a/b of about 4 for the hydrodynamic equivalent of the dimers, which is consistent with the asymmetric shape revealed by electron microscopy (see below). Because the EGF module arrays are best conserved in all Ten-m molecules (14Oohashi T. Zhou X.H. Feng K. Richter B. Morgelin M. Perez M.T., Su, W.D. Chiquet-Ehrismann R. Rauch U. Fässler R. J. Cell Biol. 1999; 145: 563-577Crossref PubMed Scopus (95) Google Scholar), they are least likely to account for biophysical differences between the subfamilies. To test whether the large COOH-terminal globular domain following the EGF module array was responsible for the differences in migratory behavior, the COOH-terminal domain of one member of each subfamily of Ten-m1 and Ten-m3 was recombinantly expressed and analyzed. As observed previously, the secreted protein products showed a different migratory behavior on SDS-PAGE. Although the migration behavior of the COOH-terminal domain of Ten-m3 (Gten-m3) was similar under reducing and nonreducing condition, the COOH-terminal region of Ten-m1 (Gten-m1) migrated considerably slower under nonreducing conditions on a 6% SDS-PAGE (Fig.4, A and B). Again, on a 3–12% gradient gel the difference in migratory behavior between the nonreduced molecules appeared decreased (Fig. 4 C). The apparent molecular masses determined from this gel were 195 kDa for Gten-m3 and 300 kDa for Gten-m1, with a second, less distinct subpopulation of Gten-m1 of about 250 kDa, most likely representing proteolytically nicked material. Thus, the observed reduced mobility of the extracellular domain of Ten-m1 on SDS-PAGE was reflected in a similarly reduced mobility of its globular COOH-terminal part. The electron microscopic analysis of Ten-m1 by negative staining revealed that the large, globular COOH-terminal domain is composed of subdomains (14Oohashi T. Zhou X.H. Feng K. Richter B. Morgelin M. Perez M.T., Su, W.D. Chiquet-Ehrismann R. Rauch U. Fässler R. J. Cell Biol. 1999; 145: 563-577Crossref PubMed Scopus (95) Google Scholar). The subdomains were especially evident in preparations of the globular COOH-terminal domains alone (Fig. 4 D). In Gten-m1 preparations we could almost always observe two globular structures, wh" @default.
• W2064424062 created "2016-06-24" @default.
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• W2064424062 creator A5012954903 @default.
• W2064424062 creator A5027751830 @default.
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• W2064424062 creator A5037516021 @default.
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• W2064424062 date "2002-07-01" @default.
• W2064424062 modified "2023-10-11" @default.
• W2064424062 title "All Four Members of the Ten-m/Odz Family of Transmembrane Proteins Form Dimers" @default.
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