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.
Highlights
Ten-m/Odz/teneurins are a new family of four distinct type II transmembrane molecules
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 [14]
The characterization of recombinant extracellular domains of the Ten-m family proteins revealed that they share the same quaternary structure, a dimer composed of two large COOHterminal domains and arrays of eight EGF modules crosslinked by two disulfide bridges (Fig. 9)
Summary
The Recombinant Expression of the Extracellular Domains of the Ten-m Proteins—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 (Gtenm; 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) [17] or APLGRGSHHHHHHGGLA (Ten-m2, Ten-m4, Gten-m1, and Gten-m3), which can be detected by the anti-RGS (H4) antibody (Qiagen). Heterodimer Analyses of Recombinant Ten-m1 and Ten-m2 or Ten-m3 and Ten-m2 in Vitro—For heterodimer analysis of recombinant Ten-m1 and Ten-m2, 1 ml of conditioned medium from the co-transfected cells was dialyzed against 50 mM NaH2PO4, pH 8, 300 mM NaCl, 0.5 mM NEM, and 1% bovine serum albumin plus 20 mM imidazole, each for 6 h for three times at 4 °C and incubated with Ni-NTA beads overnight. Molecular masses of globular protein domains from negatively stained images were estimated as described previously [23]
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