title: Functional importance of covalent homodimer of reelin protein linked via its central region.
abstract: Reelin is a 3461-residue secreted glycoprotein that plays a critical role in brain development through its action on target neurons. Although it is known that functional reelin protein exists as multimer formed by interchain disulfide bond(s) as well as through non-covalent interactions, the chemical nature of the multimer assembly has been elusive. In the present study, we identified, among 122 cysteines present in full-length reelin, the single critical cysteine residue (Cys(2101)) responsible for the covalent multimerization. C2101A mutant reelin failed to assemble into disulfide-bonded multimers, whereas it still exhibited non-covalently associated high molecular weight oligomeric states in solution. Detailed analysis of tryptic fragments produced from the purified reelin proteins revealed that the minimum unit of the multimer is a homodimeric reelin linked via Cys(2101) present in the central region and that this cysteine does not connect to the N-terminal region of reelin, which had been postulated as the primary oligomerization domain. A surface plasmon resonance binding assay confirmed that C2101A mutant reelin retained binding capability toward two neuronal receptors apolipoprotein E receptor 2 and very low density lipoprotein receptor. However, it failed to show signaling activity in the assay using the cultured neurons. These results indicate that an intact higher order architecture of reelin multimer maintained by both Cys(2101)-mediated homodimerization and other non-covalent association present elsewhere in the reelin primary structure are essential for exerting its full biological activity.
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Norihisa
authors.initials: N
authors.lastname: Yasui
authors.forename: Yu
authors.initials: Y
authors.lastname: Kitago
authors.forename: Ayako
authors.initials: A
authors.lastname: Beppu
authors.forename: Takao
authors.initials: T
authors.lastname: Kohno
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Morishita
authors.forename: Hiroki
authors.initials: H
authors.lastname: Gomi
authors.forename: Masamichi
authors.initials: M
authors.lastname: Nagae
authors.forename: Mitsuharu
authors.initials: M
authors.lastname: Hattori
authors.forename: Junichi
authors.initials: J
authors.lastname: Takagi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000091042:Reelin Protein; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D015816:Cell Adhesion Molecules, Neuronal; D004220:Disulfides; D016326:Extracellular Matrix Proteins; D009419:Nerve Tissue Proteins; D000091042:Reelin Protein; C000717127:RELN protein, human; C000717128:Reln protein, mouse; D012697:Serine Endopeptidases