title: The alpha chain of laminin-1 is independently secreted and drives secretion of its beta- and gamma-chain partners.
abstract: A mammalian recombinant strategy was established to dissect rules of basement membrane laminin assembly and secretion. The alpha-, beta-, and gamma-chain subunits of laminin-1 were expressed in all combinations, transiently and/or stably, in a near-null background. In the absence of its normal partners, the alpha chain was secreted as intact protein and protein that had been cleaved in the coiled-coil domain. In contrast, the beta and gamma chains, expressed separately or together, remained intracellular with formation of betabeta or betagamma, but not gammagamma, disulfide-linked dimers. Secretion of the beta and gamma chains required simultaneous expression of all three chains and their assembly into alphabetagamma heterotrimers. Epitope-tagged recombinant alpha subunit and recombinant laminin were affinity-purified from the conditioned medium of alphagamma and alphabetagamma clones. Rotary-shadow electron microscopy revealed that the free alpha subunit is a linear structure containing N-terminal and included globules with a foreshortened long arm, while the trimeric species has the typical four-arm morphology of native laminin. We conclude that the alpha chain can be delivered to the extracellular environment as a single subunit, whereas the beta and gamma chains cannot, and that the alpha chain drives the secretion of the trimeric molecule. Such an alpha-chain-dependent mechanism could allow for the regulation of laminin export into a nascent basement membrane, and might serve an important role in controlling basement membrane formation.
authors.affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA. yurchenc@rwja.umdnj.edu
authors.forename: P D
authors.initials: PD
authors.lastname: Yurchenco
authors.forename: Y
authors.initials: Y
authors.lastname: Quan
authors.forename: H
authors.initials: H
authors.lastname: Colognato
authors.forename: T
authors.initials: T
authors.lastname: Mathus
authors.forename: D
authors.initials: D
authors.lastname: Harrison
authors.forename: Y
authors.initials: Y
authors.lastname: Yamada
authors.forename: J J
authors.initials: JJ
authors.lastname: O'Rear
mesh_terms: D001485:Basement Membrane / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D007797:Laminin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D007797:Laminin; D011994:Recombinant Proteins