ABSTRACT
alpha-Dystroglycan (alpha-DG) is a component of the dystroglycan complex, which is involved in early development and morphogenesis and in the pathogenesis of muscular dystrophies. Here, alpha-DG was shown to serve as a Schwann cell receptor for Mycobacterium leprae, the causative organism of leprosy. Mycobacterium leprae specifically bound to alpha-DG only in the presence of the G domain of the alpha2 chain of laminin-2. Native alpha-DG competitively inhibited the laminin-2-mediated M. leprae binding to primary Schwann cells. Thus, M. leprae may use linkage between the extracellular matrix and cytoskeleton through laminin-2 and alpha-DG for its interaction with Schwann cells.
Subject(s)
Bacterial Adhesion , Cytoskeletal Proteins/metabolism , Laminin/metabolism , Membrane Glycoproteins/metabolism , Mycobacterium leprae/metabolism , Schwann Cells/microbiology , Animals , Binding Sites , Calcium/physiology , Cell Line, Transformed , Cells, Cultured , Cytoskeletal Proteins/pharmacology , Dystroglycans , Edetic Acid/pharmacology , Glycosylation , Humans , Laminin/chemistry , Membrane Glycoproteins/pharmacology , Peripheral Nerves/chemistry , Rats , Receptors, Laminin/metabolism , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Schwann Cells/metabolismABSTRACT
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.