Kunneken, K., Pohlentz, G., Schmidt-Hederich, A., Odenthal, U., Smyth, N., Peter-Katalinic, J., Bruckner, P. and Eble, J.A.
Recombinant human laminin-5 domains - Effects of heterotrimerization, proteolytic processing, and N-glycosylation on alpha(3)beta(1) integrin binding
The Journal of Biological Chemistry, 279, (7), . (doi:10.1074/jbc.M310424200).
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Human laminin-5 fragments, comprising the heterotrimeric C-terminal part of the coiled-coil (CC) domain and the globular (G) domain with defined numbers of LG subdomains, were produced recombinantly. The 3' chain with all five LG subdomains was processed proteolytically in a manner similar to the wild-type 3 chain. Conditions were established under which the proteolytic cleavage was either inhibited in cell culture or was brought to completion in vitro. The shorter chains of the laminin-5CCG molecule, 3'and 2', produced in a bacterial expression system associated into heterodimers, which then combined spontaneously with the 3' chains in vitro to form heterotrimeric laminin-5CCG molecules. Only heterotrimeric laminin-5CCG with at least subdomains LG1–3, but not the single chains, supported binding of soluble 31 integrin, proving the coiled-coil domain of laminin-5 to be essential for its interaction with 31 integrin. The N-glycosylation sites in wild-type 3 chain were mapped by mass spectrometry. Their location in a structural model of the LG domain suggested that large regions on both faces of the LG1 and LG2 domains are inaccessible by other proteins. However, neither heterotrimerization nor 31 integrin binding was affected by the loss of N-linked glycoconjugates. After the proteolytic cleavage between the subdomains LG3 and LG4, the LG4–5 tandem domain dissociated from the rest of the G domain. Further, the laminin-5CCG molecule with the 3'LG1–3 chain showed an increased binding affinity for 31 integrin, indicating that proteolytic processing of laminin-5 influences its interaction with 31 integrin.
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