Evidence for a specific mechanism of laminin assembly.
The specificity of laminin chain assembly was investigated using fragments E8 and C8-9, derived from the long arm of the molecule, whose rod-like domain consists of the alpha-helical regions of the A, B1 and B2 chains. Urea-induced chain separation and unfolding were monitored by transverse urea/polyacrylamide gel electrophoresis (PAGE) and circular dichroism. Separation of the A and disulphide-linked B1-B2 chains occurred at 3.5-4.0 M urea and by 7.0 M urea all residual alpha-helicity was lost. Removal of urea by dialysis resulted in high recoveries (87-100%) of renatured protein which in its apparent molecular mass, alpha-helix content, chain composition, degree of association and ultrastructural appearance was indistinguishable from native E8. Reduction or reduction and alkylation of the chains did not lead to a decrease in their ability to reassemble specifically. Reformation of the single interchain disulphide, linking the B1 and B2 chains, clearly demonstrates that these chains are correctly aligned in parallel and in register in E8 renatured from its reduced chains. Renaturation of E8 from its reduced and alkylated chains precludes a role for disulphide formation in determining chain alignment but suggests rather than it is involved in the stabilisation of the correctly assembled molecule. These results, together with recent sequence data, provide evidence for the interaction of the alpha-helical regions of the A, B1 and B2 chains in the formation of a triple coiled-coil within the long arm of the molecule. The highly specific nature of this interaction suggests that it is the mechanism by which laminin is assembled in vivo.
Hunter
I
I
Abteilung Biophysikalische Chemie, Biozentrum der Universität, Basel, Switzerland.
Schulthess
T
T
Bruch
M
M
Beck
K
K
Engel
J
J
D000818:Animals; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004220:Disulfides; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007797:Laminin / Q000032:analysis* / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D010446:Peptide Fragments; D011489:Protein Denaturation; D014508:Urea
D003432:Cross-Linking Reagents; D004220:Disulfides; D007797:Laminin; D010446:Peptide Fragments; D014508:Urea