Expression and Supramolecular Assembly of Recombinant α1(VIII) and α2(VIII) Collagen Homotrimers

Simon Stephan, Michael J. Sherratt, Nigel Hodson, C. Adrian Shuttleworth, Cay M. Kielty
2004 Journal of Biological Chemistry  
Collagen VIII is an extracellular matrix macromolecule comprising two polypeptide chains, ␣1(VIII) and ␣2(VIII), that can form homotrimers in vitro and in vivo. Here, recombinant collagen VIII was expressed to study its supramolecular assembly following secretion. Cells transfected with ␣1(VIII) or ␣2(VIII) assembled and secreted homotrimers that were stable in denaturing conditions and had a molecular mass of ϳ180 kDa on SDS-PAGE gels. Co-transfection with prolyl 4-hydroxylase generated
more » ... mers with stable pepsin-resistant triple-helical domains. Size fractionation of native recombinant collagen VIII molecules expressed with or without prolyl 4-hydroxylase identified urea-sensitive high molecular mass assemblies eluting in the void volume of a Superose 6HR 10/30 column and urea-resistant assemblies of ϳ700 kDa, all of which were composed of homotrimers. Immunofluorescence analysis highlighted the extracellular deposition of recombinant ␣1(VIII) 3 , ␣2(VIII) 3 , and co-expressed ␣1(VIII) 3 /␣2(VIII) 3 . Microscopy analysis of recombinant collagen VIII identified rod-like molecules of 134 nm in length that assembled into angular arrays with branching angles of ϳ114°and extensive networks. Based on these data, we propose a model of collagen VIII assembly in which four homotrimers form a tetrahedron stabilized by central interacting C-terminal NC1 trimers. Tetrahedrons may then act as building blocks of three-dimensional hexagonal lattices generated by secondary interactions involving terminal and helical sequences.
doi:10.1074/jbc.m305805200 pmid:14990571 fatcat:c5djkmt2ubhv7clssns5ovxipq