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

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... 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.