Glycobiology and Extracellular Matrices
Expression and Supramolecular Assembly of Recombinant α1(VIII) and α2(VIII) Collagen Homotrimers*

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Collagen VIII is an extracellular matrix macromolecule comprising two polypeptide chains, α1(VIII) and α2(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 α1(VIII) or α2(VIII) assembled and secreted homotrimers that were stable in denaturing conditions and had a molecular mass of ∼180 kDa on SDS-PAGE gels. Co-transfection with prolyl 4-hydroxylase generated homotrimers 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 ∼700 kDa, all of which were composed of homotrimers. Immunofluorescence analysis highlighted the extracellular deposition of recombinant α1(VIII)3, α2(VIII)3, and co-expressed α1(VIII)3/α2(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 ∼114° 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.

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This study was supported in part by the British Heart Foundation (to C. M. K., C. A. S., and S. S.), Medical Research Council (to C. M. K., C. A. S., M. J. S., and N. H.), and the UK Centre for Tissue Engineering (funded by Medical Research Council, Biotechnology and Biological Sciences Research Council, and Engineering and Physical Sciences Research Council) (to C. M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.