Expression and supramolecular assembly of recombinant alpha1(viii) and alpha2(viii) collagen homotrimers.
Collagen VIII is an extracellular matrix macromolecule comprising two polypeptide chains, alpha1(VIII) and alpha2(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 alpha1(VIII) or alpha2(VIII) assembled and secreted homotrimers that were stable in denaturing conditions and had a molecular mass of approximately 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 approximately 700 kDa, all of which were composed of homotrimers. Immunofluorescence analysis highlighted the extracellular deposition of recombinant alpha1(VIII)(3), alpha2(VIII)(3), and co-expressed alpha1(VIII)(3)/alpha2(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 approximately 114 degrees 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.
Wellcome Trust Centre for Cell Matrix Research and UK Centre for Tissue Engineering, School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Simon
S
Stephan
Michael J
MJ
Sherratt
Nigel
N
Hodson
C Adrian
CA
Shuttleworth
Cay M
CM
Kielty
D002460:Cell Line; D024102:Collagen Type VIII / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D006801:Humans; D007668:Kidney; D008958:Models, Molecular; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection; D014471:Umbilical Veins
C443712:COL8A2 protein, human; D024102:Collagen Type VIII; D011994:Recombinant Proteins