The C5 domain of the collagen VI α3(VI) chain is critical for extracellular microfibril formation and is present in the extracellular matrix of cultured cells

Collagen VI, a microfibrillar protein found in virtually all connective tissues, is composed of three distinct subunits, alpha 1(VI), alpha 2(VI), and alpha 3(VI), which associate intracellularly to form triple helical heterotrimeric monomers then dimers and tetramers. The secreted tetramers associate end-to-end to form beaded microfibrils. Although the basic steps in assembly and the structure of the tetramers and microfibrils are well defined, details of the interacting protein domains involved in assembly are still poorly understood. To explore the role of the C-terminal globular regions in assembly, alpha 3(VI) cDNA expression constructs with C-terminal truncations were stably transfected into SaOS-2 cells. Control alpha 3(VI) N6-C5 chains with an intact C-terminal globular region (subdomains C1-C5), and truncated alpha 3(VI) N6-C1, N6-C2, N6-C3, and N6-C4 chains, all associated with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers and tetramers, which were secreted. These data demonstrate that subdomains C2-C5 are not required for monomer, dimer or tetramer assembly, and suggest that the important chain selection interactions involve the C1 subdomains. In contrast to tetramers containing control alpha 3(VI) N6-C5 chains, tetramers containing truncated alpha 3(VI) chains were unable to associate efficiently end-to-end in the medium and did not form a significant extracellular matrix, demonstrating that the alpha 3(VI) C5 domain plays a crucial role in collagen VI microfibril assembly. The alpha 3(VI) C5 domain is present in the extracellular matrix of SaOS-2 N6-C5 expressing cells and fibroblasts demonstrating that processing of the C-terminal region of the alpha 3(VI) chain is not essential for microfibril formation.