The role of the α3(VI) chain in collagen VI assembly -: Expression of an α3(VI) chain lacking N-terminal modules N10-N7 restores collagen VI assembly, secretion, and matrix deposition in an α3(VI)-deficient cell line

Collagen VI is a microfibrillar protein found in the extracellular matrix of virtually all connective tissues, Three genetically distinct subunits, the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains, associate intracellularly to form triple-helical monomers, which then assemble into disulfide-bonded dimers and tetramers before secretion, Although sequence considerations suggest that collagen VI monomers composed of all three chains are the most stable isoform, the precise chain composition of collagen VI remains controversial and alternative assemblies containing only alpha 1(VI) and alpha 2(VI) chains have also been proposed, To address this question directly and study the role of the alpha 3(VI) chain in assembly, we have characterized collagen VI biosynthesis and in vitro matrix formation by a human osteosarcoma cell line (SaOS-2) that is deficient in alpha 3(VI) production, Northern analysis showed an abundance of alpha 1(VI) and alpha 2(VI) mRNAs, but no detectable alpha 3(VI) mRNA was apparent in SaOS-2 cells, By day 30 of culture, however, small amounts of alpha 3(VI) mRNA were detected, although the level of expression was still much less than alpha 1(VI) and alpha 2(VI), Collagen VI protein was not detected in SaOS-2 medium or cell layer samples until day 30 of culture, demonstrating that despite the abundant synthesis of alpha 1(VI) and alpha 2(VI), no stable collagen VI protein was produced without expression of alpha 3(VI). The alpha 1(VI) and alpha 2(VI) chains produced in the absence of alpha 3(VI) were non-helical and were largely retained intracellularly and degraded, The critical role of the alpha 3(VI) chain in collagen VI assembly was directly demonstrated after stable transfection of SaOS-2 cells with an alpha 3(VI) cDNA expression construct that lacked 4 of the 10 N-terminal type A subdomains, The transfected alpha 3(VI) N6-C5 chains associated with endogenous alpha 1(VI) and alpha 2(VI) and formed collagen VI dimers and tetramers, which were secreted and deposited into an extensive network in the extracellular matrix, These data demonstrated that alpha 3(VI) is essential for the formation of stable collagen VI molecules and subdomains N10-N7 are not required for molecular assembly.