STEM mass mapping of type VI collagen microfibrils: Implications for chain composition and alternative splicing

Abstract

Type VI collagen is a major structural interactive extracellular matrix macromolecule which forms double-beaded microfibrils in the extracellular space. These microfibrils can be isolated in native form from collagenase digest of skin fibroblast cell layers by chromatography on Sepharose CL-2B. Scanning transmission electron microscope (STEM) mass mapping showed a periodicity of 104 nm and a mass/bead of 1500 kDa. In addition, there was an uneven mass distribution along the bead. Microfibrils arise by end-to-end aggregation of tetramers which should produce microfibrils with bead mass of ≈2000 kDa and homogeneous mass distribution across the bead. Reduction in the mass across the bead implies either altered chain composition, alternative splicing or proteolytic degradation. The pattern of splicing of type VI chains in human tissues and their biological implications are poorly defined. In this study, we have examined the abundance of alternatively spliced forms of theα2(VI) andα3(VI) chains in human skin fibroblasts. BothαC2 andα2C2a variants, but not theα2C2a′ form, were expressed in these although theα2C2 form appeared more abundant. Theα3(VI) N7 domain was frequently spliced out of transcripts present in skin fibroblasts, whereas theα3(VI) N9 domain was always present. No consistent pattern of splicing was observed and splicing is unlikely to account for the reduced mass observed in type VI collagen microfibrils.