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

摘要

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. Microflbrils 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 the alpha 2(VI) and a3(VI) chains in human skin fibroblasts. Both alpha2C2 and alpha2 C2a variants, but not the alpha2C2a' form, were expressed in these although the alpha2C2 form appeared more abundant. The alpha3(VI) N7 domain was frequently spliced out of transcripts present in skin fibroblasts, whereas the a3(VI) Ng 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.