Molecular structure, processing, and tissue distribution of matrilin-4

Abstract

Matrilin-4 is the most recently identified member of the matrilin family of von Willebrand factor A-like domain containing extracellular matrix adapter proteins. Full-length matrilin-4 was expressed in 293-EBNA cells, purified using affinity tags, and subjected to biochemical characterization. The largest oligomeric form of recombinantly expressed full-length matrilin-4 is a trimer as shown by electron microscopy, SDS-polyacrylamide gel electrophoresis, and mass spectrometry. Proteolytically processed matrilin-4 species were also detected. The cleavage occurs in the short linker region between the second von Willebrand factor A-like domain and the coiled-coil domain leading to the release of large fragments and the formation of dimers and monomers of intact subunits still containing a trimeric coiled-coil. In immunoblots of calvaria extracts similar degradation products could be detected, indicating that a related proteolytic processing occurs in vivo. Matrilin-4 was first observed at day 7.5 post-coitum in mouse embryos. Affinity-purified antibodies detect a broad expression in dense and loose connective tissue, bone, cartilage, central and peripheral nervous systems and in association with basement membranes. In the matrix formed by cultured primary embryonic fibroblasts, matrilin-4 is found in a filamentous network connecting individual cells.