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

Matrilin-4 is the most recently identified member of the matrilin family of von Willebrand factor A-like domain containing extracellular matrix adapte r proteins. Full-length matrilin-4 was expressed in 293-EBNA cells, purifie d using affinity tags, and subjected to biochemical characterization. The l argest oligomeric form of recombinantly expressed full-length matrilin-1 is a trimer as shown by electron microscopy, SDS-polyacrylamide gel electroph oresis, 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 m onomers of intact subunits still containing a trimeric coiled-coil. In immu noblots 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-pur ified antibodies detect a broad expression in dense and loose connective ti ssue, bone, cartilage, central and peripheral nervous systems and in associ ation with basement membranes. In the matrix formed by cultured primary emb ryonic fibroblasts, matrilin-4 is found in a filamentous network connecting individual cells.