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.
Highlights
The matrilins are a novel family of oligomeric extracellular proteins containing von Willebrand factor A1-like domains (1)
In the matrix formed by cultured primary embryonic fibroblasts, matrilin-4 is found in a filamentous network connecting individual cells
The recombinant protein was used for production of a specific antiserum, which allowed immunohistochemical characterization of matrilin-4 expression in the mouse and an analysis of supramolecular assembly forms in the extracellular matrix formed by cultured primary embryonic fibroblasts
Summary
The matrilins are a novel family of oligomeric extracellular proteins containing von Willebrand factor A (vWFA)1-like domains (1). A unique splice variant, which does not contain the N-terminal vWFA-like domain, was identified in mouse (3). Human matrilin-4 is highly homologous to the mouse protein with an overall identity of 91% and a maximum identity of 97% in the second vWFA-like domain. Due to a mutation in the splice donor site of the third intron of the human gene, the exon, which corresponds to that specifying the first EGF-like domain in mouse, is not expressed in man. The recombinant protein was used for production of a specific antiserum, which allowed immunohistochemical characterization of matrilin-4 expression in the mouse and an analysis of supramolecular assembly forms in the extracellular matrix formed by cultured primary embryonic fibroblasts. Extraction of native matrilin-4 from mouse tissues allowed a characterization of the naturally occurring oligomers by SDSPAGE and Western blotting
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