Klatt A R, Nitsche D P, Kobbe B, Macht M, Paulsson M, Wagener R
Institute for Biochemistry and the Center for Molecular Medicine Cologne Service Laboratory, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, Germany.
J Biol Chem. 2001 May 18;276(20):17267-75. doi: 10.1074/jbc.M100587200. Epub 2001 Feb 14.
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.
基质金属蛋白酶-4是血管性血友病因子A样结构域包含细胞外基质衔接蛋白的基质金属蛋白酶家族中最近鉴定出的成员。全长基质金属蛋白酶-4在293-EBNA细胞中表达，使用亲和标签进行纯化，并进行生化特性分析。重组表达的全长基质金属蛋白酶-4的最大寡聚形式是三聚体，这通过电子显微镜、SDS-聚丙烯酰胺凝胶电泳和质谱分析得以证实。还检测到了经蛋白水解加工的基质金属蛋白酶-4物种。切割发生在第二个血管性血友病因子A样结构域和卷曲螺旋结构域之间的短连接区域，导致大片段的释放以及完整亚基二聚体和单体的形成，这些亚基仍含有三聚体卷曲螺旋结构。在颅骨提取物的免疫印迹中可以检测到类似的降解产物，表明体内发生了相关的蛋白水解加工。基质金属蛋白酶-4最早在小鼠胚胎受精后第7.5天被观察到。亲和纯化的抗体在致密和疏松结缔组织、骨骼、软骨、中枢和外周神经系统以及与基底膜相关的部位检测到广泛表达。在原代胚胎成纤维细胞培养形成的基质中，基质金属蛋白酶-4存在于连接单个细胞的丝状网络中。
