Bone morphogenetic protein‐1 cleaves prodecorin in vitro and in cellulo

Abstract

Introduction Decorin is a ubiquitously distributed small leucine‐rich protein (SLRP) with pivotal roles in collagen fibrillogenesis, regulation of cell matrix deposition as well as cell growth, adhesion and migration. It is expressed in the proform but found mainly in the processed form in various tissues. However, the role of propeptide of prodecorin is still unclear. A possible function is in the trafficking of prodecorin to specific compartment(s) in the cell where it may bind/interact with other ECM molecules prior to secretion. BMP‐1 is a metalloproteinase that is responsible for the processing of various ECM molecules including procollagens and prolysyloxidase. Probiglycan, a SLRP with 57% homology to prodecorin, has also been shown to be a substrate of BMP‐1, which led to the hypothesis that prodecorin could also be processed by the same enzyme. This would emphasize the important role of BMP‐1 in the deposition of the ECM.Materials and methods To aid detection, Flag‐tag (DYKDDDDK) was introduced into the prodecorin sequence immediately 5′ of the propeptide sequence. This was achieved via a two‐step PCR reaction. The triple‐alanine mutant was generated using the same method replacing the putative BMP‐1 cleavage site EDE (30–32) with AAA residues. Transient and stable transfectants were generated in HT1080 human fibroblast cells. These were chosen because of their ability to express and secrete the mutant prodecorin. Purification of decorin was achieved using anion exchange chromatography. BMP‐1‐Flag was expressed in, and purified from, transfected 293‐EBNA cells.Results BMP‐1 was assayed for cleavage of prodecorin in vitro. Addition of BMP‐1 to the assay resulted in removal of the prodomain. Addition of EDTA restores detection of prodecorin by anti‐Flag antibody. HT1080 cells doubly transfected with Flag‐tagged prodecorin and BMP‐1‐Flag secreted decorin in the mature form. Control cells secreted uncleaved prodecorin. HT1080 cells doubly transfected with Flag‐tagged triple‐alanine prodecorin mutant and BMP‐1‐Flag did not show cleavage of prodecorin. It was observed that the GAG‐chain addition to this mutant was affected by the mutation.Discussion BMP‐1 cleaves prodecorin in vitro and in cellulo. The cleaved bond is possibly with the E‐DE (30–32) sequence, but this will be confirmed by cleavage of prodecorin and sequencing of the mature protein. These residues are also important in the initiation and stability of the glycosaminoglycan chain of decorin, because mutation of these results in production of mainly unglycanated prodecorin. This suggests a possible role of the prodomain in the initiation of the GAG chain.