Identification of FAM20C binding proteins
FAM20 (family with sequence similarity 20) members in humans consist of FAM20A, FAM20B and FAM20C. The mutations of FAM20A in humans lead to Amelogenesis Imperfecta (AI), gingival hyperplasia and enamel renal syndrome (ERS) in humans. Mutations of FAM20B in Danio rerio result in decreased cartilage matrix production and skeletal defects. Mutations in FAM20C leads to neonatal lethal osteosclerotic bone dysplasia in humans, known as Raine syndrome. One of the mutants is FAM20C-D478A. FAM20C intracellularly functions as a Golgi casein kinase. It phosphorylates secretory pathway proteins within S-x-E motif (where S is Ser, X is any amino acid, and E is Glu). Extracellular role of FAM20C has also been suggested as a growth and differentiation factor, and the exogenous FAM20C treatment accelerates MC3T3-E1 osteoblast differentiation and mineralization in vitro. The first purpose of this study was to purify FAM20C protein. HEK 293 cells were transfected with FAM20C expression vectors, cell clones that overexpress FAM20C were isolated and FAM20C protein was collected and purified. The Western blot results of purified FAM20C showed higher bands, around 100 kDa and 170 kDa, than expected molecular weight, 66 kDa. Post-translational modification was thought to be the possible reason. Therefore, the second purpose was to find binding proteins of FAM20C by mass spectrometry protein identification analysis to check if FAM20C has other post-modifications, such as glycosylation. FAM20C-WT and FAM20C-D478A proteins were chosen to perform the study since FAM20C-D478A was investigated in previous studies of Golgi casein kinase, FAM20C, and was found to have no kinase activity. In this study, periostin was identified to bind to FAM20C and the binding of these 2 proteins was confirmed by immunoprecipitation and Western blot analysis. Since FAM20C functions as a secretory kinase, it is suggested that periostin may be a substrate for FAM20C kinase. Further investigation is needed to determine the presence of phosphorylation in periostin and its role in periosteum and periodontal ligament.