Seven proprotein convertases cleave the basic amino acid consensus sequence K/R-X-K/R↓ (where n=0， 2， 4 or 6 variable amino acids) to activate precursor proteins. Despite similarities in substrate specificity， basic amino acid-specific proprotein convertases have a distinct tissue distribution allowing for enzymatic actions on tissue-resident substrates. Proprotein convertase 5/6 (PC5/6) has two splice variants - soluble PC5/6A and membrane-bound PC5/6B - and is expressed during mouse development in many tissues including bone and tooth， but little is known about the substrates for PC5/6 therein. Osteopontin (OPN) is an abundant bone extracellular matrix protein with roles in mineralization， cell ad... More
Seven proprotein convertases cleave the basic amino acid consensus sequence K/R-X-K/R↓ (where n=0， 2， 4 or 6 variable amino acids) to activate precursor proteins. Despite similarities in substrate specificity， basic amino acid-specific proprotein convertases have a distinct tissue distribution allowing for enzymatic actions on tissue-resident substrates. Proprotein convertase 5/6 (PC5/6) has two splice variants - soluble PC5/6A and membrane-bound PC5/6B - and is expressed during mouse development in many tissues including bone and tooth， but little is known about the substrates for PC5/6 therein. Osteopontin (OPN) is an abundant bone extracellular matrix protein with roles in mineralization， cell adhesion and cell migration， and it has putative consensus sequence sites for cleavage by PC5/6， which may modify its function in bone. Since PC5/6-knockout mouse embryos show developmental abnormalities， and reduced overall mineralization， we designed this study to determine whether OPN is a substrate of PC5/6. In silico analysis of OPN protein sequences identified four potential PC5/6 consensus cleavage sites in human OPN， and three sites - including a noncanonical sequence - in mouse OPN. Ex vivo co-transfections with human OPN revealed complete OPN cleavage reducing full-length OPN (~70kDa) to an N-terminal fragment migrating at ~50kDa and two C-terminal fragments at ~18kDa and ~16kDa. Direct cleavage of OPN by PC5/6A - the predominant isoform expressed in human osteoblast cells - was confirmed by cell-free enzyme-substrate assays and by mass spectrometry. The latter was also used to investigate potential cleavage sites. Co-transfections of PC5/6 and mouse OPN showed partial cleavage of OPN into a C-terminal OPN fragment migrating at ~30kDa and an N-terminal fragment migrating at ~29kDa. Micro-computed tomography of PC5/6-knockout embryos at E18.5 confirmed a reduction in mineralized bone， and in situ hybridization performed on cryo-sections of normal mouse bone using Pcsk5 and Opn anti-sense and control-sense cRNA probes indicated the co-localization of the expression of these genes in bone cells. This mRNA expression profile was supported by semi-quantitative RT-PCR using osteoblast primary cultures， and cultured MC3T3-E1 osteoblast and MLO-Y4 osteocyte cell lines. Immunoblotting for OPN from mouse bone extracts showed altered OPN processing in PC5/6-knockout mice compared to wildtype mice. OPN fragments migrated at ~25kDa and ~16kDa in wildtype bone and were not present in PC5/6-deficient bone. In conclusion， this study demonstrates that Pcsk5 is expressed in bone-forming cells， and that OPN is a novel substrate for PC5/6. Cleavage of OPN by PC5/6 may modify the function of OPN in bone and/or modulate other enzymatic cleavages of OPN， leading to alterations in the bone phenotype.
