Identification of novel dipeptidyl peptidase 9 substrates by two‐dimensional differential in‐gel electrophoresis
Hui Emma Zhanga b, Sadiqa Maqsudic d e f, Adam Rainczukc d e f, Nadine Duffieldc d e f, Josie Lawrencec d e f, Fiona M. Keanea b, Daniela Justa‐Schuchg h, Ruth Geiss‐Friedlanderg h, Mark D. Gorrella b✉, Andrew N. Stephensc d e f i j✉
- aMolecular Hepatology Liver Injury and Cancer Group Centenary Institute Sydney Medical School University of Sydney Australia
- bMolecular Hepatology, Liver Injury and Cancer Group, Centenary Institute, Sydney Medical School, University of Sydney, Australia
- cCentre for Cancer Research Hudson Institute of Medical Research Clayton Australia
- dDepartment of Molecular and Translational Sciences Monash University Clayton Australia
- eCentre for Cancer Research, Hudson Institute of Medical Research, Clayton, Australia
- fDepartment of Molecular and Translational Sciences, Monash University, Clayton, Australia
- gDepartment of Molecular Biology Faculty of Medicine Georg‐August‐University of Goettingen Germany
- hDepartment of Molecular Biology, Faculty of Medicine, Georg-August-University of Goettingen, Germany
- iEpworth Research Institute Epworth HealthCare Richmond Victoria Australia
- jEpworth Research Institute, Epworth HealthCare, Richmond, Victoria, Australia
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Abstract
Dipeptidyl peptidase 9 (DPP9) is a member of the S9B/DPPIV (DPP4) serine protease family, which cleaves N-terminal dipeptides at an Xaa-Pro consensus motif. Cytoplasmic DPP9 has roles in epidermal growth factor signalling and in antigen processing, whilst the role of the recently discovered nuclear form of DPP9 is unknown. Mice lacking DPP9 proteolytic activity die as neonates. We applied a modified 2D differential in-gel electrophoresis approach to identify novel DPP9 substrates, using mouse embryonic fibroblasts lacking endogenous DPP9 activity. A total of 111 potential new DPP9 substrates were identified, with nine proteins/peptides confirmed as DPP9 substrates by MALDI-TOF or immunoblotting. Moreover, we also identified the dipeptide Val-Ala as a consensus site for DPP9 cleavage that was not recognized by DPP8, suggesting different in vivo roles for these closely related enzymes. The relative kinetics for the cleavage of these nine candidate substrates by DPP9, DPP8 and DPP4 were determined. This is the first identification of DPP9 substrates from cells lacking endogenous DPP9 activity. These data greatly expand the potential roles of DPP9 and suggest different in vivo roles for DPP9 and DPP8.
TL;DRThis work identified the dipeptide Val‐Ala as a consensus site for DPP9 cleavage that was not recognized by DPP8, suggesting different in vivo roles for these closely related enzymes. (Semantic Scholar)