title: Identification of novel dipeptidyl peptidase 9 substrates by two-dimensional differential in-gel electrophoresis.
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.
authors.affiliation: Molecular Hepatology, Liver Injury and Cancer Group, Centenary Institute, Sydney Medical School, University of Sydney, Australia.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Australia.|Department of Molecular and Translational Sciences, Monash University, Clayton, Australia.
authors.forename: Sadiqa
authors.initials: S
authors.lastname: Maqsudi
authors.affiliation: Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Australia.|Department of Molecular and Translational Sciences, Monash University, Clayton, Australia.
authors.forename: Adam
authors.initials: A
authors.lastname: Rainczuk
authors.affiliation: Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Australia.|Department of Molecular and Translational Sciences, Monash University, Clayton, Australia.
authors.forename: Nadine
authors.initials: N
authors.lastname: Duffield
authors.affiliation: Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Australia.|Department of Molecular and Translational Sciences, Monash University, Clayton, Australia.
authors.forename: Josie
authors.initials: J
authors.lastname: Lawrence
authors.affiliation: Molecular Hepatology, Liver Injury and Cancer Group, Centenary Institute, Sydney Medical School, University of Sydney, Australia.
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Keane
authors.affiliation: Department of Molecular Biology, Faculty of Medicine, Georg-August-University of Goettingen, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Justa-Schuch
authors.affiliation: Department of Molecular Biology, Faculty of Medicine, Georg-August-University of Goettingen, Germany.
authors.forename: Ruth
authors.initials: R
authors.lastname: Geiss-Friedlander
authors.affiliation: Molecular Hepatology, Liver Injury and Cancer Group, Centenary Institute, Sydney Medical School, University of Sydney, Australia.
authors.forename: Mark D
authors.identifier: http://orcid.org/0000-0002-0528-2604
authors.initials: MD
authors.lastname: Gorrell
authors.affiliation: Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Australia.|Department of Molecular and Translational Sciences, Monash University, Clayton, Australia.|Epworth Research Institute, Epworth HealthCare, Richmond, Victoria, Australia.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Stephens
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002232:Carbocyanines / Q000737:chemistry; D002478:Cells, Cultured; D054357:Chemokine CXCL10 / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D002232:Carbocyanines; D054357:Chemokine CXCL10; C485433:Cxcl10 protein, mouse; D004151:Dipeptides; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C085321:cyanine dye 5; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C585325:dipeptidyl peptidase 8, mouse; C585326:dipeptidyl peptidase 9, mouse
keywords: DIGE; dipeptidyl peptidase; function; mass spectrometry; substrates