Identification of novel dipeptidyl peptidase 9 substrates by two‐dimensional differential in‐gel electrophoresis

• Published in: The FEBS journal Vol. 282; no. 19; pp. 3737 - 3757
• Main Authors: Zhang, Hui, Maqsudi, Sadiqa, Rainczuk, Adam, Duffield, Nadine, Lawrence, Josie, Keane, Fiona M, Justa‐Schuch, Daniela, Geiss‐Friedlander, Ruth, Gorrell, Mark D, Stephens, Andrew N
• Format: Journal Article
• Language: English
• Published: England Published by Blackwell Pub. on behalf of the Federation of European Biochemical Societies 01.10.2015; Blackwell Publishing Ltd
• Subjects: Amino Acid Sequence; Animals; antigens; Capillary electrophoresis; Carbocyanines - chemistry; Cells, Cultured; Chemokine CXCL10 - metabolism; consensus sequence; DIGE; dipeptides; Dipeptides - metabolism; dipeptidyl peptidase; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - genetics; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - metabolism; electrophoresis; Electrophoresis, Gel, Two-Dimensional - methods; Enzymes; epidermal growth factor; Fibroblasts; Fibroblasts - metabolism; Fluorescent Dyes - chemistry; function; immunoblotting; mass spectrometry; mice; Mice, Knockout; Molecular biology; Molecular Sequence Data; neonates; Peptides; proteins; proteolysis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reproducibility of Results; serine proteinases; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; substrates; substrates; mass spectrometry; DIGE; dipeptidyl peptidase; function
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.13371

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.