Investigating the biological importance of Dipeptidyl Peptidase 9 enzymatic activity using a mouse model

Dipeptidyl peptidase 9 (DPP9) is an atypical post-proline serine protease of the DPP4 enzyme family which has ubiquitous expression and DPP4-like enzymatic activity. It is localised intracellularly and has roles in antigen processing and epidermal growth factor signalling. It interacts with H-Ras and small ubiquitin-like modifier-1 and influences cellular interactions with extracellular matrix. Our lab made the first DDP9 gene knock-in (gki) mouse with a DPP9 active site (S729A) mutation which results in a lack of DPP9 enzymatic activity. This thesis investigated the biological properties of DPP9 using this genetically modified mouse model. Mice lacking DPP9 proteolytic activity die in the early neonatal stage. This study validated that the DPP9 protein, while present in the gki mouse, is enzyme-inactive and the DPP9-S729A protein and wild-type DPP9 have similar subcellular localisation. A range of investigations were undertaken to determine if obvious histological and/or physiological differences exist between early neonate DPP9S729A/S729A homozygotes from their heterozygous and wild-type littermates and involved analysis of organ morphology, histology and function and investigation of autophagy. The novel findings of this study show that DPP9 enzymatic activity is essential for early neonatal survival in mice and suggest that there is dysregulated autophagy in the DPP9-enzyme-activity-deficient neonatal mice that may contribute to the lethal phenotype. Other members of the DPP4 protein family are implicated in wound healing with fibroblast activation protein expressed in the granulation tissue of healing wounds and DPP4-positive T cells involved in regulation of granulation tissue formation. As there is a heterozygous effect on survival to weaning and DPP9 immunoreactivity was shown in the skin of both WT and DPP9S729A/S729A neonates, the skin of adult mice was studied using a model of cutaneous wound healing. This involved analysis of wound closure rates, tensile strength and collagen levels of both wounded and steady state tissue in adult DPP9wt/S729A mice and their wild-type littermates. It was found that adult heterozygous DPP9-GKI mice display reduced skin collagen levels compared to their WT littermates, but that the rate of skin wound healing appeared unaffected. DPP9 has in vivo expression in normal immunological tissues and major lymphocyte populations that alters with chronic liver injury, suggesting a role in immune function. To investigate the importance of DPP9 in immune regeneration and function, primary and secondary mixed chimeric mice were created using DPP9S729A/S729A and WT fetal liver cells and adult bone marrow, respectively, injected into irradiated recipients. The process of regeneration of the immune system in these mice was assessed at several time-points and the immune cells present in the regenerated immune systems were compared between DPP9S729A/S729A-origin and WT-origin chimeras. These chimeric mice were then subjected to an immune challenge by influenza virus infection. The novel findings of this study suggest that DPP9-enzyme-activity-deficient secondary chimeric mice may have an enhanced ability to recover from an immune challenge with influenza virus. Therefore, DPP9 appears to influence immune function in that viral infection. Together, these studies underline the biological significance of DPP9 in vivo in both neonatal and adult mice. These data contribute to the expanding knowledge of the role of DPP9, building upon recent discoveries in vitro of its involvement in multiple biological processes. By providing physiologically relevant results, this study has enhanced the knowledge of DPP9 enzyme function and the overall biological roles of DPP9