dc.description.abstract | The family of prolyl dipeptidases has attracted extensive investigation in recent years because of their unique ability to cleave the peptide bond after a penultimate proline residue. It includes dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5), FAP (fibroblast activation protein), DPP2 (E.C. 3.4.14.2), DPP8 and DPP9. DPP-IV is the most extensively studied, and the functions for other members are not known so far. DPP9 and DPP8 are highly homologous proteases with 58% sequence identity at the amino acid level. Both the structure and function of these two proteases are not known.
In this thesis, we first characterized the biochemical property of DPP9 including its enzymatic activity, quaternary structure, substrate specificity and pH optimum. DPP9 was expressed and purified from the baculovirus-infected insect cells using Strep‧TactinTM purification system. The yield is significantly higher than what was reported in the literature. DPP9 has similar enzymatic activity, substrate specificity and pH optimum as DPP8. Both of them are homodimeric. Single site mutation at the C-terminal loop (F842A), one of the dimer interfaces, results in dimeric DPP9 with little enzymatic activity. The results indicate that the interaction mode of dimerization is similar to that of DPP8, reported previously from our lab. Therefore, the biochemical property of DPP9 we discovered so far is almost identical to that of DPP8. We speculate that DPP9 and DPP8 carry out overlapping functions in vivo.
We have also determined the expression profile of DPP9. DPP9 is ubiquitously expressed in different cell types including fibroblasts, epithelial and blood cells. Surprisingly, contrary to previous report, we found that the expression levels of DPP8 and DPP9 do not change upon the activation of T-cells such as PBMC and Jurkat cells. Because DPP8 and DPP9 are ubiquitously expressed, whether they involve in immunological function as speculated awaits further studies. The characterization of DPP9 reported here lays the foundation for revealing its function in the future
DPP-IV is a validated drug target for human type II diabetes. DPP-IV inhibitors without DPP8/9 inhibitory activity have been sought because a possible association was reported between a “DPP8/9 inhibitor” and severe toxicity in animals. However, at present, it is not known whether the observed toxicity is associated with DPP8/9 inhibition, or an off-target effect induced by the compound. We investigated whether the inhibition of DPP8/9 is the cause of the severe toxicity in animals using a very potent and selective DPP8/9 inhibitor with different pharmacophore, 1G244. By Ki measurement, 1G244 is 15- and 8-fold more potent against DPP8 and DPP9, respectively, than the “DPP8/9 inhibitor”. Strikingly, the “DPP8/9 inhibitor” does not penetrate the plasma membrane but remains outside the cells, whereas 1G244 readily enters the cells, even at low doses. By repeatedly exposing Strague-Dawley rats to 1G244 by intravenous injection for a period of 14 days, we observed no significant toxicological symptoms associated with 1G244. Blood and serum chemistry parameters were all within the normal ranges for the treated animals. Because of the high potency, good membrane penetration and adequate tissue distribution of 1G244, the mild symptoms observed are probably associated with DPP8/9 inhibition. Our results demonstrate that there is no direct causal effect of DPP8/9 inhibition with toxicity in animals.
Finally, we have examined the contribution of the propeller loop to the enzymatic activity and quaternary structure of DPP9 and DPP-IV. The propeller loop is one of the two dimer interfaces and its function for the structure and activity of prolyl dipeptidase family is not known. Because the crystal structure of DPP9 has not been resolved, we have identified the sequence corresponding to the propeller loop of DPP9 based on the sequence alignment with other members of prolyl dipeptidases, and computer modeling of DPP8 reported in the literature. The conserved residues or the corresponding residues whose mutations have drastic effects on DPP-IV structure and activity were chosen to be mutated. The mutant proteins were expressed and purified from insect cells. For DPP9, five mutations located on the propeller loop were generated, which include a complete deletion of the propeller loop (DEL), V319A, E325A, K328A and Y334A. Among them, the dimeric structure and enzymatic activity of V319A, E325A and K328A mutant proteins were similar to those of wild type DPP9. In contrast, Y334A and DEL fail to disrupt DPP9 dimers to monomers. However, the mutant dimers are inactive with kcat significantly decreased and Km increased.
Interestingly, differential effects on the structure and activity of DPP-IV were discovered with mutations on the propeller loop. Based on the crystal structure of DPP-IV, we have identified two groups of residues on the propeller loop that are involved in inter-molecular and intra-molecular interactions, Y248 involved in intermolecular interaction, and Y238 and Y256 involved in intramolecular interaction. We have introduced single site mutation to these residues resulting in Y248F, Y248A, Y248T, Y238A and Y256A, respectively. We also generated a deletion mutation, called Del, by deleting the whole propeller loop. We demonstrated that phenyl group of Y248 is essential for dimer formation. Lack of phenyl group, such as Y248T, Y248A and Del, results in monomeric DPP-IVs with very low or no activities. Specifically, Y248A and deletion mutants result in monomers with no activity detectable, while monomeric Y248T has a low kcat and an unchanged Km. Difference on Km effects suggests that the hydroxyl group might be important for the integrity of the substrate binding pocket. Y238A and Y256A mutations result in a mixture of monomers and dimers. Intriguingly, the monomers of Y238A and Y256A were fully active as the dimers. This is drastically different from all the monomeric mutations we generated previously. Further work will be required to fully understand the underlying mechanism of these active monomeric DPP-IVs. In summary, our results demonstrate that the propeller loop exerts differential effect on the structure and activity of DPP-IV and DPP9. Understanding how propeller loop affects the structure and activity of prolyl dipeptidases will help the understanding of the biogenesis and folding of homomeric proteins in the future.
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