Vesicular localization and characterization of a novel post-proline-cleaving aminodipeptidase, quiescent cell proline dipeptidase.

A large number of chemokines, cytokines, and signal peptides share a highly conserved X-Pro motif on the N-terminus. The cleavage of this N-terminal X-Pro dipeptide results in functional alterations of chemokines such as RANTES, stroma-derived factor-1, and macrophage-derived chemokine. Until recently, CD26/DPPIV was the only known protease with the ability to cleave N-terminal X-Pro motifs at neutral pH. We have isolated and cloned a novel serine protease, quiescent cell proline dipeptidase (QPP), with substrate specificity similar to that of CD26/DPPIV. In this paper we show that QPP, like CD26/DPPIV, is synthesized with a propeptide and undergoes N:-glycosylation. Interestingly, this glycosylation is required for QPP enzymatic activity, but not for its localization. Unlike the cell surface molecule, CD26/DPPIV, QPP is targeted to intracellular vesicles that are distinct from lysosomes. Proteinase K treatment of intact vesicles indicates that QPP is located within the vesicles. These vesicles appear to have a secretory component, as QPP is secreted in a functionally active form in response to calcium release. The presence of QPP in the vesicular compartment suggests that molecules bearing the N-terminal X-Pro motif can be cleaved at multiple sites within and outside the cell. These results expand the potential site(s) and scope of a process that appears to be an important mechanism of post-translational regulation.

Homodimerization via a leucine zipper motif is required for enzymatic activity of quiescent cell proline dipeptidase.

Quiescent cell proline dipeptidase (QPP) is an intracellular serine protease that is also secreted upon cellular activation. This enzyme cleaves N-terminal Xaa-Pro dipeptides from proteins, an unusual substrate specificity shared with dipeptidyl peptidase IV (CD26/DPPIV). QPP is a 58-kDa protein that elutes as a 120-130-kDa species from gel filtration, indicating that it forms a homodimer. We analyzed this dimerization with in vivo co-immunoprecipitation assays. The amino acid sequence of QPP revealed a putative leucine zipper motif, and mutational analyses indicated that this leucine zipper is required for homodimerization. The leucine zipper mutants showed a complete lack of enzymatic activity, suggesting that homodimerization is important for QPP function. On the other hand, an enzyme active site mutant retained its ability to homodimerize. These data are the first to demonstrate a role for a leucine zipper motif in a proteolytic enzyme and suggest that leucine zipper motifs play a role in mediating dimerization of a diverse array of proteins.