Cloning and functional expression of rat kidney dipeptidyl peptidase II

Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4 mu mol/min per mg of protein for Lys-Ala-bet a -naphthylamide, The N-terminal and partial amino acid sequences of the en zyme were determined. The peptide sequences were used to identify expressed sequence tag (EST) clones. By using the cDNA fragment of one of the EST cl ones as a probe, we isolated a cDNA clone with 1710 bp encoding DPP II from a rat kidney cDNA library. The cDNA of rat DPP II contained an open readin g frame of 1500 bp, coding for a protein of 500 amino acids. The first 10 r esidues of the purified enzyme matched the deduced protein sequence startin g with residue 37, suggesting the presence of a signal peptide. The mature enzyme (464 residues) had a calculated molecular mass of 51 400 Da, which w as lower than the value (about 60 000 Da) determined by SDS/PAGE; and the d educed amino acid sequence showed six potential N-glycosylation sites. The deduced amino acid sequence of rat DPP II shared high similarity with quies cent-cell proline dipeptidase (78% identity) and prolyl carboxypeptidase (3 8% identity) and bore the putative catalytic triad (Ser, Asp, His) conserve d in serine peptidase families. We transiently transfected COS-7 cells with pcDNA3.1 containing the cloned cDNA and obtained the overexpression of an immunoreactive protein (of about 60 000 Da). The transfected cells showed L ys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher th an the control cells.