Cloning and functional expression of rat kidney dipeptidyl peptidase II

Abstract

Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4 micromol/min per mg of protein for Lys-Ala-beta-naphthylamide. The N-terminal and partial amino acid sequences of the enzyme were determined. The peptide sequences were used to identify expressed sequence tag (EST) clones. By using the cDNA fragment of one of the EST clones 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 reading frame of 1500 bp, coding for a protein of 500 amino acids. The first 10 residues of the purified enzyme matched the deduced protein sequence starting with residue 37, suggesting the presence of a signal peptide. The mature enzyme (464 residues) had a calculated molecular mass of 51400 Da, which was lower than the value (about 60000 Da) determined by SDS/PAGE; and the deduced amino acid sequence showed six potential N-glycosylation sites. The deduced amino acid sequence of rat DPP II shared high similarity with quiescent-cell proline dipeptidase (78% identity) and prolyl carboxypeptidase (38% identity) and bore the putative catalytic triad (Ser, Asp, His) conserved 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 60000 Da). The transfected cells showed Lys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher than the control cells.

Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell proline dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7)

“…The natural DPPII from human seminal plasma appeared to be a homodimer of 120 kDa composed of 60 kDa subunits. These data are in good agreement with the molecular masses determined for DPPII from other sources [9,10,12,14,45] and for human QPP [24,26]. In contrast, Huang et al [17] reported that the DPPII purified from porcine seminal plasma was composed of three identical subunits.…”

“…QPP was able to cleave substrate molecules as efficiently at acidic pH as at neutral pH. Fukasawa et al [10] mentioned a similar high activity of rat kidney DPPII at neutral pH using Gly-Pro-β-naphthylamide as the substrate. DPPII from other sources only showed an acidic pH optimum for dipeptide-derived substrate cleavage [6][7][8][9][11][12][13][14][15][16][17]41].…”

“…From then on, DPPII (E.C. 3.4.14.2) has been demonstrated in various mammalian tissues [6][7][8][9][10][11][12][13][14][15][16][17]. However, a profound enzymological and molecular characterization, especially of human natural DPPII is missing.…”

“…Since DPPII is glycosylated [10,14,15,19], we preferred to use the natural enzyme for biochemical and enzymological characterization. In the present study, we introduce a protocol that can be used for high-yield purification of DPPII from human seminal plasma.…”

Cloning and characterization of dipeptidyl peptidase 10, a new member of an emerging subgroup of serine proteases

“…DASH genes with sequence homology to DPP4 include fibroblast activation protein (FAP) [10][11][12][13][14], DPP6/DDPX [15] and the recently cloned DPP8 [16]. Cloned DASH proteins with DPP4-like substrate specificity include DPP7 (human quiescent cell proline dipeptidase, the probable orthologue of DPPII [17,18]) and N-acetylated α-linked acidic dipeptidases I, II and L [19,20]. Purified DASH proteins, with enzymic activities similar to DPP4, include DPP4β [21][22][23] and attractin/mahogany protein [24].…”