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)

Abstract

The presence of DPPII (dipeptidyl peptidase II; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40%) purification scheme, including an inhibitor-based affinity chromatographic step. The inhibitor lysyl-piperidide (K(i) approximately 0.9 microM at pH 5.5) was chosen, as it provided a favourable affinity/recovery ratio. The human enzyme appeared as a 120 kDa homodimer. Mass spectrometric analysis after tryptic digestion together with a kinetic comparison indicate strongly its identity with QPP (quiescent cell proline dipeptidase), also called dipeptidyl peptidase 7. pH profiles of both kcat and kcat/K(m) clearly demonstrated that DPPII/QPP possesses an acidic and not a neutral optimum as was reported for QPP. Kinetic parameters of the human natural DPPII for dipeptide-derived chromogenic [pNA (p-nitroanilide)] and fluorogenic [4Me2NA (4-methoxy-2-naphthylamide)] substrates were determined under different assay conditions. DPPII preferred the chromogenic pNA-derived substrates over the fluorogenic 4Me2NA-derived substrates. Natural human DPPII showed high efficiency towards synthetic substrates containing proline at the P1 position and lysine at P2. The importance of the P1' group for P2 and P1 selectivity was revealed, explaining many discrepancies in the literature. Furthermore, substrate preferences of human DPPII and dipeptidyl peptidase IV were compared based on their selectivity constants (kcat/K(m)). Lys-Pro-pNA (k(cat)/K(m) 4.1x10(6) s(-1) x M(-1)) and Ala-Pro-pNA (kcat/K(m) 2.6x10(6) s(-1) x M(-1)) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (kcat/K(m) 0.4x10(6) s(-1) x M(-1)).

Figure 1. Selective DPPII inhibitors

1, Lys-Pip; 2, N-(4-chlorobenzyl)-4-oxo-4-(1-piperidinyl)-1,3-(S)-butane-diamine.

Figure 2. SDS/PAGE analysis of purified human DPPII

The purified enzyme was resolved by SDS/10% PAGE under reducing conditions. Lanes 1 and 2 contain 0.3 and 0.9 μg of purified DPPII respectively. Molecular-mass standards (sizes indicated in kDa) are shown in lane 3. Proteins were visualized using silver staining. The molecular mass estimated for DPPII was approx. 60 kDa.

Figure 3. MS analysis of silver-stained SDS/PAGE band

(A) The nano-electrospray MS spectrum in the m/z region from 400 to 1000 of the peptides generated by tryptic digestion of the silver-stained band. Five multiple-charged peptides were chosen for fragmentation. These precursor ions are shown in the spectrum. The five multiple-charged peptides are 556.75(+2), 596.26(+2), 647.26(+2), 699.33(+2) and 732.86(+2). (B) The electrospray MS/MS spectrum for the double-charged ion at m/z 596.26 is shown in b and y series, and inspected amino acid sequence are indicated (one-letter amino acid codes are used).

Figure 4. Alignment of the amino acid sequences of QPP and DPPII, and sequence coverage of the identified tryptic peptides of the purified human DPPII

(A) ClustalW multiple sequence alignment of the N-terminal sequences of human QPP (hQPP), and human and pig DPPII (hDPPII and pDPPII respectively). (B) ClustalW multiple sequence alignment of human and mouse QPP (hQPP and mQPP respectively), and rat DPPII (rDPPII). Residues most probably forming the catalytic triad are labelled with an asterisk. The consensus sequence for the active site serine residue of serine-type proteases (Gly-Xaa-Ser-Xaa-Gly) is underlined the leucine zipper motif is marked in bold. The tryptic peptide sequences of the purified human DPPII identified by MS are marked with grey boxes in the human QPP sequence. GenBank® accession numbers are as follows: hQPP, AAF12747; hDPPII, S77568; pDPPII, S70349; mQPP, AAG01154; rDPPII, Q9EPB1.

Figure 5. pH profiles of purified DPPII and recombinant QPP with different substrates

Measurements were carried out using the same reagent dilutions for both purified DPPII (△) and recombinant QPP (○) in 0.025 M ethanoic acid, 0.025 M cacodylic acid and 0.025 M Hepes buffer, containing 0.1 M NaCl (titrated with NaOH or HCl to the desired pH) at 37 °C as described under the Materials and methods section, with (A) Lys-Ala-pNA and (B) Ala-Pro-pNA. The initial rate was proportional to k_cat. The experimental data were fitted to a double ionization equation (eqn 1) that results in a bell-shaped curve using GraFit software version 5 [38].

Figure 6. Effects of increasing NaCl concentrations on the steady-state kinetic parameters

Initial velocities of substrate cleavage were measured with different concentrations of substrate at pH 5.5 and pH 7.0 in buffer containing (○) 0 M NaCl, (□) 0.15 M NaCl or (△) 0.75 M NaCl. The Michaelis–Menten plot of the experimental data was generated using GraFit version 5 [38]. (A) Lys-Ala-pNA in 0.05 M cacodylic acid buffer, pH 5.5. (B) Lys-Ala-pNA in 0.05 M Hepes buffer, pH 7.0. (C) Ala-Pro-pNA in 0.05 M cacodylic acid buffer, pH 5.5. (D) Ala-Pro-pNA in 0.05 M Hepes buffer, pH 7.0.

Figure 7. Comparison of the substrate preferences of human DPPII and human DPPIV

Selectivity constants k_cat/K_m (×10⁶ M⁻¹·s⁻¹) of human DPPII and human DPPIV were compared for substrates of the type P₁-P₂-pNA in 50 mM cacodylic acid, pH 5.5, and 50 mM Tris/HCl, pH 8.3, respectively.