A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2.

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

Hematopoietic cell- versus enterocyte-derived dipeptidyl peptidase-4 differentially regulates triglyceride excursion in mice.

Postprandial triglycerides (TGs) are elevated in people with type 2 diabetes (T2D). Glucose-lowering agents, such as glucagon-like peptide-1 (GLP-1) receptor agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors, also reduce postprandial TG excursion. Although the glucose-lowering mechanisms of DPP-4 have been extensively studied, how the reduction of DPP-4 activity improves lipid tolerance remains unclear. Here, we demonstrate that gut-selective and systemic inhibition of DPP-4 activity reduces postprandial TG excursion in young mice. Genetic inactivation of Dpp4 simultaneously within endothelial cells and hematopoietic cells using Tie2-Cre reduced intestinal lipoprotein secretion under regular chow diet conditions. Bone marrow transplantation revealed a key role for hematopoietic cells in modulation of lipid responses arising from genetic reduction of DPP-4 activity. Unexpectedly, deletion of Dpp4 in enterocytes increased TG excursion in high-fat diet-fed (HFD-fed) mice. Moreover, chemical reduction of DPP-4 activity and increased levels of GLP-1 were uncoupled from TG excursion in older or HFD-fed mice, yet lipid tolerance remained improved in older Dpp4-/- and Dpp4EC-/- mice. Taken together, this study defines roles for specific DPP-4 compartments, age, and diet as modifiers of DPP-4 activity linked to control of gut lipid metabolism.

Purification and biochemical characterization of a novel secretory dipeptidyl peptidase IV from porcine serum.

Purification of DPP-IV enzyme from porcine serum, is presented in this study for the first time. The high molecular weight DPP-IV from porcine serum was fractioned using Sephadex G-75 gel filtration followed by DEAE Sephadex anion exchange and Sephadex G-100 gel filtration chromatography columns with a final yield of 11.25%. The SDS-PAGE of the purified sample showed a single band of molecular mass nearing 160 kDa. Distinct single band was observed after PAS staining confirmed it to be a glycoprotein. The purified enzyme showed an optimum pH and temperature of 8 and 37 °C, respectively. The enzyme effectively cleaved fluorogenic substrate Gly-Pro-AMC with Km and Vmax of 4.578 µM and 90.84 nmoles/min, respectively. Purified DPP-IV activity was inhibited by Diprotin A with an IC50 value of 8.473 µM. Among the three plant extracts used to study DPP-IV inhibition, the aqueous hot extract of Terminalia chebula showed the highest inhibition of 87.19%, followed by the aqueous cold extract of Momordica carantia, ( 31.6%) and Azadirachta indica (34.16%) at the concentration of 25 µg.

Flow Cytometry Assessment of CD26+ Leukemic Stem Cells in Peripheral Blood: A Simple and Rapid New Diagnostic Tool for Chronic Myeloid Leukemia.

BACKGROUND: Recent investigations in chronic myeloid leukemia (CML) have focused on the identification and characterization of leukemic stem cells (LSCs). These cells reside within the CD34+ /CD38─ /Lin─ fraction and score positive for CD26 (dipeptidylpeptidase IV) a marker, expressed in both bone marrow (BM) and peripheral blood (PB) samples, that discriminates CML cells from normal hematopoietic stem cells (HSCs) or from LSCs of other myeloid neoplasms. CD26 evaluation could be a useful tool to improve the identification of CML LCSs by using flow-cytometry assay. METHODS: CD26+ LSCs have been isolated from EDTA PB and BM samples of patients with leucocytosis suspected for CML. Analysis of LSCs CML has been performed by using custom-made lyophilized pre-titrated antibody mixture test and control tube and a CD45+ /CD34+ /CD38- /CD26+ panel as a strict flow cytometric gating strategy. RESULTS: The expression of CD26 on CD34+ /CD38- population was detectable in 211/211 PB and 84/84 BM samples of subsequently confirmed BCR-ABL+ CP-CML patients. None of the 32 samples suspicious for CML but scoring negative for circulating CD26+ LSCs were diagnosed as CML after conventional cytogenetic and molecular testing. To validate our results, we checked for PB CD26+ LSCs in patients affected by other hematological disorders and they all scored negative for CD26 expression. CONCLUSIONS: We propose flow cytometry evaluation of CD26 expression on PB CD34+ /CD38- population as a new rapid, reproducible, and powerful diagnostic tool for the diagnosis of CML. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Periplasmic form of dipeptidyl aminopeptidase IV from Pseudoxanthomonas mexicana WO24: purification, kinetic characterization, crystallization and X-ray crystallographic analysis.

Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) from Pseudoxanthomonas mexicana WO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1'-P2'…]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced in Escherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90 Šresolution were collected from a triclinic crystal form belonging to space group P1, with unit-cell parameters a = 88.66, b = 104.49, c = 112.84 Å, α = 67.42, ß = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method using Stenotrophomonas maltophilia DPP IV (PDB entry 2ecf) as a template and refinement of the structure is in progress.

Anagliptin, a potent dipeptidyl peptidase IV inhibitor: its single-crystal structure and enzyme interactions.

The single-crystal structure of anagliptin, N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide, was determined. Two independent molecules were held together by intermolecular hydrogen bonds, and the absolute configuration of the 2-cyanopyrrolidine ring delivered from l-prolinamide was confirmed to be S. The interactions of anagliptin with DPP-4 were clarified by the co-crystal structure solved at 2.85 Å resolution. Based on the structure determined by X-ray crystallography, the potency and selectivity of anagliptin were discussed, and an SAR study using anagliptin derivatives was performed.

Identification of protein receptors for coronaviruses by mass spectrometry.

As obligate intracellular parasites, viruses need to cross the plasma membrane and deliver their genome inside the cell. This step is initiated by the recognition of receptors present on the host cell surface. Receptors can be major determinants of tropism, host range, and pathogenesis. Identifying virus receptors can give clues to these aspects and can lead to the design of intervention strategies. Interfering with receptor recognition is an attractive antiviral therapy, since it occurs before the viral genome has reached the relative safe haven within the cell. This chapter describes the use of an immunoprecipitation approach with Fc-tagged viral spike proteins followed by mass spectrometry to identify and characterize the receptor for the Middle East respiratory syndrome coronavirus. This technique can be adapted to identify other viral receptors.

In vitro evaluation of caffeoyl and cinnamoyl derivatives as potential prolyl oligopeptidase inhibitors.

A screening of the natural product chlorogenic acid, isolated from the Brazilian medicinal plant Hypericum brasiliense, caffeic acid, cinnamic acid, and p-methoxycinnamic acid, and derivatives of caffeoylquinic, caffeoyl, and cinnamoyl against the enzymes prolyl oligopeptidase and dipeptidyl peptidase IV was carried out. Caffeoylquinic, caffeoyl, and cinnamoyl derivatives were prepared using simple derivatization procedures and through coupling reactions with the amino acid proline. The dipeptidyl peptidase IV assay showed inhibitory activity of the tested compounds at a high concentration (500 µM) in the range of 81.5-7.2 %. In contrast, the derivatives methyl ester and 1,7-acetonide obtained from chlorogenic acid, and caffeic acid and its methyl ester derivative showed selectivity and activity as prolyl oligopeptidase inhibitors, with IC50 values of 3 to 14 mM.

Effect of zinc and calcium ions on the rat kidney membrane-bound form of dipeptidyl peptidase IV.

Dipeptidyl peptidase IV (DPP-IV) is an ectopeptidase with many roles, and a target of therapies for different pathologies. Zinc and calcium produce mixed inhibition of porcine DPP-IV activity. To investigate whether these results may be generalized to mammalian DPP-IV orthologues, we purified the intact membrane-bound form from rat kidney. Rat DPP-IV hydrolysed Gly-Pro-p-nitroanilide with an average Vmax of 0.86 +/- 0.01 meu mol min-1mL-1 and KM of 76 +/- 6 meu M. The enzyme was inhibited by the DPP-IV family inhibitor L-threo-Ile-thiazolidide (Ki=64.0 +/- 0.53 nM), competitively inhibited by bacitracin (Ki=0.16 +/- 0.01 mM) and bestatin (Ki=0.23 +/- 0.02 mM), and irreversibly inhibited by TLCK (IC50 value of 1.20 +/- 0.11 mM). The enzyme was also inhibited by divalent ions like Zn2+ and Ca2+, for which a mixed inhibition mechanism was observed (Ki values of the competitive component: 0.15 +/- 0.01 mM and 50.0 +/- 1.05 mM, respectively). According to bioinformatic tools, Ca2+ ions preferentially bound to the beta-propeller domain of the rat and human enzymes, while Zn2+ ions to the alpha-beta hydrolase domain; the binding sites were essentially the same that were previously reported for the porcine DPP-IV. These data suggest that the cationic susceptibility of mammalian DPP-IV orthologues involves conserved mechanisms.

Production and separation of dipeptidyl peptidase IV from Lactococcus lactis: scale up for industrial production.

Lactococcus lactis spp. lactis and Lactococcus lactis spp. cremoris are widely used in the manufacture of fermented milk. These strains were compared for production of Dipeptidyl Peptidase IV (DPP IV) enzyme in terms of enzyme activity, specific growth rates and productivity. Lactococcus lactis spp. lactis was produced in 3 L bioreactor and scaled up to 30 and 150 L stirred tank bioreactors, and the enzyme activities were found as 110, 110 and 122 mU mL(-1), respectively. After 8 h of production, separation steps were performed. While purification fold was 127 and yield was 2.69 %, the molecular weight of the enzyme was estimated as 68 kDa. Partially purified enzyme was enteric coated with capsules and a 95.5 % of DPP IV enzyme passed into the artificial intestine. Results show that production of DPP IV enzyme by Lactococcus lactis spp. lactis strain in submerged culture is comparable with the productions by commercial strains, mostly Aspergillus, in solid state fermentations based on productivity.

Functional expression and characterization of dipeptidyl peptidase IV from the black-bellied hornet Vespa basalis in Sf21 insect cells.

The maturation of mastoparan B, the major toxin peptide in the venom of Vespa basalis, requires enzymatic cleavage of its prosequence presumably via sequential liberation of dipeptides. The putative processing enzyme, dipeptidyl peptidase IV, was expressed as a glycosylated His-tag fusion protein (rDPP-IV) via the baculovirus expression system. rDPP-IV purified by one-step nickel-affinity chromatography was verified by Western blot and LC-MS/MS analysis. The k(cat)/K(m) of rDPP-IV was determined to be in the range of 10-500 mM(-1)·S(-1) for five synthetic substrates. The optimal temperature and pH for rDPP-IV were determined to be 50 °C and pH 9. Enzymatic activity of rDPP-IV was significantly reduced by 80 and 60% in the presence of sitagliptin and phenylmethylsulfonyl fluoride respectively.

Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization.

Abstract In the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4-6, activity staining resulted in at least 17 isoforms between pH 4.8-6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5-6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.

Effect of divalent cations on the porcine kidney cortex membrane-bound form of dipeptidyl peptidase IV.

Dipeptidyl peptidase IV is an ectopeptidase with multiple physiological roles including the degradation of incretins, and a target of therapies for type 2 diabetes mellitus. Divalent cations can inhibit its activity, but there has been little effort to understand how they act. The intact membrane-bound form of porcine kidney dipeptidyl peptidase IV was purified by a simple and fast procedure. The purified enzyme hydrolyzed Gly-Pro-p-nitroanilide with an average V(max) of 1.397±0.003 µmol min(-1) mL(-1), k(cat) of 145.0±1.2 s(-1), K(M) of 0.138±0.005 mM and k(cat)/K(M) of 1050 mM(-1) s(-1). The enzyme was inhibited by bacitracin, tosyl-L-lysine chloromethyl ketone, and by the dipeptidyl peptidase IV family inhibitor L-threo-Ile-thiazolidide (K(i) 70 nM). The enzyme was inhibited by the divalent ions Ca(2+), Co(2+), Cd(2+), Hg(2+) and Zn(2+), following kinetic mechanisms of mixed inhibition, with K(i) values of 2.04×10(-1), 2.28×10(-2), 4.21×10(-4), 8.00×10(-5) and 2.95×10(-5) M, respectively. According to bioinformatic tools, Ca(2+) ions preferentially bound to the ß-propeller domain of the porcine enzyme, while Zn(2+) ions to the α-ß hydrolase domain; the binding sites were strikingly conserved in the human enzyme and other homologues. The functional characterization indicates that porcine and human homologues have very similar functional properties. Knowledge about the mechanisms of action of divalent cations may facilitate the design of new inhibitors.

Histoplasma capsulatum encodes a dipeptidyl peptidase active against the mammalian immunoregulatory peptide, substance P.

The pathogenic fungus Histoplasma capsulatum secretes dipeptidyl peptidase (Dpp) IV enzyme activity and has two putative DPPIV homologs (HcDPPIVA and HcDPPIVB). We previously showed that HcDPPIVB is the gene responsible for the majority of secreted DppIV activity in H. capsulatum culture supernatant, while we could not detect any functional contribution from HcDPPIVA. In order to determine whether HcDPPIVA encodes a functional DppIV enzyme, we expressed HcDPPIVA in Pichia pastoris and purified the recombinant protein. The recombinant enzyme cleaved synthetic DppIV substrates and had similar biochemical properties to other described DppIV enzymes, with temperature and pH optima of 42 degrees C and 8, respectively. Recombinant HcDppIVA cleaved the host immunoregulatory peptide substance P, indicating the enzyme has the potential to affect the immune response during infection. Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism. However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis. This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P.

In vivo profiling of DPP4 inhibitors reveals alterations in collagen metabolism and accumulation of an amyloid peptide in rat plasma.

Dipeptidyl peptidase 4 (DPP4) inhibitors represent a novel class of oral anti-hyperglycemic agents. The complete pharmacological profile of these protease inhibitors remains unclear. In order to gain deeper insight into the in vivo effects caused by DPP4 inhibition, two different DPP4 inhibitors (vildagliptin and AB192) were analyzed using differential peptide display. Wistar rats were treated with the DPP4 inhibitors (0.3mgkg(-1); 1mgkg(-1) or 3mgkg(-1) body weight) and DPP4 activity was measured before and at the end of the experiment. One hour after compound administration, blood plasma samples were collected to generate peptide displays and to subsequently identify differentially regulated peptides. A dose-dependent decrease in blood plasma DPP4 activity was measured for both inhibitors. DPP4 inhibition influenced collagen metabolism leading to depletion of collagen derived peptides (e.g. collagen alpha 1 (III) 521-554) and accumulation of related N-terminally extended collagen derived peptides (e.g. collagen alpha 1 (III) 519-554). Furthermore, the intact amyloid rat BRI (1-23) peptide was detected in plasma following in vivo DPP4 inhibition. DPP4 catalyzed cleavage kinetics of the BRI peptide were determined in vitro. The k(cat) and K(m) for cleavage by DPP4 were 5.2s(-1) and 14microM, respectively, resulting in a specificity constant k(cat)/K(m) of 0.36 x 10(6)s(-1)M(-1). Our results demonstrate that differential peptide analysis can be applied to monitor action of DPP4 inhibition in blood plasma. For the first time effects on basal collagen metabolism following DPP4 inhibition in vivo were demonstrated and the BRI amyloid peptide was identified as a novel DPP4 substrate.

Dipeptide boronic acid inhibitors of dipeptidyl peptidase IV: determinants of potency and in vivo efficacy and safety.

Dipeptidyl peptidase IV (DPP-IV; E.C. 3.4.14.5), a serine protease that degrades the incretin hormones GLP-1 and GIP, is now a validated target for the treatment of type 2 diabetes. Dipeptide boronic acids, among the first, and still among the most potent DPP-IV inhibitors known, suffer from a concern over their safety. Here we evaluate the potency, in vivo efficacy, and safety of a selected set of these inhibitors. The adverse effects induced by boronic acid-based DPP-IV inhibitors are essentially limited to what has been observed previously for non-boronic acid inhibitors and attributed to cross-reactivity with DPP8/9. While consistent with the DPP8/9 hypothesis, they are also consistent with cross-reactivity with some other intracellular target. The results further show that the potency of simple dipeptide boronic acid-based inhibitors can be combined with selectivity against DPP8/9 in vivo to produce agents with a relatively wide therapeutic index (>500) in rodents.

Purification and characterization of dipeptidyl peptidase IV-like enzymes from bovine testes.

Until now, only recombinant forms of dipeptidyl peptidase (DPP) 8 and 9 have been characterized. We purified non DPPII-non DPPIV enzymes from a natural source. A first DPP8/9-like enzyme was enriched 1160-fold from bovine testes and identified as 'DPP9-like enzyme' by using an anti-DPP9 antibody. A second 576-fold enriched preparation ('DPP enriched peak 3') also showed DPP8/9-like activity. SDS-PAGE analysis showed that the DPP9-like enzyme had a monomeric molecular mass of approx. 100 kDa. Size exclusion chromatography generated a native molecular mass of 164 kDa for the DPP9-like enzyme and one of 234 kDa for the DPP enriched peak 3, suggesting that both proteins appeared to be dimeric. Both enriched preparations and rDPP8 showed roughly similar substrate specificity and inhibitor profiles. The DPP9-like enzyme and the DPP enriched peak 3 possessed a neutral pH optimum and were stable at -80 degrees C. We can conclude that the natural DPP9-like enzyme and the DPP enriched peak 3 are closely related to the recombinant forms of human DPP9 and DPP8.

Stromal cell-derived factors 1alpha and 1beta, inflammatory protein-10 and interferon-inducible T cell chemo-attractant are novel substrates of dipeptidyl peptidase 8.

N-terminal truncation of chemokines by proteases including dipeptidyl peptidase (DP) IV significantly alters their biological activity; generally ablating cognate G-protein coupled receptor engagement and often generating potent receptor antagonists. DP8 is a recently recognised member of the prolyl oligopeptidase gene family that includes DPIV. Since DPIV is known to process chemokines we surveyed 27 chemokines for cleavage by DP8. We report DP8 cleavage of the N-terminal two residues of IP10 (CXCL10), ITAC (CXCL11) and SDF-1 (CXCL12). This has implications for DP8 substrate specificity. Chemokine cleavage and inactivation may occur in vivo upon cell lysis and release of DP8 or in the inactivation of internalized chemokine/receptor complexes.

Crystal structures of DPP-IV (CD26) from rat kidney exhibit flexible accommodation of peptidase-selective inhibitors.

Dipeptidyl peptidase IV (DPP-IV) belongs to a family of serine peptidases, and due to its indirect regulatory role in plasma glucose modulation, DPP-IV has become an attractive pharmaceutical target for diabetes therapy. DPP-IV inactivates the glucagon-like peptide (GLP-1) and several other naturally produced bioactive peptides that contain preferentially a proline or alanine residue in the second amino acid sequence position by cleaving the N-terminal dipeptide. To elucidate the details of the active site for structure-based drug design, we crystallized a natural source preparation of DPP-IV isolated from rat kidney and determined its three-dimensional structure using X-ray diffraction techniques. With a high degree of similarity to structures of human DPP-IV, the active site architecture provides important details for the design of inhibitory compounds, and structures of inhibitor-protein complexes offer detailed insight into three-dimensional structure-activity relationships that include a conformational change of Tyr548. Such accommodation is exemplified by the response to chemical substitution on 2-cyanopyrrolidine inhibitors at the 5 position, which conveys inhibitory selectivity for DPP-IV over closely related homologues. A similar conformational change is also observed in the complex with an unrelated synthetic inhibitor containing a xanthine core that is also selective for DPP-IV. These results suggest the conformational flexibility of Tyr548 is unique among protein family members and may be utilized in drug design to achieve peptidase selectivity.

Use of short monolithic columns for isolation of low abundance membrane proteins.

Convective interaction media (CIM) monoliths provide a stationary phase with a high binding capacity for large molecules and are capable of high flow rates at a very low pressure drop. Used as anion- and cation-exchangers or with affinity ligands such as antibodies, these columns have the potential for processing large volumes of complex biological mixtures within a short time. In the present report, monoclonal antibodies against several rat liver plasma membrane proteins were bound and cross-linked to protein A or protein G CIM affinity columns with a bed volume of only 60 microL. Antigens recognized by bound antibodies and co-eluting (interacting) proteins were rapidly isolated in a single step from either total plasma membrane extracts or subfractions isolated using anion-exchange CIM disk-shaped columns. The isolated antigens and co-eluting proteins were subsequently identified by immunoblot or by LC-MS/MS.