Contribution of amino acids in the active site of dipeptidyl peptidase 4 to the catalytic action of the enzyme.

Dipeptidyl peptidase 4 (DP4)/CD26 regulates the biological function of various peptide hormones by releasing dipeptides from their N-terminus. The enzyme is a prominent target for the treatment of type-2 diabetes and various DP4 inhibitors have been developed in recent years, but their efficacy and side effects are still an issue. Many available crystal structures of the enzyme give a static picture about enzyme-ligand interactions, but the influence of amino acids in the active centre on binding and single catalysis steps can only be judged by mutagenesis studies. In order to elucidate their contribution to inhibitor binding and substrate catalysis, especially in discriminating the P1 amino acid of substrates, the amino acids R125, N710, E205 and E206 were investigated by mutagenesis studies. Our studies demonstrated, that N710 is essential for the catalysis of dipeptide substrates. We found that R125 is not important for dipeptide binding but interacts in the P1`position of the peptide backbone. In contrast to dipeptide substrates both amino acids play an essential role in the binding and arrangement of long natural substrates, particularly if lacking proline in the P1 position. Thus, it can be assumed that the amino acids R125 and N710 are important in the DP4 catalysed substrate hydrolysis by interacting with the peptide backbone of substrates up- and downstream of the cleavage site. Furthermore, we confirmed the important role of the amino acids E205 and E206. However, NP Y, displaying proline in P1 position, is still processed without the participation of E205 or E206.

Controlled release minocycline-lipid-complex extrudates for the therapy of periodontitis with enhanced flexibility.

We describe the development of flexible minocycline-lipid-complex extrudates with optimized mechanical and drug release properties. These extrudates contain a minocycline - magnesium stearate chelate complex with a higher stability in aqueous media, which has now been incorporated in a PEG-PLGA (polyethylene glycol - poly(lactic-co-glycolic acid)) matrix. PEG 1500 has been utilized in different concentrations to serve as plasticizer. The novel formulations have been characterized by texture analysis, X-Ray powder diffraction (XRPD) and differential scanning calorimetry (DSC). Extrudates with a reduced diameter of 300 µm (previously 600 µm) were introduced, and a more sensitive quantification method with a tandem-mass spectrometry detector was developed. From all tested formulations, the extrudates consisting of Expansorb DLG 50 - 6P (PEG-PLGA, molar weight 30-60 kDa) paired with 10% PEG 1500 emerged as best formulation. These extrudates feature a drug content of 11.5% and a controlled release over at least 42 days. The release profile is without a lag time and shows initially a slightly higher release rate, which is desired. Compared to previous developments, the extrudates now offer a high flexibility combined with a large mechanical resilience, which will ease the handling and administration.

Simple Purification of Nicotiana benthamiana-Produced Recombinant Colicins: High-Yield Recovery of Purified Proteins with Minimum Alkaloid Content Supports the Suitability of the Host for Manufacturing Food Additives.

Colicins are natural non-antibiotic bacterial proteins with a narrow spectrum but an extremely high antibacterial activity. These proteins are promising food additives for the control of major pathogenic Shiga toxin-producing E. coli serovars in meats and produce. In the USA, colicins produced in edible plants such as spinach and leafy beets have already been accepted by the U. S. Food and Drug Administration (FDA) and U. S. Department of Agriculture (USDA) as food-processing antibacterials through the GRAS (generally recognized as safe) regulatory review process. Nicotiana benthamiana, a wild relative of tobacco, N. tabacum, has become the preferred production host plant for manufacturing recombinant proteins-including biopharmaceuticals, vaccines, and biomaterials-but the purification procedures that have been employed thus far are highly complex and costly. We describe a simple and inexpensive purification method based on specific acidic extraction followed by one chromatography step. The method provides for a high recovery yield of purified colicins, as well as a drastic reduction of nicotine to levels that could enable the final products to be used on food. The described purification method allows production of the colicin products at a commercially viable cost of goods and might be broadly applicable to other cost-sensitive proteins.

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.

Isoaspartate residues dramatically influence substrate recognition and turnover by proteases.

Posttranslational modifications influence the structure, stability and biological activity of proteins. Most of the reactions are enzyme-catalyzed, but some, such as asparagine (Asn) and glutamine (Gln) deamidation and the isoaspartate (isoAsp) formation within peptide chains, occur spontaneously. It has been previously shown that certain peptide sequences form isoAsp quite fast if the Asp stretches are exposed to the protein surface, thereby potentially changing susceptibility to proteolysis at these sites. This tempted us to investigate the activity of exo- and endopeptidases against Asp- or isoAsp-containing substrates. Members of the prolyl oligopeptidase family were unable to cleave substrates after proline if isoAsp was placed in the P2-position. Caspases, usually accepting Asp at P1-position of their substrates, did not cleave isoAsp-containing sequences. Similarly, the metal-dependent aminopeptidase amino peptidase N did not turnover N-terminal isoAsp-containing substrates, nor could the endopeptidase matrix metalloproteinase 3 (MMP 3) hydrolyze a serum amyloid A protein-like substrate if the sequence contained isoAsp instead of Asp. Also, the highly specific enterokinase, usually clipping after a stretch of four Asp residues and a lysine in the P1 position, could not turnover substrates if the P2 amino acid was replaced by isoAsp. In contrast, acylamino acid-releasing enzyme and dipeptidyl peptidases 1, 2 and 4 hydrolyzed substrates containing the isoAsp-Ala motif.

Simultaneous determination of incretin hormones and their truncated forms from human plasma by immunoprecipitation and liquid chromatography-mass spectrometry.

The incretins, glucose-dependent insulinotropic peptide (GIP(1-42)) and glucagon-like peptide 1 (GLP-1(7-36)), are involved in regulation of gastric emptying, glucose homeostasis, body fat regulation and the glucose-induced insulin secretion from the endocrine pancreas. After release in the circulation both peptides are rapidly degraded by the exopeptidase dipeptidyl peptidase IV (DP IV) to the inactive polypeptides GIP(3-42) and GLP-1(9-36). In vivo stabilization of the active incretins by orally available DP IV-inhibitors is now widely accepted as a new therapeutic approach in antidiabetic treatment. In order to demonstrate the pharmacodynamic effect of DP IV-inhibitors, it is necessary to measure the plasma levels of active and inactive forms of GIP and GLP-1. We previously described an immunoprecipitation method as sample preparation and concentration in combination with a LC-MS analysis for determination of active and inactive GIP. We could improve the efficiency and suitability of this method by reduction of the necessary sample volume to 1.0 ml and simultaneous measurement of GIP(1-42), GIP(3-42) and GLP-1(7-36), GLP-1(9-36), without loss of sensitivity. An LOQ of approximately 5 and 11 pmol/l was maintained for GIP and GLP-1, respectively.

Structure-activity relationships of glucose-dependent insulinotropic polypeptide (GIP).

Six GIP(1-NH2) analogs were synthesized with modifications (de-protonation, N-methylation, reversed chirality, and substitution) at positions 1, 3, and 4 of the N-terminus, and additionally, a cyclized GIP derivative was synthesized. The relationship between altered structure to biological activity was assessed by measuring receptor binding affinity and ability to stimulate adenylyl cyclase in CHO-K1 cells transfected with the wild-type GIP receptor (wtGIPR). These structure-activity relationship studies demonstrate the importance of the GIP N-terminus and highlight structural constraints that can be introduced in GIP analogs. These analogs may be useful starting points for design of peptides with enhanced in vivo bioactivity.

Heterologous expression and characterization of human glutaminyl cyclase: evidence for a disulfide bond with importance for catalytic activity.

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues from glutamine at the N-terminus of peptides and proteins. In the current study, human QC was functionally expressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purification from the supernatant of a 5 L fermentation. Initial characterization studies of the recombinant QC using MALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at both potential sites with similar 2 kDa extensions. CD spectral analysis indicated a high alpha-helical content, which contrasts with plant QC from Carica papaya. The kinetic parameters for conversion of H-Gln-Tyr-Ala-OH by recombinant human QC were almost identical to those previously reported for purified bovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, and H-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularly in E. coli. Expression of QC in E. coli showed that approximately 50% of the protein did not contain a disulfide bond that is present in the entire QC expressed in P. pastoris. Further, the enzyme was consistently inactivated by treatment with 15 mM DTT, whereas deglycosylation had no effect on enzymatic activity. Analysis of the fluorescence spectra of the native, reduced, and unfolded human QC point to a conformational change of the protein upon treatment with DTT. In terms of the different enzymatic properties, the consequences of QC expression in different environments are discussed.