Characterisation of Human Dipeptidyl Peptidase IV Expressed in Pichia pastoris. A Structural and Mechanistic Comparison between the Recombinant Human and the Purified Porcine Enzyme

Abstract

Dipeptidyl peptidase IV/CD26 (DP IV) is a multifunctional serine protease cleaving off dipeptides from the N-terminus of peptides. The enzyme is expressed on the surface of epithelial and endothelial cells as a type II transmembrane protein. However, a soluble form of DP IV is also present in body fluids. Large scale expression of soluble human recombinant His(6)-37-766 DP IV, using the methylotrophic yeast Pichia pastoris, yielded 1.7 mg DP IV protein per litre of fermentation supernatant. The characterisation of recombinant DP IV confirmed proper folding and glycosylation similar to DP IV purified from porcine kidney. Kinetic comparison of both proteins using short synthetic substrates and inhibitors revealed similar characteristics. However, interaction analysis of both proteins with the gastrointestinal hormone GLP-1(7-36) resulted in significantly different binding constants for the human and the porcine enzyme (Kd = 153.0 +/- 17.0 microM and Kd = 33.4 +/- 2.2 microM, respectively). In contrast, the enzyme adenosine deaminase binds stronger to human than to porcine DP IV (Kd = 2.15 +/- 0.18 nM and Kd = 7.38 +/- 0.54 nM, respectively). Even though the sequence of porcine DP IV, amplified by RT-PCR, revealed 88% identity between both enzymes, the species-specific variations between amino acids 328 to 341 are likely to be responsible for the differences in ADA-binding.

Dipeptidyl Peptidase IV as a Potential Target for Selective Prodrug Activation and Chemotherapeutic Action in Cancers

“…Porcine kidney DPPIV was used in the studies due to its commercial availability and its close alignment with human DPPIV (88% homology). 34 Stock solutions of porcine DPPIV were prepared by reconstituting lyophilized powder (0.87 unit, 23 units/mg) in 1 mL of 0.1 M Tris-HCl (pH 8.0) and stored at −80 °C. Solutions of the various substrates glycyl-prolyl- p -nitroanilide (GP- p NA), glycyl-phenylalaninyl- p -nitroanilide (GF- p NA), or glycyl-argininyl- p -nitroanilide (GR- p NA) at 0.4 mM were mixed with appropriate amounts of diluted DPPIV solution (final DPPIV concentration in mixture, 0.473 μg/mL) and incubated at 37 °C for 15–30 min in flat-bottom 96-well plates.…”

Sitagliptin (MK0431) Inhibition of Dipeptidyl Peptidase IV Decreases Nonobese Diabetic Mouse CD4+ T-Cell Migration Through Incretin-Dependent and -Independent Pathways

“…Cell migration was assayed using Transwell chambers (Corning) in media ± purified porcine kidney DPP-IV (18) (32.1 units/mg; 100 mU/ml final concentration) ± sitagliptin (100 μmol/l) or human GIP (100 nmol/l) or human GLP-1 (100 nmol/l), all kindly provided by Dr. Hans-Ulrich Demuth (Probiodrug, Halle/Saale, Germany). After 1 h, cells on the upper surface were removed mechanically and migrated cells in the lower compartment were counted.…”

Multifunctional peptides for the prevention of cardiovascular disease: A new concept in the area of bioactive food-derived peptides

“…Most in vitro studies investigating the effects on DPP-IV are conducted using the porcine enzyme (Tables 2 and 4), although the human enzyme is commercially available (Table 1). Though the sequence is highly conserved among mammalian species, human and porcine DPP-IV enzymes have only an 88% sequence identity and there are evidences that porcine and human DPP-IV differ in their susceptibility to inhibition by food-derived peptides (Bär, Weber, Hoffmann, Stork, Wermann, Wagner, et al, 2003;Lacroix & Li-Chan, 2015). Since the inhibition is stronger on the porcine DPP-IV enzyme than the human one, the employment of the former may lead to an overestimation of the actual potency or effectiveness of a substance (Lacroix & Li-Chan, 2015).…”