Purification, crystal structural characterization of porcine kidney dipeptidyl peptidase IV (PkDPP-IV) and its interaction with oyster derived inhibitory peptide ILAPPER

Dipeptidyl peptidase IV (DPP-IV) is an important target enzyme for the treatment of type 2 diabetes mellitus (T2DM). Increasing researchers try to screen DPP-IV inhibitory peptides while the cost of DPP-IV is high. In this study, PkDPP-IV was efficiently purified by acid precipitation, ammonium sulfate salting out and gel filtration chromatography with a purification of 283.5 folds and 16.5 % yield. PkDPP-IV is a glycoprotein with molecular weight of 110 kDa and optimal activity at pH 7.0 and 40 °C. Crystal structure indicated that PkDPP-IV is composed of an α/β hydrolase domain and a β-propeller domain, which is highly similar to that of human DPP-IV. A peptide ILAPPER derived from oyster exhibited high inhibitory activity with K_i value of 0.131 μM against PkDPP-IV. The crystal structure of the PkDPP-IV + ILAPPER complex revealed that ILAPPER stably occupy the S1 and S2 catalytic pockets of PkDPP-IV by forming three hydrogen bonds with Tyr-547, Ser-630, and Tyr-662, thereby inhibiting enzyme activity. Analysis of transmembrane transport pathway suggested that ILAPPER is transported by the Caco-2 cell monolayer via the paracellular pathway. All the results provide a new approach for rapid preparation of natural PkDPP-IV, and the potential application of ILAPPER as an antihyperglycemic peptide in functional foods.