The dimeric transmembrane domain of prolyl dipeptidase DPP‐IV contributes to its quaternary structure and enzymatic activities

Abstract

Dipeptidyl peptidase IV (DPP-IV) is a drug target in the treatment of human type II diabetes. It is a type II membrane protein with a single transmembrane domain (TMD) anchoring the extracellular catalytic domain to the membrane. DPP-IV is active as a dimer, with two dimer interacting surfaces located extracellularly. In this study, we demonstrate that the TM of DPP-IV promotes DPP-IV dimerization and rescues monomeric DPP-IV mutants into partial dimers, which is specific and irreplaceable by TMs of other type II membrane proteins. By bioluminescence resonance energy transfer (BRET) and peptide electrophoresis, we found that the TM domain of DPP-IV is dimerized in mammalian cells and in vitro. The TM dimer interaction is very stable, based on our results with TM site-directed mutagenesis. None of the mutations, including the introduction of two prolines, resulted in their complete disruption to monomers. However, these TM proline mutations result in a significant reduction of DPP-IV enzymatic activity, comparable to what is found with mutations near the active site. A systematic analysis of TM structures deposited in the Protein Data Bank showed that prolines in the TM generally produce much bigger kinking angles than occur in nonproline-containing TMs. Thus, the proline-dependent reduction in enzyme activity may result from propagated conformational changes from the TM to the Abbreviations: Ala-Pro-AFC, H-Ala-Pro-7-amino-4-trifluoromethylcoumarin; BRET, bioluminescence resonance energy transfer; DPP, dipeptidyl peptidase; DPP-IV, dipeptidyl peptidase IV; EOM, enzyme overlay membrane; FBS, fetal bovine serum; GFP, green fluorescent protein; Gly-Pro-AMC, H-Gly-Pro-7-amino-4-methylcoumarin; GPCR, G-Protein coupled receptor; PBS, phosphate buffered saline; PDB, protein data bank; PDBTM, protein data bank of transmembrane proteins; PFO, perfluorooctanoate; Rluc, renilla luciferase; SI, sucrase isomaltase; TM, transmembrane; TMD, transmembrane domain; WT, wild-type.Additional Supporting Information may be found in the online version of this article.Kuei-Min Chung and Jai-Hong Cheng contributed equally to this article. Our results demonstrate that TM dimerization and conformation contribute significantly to the structure and activity of DPP-IV. Optimal enzymatic activity of DPP-IV requires an optimal interaction of all three dimer interfaces, including its TM.

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“…Iwaki-Egawa and colleagues reported that the N-terminal intracellular domain contributes to enzymatic activity, 16 and it was later demonstrated by the Chung group that the transmembrane region is responsible for the main enzymatic activity. 17 In addition to the transmembrane domain, DPP-IV presents as a soluble form (called soluble CD26) in the plasma to exhibit its enzymatic activity, which is the extracellular domain of the peptide considered to be cut down from the cell plasma membrane.…”

The amino terminus extension in the long dipeptidyl peptidase 9 isoform contains a nuclear localization signal targeting the active peptidase to the nucleus

“…Four active members of this family are known so far: the two membrane-bound cell-surface members dipeptidyl peptidase IV (DPPIV) and the Fibroblast activation protein alpha (FaP), as well as the two soluble members DPP8 and 1 3 DPP9 (reviewed in [3][4][5][6]). Dimerization is a common feature for members of the S9B/DPPIV family [6][7][8][9][10][11] and is essential for enzymatic activity [12][13][14][15][16].…”

Functional Expression and Characterization of Dipeptidyl Peptidase IV from the Black-Bellied HornetVespa basalisin Sf21 Insect Cells

“…8,19,20) In this study, we found that purified His-tagged V. basalis DPP-IV is mainly monomeric form, but the dimeric form (190 kDa) was also weakly detected by the anti-6His antibody (Fig. 4A).…”