ADABP;  CD26;  DPP4;  DPPIV

Nature has evolved a number of regulatory, neuronal, and immune peptides with a proline residue at the penultimate position determining their structural conformation and biological activity. Generally, the proline peptide bonds are resistant to proteolytic cleavage, yet an exclusive number of postproline-specific peptidases have emerged to regulate these peptides. The best-characterized one is dipeptidyl peptidase 4 (DPP4), though additional functional homologues of DPP4-like enzymes have been discovered, some structurally related, others without any structural homology. Since DPP4 is involved in glucose homeostasis and immune response, it is of medical and pharmaceutical interest to distinguish between these enzymes (Klemann et al. 2016; Lambeir et al. 2003; Wagner et al. 2016b).

DPP4 [EC 3.4.14.5] was first discovered in 1966 by Hopsu-Havu and Glenner and denoted with glycyl-prolyl-β-naphtylamidase. Other names include postproline dipeptidyl aminopeptidase IV, glycoprotein gp110, peptide Hep105, glycoprotein gp108, adenosine deaminase binding protein ADAbp, and T cell activation antigen CD26. The enzyme is a serine protease that hydrolyses proline, hydroxyproline, dehydroproline, to a lesser extent alanine, and at slow rates serine, glycine, threonine, valine, and leucine at the penultimate position. To allow proteolysis, the N-terminus must be protonated, P1 and P2 in trans configuration, amino acids at P2 neither be phosphorylated nor in D-configuration, whereas P′1 position must not be occupied by proline or hydroxyproline (Fig. 1) (Klemann et al. 2016; Lambeir et al. 2003).

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DPP4 [EC 3.4.14.5] belongs to the serine peptidase clan SC, subfamily 9B referred to as the DPP4-gene family, that includes dipeptidyl peptidase 4 (DPP4), fibroblast activation protein alpha (FAP), dipeptidyl peptidase 8 (DPP8), and dipeptidyl peptidase 9 (Klemann et al. 2016; Yu et al. 2010).

DPP4 as representative member of the DPP4 gene family is the best-characterized postproline-dipeptidyl peptidase with most known in vivo substrates (Table 1) (Klemann et al. 2016; Lambeir et al. 2003). The human gene of DPP4 is localized at 2q24.2, encompassing 81.8 kb, spanning 26 exons, that code for two mRNAs of 2.8 kb and 4.2 kb, respectively. Interestingly, the nucleotides coding for the residues of the catalytic triad are found on three different exons. The resulting protein has 766 amino acids and the primary structure consists of a short six amino acid cytoplasmic tail, a 22 amino acid transmembrane, a 738 amino acid extracellular portion comprised of a flexible stalk, glycosylation-rich region, cysteine-rich region, and catalytic region with the catalytic triad. Although DPP4 is a type II trans-membrane glycoprotein, a soluble shedded form is also found in the blood circulation. The human crystal structure of DPP4 reveals two domains, an eight bladed β-propeller and a catalytic α/β-hydrolase domain. The active site is composed of the catalytic triad Ser630, Asp708, and His740, two anchoring residues Glu204 and Glu205, as well as substrate stabilizing residues Arg125, Asn710, and Tyr457. The β-propeller is open and consists of two subdomains made up of blades II–V and VI–VIII, I, and represents the glycosylation- and cysteine-rich regions, respectively. Each blade has four antiparallel β-sheets, except for blade IV that has an additional α-helix and two β-sheets forming an extended arm for dimerization. Almost all binding partners of DPP4 and monoclonal antibodies bind to the β-propeller domain, with adenosine deaminase and Caveolin-1 (Cav-1) binding to the glycolsylation-rich region and HIV-gp120, collagen, fibronectin (FN), streptokinase (SK), and plasminogen receptor (PgR) to the cysteine-rich region. Cav-1 and HIV-TAT also bind to the active site (Klemann et al. 2016; Weihofen et al. 2004; Weihofen et al. 2005). There are two openings, a side opening and a propeller tunnel (Rasmussen et al. 2003). The substrate NPY is suggested to enter via the side opening (Aertgeerts et al. 2004). Tetramerization has also been described in porcine DPP4 (Engel et al. 2003) (Fig. 2).

Dipeptidyl Peptidase 4, Table 1

Summary of DPP4-substrates, receptor-binding after DPP4-truncation and the subsequent response (De Meester et al. 2002; Harmar 2001; Klemann et al. 2016; Lambeir et al. 2003; Mortier et al. 2016; Sah et al. 2007; Sherwood et al. 2000; Wagner et al. 2016a). ⇑, increased receptor binding; ⇓, decreased receptor binding; ≈, receptor binding unchanged; neuropeptide Y, NPY; peptide YY, PYY; GALP, galanin-like-peptide; substance P, SP; vasoactive intestinal peptide, VIP; Endomorphin- 1/2, EM-1/2; Tyr-Melanocyte-inhibiting factor-1, Tyr-MIF-1; glucagon-like peptide 1/2, GLP-1/2; glucose-dependent insulinotropic peptide, GIP; pituitary adenylate cyclase activating peptide, PACAP; growth hormone releasing hormone, GHRH; insulin-like growth factor-1, IGF-1; gastrin releasing peptide, GRP; Brain natriuretic peptide, BNP; CXCL2, GRO-β; CXCL6, GCP-2; CXCL9, Mig; CXCL10, IP-10; CXCL11, I-TAC; CXCL12α; SDF-1α; CXCL12β, SDF-1β; CCL3L1, LD78β; CCL4, MIP-1α; CCL5, RANTES; CCL11, Eotaxin; CCL14, HCC-1; CCL22, MDC

DPP4-substrate	Receptors binding after DPP4-truncation	Physiological/pathophysiological output
Neuropeptides
NPY	≈ Y2-R, ≈Y5-R, ⇓Y1-R, ⇑ GIR	⇑ anxiety, ⇑ depression, ⇑ psychosis, ⇓ food intake, ⇑ angiogenesis, ⇓ vasoconstriction/vascular stress
PYY	≈ Y2-R, ≈Y5-R, ⇓Y1-R	⇓ food intake, ⇑ energy expenditure
GALP32	≈ Gal1-R, ≈Gal2-R, ⇑ Gal3-R	⇑ anxiety, ⇑ depression, ⇑ stress, ⇑ alcohol consumption
SP	≈ NK-R1, ⇑ NK-R2, ⇑ NK-R3	⇑ nociception, ⇑ anxiety, ⇑ depression, ⇑ psychosis, ⇑ vasodilation
VIP	⇓ VPAC1-R ⇓ VPAC2-R	⇑ sleep, ⇓ vasorelexant of vascular/non-vascular smooth muscles; ⇓ insulin/glucagon secretion depending on plasma glucose level
EM-1	⇓ μ-opioid-R.	⇑ nociception
EM-2	⇓ μ-opioid-R	⇑ nociception
Tyr-MIF-1	⇓ μ-opioid-R	⇑ nociception
Spinorphin	?	⇑ nociception, ⇓ ACE-, APN-, NEP-, DPPIII-, DPP4-inhibition
Peptide Hormones
GLP-1	⇓ GLP1-R	⇓ insulin secretion
GIP	⇓ GIP-R	⇓ insulin secretion
Glucagon	⇓ GCG-R	⇓ glucagon secretion
PACAP	⇓ PAC1-R, ⇓ VPAC1-R, ⇓ VPAC2-R	⇓ insulin/glucose secretion in glucose dependent manner, ⇓sleep, ⇓ learning/memory, ⇑ anxiety, ⇓ aggression
GLP-2	⇓ GLP2-R	⇓ intestinal adaptation, ⇓ intestinal growth
Sectretin	⇓ SCTR	⇓secretion of HCO3−, ⇓ enzymes
GHRH	⇓ GHRH-R	⇓ growth hormone secretion
IGF-1	⇓ IGF1R	⇓ cell proliferation
GRP	⇓ GRP-R	⇓ secretion of PACAP/VIP, ⇓ secretion of gastric hormones
BNP	⇓ NPRA, ⇓ NPRB	⇓ vasodilation, ⇓ natriuresis,
Chemokines
CXCL2	? (CXCR2)	? polymorphonuclear leukocytes, hematopoietic stem cells
CXCL6	? (CXCR1, CXCR2)	≈ Neutrophil trafficking
CXCL9	⇓ CXCR3	⇓ Th1 response, ⇓ Th1, CD8 + NK trafficking
CXCL10	⇓ CXCR3	⇓ Th1 response, ⇓ Th1, CD8 + NK trafficking
CXCL11	⇓ CXCR3	⇓ Th1 response, ⇓ Th1, CD8 + NK trafficking
CXCL12α	⇓ CXCR4	⇓ Bone marrow homing
CXCL12β	⇓ CXCR4	⇓ Bone marrow homing
CCL3L1	⇑ CCR1, ⇓ CCR3, ⇑ CCR5	⇑ Macrophage-NK migration; ⇑ T cell/DC interaction; ⇓ eosinophiles
CCL4	⇑ CCR1, ⇑ CCR2, ≈ CCR5	⇑ Monocytes, ⇑ peripheral lymphocytes, ≈ T cell/DC interaction, ⇓ hematopoietic progenitor cells proliferation
CCL5	⇓CCR1, ⇓ CCR3, ⇑ CCR5	⇑ Macrophage-NK migration; ⇑ T cell/DC interaction; ⇓ eosinophiles
CCL11	⇓ CCR3	⇓ Eosinophil and basophil migration
CCL14	⇓CCR1, ⇓ CCR3, ⇓ CCR5	⇓ Monocyte activation
CCL22	⇓ CCR4	⇓Th2 response, ⇓ Th2 cell migration, ⇓ T reg migration

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In addition, DPP4 is reported to be a homodimer with glycosylation contributing to 23% of the molecular weight of 110 kDa per subunit (Klemann et al. 2016). Post-translational modifications include nine N-terminal glycolsylation sites and five disulfide bonds, though O-glycosylation and phosphorylation have also been reported (Klemann et al. 2016).

DPP4 is ubiquitously distributed with the highest expression in kidney, lung, liver, and small intestine, whereas low expression is found in brain, heart, and skeletal muscle. It is predominantly found on endothelial and epithelial cells throughout the body, but also found on immune cells like T cells, activated B-, activated natural killer (NK) cells, and myeloid cells (Klemann et al. 2016; Lambeir et al. 2003; Wagner et al. 2016b; Waumans et al. 2015). DPP4 contains neither TATAA nor CCAAT box as a promoter, but has a C- and G-rich region, containing several consensus binding sites for transcriptional factors like NFκB, SP-1, EGFR, and AP-1 factor NF-1 (Bohm et al. 1995; Lambeir et al. 2003; Rohrborn et al. 2015). Expression is regulated at RNA level and is organ specific. Within an organ, it is dependent on cell type, differentiation state, and activation state. Several cytokines such as IL-12 are known to regulate DPP4 expression in a cell-type-specific manner. In some pathogenic tissues, the binding of the transcription factors is enhanced by certain cytokines like STAT1α by interferons, α, β, and γ in lymphocytic leukemia cells (Frerker et al. 2007; Klemann et al. 2016; Lambeir et al. 2003; Rohrborn et al. 2015). Furthermore, expression of DPP4/CD26 is modulated on malignant hematologic or solid tumor cells and proposed as potential diagnostic biomarker (Mortier et al. 2016; Wagner et al. 2016b; Yu et al. 2010).

In addition, CD26/ DPP4 exists in a soluble active form, shed from the membrane into plasma by MMPs (MMP1, MMP2, MMP9, MMP14) (Rohrborn et al. 2014). Recently, the bone marrow – but not the kidney – could be determined as one of the sources of soluble serum CD26/DPP4 in rats (Wang et al. 2014). Alterations of DPP4 activity and CD26/DPP4 concentrations in human serum have been reported in numerous diseases (Klemann et al. 2016). High levels of soluble DPP4 are also found in seminal fluids, whereas only small levels are detected in CSF and urine (Lambeir et al. 2003).

DPP4 has been identified as a therapeutic target for type-2 diabetes mellitus due to its ability to cleave and inactivate insulinotrophic incretins such as GIP and GLP-1 as well as PACAP, VIP, GHRH, and GRP as illustrated in Fig. 6. The incretins are released into the blood circulation from the intestinal jejunum upon glucose intake and enhance the insulin secretion in β-cells from the Langerhans’ islets. Their half-life of a few minutes is strictly dependent upon DPP4-like enzymatic activity. Furthermore, incretins exhibit positive effects on pancreatic β cells in the islets, including upregulation of insulin expression by stimulation its gene transcription. Once released into the blood circulation, GIP and GLP are degraded rapidly by DPP4 and thus DPP4-inhibition prolongs GIP/GLP half-life and has insulinotrophic effect (Klemann et al. 2016; Rohrborn et al. 2015). Currently, there are nine DPP4 inhibitors commercially available on the market, with sitagliptin Januvia® (Merck & Co., Inc., Kenilworth, NJ, USA), saxagliptin Onglyza® (Bristol Myers Squibb, New York, NY, USA), and linagliptin TradjentaTM (Böhringer Ingelheim, Ingelheim, Germany) being approved by the FDA and EMA. Vildagliptin Galvus® (Norvatis, Basel, Switzerland) is only approved by the EMA, whereas alogliptin Nesina® (Takeda Pharmaceuticals, London, UK) only by the FDA. Anagliptin Suiny® (Sanwa Kagaku Kenkyusho Company Ltd. and Kowa Company Ltd., Nagoya, Japan), teneligliptin Tenelia® (Mitsubishi Tanabe Pharma and Daiichi Sankyo, Dusseldorf, Germany), trelagliptin Zafatek® (Takeda Pharmaceuticals), and omarigliptin Marizev® (Merck & Co., Inc.) being approved in Japan. All of them are administered orally and taken daily, except for omarigliptin, which has weekly doses.

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Generally, DPP4 inhibitors reduce DPP4 activity at approximately 70–90% of baseline and also lower the hemoglobin A1c (HbA1c) 0.74%. All DPP4 inhibitors are excreted via the renal route except for linagliptin, which is eliminated via the biliary route. DPP4-inhibitors are weight neutral and have no hypoglycemic effect as they are glucose-dependent and are overall well tolerated, with mild side effects (Klemann et al. 2016; Rohrborn et al. 2015). However, DPP4-dependent glucose-homeostasis is rather complex as insulin secretion is regulated by many DPP4-substrates, involved in the endocrine, paracrine, and neuronal systems (Fig. 6) (Lambeir et al. 2003).

CD26/DPP4 is expressed on only a fraction of resting CD4+CD45RO+ memory T cells, but is strongly upregulated upon T cell activation. Also, CD26/DPP4 has been described as a negative selection marker for human regulatory T cells (Tregs), whereas human T helper type 17 (Th17) cells showed high expression of CD26/DPP4. Recently, mucosal-associated invariant T cells (MAITs) have also been shown to express high levels of CD26/DPP4 in humans (Klemann et al. 2016).

DPP4 is known to stimulate T-cell activation as CD26, and it could recently be shown to trigger T-cell activation and proliferation directly via CARMA1-mediated nuclear factor (NF)-κB. Additionally, CD26/DPP4 on T-cells interacts directly with antigen-presenting cells (APCs) via caveolin-1 that binds to the glycosylation-rich region of the β-propeller as well as the active Ser630 of DPP4. Upon linkage, Tollip and interleukin-1 receptor-associated kinase 1 (IRAK-1) disengage from caveolin-1 leading to subsequent IRAK-1 phosphorylation. As illustrated in Fig. 8, this results in an upregulation of the co-stimulatory molecule CD86, which enhances the bond of the immunological synapse (Klemann et al. 2016; Ohnuma et al. 2008a, b).

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CD26 binds at the intracellular PTP2 domain of CD45RO upon internalization, thereby causing recruitment of both enzymes to the lipid rafts. Association of CD26 and CD45RO as well as their compartmentation to lipid rafts is IL-12 dependent and results in signal transduction, by inducing tyrosine phosphorylation of Erk1/2, TCRζ, ZAP70, and p56lck (Ishii et al. 2001; Salgado et al. 2003).

Although CD26/DPP4 is mainly upregulated in T helper type 1 (Th1) cells, T-cell activation in T helper type 2 (Th2) cells is mediated by binding of mannose-6 phosphate and insulin-like growth factor II receptor (M6P/IGFRII) to CD26, followed by subsequent internalization (Ikushima et al. 2000).

Binding of extracellular ADA to AB2 receptor on dentritic APC cells and CD26 on T cells to form a ternary complex results in co-stimulation of T cells, T cell proliferation, and T cell protection (Pacheco et al. 2005).

CD26/DPP4 is only found on activated B-cells and animal models deficient of CD26/DPP4 point to impaired cytokine production and B-cell numbers (Klemann et al. 2016). NK cells usually express only low amounts of CD26/DPP4, but surface expression increases significantly up to 30% after IL-2, IL-12, or IL-15 stimulation. Upregulation CD26/DPP4 on NK cells appears to correlate with increased CD16-dependent lysis. This may be caused by the mediation of protein tyrosine phosphorylation and an involvement of CD26/DPP4 in the production of cytokines by NK cells (Klemann et al. 2016; Wagner et al. 2016b). Additionally, the capacity of single NK cells to lyse tumor target cells is reduced in a congenic rat model, suggesting that CD26/DPP4 enzymatic activity sustains NK cytotoxicity. NK cells exert their cytotoxicity via secretory lysosomes, and CD26/DPP4 was identified on the membrane of secretory lysosomes in NK cells by proteomic analysis. Concerning the NK cell maturation, the percentage of NK cells in DPP4-deficient animals was increased significantly, while total leucocyte numbers were decreased in a congenic DPP4-deficient rat model, as well as in knock-out mice (Frerker et al. 2009; Klemann et al. 2016; Waumans et al. 2015).

CD26/DPP4 was shown to be chemorepellent for human and murine neutrophils, whereas DPP4 truncation affected recruitment of eosinophils via its substrate eotaxin (CCL11). Expression of CD26/DPP4 is increased upon activation on dendritic cells and monocytes/macrophages as well as in Kupffer- and microglia cells, where it is localized in lysosomes (Klemann et al. 2016; Waumans et al. 2015, 2016).

However, enzymatic activity of DPP4/CD26 also influence immune response, and therefore, it has not been surprising that one of the side effects of DPP4-inhibitors includes increase of infection such as SP in rhinosinusitis and angioedema, bioactive SDF-α in arthritis and NPY/PYY, as well as SP in blood pressure. Furthermore, DPP4/CD26 was also shown to regulate HIV infection by truncating CCL5 (RANTES) and CXCL12α (SDF-1α), respectively. Yet, while truncation of CCL5 (RANTES) by DPP4 results in protection of the M-tropic virus via CCR5, truncation of CXCL12α by DPP4 enhances entry of the T-trophic virus via CXCR4 (De Meester et al. 1999; Mortier et al. 2016). Apart from type-2-diabetes mellitus and HIV-infection, DPP4/CD26 is also involved in arthritis, atherosclerosis, inflammatory bowel disease, multiple sclerosis, stress-related disease, asthma, atopic dermatitis, and cancer as recently reviewed (Klemann et al. 2016; Ohnuma et al. 2011; Wagner et al. 2016b; Waumans et al. 2015; Yu et al. 2010).

DPP4/CD26 is a multifunctional protein that is involved in signal transduction by means of its enzymatic activity and binding partners. As a postproline dipeptidyl peptidase, it regulates the bioactivity of its substrates, resulting either in modulation of receptor selectivity such as in case of NPY, PYY, CCL3L1, CCL4, CCL5, loss of receptor selectivity like SP or lower/loss of receptor binding such as GLP-1, GIP, and PACAP. Association of CD26 on T-cells with caveolin-1 on antigen-presenting cells results in T-cell activation mediated by NF-κB and subsequent upregulation of CD86. Binding of CARMA1 to cytoplasmic tail of CD26 causes T-cell proliferation mediated by NF-κB and secretion of IL-2. Binding of CD26 to ADA, CD45RO, and M6P/IGFRII also leads to T-cell activation, T-cell proliferation, and T-cell protection. Thus, based on its multifunctional roles, DPP4/CD26 is involved in many physiological and pathological conditions including glucose-homeostasis and neuroendocrine-, cardiovascular- and immune-systems.