Crystal structure of human dipeptidyl peptidase IV in complex with a decapeptide reveals details on substrate specificity and tetrahedral intermediate formation

Dipeptidyl peptidase IV (DPPIV) is a member of the prolyl oligopeptidase family of serine proteases. DPPIV removes dipeptides from the N terminus of substrates, including many chemokines, neuropeptides, and peptide hormones. Specific inhibition of DPPIV is being investigated in human trials for the treatment of type II diabetes. To understand better the molecular determinants that underlie enzyme catalysis and substrate specificity, we report the crystal structures of DPPIV in the free form and in complex with the first 10 residues of the physiological substrate, Neuropeptide Y (residues 1-10; tNPY). The crystal structure of the free form of the enzyme reveals two potential channels through which substrates could access the active site-a so-called propeller opening, and side opening. The crystal structure of the DPPIV/tNPY complex suggests that bioactive peptides utilize the side opening unique to DPPIV to access the active site. Other structural features in the active site such as the presence of a Glu motif, a well-defined hydrophobic S1 subsite, and minimal long-range interactions explain the substrate recognition and binding properties of DPPIV. Moreover, in the DPPIV/tNPY complex structure, the peptide is not cleaved but trapped in a tetrahedral intermediate that occurs during catalysis. Conformational changes of S630 and H740 between DPPIV in its free form and in complex with tNPY were observed and contribute to the stabilization of the tetrahedral intermediate. Our results facilitate the design of potent, selective small molecule inhibitors of DPPIV that may yield compounds for the development of novel drugs to treat type II diabetes.     

CD26/DPPIV cell membrane expression and DPPIV activity in plasma of patients with acute leukemia

CD26/DPPIV (dipeptidil peptidase IV) displays an array of diverse functional properties, with a role in the development of several human cancers. This enzyme is found mainly anchored in the membrane of cells although it also has an enzymatically active plasma isoform. The regulation of biological activities of cytokines by DPP IV activity has a potential role in the homeostatic regulation of hematopoiesis. In this study, we analyzed the CD26 antigen cell membrane expression by flow cytometry and the DPPIV activity in plasma of patients of acute leukemia. The results showed that the plasma DPPIV activity is significantly higher in leukemia patients and could be 100% inhibited by Januvia? (Merck Sharp & Dohme) a selective DPPIV inhibitor. Although CD26 expression on immune cells were not leukemia-dependent the analysis of the correlation between CD26 expression and the DPPIV plasma activity were statistically significant (p < 0.01) in acute lymphoid leukemia (B-ALL and T-ALL).     

N-linked glycosylation of dipeptidyl peptidase IV (CD26): effects on enzyme activity, homodimer formation, and adenosine deaminase binding

The type II transmembrane serine protease dipeptidyl peptidase IV (DPPIV), also known as CD26 or adenosine deaminase binding protein, is a major regulator of various physiological processes, including immune, inflammatory, nervous, and endocrine functions. It has been generally accepted that glycosylation of DPPIV and of other transmembrane dipeptidyl peptidases is a prerequisite for enzyme activity and correct protein folding. Crystallographic studies on DPPIV reveal clear N-linked glycosylation of nine Asn residues in DPPIV. However, the importance of each glycosylation site on physiologically relevant reactions such as dipeptide cleavage, dimer formation, and adenosine deaminase (ADA) binding remains obscure. Individual Asn-->Ala point mutants were introduced at the nine glycosylation sites in the extracellular domain of DPPIV (residues 39-766). Crystallographic and biochemical data demonstrate that N-linked glycosylation of DPPIV does not contribute significantly to its peptidase activity. The kinetic parameters of dipeptidyl peptidase cleavage of wild-type DPPIV and the N-glycosylation site mutants were determined by using Ala-Pro-AFC and Gly-Pro-pNA as substrates and varied by <50%. DPPIV is active as a homodimer. Size-exclusion chromatographic analysis showed that the glycosylation site mutants do not affect dimerization. ADA binds to the highly glycosylated beta-propeller domain of DPPIV, but the impact of glycosylation on binding had not previously been determined. Our studies indicate that glycosylation of DPPIV is not required for ADA binding. Taken together, these data indicate that in contrast to the generally accepted view, glycosylation of DPPIV is not a prerequisite for catalysis, dimerization, or ADA binding.     

Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3

Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.     

Protease-mediated processing of Argonaute proteins controls small RNA association

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Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV

Human dipeptidylpeptidase IV (hDPPIV) is an enzyme that is in hydrolase class and has various roles in different parts of human body. Its deficiency may cause some disorders in the gastrointestinal, neurologic, endocrinological and immunological systems of humans. In the present study, hDPPIV enzyme was expressed on Spodoptera frugiperda (Sf9) cell lines as a host cell, and the expression of hDPPIV was obtained by a baculoviral expression system. The enzyme production, optimum multiplicity of infection, optimum transfection time, infected and uninfected cell size and cell behavior during transfection were also determined. For maximum hDPPIV (269 mU mL⁻¹) enzyme, optimum multiplicity of infection (MOI) and time were 0.1 and 72 h, respectively. The size of infected cells increased significantly (P < 0.001) after 24 h post infection. The results indicated that Sf9 cell line was applicable to the large scale for hDPPIV expression by using optimized parameters (infection time and MOI) because of its high productivity (4.03 mU m L⁻¹ h⁻¹).     

Uric acid inhibition of dipeptidyl peptidase IV in vitro is dependent on the intracellular formation of triuret

Uric acid affects endothelial and adipose cell function and has been linked to diseases such as hypertension, metabolic syndrome, and cardiovascular disease. Interestingly uric acid has been shown to increase endothelial progenitor cell (EPC) mobilization, a potential mechanism to repair endothelial injury. Since EPC mobilization is dependent on activity of the enzyme CD26/dipeptidyl peptidase (DPP)IV, we examined the effect uric acid will have on CD26/DPPIV activity. Uric acid inhibited the CD26/DPPIV associated with human umbilical vein endothelial cells but not human recombinant (hr) CD26/DPPIV. However, triuret, a product of uric acid and peroxynitrite, could inhibit cell associated and hrCD26/DPPIV. Increasing or decreasing intracellular peroxynitrite levels enhanced or decreased the ability of uric acid to inhibit cell associated CD26/DPPIV, respectively. Finally, protein modeling demonstrates how triuret can act as a small molecule inhibitor of CD26/DPPIV activity. This is the first time that uric acid or a uric acid reaction product has been shown to affect enzymatic activity and suggests a novel avenue of research in the role of uric acid in the development of clinically important diseases.     

Toxic peptides in Frazer's fraction interact with the actin cytoskeleton and affect the targeting and function of intestinal proteins

Celiac disease (CD) is a multisystemic autoimmune inflammation of the intestinal tract induced by wheat gluten and related cereals in HLA-DQ2/8 positive individuals. An essential role in the pathogenesis of CD is played by a fraction of the peptic-tryptic digest of gluten, Frazer's Fraction (FF). Here, we investigate the effects of FF on the integrity of intestinal cells with particular emphasis on brush border membrane (BBM) components, their subsequent trafficking and endocytosis.Caco-2 cells were incubated with FF at different concentrations. Thereafter, several protein and lipid components of treated and untreated cells were analysed at the molecular, functional and cellular levels. The control employed tryptic-peptic digests of ovalbumin.Our results show that FF directly interacts with actin in an alternating manner eliciting substantial alterations in its integrity and extent in the BBM. These alterations lead to an impaired trafficking of SI to the apical membrane and reduction in its enzymatic function. ApN and DPPIV follow a transcytotic pathway and are only partly affected by FF. By contrast, the trafficking of LPH remains unaffected concomitant with its actin-independent trafficking pattern. Finally, the endocytic pathway is substantially blocked in FF-treated cells leading to an accumulation of cholesterol, and sphingolipids in the BBM.We conclude that FF deteriorates the actin cytoskeleton in Caco-2 leading to reduced protein sorting and hampered endocytic events with subsequent alterations in the protein and lipid composition of the BBM. The reduced levels of the disaccharidase SI in the BBM suggest a potential pathomechanism of carbohydrate malabsorption in CD.     

Xanthine mimetics as potent dipeptidyl peptidase IV inhibitors

A series of xanthine mimetics containing 5,5 and 5,6 heterocycle fused imidazoles were synthesized as dipeptidyl peptidase IV inhibitors. Compound 7 is potent (h-DPPIV K_i = 2 nM) and exhibits excellent selectivity and no species specificity against rat and human enzymes. The X-ray structure confirms that the binding mode of 7 to rat DPPIV is similar to the parent xanthines.