Anne-Marie Lambeir

Dipeptidyl-Peptidase IV from Bench to Bedside: An Update on Structural Properties, Functions, and Clinical Aspects of the Enzyme DPP IV

Dipeptidyl-peptidase IV/CD26 (DPP IV) is a cell-surface protease belonging to the prolyloligopeptidase family. It selectively removes the N-terminal dipeptide from peptides with proline or alanine in the second position. Apart from its catalytic activity, it interacts with several proteins, for instance, adenosine deaminase, the HIV gp120 protein, fibronectin, collagen, the chemokine receptor CXCR4, and the tyrosine phosphatase CD45. DPP IV is expressed on a specific set of T lymphocytes, where it is up-regulated after activation. It is also expressed in a variety of tissues, primarily on endothelial and epithelial cells. A soluble form is present in plasma and other body fluids. DPP IV has been proposed as a diagnostic or prognostic marker for various tumors, hematological malignancies, immunological, inflammatory, psychoneuroendocrine disorders, and viral infections. DPP IV truncates many bioactive peptides of medical importance. It plays a role in glucose homeostasis through proteolytic inactivation of the incretins. DPP IV inhibitors improve glucose tolerance and pancreatic islet cell function in animal models of type 2 diabetes and in diabetic patients. The role of DPP IV/CD26 within the immune system is a combination of its exopeptidase activity and its interactions with different molecules. This enables DPP IV/CD26 to serve as a co-stimulatory molecule to influence T cell activity and to modulate chemotaxis. DPP IV is also implicated in HIV-1 entry, malignant transformation, and tumor invasion. Referee: Dr. Albert Adam, Faculté de Pharmace, Université de Montréal, 2900 Blvd. Edouard—Montpetit, CP succursale Centreville, Montreal, Quebec H3C 3J7, Canada

Kinetic study of the processing by dipeptidyl-peptidase IV/CD26 of neuropeptides involved in pancreatic insulin secretion

Dipeptidyl‐peptidase IV (DPPIV/CD26) metabolizes neuropeptides regulating insulin secretion. We studied the in vitro steady‐state kinetics of DPPIV/CD26‐mediated truncation of vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase‐activating peptide (PACAP27 and PACAP38), gastrin‐releasing peptide (GRP) and neuropeptide Y (NPY). DPPIV/CD26 sequentially cleaves off two dipeptides of VIP, PACAP27, PACAP38 and GRP. GRP situates between the best DPPIV/CD26 substrates reported, comparable to NPY. Surprisingly, the C‐terminal extension of PACAP38, distant from the scissile bond, improves both PACAP38 binding and turnover. Therefore, residues remote from the scissile bond can modulate DPPIV/CD26 substrate selectivity as well as residues flanking it.

Amino-terminal Truncation of Chemokines by CD26/Dipeptidyl- peptidase IV

Chemokines are key players in inflammation and infection. Natural forms of the C-X-C chemokine granulocyte chemotactic protein-2 (GCP-2) and the C-C chemokine regulated on activation normal T cell expressed and secreted (RANTES), which miss two NH2-terminal residues, including a Pro in the penultimate position, have been isolated from leukocytes or tumor cells. In chemotaxis and intracellular calcium mobilization assays, the truncation caused a reduction in the specific activity of RANTES but not of GCP-2. The serine protease CD26/dipeptidyl-peptidase IV (CD26/DPP IV) could induce this observed NH2-terminal truncation of GCP-2 and RANTES but not that of the monocyte chemotactic proteins MCP-1, MCP-2 and MCP-3. No significant difference in neutrophil activation was detected between intact and CD26/DPP IV-truncated GCP-2. In contrast to intact natural RANTES(1–68), which still chemoattracts monocytes at 10 ng/ml, CD26/DPP IV-truncated RANTES(3–68) was inactive at 300 ng/ml and behaved as a natural chemotaxis inhibitor. Compared with intact RANTES, only a 10-fold higher concentration of RANTES(3–68) induced a significant Ca2+ response. Furthermore, RANTES(3–68) inhibited infection of mononuclear cells by an M-tropic HIV-1 strain 5-fold more efficiently than intact RANTES. Thus, proteolytic processing of RANTES by CD26/DPP IV may constitute an important regulatory mechanism during anti-inflammatory and antiviral responses.

Use of immobilized adenosine deaminase (EC 3.5.4.4) for the rapid purification of native human CD26/dipeptidyl peptidase IV (EC 3.4.14.5)☆

The leukocyte differentiation antigen CD26 identified as dipeptidyl peptidase IV.(EC 3.4.14.5), cleaves off N-terminal dipeptides from peptides when a proline or alanine is located at the penultimate position. Seminal plasma and especially prostasomes, prostate-derived organelles which occur freely in seminal plasma, contain high amounts of CD26/dipeptidyl peptidase IV and therefore are suitable sources for the purification of the protein. The use of adenosine deaminase (EC 3.5.4.4) affinity chromatography for its purification is described. CD26/dipeptidyl peptidase IV was purified from human seminal plasma and prostasomes by a two step procedure. Ion exchange chromatography on DEAE-Sepharose, followed by affinity chromatography on adenosine deaminase-Sepharose resulted in the pure, native protein with an overall yield ranging from 35 to 55%. The N-terminal sequence of the amphiphilic enzyme purified from human prostasomes was determined to be Met-Lys-Thr-Pro-Trp-Lys-Val-Leu. The preparation obtained was free of contaminating aminopeptidase activity and proved to be very stable (up to 1 month at 37 degrees C). The calf intestinal adenosine deaminase we used is commercially available and can be employed for the purification of human, bovine and rabbit CD26/dipeptidyl peptidase IV. High affinity binding of porcine dipeptidyl peptidase IV was not observed. The availability of a source with high specific activity and the introduction of adenosine deaminase affinity chromatography permits the rapid purification of milligram quantities of natural mammalian CD26/dipeptidyl peptidase IV.

Carboxypeptidase M Expressed by Human Bone Marrow Cells Cleaves the C-Terminal Lysine of Stromal Cell-Derived Factor-1α: Another Player in Hematopoietic Stem/Progenitor Cell Mobilization?

Carboxypeptidase M (CPM) is a membrane‐bound zinc‐dependent protease that cleaves C‐terminal basic residues, such as arginine or lysine, from peptides/proteins. We examined whether CPM is expressed by hematopoietic and stromal cells and could degrade stromal cell‐derived factor (SDF)‐1α, a potent chemoattractant for hematopoietic stem/progenitor cells (HSPC). We found that (a) CPM transcript is expressed by bone marrow (BM) and mobilized peripheral blood CD34+ cells, myeloid, erythroid, and megakaryocytic cell progenitors, mononuclear cells (MNC), polymorphonuclear cells (PMN), and stromal cells, including mesenchymal stem cells; and that (b) granulocyte‐colony‐stimulating factor (G‐CSF) significantly increases its expression at the gene and protein levels in MNC and PMN. Moreover, we found that recombinant CPM cleaves full‐length SDF‐1α (1–68) rapidly, removing the C‐terminal lysine and yielding des‐lys SDF‐1α (1–67). We demonstrated that such CPM treatment of SDF‐1α reduced the in vitro chemotaxis of HSPC, which, however, was preserved when the CPM was exposed to the carboxypeptidase inhibitor dl‐2‐mercaptomethyl‐3‐guanidino‐ethylthiopropanoic acid. Thus, we present evidence that CPM is expressed by cells occurring in the BM microenvironment and that the mobilizing agent G‐CSF strongly upregulates it in MNC and PMN. We suggest that cleavage of the C‐terminal lysine residue of SDF‐1α by CPM leads to attenuated chemotactic responses and could facilitate G‐CSF‐induced mobilization of HSPC from BM to peripheral blood.

Prolyl oligopeptidase stimulates the aggregation of α-synuclein

Despite its thorough enzymological and biochemical characterization the exact function of prolyl oligopeptidase (PO, E.C. 3.4.21.26) remains unclear. The positive effect of PO inhibitors on learning and memory in animal models for amnesia, enzyme activity measurements in patient samples and (neuro)peptide degradation studies link the enzyme with neurodegenerative disorders. The brain protein alpha-synuclein currently attracts much attention because of its proposed role in the pathology of Parkinsons disease. A fundamental question concerns how the essentially disordered protein is transformed into the highly organized fibrils that are found in Lewy bodies, the hallmarks of Parkinsons disease. Using gel electrophoresis and MALDI TOF/TOF mass spectrometry we investigated the possibility of alpha-synuclein as a PO substrate. We found that in vitro incubation of the protein with PO did not result in truncation of full-length alpha-synuclein. Surprisingly, however, we found an acceleration of the aggregation process of alpha-synuclein using turbidity measurements that was reversed by specific inhibitors of PO enzymatic activity. If PO displays this activity also in vivo, PO inhibitors might have an effect on neurodegenerative disorders through a decrease in the aggregation of alpha-synuclein.

A prolyl oligopeptidase inhibitor, KYP‐2047, reduces α‐synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease

The aggregation of α‐synuclein is connected to the pathology of Parkinsons disease and prolyl oligopeptidase (PREP) accelerates the aggregation of α‐synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP‐2047, on α‐synuclein aggregation in cell lines overexpressing wild‐type or A30P/A53T mutant human α‐syn and in the brains of two A30P α‐synuclein transgenic mouse strains.

Dipeptidyl peptidase 8/9-like activity in human leukocytes.

The proline‐specific dipeptidyl peptidases (DPPs) are emerging as a protease family with important roles in the regulation of signaling by peptide hormones. Inhibitors of DPPs have an intriguing, therapeutic potential, with clinical efficacy seen in patients with diabetes. Until now, only recombinant forms of DPP8 and DPP9 have been characterized. Their enzymatic activities have not been demonstrated in or purified from any natural source. Using several selective DPP inhibitors, we show that DPP activity, attributable to DPP8/9 is present in human PBMC. All leukocyte types tested (lymphocytes, monocytes, Jurkat, and U937 cells) were shown to contain similar DPP8/9‐specific activities, and DPPII‐ and DPPIV‐specific activities varied considerably. The results were confirmed by DPPIV/CD26 immunocapture experiments. Subcellular fractionation localized the preponderance of DPP8/9 activity to the cytosol and DPPIV in the membrane fractions. Using Jurkat cell cytosol as a source, a 30‐fold, enriched DPP preparation was obtained, which had enzymatic characteristics closely related to the ones of DPP8 and/or ‐9, including inhibition by allo‐Ile‐isoindoline and affinity for immobilized Lys‐isoindoline.

Pyrrolidides: synthesis and structure-activity relationship as inhibitors of dipeptidyl peptidase IV

Summary Dipeptidyl peptidase IV cleaves specifically the peptide bond at the carboxyl side of a proline at the penultimate N-terminal position of a peptide. It is thought to be important for the regulation of biologically active peptides. Moreover, it has been identified as an activation marker of T-lymphocytes (CD26). Pyrrolidides and thiazolidides are known as reversible inhibitors of DPP IV. Several homologues, unsaturated, open and 3-substituted analogues were synthesized in order to determine the structure-activity relationship of the P-1 site. l -Isoleucine was taken as P-2 amino acid. 1-( l -Isoleucyl)-3( S )-fluoropyrrolidine is about as active as the non-fluorinated compound and behaves as a competitive inhibitor. Other changes decrease or abolish the activity.

A prediction of DPP IV/CD26 domain structure from a physico-chemical investigation of dipeptidyl peptidase IV (CD26) from human seminal plasma

Human DPP IV, isolated from seminal plasma by means of immobilised adenosine deaminase, occurs in different forms which are distinguishable by net charge and native molecular weight. Charge differences arise primarily from different degrees of glycosylation containing various amounts of sialic acid. The majority of DPP IV isolated from total seminal plasma consists of the extracellular part of the protein starting at Gly-31. It is a very stable protein resisting high concentrations of denaturant. Unfolding experiments under reducing conditions are indicative of the existence of at least two domains which function independently. One of these domains is highly stabilised by disulfide bonds. Disruption of the disulfide bonds does not affect the activity, the dimeric state nor the adenosine deaminase binding properties of the protein but renders it more susceptible to proteolysis. The low-angle X-ray scattering spectrum is consistent with a model for a protein containing two subunits, each composed of three domains linked by flexible regions with low average mass. The secondary structure composition, determined by FTIR spectrometry, indicates that 45% of the protein consists of beta-sheets, which is higher than expected from computed secondary structure predictions. Our results provide compelling experimental evidence for the three-domain structure of the extracellular part of DPP IV.