I. De Meester

Proline motifs in peptides and their biological processing.

Many biologically important peptide sequences contain proline. It confers unique conformational constraints on the peptide chain in that the side‐chain is cyclized back onto the backbone amide position. Inside an a‐helix the possibility of making hydrogen bonds to the preceding turn is lost and a kink will be introduced. The conformational restrictions imposed by proline motifs in a peptide chain appear to imply important structural or biological functions as can be deduced from their often remarkably high degree of conservation as found in many proteins and peptides, especially cytokines, growth factors, G‐protein‐coupled receptors, V3 loops of the HIV envelope glycoprotein gpl20, and neuro‐ and vasoactive peptides. Only a limited number of peptidases are known to be able to hydrolyze proline adjacent bonds. Their activity is influenced by the isomeric state (cis‐trans) as well as the position of proline in the peptide chain. The three proline specific metallo‐peptidases (aminopeptidase P. car‐boxroeptidase P and prolidase) are activated by Mn2+, whereas the three serine type peptidases cleaving a post proline bond (prolyl oligopeptidase, dipep‐tidyl peptidase IV, and prolylcarboxypeptidase) share the sequential order of the catalytic Ser‐Asp‐His triade, which differentiates them from the chy‐motrypsin (His‐Asp‐Ser) and subtilisin (Asp‐His‐Ser) families. An endo or C terminal Pro‐Pro bond and an endo pre‐Pro peptide bond possess a high degree of resistance to any mammalian proteolytic enzyme.—Vanhoof, G., Goossens, F., De Meester, I., Hendrike, D., Schärpé, S. Proline motifs in peptides and their biological processing. FASEB J. 9, 736‐744 (1995)

Inhibition of CD26/dipeptidyl peptidase IV activity in vivo prolongs cardiac allograft survival in rat recipients

The CD26 antigen, one of the major costimulatory molecules in T cell activation, was shown to possess dipeptidyl peptidase IV (DPP IV) activity. Previously, we demonstrated that immunosuppressed kidney transplant patients exhibit lower DPP IV serum activity as compared with healthy individuals. In the present study, we analyzed the role of CD26/DPP IV in the immune cascade triggered by organ transplantation and leading to acute rejection of cardiac allografts in rat recipients. Transplantation of hearts from (Lewis x Brown Norway)F1 donors into Lewis hosts resulted in an early (24 hr) increase in cellular CD26 expression, followed by a rise in DPP IV serum activity, which peaked at day 6, i.e., before the time of actual graft loss. Specific targeting of DPP IV activity with a novel, low-molecular-weight inhibitor of the diphenyl-phosphonate group (prodipine) abrogated acute rejection and prolonged cardiac allograft survival to 14.0+/-0.9 days (P<0.0001). Prodipine treatment prevented the early peak of cellular CD26 expression and thoroughly suppressed systemic DPP IV activity. The inhibition of DPP IV was associated with severely impaired host cytotoxic T lymphocyte responses in vitro. These results demonstrate the role of CD26/DPP IV in alloantigen-mediated immune regulation in vivo and provide the first direct evidence that CD26/DPP IV plays an important role in the mechanism of allograft rejection. The model of targeting CD26/DPP IV may reveal essential interactions on the level of costimulatory alternate T cell activation pathways, allowing a more subtle approach for more selective immunosuppression in transplant recipients.

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.

Natural substrates of dipeptidyl peptidase IV

During the last decade it has become clear that DPP IV may have various substrates in vivo and that the preferred peptide will depend on the localization and physiological circumstances. It is at present impossible to depict a certain chain length as the maximal acceptable substrate size as it turns out that the immediate surrounding and surface accessibility of the NH2-terminal dipeptide are determining the susceptibility for cleavage of a peptide.

Distribution of proline-specific aminopeptidases in human tissues and body fluids.

The proline-specific peptidases, aminopeptidase P (EC 3.4.11.9) and dipeptidyl peptidase IV (EC 3.4.14.5), were measured in human tissue homogenates and physiological fluids. All tissues examined contained measurable aminopeptidase P and dipeptidyl peptidase IV activities. High specific activities for both enzymes under study were found in benign prostatic hypertrophy. Normal prostate and prostatic adenocarcinoma had a much lower activity. This difference, however, is not reflected in the serum values of the patients. The most striking finding is the extremely high activity of dipeptidyl peptidase IV in prostatosomes, prostate-derived organelles, which occur freely in human seminal plasma, and which are important for enhancement of sperm forward motility.

Decreased serum dipeptidyl peptidase IV activity in major depression

It has been recently shown that severe depression is characterized by immune dysfunctions such as blunted mitogen-induced blast transformation, which is linked to interleukin-2 (IL-2) mechanisms, and to autoimmune responses. In order to explore one of the putative pathophysiological mechanisms underlying both factors, we have measured the predexamethasone and postdexamethasone serum dipeptidyl-peptidase IV (DPP IV) activity in depressed inpatients and normal controls. This enzyme is an important mediator of IL-2-related blast proliferation, and it may play a role in autoimmunity. We found significantly lower DPP IV levels in major depressives as compared with healthy controls, and melancholics exhibited significantly lower enzyme activity than minor depressives. There was a significant negative correlation between serum DPP IV activity and the severity of illness. However, we were unable to detect any significant relationships between DPP IV on the one hand, and mitogen-induced blast transformation, soluble IL-2 receptor accumulation in PHA culture supernatant, total number of leukocytes and lymphocytes, T lymphocytes, CD4+ and CD25+ cells, on the other. Men exhibited significantly higher serum DPP IV levels than women.

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.

Alterations in plasma dipeptidyl peptidase IV enzyme activity in depression and schizophrenia: effects of antidepressants and antipsychotic drugs.

Maes M, De Meester I, Scharpé S, Desnyder R, Ranjan R, Meltzer HY. Alterations in plasma dipeptidyl peptidase IV enzyme activity in depression and schizophrenia effects of antidepressants and antipsychotic drugs. Acta Psychiatr Scand 1996: 93: 1–8. ©Munksgaard 1996.

Dipeptide-derived diphenyl phosphonate esters: mechanism-based inhibitors of dipeptidyl peptidase IV

A number of dipeptide diphenyl phosphonate esters were studied as inhibitors of dipeptidyl peptidase IV, focusing on the role of the P2 residue in the inactivation process. The active compounds were slow irreversible inhibitors of the catalytic activity of the enzyme. With proline (or alanine) in the P1 position, the rate constants of inactivation correlated with the acylation rate constants reported for homologous dipeptide derived substrates. The kinetic data indicate that the mechanism of inhibition consists of the formation of a fairly weak initial complex, followed by a slow irreversible inactivation step. This indicates that, as in the case of trypsin-like proteinases, dipeptide diphenyl phosphonate esters form a covalent adduct with the catalytic site of DPP IV, even though this enzyme belongs to a completely distinct class of serine peptidases. Enantioselectivity and secondary specificity further support the evidence that diphenyl phosphonate esters are mechanism-based inhibitors. The dipeptide diphenyl phosphonate esters had a half-life of 3-10 h at 37 degrees C in Tris buffer. The inhibitors were degraded in human plasma, depending on the type of amino-terminal amino acid. The compound with proline in the P2 position was the most resistant to degradation in plasma. Due to their stability and the irreversible nature of the inhibition, the diphenyl phosphonate esters promise to be useful tools in the continuing investigation of the physiological function of dipeptidyl peptidase IV.

DISTURBANCES IN DEXAMETHASONE SUPPRESSION TEST AND LOWER AVAILABILITY OF L-TRYPTOPHAN AND TYROSINE IN EARLY PUERPERIUM AND IN WOMEN UNDER CONTRACEPTIVE THERAPY

This study investigates the function of the hypothalamic-pituitary-adrenal (HPA)-axis and the availability of L-tryptophan and tyrosine to the brain in postpartum women and in women taking long-term oral contraceptives. To this end, we have measured the following parameters in 50 women (i.e. 9 normal controls, 10 women taking oral contraceptives, and 31 postpartum females): plasma cortisol, L-tryptophan, tyrosine and the amino acids (CAA) known to compete with them for transport through the blood-brain barrier. We have determined the effects of 1 mg of dexamethasone on the above-mentioned biological markers in postpartum females. Plasma cortisol and tyrosine were significantly higher and lower, respectively, in puerperium and in women under contraceptive therapy as opposed to normal controls. L-Tryptophan was significantly lower in postpartum females, whilst the L-tryptophan/CAA ratio did not differ across the three study groups. Postpartum females revealed a significant negative relationship between the availability of L-tryptophan to the brain and postpartum mood, as measured by Zungs Depression and Anxiety Scales and State Anxiety Inventory. Dexamethasone had a significant suppressive effect on L-tryptophan/CAA and tyrosine/CAA ratios, with cortisol nonsuppression appearing in 82% of the postpartum females.