F. Goossens

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)

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.

Proteolytic activation of purified human procarboxypeptidase U.

Carboxypeptidase U (CPU, EC 3.4.17.20) is a recently described basic carboxypeptidase which circulates in plasma as an enzymatically inactive precursor procarboxypeptidase U (proCPU), also known as plasma carboxypeptidase B precursor or thrombin activatable fibrinolysis inhibitor (TAFI). The activation of the zymogen proceeds through a proteolytic cleavage at Arg-92. The active form - CPU - is able to retard the initial phase of fibrinolysis by cleaving C-terminal lysine residues exposed on fibrin partially degraded by the action of plasmin. These C-terminal lysine residues are essential for the high affinity binding of plasminogen to fibrin and the subsequent activation to plasmin. In this report, the activation of purified human proCPU was studied using trypsin and some key proteases of the coagulation and fibrinolytic cascade, i.e., kallikrein, plasmin and thrombin. The most efficient activation is obtained in the presence of thrombin in complex with thrombomodulin. After in vitro activation, CPU is unstable at 37 degrees C (T(1/2)=15 min). Its stability can be improved dramatically using lower temperatures.

Lower serum activity of prolyl endopeptidase in fibromyalgia is related to severity of depressive symptoms and pressure hyperalgesia.

BACKGROUND The aims of the present study were to examine serum activities of peptidases, i.e. prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DPP IV), in patients with fibromyalgia and to examine the effects of subchronic treatment with sertraline on these variables. METHOD Serum PEP and DPP IV activity were measured in 28 normal volunteers and 21 fibromyalgia patients, classified according to the American College of Rheumatology criteria. Tenderness at tender points was evaluated by means of dolorimetry. Fibromyalgia patients had repeated measurements of serum PEP and DPP IV both before and after repeated administration of sertraline or placebo for 12 weeks. RESULTS Patients with fibromyalgia had significantly lower serum PEP activity than normal volunteers. There were significantly negative correlations between serum PEP activity and severity of pressure hyperalgesia and the non-somatic, cognitive symptoms of the Hamilton Depression Rating Scale. Fibromyalgia patients with severe pressure hyperalgesia had significantly lower PEP activity than normal controls and fibromyalgia patients with less severe hyperalgesia. Fibromyalgia patients with severe non-somatic depressive symptoms had significantly lower serum PEP activity than normal volunteers. There were no significant changes in serum DPP IV activity in fibromyalgia. There were no significant effects of repeated administration of sertraline on serum PEP and DPP IV activity in patients with fibromyalgia. CONCLUSIONS The results show that fibromyalgia, and aberrant pain perception and depressive symptoms in fibromyalgia are related to lower serum PEP activity. It is hypothesized that lower serum PEP activity may play a role in the biophysiology of fibromyalgia through diminished inactivation of algesic and depression-related peptides.

Subregional mapping of the human lymphocyte prolyl oligopeptidase gene (PREP) to human chromosome 6q22

Prolyl oligopeptidase is a large monomeric proline specific serine endopeptidase, the activity of which correlates well with different stages of depression. We have subregionally mapped human lymphocytic prolyl oligopeptidase (PREP) by FISH using a cosmid probe. The probe mapped to the long arm of chromosome 6, and the signal clustered in band q22.

Fast homogeneous assay for plasma procarboxypeptidase U.

Abstract Carboxypeptidase U (EC 3.4.17.20, CPU, TAFIa) is a novel determinant of the fibrinolytic rate. It circulates as an inactive zymogen, procarboxypeptidase U, which becomes active during the process of coagulation. We developed a high throughput method on microtiter plates for the determination of the procarboxypeptidase U concentration in human plasma samples. Following activation of procarboxypeptidase U by thrombin-thrombomodulin, the resulting enzyme activity cleaves p-OH-Hip-Arg and the generated p-OH-hippuric acid is converted by hippuricase to p-hydroxybenzoic acid and glycine. Finally, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine by NaIO4 forms the quinoneimine dye. The absorbance of the latter dye is determined at 506 nm in a microtiter plate reader. A mean value of 620 U/l was found, with a CV of 3.0% within-run and 4.3% between-run. The assay showed a good correlation with the activities observed using a HPLC assay as reference method (n = 25, r = 0.979). The presented method enables the routine analysis of large sample pools in clinical setting.

Isolation and sequence analysis of a human cDNA clone (XPNPEPL) homologous to X-prolyl aminopeptidase (aminopeptidase P)

A novel human cDNA (XPNPEPL) encoding a protein of 623 amino acids exhibiting 44% sequence identity and 62% sequence similarity to pig kidney X-prolyl aminopeptidase (aminopeptidase P; EC 3.4.11.9) was obtained by reverse transcription/polymerase chain reaction of phytohemagglutinin-stimulated lymphocyte mRNA. Conserved sequences were found with the prokaryotic X-prolyl aminopeptidase encoding gene (pepP). The human gene translation product exhibits a high sequence homology to the Schizosaccharomyces pombe chromosome I hypothetical protein C22G7.01c and to the S. cerevisiae ORF y11029w. Northern blot analysis indicates an ubiquitous expression of the human XPNPEPL sequence.

Synthesis and evaluation of azaproline peptides as potential inhibitors of dipeptidylpeptidase IV and prolyl oligopeptidase

A series of azaproline dipeptides with various N-substituents were synthesized as possible active-site-directed inhibitors of two proline-specific serine proteases, dipeptidyl peptidase IV and prolyl oligopeptidase. Compounds with semicarbazide, carbazate, acylhydrazine and sulphonylhydrazine structures were tested. Some compounds show moderate activity, i.e., in the millimolar range.

Human lymphocyte X-prolyl aminopeptidase (aminopeptidase P)-like protein. A new member of the proline peptidase family?

Peptidases are grouped according to their mechanism of catalysis in serine-type, cysteine-type, aspartic-type and metallo-type peptidases. Amongst these groups, the metallopeptidases represent the most diverse group, comprising 25 different families (1,2). Most of the Zn-binding metallopeptidases, named zincins, have the HEXXH motif for Zn-binding. Some other Zn-binding motifs have been defined, as there are the HXXEH, the HXXE, and the HXH motif (3). X-prolyl aminopeptidase (aminopeptidase P, EC 3.4.11.9), is an aminopeptidase that has been reported to bind zinc, and is activated by manganese ions, but does not contain any of these Zn-binding motifs. Aminopeptidase P from Escherichia coli was classified in the peptidase family M24, a family of metallopeptidases in which the ligands for metal ion binding are predominantly carboxylic acids (2). Apart from E. coli aminopeptidase P, this family comprises E. coli methionyl aminopeptidase (EC 3.4.11.18) and E. coli and human proline dipeptidase (EC 3.4.13.9). We have been studying the soluble form of aminopeptidase P in human lymphocytes and platelets (4,5), and were interested in determining the nucleotide sequence that would provide useful information for the development of potent and specific inhibitors. Aminopeptidase P is a proline-specific metallo-aminopeptidase that catalyses specifically the removal of any unsubstituted N-terminal amino acid that is adjacent to a penultimate proline residue. Due to its specificity towards proline, it has been suggested that aminopeptidase P is important