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PubMed1984

Chemical modification of dipeptidyl peptidase iv: involvement of an essential tryptophan residue at the substrate binding site.

Inactivation of pig kidney dipeptidyl peptidase IV (EC 3.4.14.5) by photosensitization in the presence of methylene blue at pH 7.5 was observed to have pseudo-first-order kinetics. During the process, until over 95% inactivation was achieved, the histidine and tryptophan residues were decreased from 14.0 to 2.7 and 12.6 to 7.1, respectively, per 94,000-Da subunit, without any detectable changes in other photosensitive amino acids. Modification of four histidine residues per subunit using diethylpyrocarbonate resulted in only 30% inactivation of the enzyme, while N-bromosuccinimide almost completely inactivated the enzyme with the modification of only one tryptophan residue per subunit, as determined by absorption spectrophotometry at 280 nm. The protective action of the substrate and inhibitors such as Ala-Pro-Ala and Pro-Pro against the modification of tryptophan residues with N-bromosuccinimide was observed both fluorometrically and by measurement of activity. On the basis of these results it is suggested that one of the tryptophan residues in the enzyme subunit is essential for the functioning of the substrate binding site of pig kidney dipeptidyl peptidase IV.

Animals↗
PubMed1985

Reconstitution of purified dipeptidyl peptidase IV. A comparison with aminopeptidase N with respect to morphology and influence of anchoring peptide on function.

The pig small intestinal dipeptidyl peptidase IV was asymmetrically integrated into egg phosphatidylcholine and microvillar lipid vesicles prepared by a beta-octylglucoside dialysis method. The enzyme molecules appeared dumbell-shaped ((11.0-11.5) X (5.0-5.5)nm) and were separated from the liposomal membrane by a stain-filled gap of about 2.5 nm, representing the 'junctional segment'. The influence of lipid bilayer and detergents on the kinetic parameters of amphiphilic and hydrophilic forms of aminopeptidase N and dipeptidyl peptidase IV was studied. Since the lipid bilayer and detergents, which interact only with the anchoring root, had no crucial effect on the kinetic parameters of the different forms of the enzymes, it is concluded that the anchoring roots exert little effect on the catalytic domain of the stalked integral membrane proteins.

Aminopeptidases↗
PubMed1991

Aminopeptidase P and dipeptidyl peptidase IV activity in human leukocytes and in stimulated lymphocytes.

Human white blood cells were shown to contain high aminopeptidase P activity. The specific activities found in the high-speed supernatant of the extracts of granulocytes, lymphocytes and monocytes ranged from 30 to 70 units per mg protein. Culturing lymphocytes during 7 days in the presence of phytohaemagglutinin resulted in a 70-200% increase in the specific aminopeptidase P activity and a 200% increase in the specific activity of dipeptidyl peptidase IV. The time-course of the activity of both aminopeptidase P and dipeptidyl peptidase IV during the stimulation of human T-lymphocytes by phytohaemagglutinin indicates an involvement of these two enzymes in the proliferative process of these immunocompetent cells. Due to their substrate specificity their potential substrates must have the N-terminal Xaa-Pro sequence known to be present in several immunologically important polypeptides.

Aminopeptidases↗
PubMed1989

Expression of dipeptidyl peptidase IV in rat tissues is mainly regulated at the mRNA levels.

Dipeptidyl peptidase IV (DPPIV) is a serine peptidase that cleaves N-terminal dipeptides from polypeptides when the second residue is a proline or an alanine. We have recently cloned cDNAs for rat gp110, a membrane glycoprotein with Mr of 110,000 isolated initially from rat liver. Studies reported here establish that the gp110 for which we have cloned cDNAs is DPPIV. Using the antibodies against and cDNA for DPPIV, we have assessed the tissue distribution of DPPIV by molecular approaches. Immunoblot analysis demonstrated that DPPIV is present in the kidney, lung, and small intestine at high levels, in the liver and spleen at moderate levels, and in the heart at low levels. The highest levels of mRNA for DPPIV were detected in the kidney and small intestine as compared to moderate levels found in the lung, liver, and spleen. The lowest levels of DPPIV mRNA were found in the stomach, testis, and heart. No detectable DPPIV protein and mRNA were found in brain or muscle. LDPPIV protein and mRNA are present at much lower levels in fetal livers as compared to the adult liver. Indirect immunofluorescence microscopy demonstrated that DPPIV is localized in the bile canaliculus of hematocytes and in the apical membrane domains of kidney tubule and small intestine. Further studies by Southern blot analysis indicate that DPPIV is encoded by a single gene.

Animals↗
PubMed1994

Transcripts encoding a neural membrane CD26 peptidase-like protein are stimulated by synaptic activity.

We isolated a cDNA clone, named BSPL, that encodes a brain-specific dipeptidyl peptidase-like protein with 30% identity and 50% similarity to CD26, a lymphocyte membrane antigen involved in T-cell activation. BSPL lacks, however, the catalytic residue responsible for peptidase activity. The expression of BSPL is widespread throughout the CNS but restricted to neurons under normal conditions. Twenty-four hours after injection of kainic acid into the hippocampus, a dramatic increase in the concentration of BSPL mRNA was detected by in situ hybridization in the CA3 region of the injected hemisphere as compared with the contralateral hemisphere or sham-injected animals. An increase in the steady-state level of BSPL mRNA concentration was also found following tetanic stimulation of the perforant path to produce LTP in granule cells of the dentate gyrus. Hybridization signals could be detected in dendritic processes of pyramidal neurons and in some glial cells upon either type of stimulation. These data suggest that BSPL may be involved in synaptic plasticity.

Amino Acid Sequence↗
PubMedDate not supplied

Differential processing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidase IV, aminopeptidase M and angiotensin converting enzyme.

In addition to plasma metabolism of substance P (SP) by angiotensin converting enzyme (ACE; EC 3.4.15.1) (less than 1.0 nmol/min/ml), the majority of SP hydrolysis by rat and human plasma was due to dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) (3.15-5.91 nmol/min/ml), which sequentially converted SP to SP(3-11) and SP(5-11). In turn, the SP(5-11) metabolite was rapidly hydrolyzed by rat and human plasma aminopeptidase M (AmM; EC 3.4.11.2) (24.2-25.5 nmol/min/ml). The Km values of SP for DAP IV and of SP(5-11) for AmM ranged from 32.7 to 123 microM. In contrast, neurokinin A (NKA) was resistant to both ACE and DAP IV but was subject to N-terminal hydrolysis by AmM (3.76-10.8 nmol/min/ml; Km = 90.7 microM). These data demonstrate differential processing of SP and NKA by specific peptidases in rat and human plasma.

Aminopeptidases↗
PubMed2005

Ser475, Glu272, Asp276, Asp327, and Asp360 are involved in catalytic activity of human tripeptidyl-peptidase I.

Tripeptidyl-peptidase I (TPP I) is a lysosomal aminopeptidase that sequentially removes tripeptides from small polypeptides and also shows a minor endoprotease activity. Mutations in TPP I are associated with a fatal lysosomal storage disorder--the classic late-infantile form of neuronal ceroid lipofuscinoses. In the present study, we analyzed the catalytic mechanism of the human enzyme by using a site-directed mutagenesis. We demonstrate that apart from previously identified Ser475 and Asp360, also Glu272, Asp276, and Asp327 are important for catalytic activity of the enzyme. Involvement of serine, glutamic acid, and aspartic acid in the catalytic reaction validates the idea, formulated on the basis of significant amino acid sequence homology and inhibition studies, that TPP I is the first mammalian representative of a growing family of serine-carboxyl peptidases.

Amino Acid Sequence↗
PubMed1987

Dipeptidyl peptidase IV (DP IV), a functional marker of the T lymphocyte system.

Dipeptidyl peptidase IV (DP IV; E.C. 3.4.14.5), a plasma membrane structure of human T lymphocytes has been shown to be an important enzyme in the process of activation and proliferation of lymphocytes. In presence of specific inhibitors and antibodies against DP IV different functions of lymphocytes in vitro were found to be impaired. This holds true for mitogen and alloantigen induced DNA synthesis, immunoglobulin production and secretion, and interleukin-2 as well as interferon-gamma production. Studies of mitogen-induced expression of different activation markers (HLA class II antigen, 4F2, Tac) suggested that one of the functions of DP IV lies in overriding the cell cycle restriction point at G1. These data, together with other features of the DP IV, support the notion that this enzyme plays a key role in the modulation of lymphokine action by X-Pro- or X-Ala-directed limited proteolysis. Moreover the high frequency of DP IV susceptible bonds in different growth factors (e.g. IL-1, IL-2) and other biologically active peptides leads to the speculation that this peptidase is of more general significance to the regulation of cell growth.

B-Lymphocytes↗
PubMed2002

Tripeptidyl peptidase-I is essential for the degradation of sulphated cholecystokinin-8 (CCK-8S) by mouse brain lysosomes.

Tripeptidyl peptidase-I (TPP-I) is a lysosomal exopeptidase which removes tripeptides from the N-terminus of small proteins. Mutations in the TPP-I gene result in a lethal neurodegenerative disease, late infantile neuronal ceroid lipofuscinosis. The pathological consequences of loss of activity are only manifested in neuronal cells suggesting that TPP-I may be involved in the lysosomal degradation of neuropeptides. We have investigated the degradation of the C-terminal octapeptide of sulphated cholecystokinin (CCK-8S) by a lysosomal fraction purified from mouse brain. Degradation products were characterised by reversed phase HPLC and mass spectrometry. Incubation of CCK-8S with brain lysosomes results in the sequential removal of the tripeptides DY(SO(3)H)M and Glycl-Tryptophanyl-Methionine from the N-terminus of CCK-8S. Degradation of CCK-8S in the isolated lysosomal fraction is completely prevented by Ala-Ala-Phe-chloromethyl ketone, an inhibitor of TPP-I. Butabindide, a specific inhibitor of TPP-II, a cell surface peptidase which also cleaves CCK-8S, inhibits TPP-I but kinetic studies indicate that the Ki for inhibition of TPP-I is 1000-fold higher than the Ki for the inhibition of TPP-II. Consequently, higher concentrations of butabindide are required for the inhibition of CCK-8S degradation by TPP-I than by TPP-II. These results indicate that whereas cell surface TPP-II is responsible for regulating extracellular CCK-8S levels, lysosomal TPP-I is largely responsible for the degradation of CCK-8S which enters the cell by receptor-mediated endocytosis.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Identification and characterization of human DPP9, a novel homologue of dipeptidyl peptidase IV.

We used an in silico approach to identify new cDNAs with homology to dipeptidyl peptidase IV (DPP IV). DPP IV (EC 3.4.14.5) is a serine protease with a rare enzyme activity having an important role in the regulation of various processes, such as blood glucose control and immune responses. Here, we report the identification and characterization of a novel DPP IV-like molecule, termed dipeptidyl peptidase-like protein 9 (DPP9). The deduced amino acid sequence of DPP9 has a serine protease motif, GWSYG, identical to that found in DPP IV. The presence of this motif, together with a conserved order and spacing of the Ser, Asp, and His residues that form the catalytic triad in DPP IV, places DPP9 in the "DPP IV gene family". Northern blots showed that DPP9 is ubiquitously expressed, with the highest expression levels in skeletal muscle, heart, and liver, and the lowest in brain. In vitro translation of the cloned full-length DPP9 sequence resulted in a DPP9 protein product that migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis at a position similar to the predicted protein size of 98 kDa. Consistent with the lack of predicted transmembrane domains and a signal sequence, DPP9 was found in a soluble, putative cytosolic form. A DPP9 orthologue in mice was identified by expressed sequence tag database searches and verified by cDNA cloning.

5' Flanking Region↗
PubMed2003

Cloning and characterization of dipeptidyl peptidase 10, a new member of an emerging subgroup of serine proteases.

Two dipeptidyl peptidase IV (DPPIV, DPP4)-related proteins, DPP8 and DPP9, have been identified recently [Abbott, Yu, Woollatt, Sutherland, McCaughan, and Gorrell (2000) Eur. J. Biochem. 267, 6140-6150; Olsen and Wagtmann (2002) Gene 299, 185-193; Qi, Akinsanya, Riviere, and Junien (2002) Patent application WO0231134]. In the present study, we describe the cloning of DPP10, a novel 796-amino-acid protein, with significant sequence identity to DPP4 (32%) and DPP6 (51%) respectively. We propose that DPP10 is a new member of the S9B serine proteases subfamily. The DPP10 gene is located on the long arm of chromosome 2 (2q12.3-2q14.2), close to the DPP4 (2q24.3) and FAP (2q23) genes. The active-site serine residue is replaced by a glycine residue in DPP10, resulting in the loss of DPP activity. The serine residue is also replaced in DPP6, which lacks peptidase activity. DPP8 and DPP9 share an identical active site with DPP4 (Gly-Trp-Ser-Tyr-Gly). In contrast with the previous results suggesting that DPP9 is inactive, we show that DPP9 is a DPP, hydrolysing Ala-Pro-(7-amino-4-methyl-coumarin) with similar pH-specificity and protease-inhibitor-sensitivity to those of DPP4 and DPP8. Northern-blot analysis shows that whereas DPP8 and DPP9 are widely expressed, DPP10 is expressed mainly in the brain and pancreas. DPP6, which has the highest amino acid identity with DPP10, has been shown previously [Nadal, Ozaita, Amarillo, de Miera, Ma, Mo, Goldberg, Misumi, Ikehara, Neubert et al. (2003) Neuron 37, 449-461] to associate with A-type K(+) channel subunits, modulating their transport and function in somatodendritic compartments of neurons. It is possible that DPP10 is involved in similar functions in the brain. Elucidation of the physiological or pathophysiological role of DPP8, DPP9 and DPP10 and characterization of their structure-function relationships will add impetus to the development of inhibitor molecules for pharmacological or therapeutic use.

Amino Acid Sequence↗
PubMed2005

Dipeptidyl peptidase IV and related enzymes in cell biology and liver disorders.

DP (dipeptidyl peptidase) IV is the archetypal member of its six-member gene family. Four members of this family, DPIV, FAP (fibroblast activation protein), DP8 and DP9, have a rare substrate specificity, hydrolysis of a prolyl bond two residues from the N-terminus. The ubiquitous DPIV glycoprotein has proved interesting in the fields of immunology, endocrinology, haematology and endothelial cell and cancer biology and DPIV has become a novel target for Type II diabetes therapy. The crystal structure shows that the soluble form of DPIV comprises two domains, an alpha/beta-hydrolase domain and an eight-blade beta-propeller domain. The propeller domain contains the ADA (adenosine deaminase) binding site, a dimerization site, antibody epitopes and two openings for substrate access to the internal active site. FAP is structurally very similar to DPIV, but FAP protein expression is largely confined to diseased and damaged tissue, notably the tissue remodelling interface in chronically injured liver. DPIV has a variety of peptide substrates, the best studied being GLP-1 (glucagon-like peptide-1), NPY (neuropeptide Y) and CXCL12. The DPIV family has roles in bone marrow mobilization. The functional interactions of DPIV and FAP with extracellular matrix confer roles for these proteins in cancer biology. DP8 and DP9 are widely distributed and indirectly implicated in immune function. The DPL (DP-like) glycoproteins that lack peptidase activity, DPL1 and DPL2, are brain-expressed potassium channel modulators. Thus the six members of the DPIV gene family exhibit diverse biological roles.

Antigens, Neoplasm↗
PubMed2001

The substrate range of tripeptidyl-peptidase I.

Tripeptidyl-peptidase I (TPP-I) is an exopeptidase which removes tripeptides from the N-terminus of peptides. Mutations in TPP-I are responsible for late infantile neuronal ceroid lipofuscinosis (CLN2). The nature of the physiological substrates and the range and specificity of the enzyme are unclear. Previous experiments suggest that the enzyme can degrade small peptides but not proteins. Digestion of a range of peptides of different size by TTP-I suggests that the enzyme will degrade small peptides with an extended N-terminal domain but not structured peptides. In general, this cut-off occurs between masses of 4.5 kDa and 6 kDa. Reference to the structures of other peptidases suggests a mechanism for this size selectivity.

Aminopeptidases↗
PubMed1994

Enhancement of antigen-induced T-cell proliferation by soluble CD26/dipeptidyl peptidase IV.

The addition of a soluble recombinant CD26 (sCD26) enhanced proliferation of peripheral blood lymphocytes induced by the recall antigen tetanus toxoid. sCD26 itself did not provide a mitogenic signal and did not augment the proliferative response of T cells to other mitogenic stimuli such as phytohemagglutinin and anti-CD3. Dipeptidyl peptidase IV-negative sCD26 did not have this enhancement effect, implying a requirement for enzyme activity. It was found that there exists a large variation in the levels of human plasma sCD26/dipeptidyl peptidase IV in vivo which may regulate T-cell activity. Peripheral blood lymphocytes from individuals whose plasma sCD26 was high and responded strongly to tetanus toxoid stimulation were insensitive to the enhancing effects of exogenously added sCD26. This suggests that plasma sCD26 had modulated the responsiveness of T cells of these individuals in vivo and that the endogenous plasma sCD26 regulates immune responses by allowing antigen-specific T cells to exert a maximal response to their specific antigen.

Adjuvants, Immunologic↗
PubMed2003

N-glycosylation is crucial for folding, trafficking, and stability of human tripeptidyl-peptidase I.

Tripeptidyl-peptidase I (TPP I) is a lysosomal serine-carboxyl peptidase that sequentially removes tripeptides from polypeptides. Naturally occurring mutations in TPP I are associated with the classic late infantile neuronal ceroid lipofuscinosis. Human TPP I has five potential N-glycosylation sites at Asn residues 210, 222, 286, 313, and 443. To analyze the role of N-glycosylation in the function of the enzyme, we obliterated each N- glycosylation consensus sequence by substituting Gln for Asn, either individually or in combinations, and expressed mutated cDNAs in Chinese hamster ovary and human embryonic kidney 293 cells. Here, we demonstrate that human TPP I in vivo utilizes all five N-glycosylation sites. Elimination of one of these sites, at Asn-286, dramatically affected the folding of the enzyme. However, in contrast to other misfolded proteins that are retained in the endoplasmic reticulum, only a fraction of misfolded TPP I mutant expressed in Chinese hamster ovary cells, but not in human embryonic kidney 293 cells, was arrested in the ER, whereas its major portion was secreted. Secreted proenzyme formed non-native, interchain disulfide bridges and displayed only residual TPP I activity upon acidification. A small portion of TPP I missing Asn-286-linked glycan reached the lysosome and was processed to an active species; however, it showed low thermal and pH stability. N-Glycans at Asn-210, Asn-222, Asn-313, and Asn-443 contributed slightly to the specific activity of the enzyme and its resistance to alkaline pH-induced inactivation. Phospholabeling experiments revealed that N-glycans at Asn-210 and Asn-286 of TPP I preferentially accept a phosphomannose marker. Thus, a dual role of oligosaccharide at Asn-286 in folding and lysosomal targeting could contribute to the unusual, but cell type-dependent, fate of misfolded TPP I conformer and represent the molecular basis of the disease process in subjects with naturally occurring missense mutation at Asn-286.

Aminopeptidases↗
PubMed1992

Dipeptidyl peptidase IV as a differentiation marker of the human endometrial glandular cells.

To investigate the involvement of membrane-bound peptidases in the human endometrial function, we examined the expression of dipeptidyl peptidase (DPP) IV and its enzyme activity. Immunohistological studies revealed that DPP IV was detected on human endometrial glandular cells and endometrial surface epithelium, but not on endometrial stromal cells or decidual cells in the first trimester of pregnancy. DPP IV expression on glandular cells and surface epithelium was weak in the proliferative phase, began to increase gradually in the early secretory phase, and was strong in mid-to late secretory phase and in the first trimester of pregnancy. DPP IV enzyme activity was detected histochemically in glandular cells and surface epithelium in the mid-secretory phase, and became stronger in the late secretory phase, but was rarely detected in the proliferative phase and early secretory phase. During the first trimester of pregnancy DPP IV enzyme activity in glandular cells and surface epithelium was slightly weaker than in the late secretory phase. Endometrial stromal cells and decidual cells, however, had no detectable DPP IV enzyme activity at any time throughout the menstrual cycle or during the first trimester of pregnancy. These findings indicate that DPP IV is a differentiation marker for glandular cells and surface epithelium and that active DPP IV is present in both areas during the peri-implantation period and thereafter.

Adult↗
PubMed1987

Role of dipeptidyl peptidase IV in uptake of peptide nitrogen from beta-casomorphin in rabbit renal BBMV.

We examined the handling of radiolabeled beta-casomorphin, Tyr-Pro-[3H]Phe-Pro-Gly, by rabbit renal brush-border membrane vesicles (BBMV). The uptake of radiolabel into the vesicles was Na+-independent, but an inward-directed H+ gradient stimulated the uptake. The H+ gradient-dependent uptake was further accelerated by an interior-negative membrane potential, but inhibited in the presence of a protonophore. Treatment of the membrane vesicles with diisopropylfluorophosphate (DFP) greatly reduced the uptake of the radiolabel. Control as well as DFP-treated vesicles exhibited H+ gradient-dependent Gly-Sar uptake. Unlabeled beta-casomorphin inhibited Gly-Sar uptake in control vesicles, but the inhibition was significantly reduced in DFP-treated vesicles. DFP inhibited the activity of dipeptidyl peptidase IV in these vesicles and there was a direct correlation between the activity of the enzyme and the capacity of beta-casomorphin to inhibit Gly-Sar uptake. Many di- and tripeptides reduced the uptake of Gly-Sar and the uptake of radiolabel from beta-[3H]casomorphin to a similar extent. We conclude that beta-casomorphin is hydrolyzed by dipeptidyl peptidase IV and the products are transported into the vesicles by the H+ gradient-driven peptide transport system. This conclusion is supported by the results from the analysis of the incubation medium by high-performance liquid chromatography that showed rapid hydrolysis of the pentapeptide by brush-border membranes to di- and tripeptides.

Animals↗
PubMed2003

Hematopoietic stimulation by a dipeptidyl peptidase inhibitor reveals a novel regulatory mechanism and therapeutic treatment for blood cell deficiencies.

In hematopoiesis, cytokine levels modulate blood cell replacement, self-renewal of stem cells, and responses to disease. Feedback pathways regulating cytokine levels and targets for therapeutic intervention remain to be determined. Amino boronic dipeptides are orally bioavailable inhibitors of dipeptidyl peptidases. Here we show that the high-affinity inhibitor Val-boro-Pro (PT-100) can stimulate the growth of hematopoietic progenitor cells in vivo and can accelerate neutrophil and erythrocyte regeneration in mouse models of neutropenia and acute anemia. Hematopoietic stimulation by PT-100 correlated with increased cytokine levels in vivo. In vitro, PT-100 promoted the growth of primitive hematopoietic progenitor cells by increasing granulocyte-colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and IL-11 production by bone marrow stromal cells. Two molecular targets of PT-100 are expressed by stromal cells- CD26/DPP-IV and the closely related fibroblast activation protein (FAP). Because PT-100 was active in the absence of CD26, FAP appears to be the hematopoietic target for PT-100. Interaction of PT-100 with the catalytic site seems to be required because amino-terminal acetylation of PT-100 abrogated enzyme inhibition and hematopoietic stimulation. PT-100 is a therapeutic candidate for the treatment of neutropenia and anemia. The data support increasing evidence that dipeptidyl peptidases can regulate complex biologic systems by the proteolysis of signaling peptides.

Anemia↗