Isolation and Characterization of Dipeptidyl Peptidase IV from Human Placenta

Püschel, Gerd; Mentlein, Rolf; Heymann, Eberhard

doi:10.1111/j.1432-1033.1982.tb06788.x

Cited by 142 publications

(75 citation statements)

References 26 publications

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“…In addition, previous studies showed that purified DPP-IV proteins from various sources migrated at sizes corresponding to either dimer or tetramer/oligomer according to gel filtration experiments (19,(27)(28)(29)(30). To determine the physiologically relevant oligomerization state of DPP-IV, we performed chemical cross-linking in the DPP-IV-containing Caco-2 cells.…”

Section: Human Dpp-iv Protein Is a Dimer In Intact Cells And Inmentioning

confidence: 99%

One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins

Chien

Huang

Chou

et al. 2004

Journal of Biological Chemistry

103

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DPP-IV is a prolyl dipeptidase, cleaving the peptide bond after the penultimate proline residue. It is an important drug target for the treatment of type II diabetes. DPP-IV is active as a dimer, and monomeric DPP-IV has been speculated to be inactive. In this study, we have identified the C-terminal loop of DPP-IV, highly conserved among prolyl dipeptidases, as essential for dimer formation and optimal catalysis. The conserved residue His 750 on the loop contributes significantly for dimer stability. We have determined the quaternary structures of the wild type, H750A, and H750E mutant enzymes by several independent methods including chemical crosslinking, gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Wild-type DPP-IV exists as dimers both in the intact cell and in vitro after purification from human semen or insect cells. The H750A mutation results in a mixture of DPP-IV dimer and monomer. H750A dimer has the same kinetic constants as those of the wild type, whereas the H750A monomer has a 60-fold decrease in k cat . Replacement of His 750 with a negatively charged Glu (H750E) results in nearly exclusive monomers with a 300-fold decrease in catalytic activity. Interestingly, there is no dynamic equilibrium between the dimer and the monomer for all forms of DPP-IVs studied here. This is the first study of the function of the C-terminal loop as well as monomeric mutant DPP-IVs with respect to their enzymatic activities. The study has important implications for the discovery of drugs targeted to the dimer interface.

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Section: Human Dpp-iv Protein Is a Dimer In Intact Cells And Inmentioning

confidence: 99%

One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins

Chien

Huang

Chou

et al. 2004

Journal of Biological Chemistry

103

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“…Assays were performed in 50 mM triethanolamineMC1, pH 7.8, at 37°C. Values of k,, were calculated using a molecular mass of 120 kDa for one identical subunit of the human placental DPP IV dimer (Piischel et al, 1982). GLP-1(7-36)amide shows substrate inhibition above 50 pM, catalytic constants ( 2 SD) were calculated from the linear ranges of Lineweaver-Burk plots.…”

Section: Separation Of Dpp IV Cleavage Products Of Gastric Inhibitorymentioning

confidence: 99%

“…Lys-pyrrolidide was a gift from Dr. Mike Schutkowski, Martin-Luther-Universitat Halle/ Saale, Germany. Dipeptidyl-peptidase IV was purified from human placenta and free of contaminating proteases (Piischel et al, 1982).…”

Section: Peptides Inhibitors and Enzymesmentioning

confidence: 99%

Dipeptidyl‐peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon‐like peptide‐1(7–36)amide, peptide histidine methionine and is responsible for their degradation in human serum

Mentlein

Gallwitz

Schmidt

1993

European Journal of Biochemistry

Self Cite

1,078

655

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Anatomisches Institut andPeptides of the glucagodvasoactive-intestinal-peptide (VIP) peptide family share a considerable sequence similarity at their N-terminus. They either start with Tyr-Ala, His-Ala or His-Ser which might be in part potential targets for dipeptidyl-peptidase IV, a highly specialized aminopeptidase removing dipeptides only from peptides with N-terminal penultimate proline or alanine. Growthhormone-releasing factor(1-29)amide and gastric inhibitory peptidefglucose-dependent insulinotropic peptide (GIP) with terminal Tyr-Ala as well as glucagon-like peptide-1 (7 -36)amidehnsulinotropin [GLP-l(7 -36)amidel and peptide histidine methionine (PHM) with terminal His-Ala were hydrolysed to their des-Xaa-Ala derivatives by dipeptidyl-peptidase IV purified from human placenta. VIP with terminal His-Ser was not significantly degraded by the peptidase. The kinetics of the hydrolysis of GIP, GLP-1(7-36)amide and PHM were analyzed in detail. For these peptides K, values of 4-34 pM and V,,, values of 0.6-3.8 pmol . min-' . mg protein-' were determined for the purified peptidase which should allow their enzymic degradation also at physiological, nanomolar concentrations. When human serum was incubated with GIP or GLP-1(7-36)amide the same fragments as with the purified dipeptidyl-peptidase IV, namely the des-Xaa-Ala peptides and Tyr-Ala in the case of GIP or His-Ala in the case of GLP-1(7-36)amide, were identified as the main degradation products of these peptide hormones. Incorporation of inhibitors specific for dipeptidylpeptidase IV, 1 mM Lys-pyrrolidide or 0.1 mM diprotin A (Ile-Pro-Ile), completely abolished the production of these fragments by serum. It is concluded that dipeptidyl-peptidase IV initiates the metabolism of GIP and GLP-1(7-36)amide in human serum. Since an intact N-terminus is obligate for the biological activity of the members of the glucagonNIP peptide family [e. g. ) is known to be inactive to release insulin in the presence of glucose as does intact GIP], dipeptidylpeptidase-IV action inactivates these peptide hormones. The relevance of this finding for their inactivation and their determination by immunoassays is discussed.

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“…The C-terminal serine protease domain is homologous to ␣͞␤-hydrolases. Dimerization was found to be a prerequisite for enzyme activity (6), albeit enzymatically active heterodimers with the fibroblast activation protein ␣ (FAP ␣) are observed (7). DP IV is a serine aminopeptidase that cleaves preferentially Xaa-Pro dipeptides from oligopeptides with typical length of Ͻ30 aa, but tolerates other small residues at P 1 -position (8).…”

mentioning

confidence: 99%

The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism

Engel

Hoffmann

Wagner

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

292

251

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The membrane-bound glycoprotein dipeptidyl peptidase IV (DP IV, CD26) is a unique multifunctional protein, acting as receptor, binding and proteolytic molecule. We have determined the sequence and 1.8 Å crystal structure of native DP IV prepared from porcine kidney. The crystal structure reveals a 2-2-2 symmetric tetrameric assembly which depends on the natively glycosylated ␤-propeller blade IV. The crystal structure indicates that tetramerization of DP IV is a key mechanism to regulate its interaction with other components. Each subunit comprises two structural domains, the N-terminal eight-bladed ␤-propeller with open Velcro topology and the C-terminal ␣͞␤-hydrolase domain. Analogy with the structurally related POP and tricorn protease suggests that substrates access the buried active site through the ␤-propeller tunnel while products leave the active site through a separate side exit. A dipeptide mimicking inhibitor complexed to the active site discloses key determinants for substrate recognition, including a Glu-Glu motif that distinguishes DP IV as an aminopeptidase and an oxyanion trap that binds and activates the P 2-carbonyl oxygen necessary for efficient postproline cleavage. We discuss active and nonactive site-directed inhibition strategies of this pharmaceutical target protein.serine protease ͉ oxyanion hole ͉ substrate channeling ͉ drug design ͉ diabetes mellitus

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Isolation and Characterization of Dipeptidyl Peptidase IV from Human Placenta

Cited by 142 publications

References 26 publications

One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins

One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins

Dipeptidyl‐peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon‐like peptide‐1(7–36)amide, peptide histidine methionine and is responsible for their degradation in human serum

The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism

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