Membrane-bound Dipeptidase Research Articles

Tamrintamab pamozirine (SC-003) in patients with platinum-resistant/refractory ovarian cancer: Findings of a phase 1 study

ObjectiveEpithelial ovarian carcinoma (EOC) is diagnosed at advanced stage in the majority of women and, despite being initially chemosensitive, eventually recurs and develops resistance to known therapies. SC-003 is a pyrrolobenzodiazepine-based antibody-drug conjugate targeting dipeptidase 3 (DPEP3), a membrane-bound dipeptidase associated with tumor-initiating cells in patient-derived EOC xenograft models. This first-in-human phase 1a/1b study evaluated the safety/tolerability, pharmacokinetics, and preliminary antitumor activity of SC-003 alone or in combination with budigalimab (formerly ABBV-181), an antibody targeting PD-1, in patients with platinum-resistant/refractory EOC (NCT02539719). MethodsPatients received SC-003 at 1 of 6 dose levels (0.025–0.4 mg/kg) every 3 weeks (Q3W), utilizing a standard 3 + 3 design (dose-limiting toxicity [DLT] period: 21 days). Patients with DPEP3-positive tumors were enrolled in the dose-expansion phase of the study and treated with SC-003 monotherapy or in combination with budigalimab. ResultsSeventy-four patients (n = 29, dose escalation; n = 45, dose expansion; n = 3 budigalimab combination) were enrolled and received ≥1 dose of study drug. One DLT occurred (grade 3 ileus) but was considered unrelated to study drug. The MTD for the Q3W schedule was 0.3 mg/kg and the SC-003 doses selected for the dose-expansion phase of the study were 0.3 mg/kg and 0.2 mg/kg. The most common treatment-emergent adverse events were fatigue, nausea, decreased appetite, pleural effusion, abdominal pain, and peripheral edema. The overall response rate was low (4%), and responses were not durable. Post-hoc examination of antitumor activity suggested a higher response rate in patients with higher DPEP3 expression. ConclusionsSC-003 lacked the requisite safety profile and antitumor activity to warrant further development.

Molecular characterization and expression of dipeptidase 3, a testis-specific membrane-bound dipeptidase: complex formation with TEX101, a germ-cell-specific antigen in the mouse testis

We previously established an anti-sperm head auto-monoclonal antibody designated Ts4. The immunoreactivity of this antibody was also observed in other reproduction-related cells, such as testicular germ cells and early embryos, suggesting that the Ts4-recognized molecules might play a role in the reproductive process. However, the molecular characteristics and functions of the antigens warrant further clarification. In this study, we primarily attempted identification of the mAb-recognized molecules within the mouse testis. An immunoprecipitation method, together with liquid chromatography-tandem mass spectrometry, revealed that the testicular immunoprecipitants with Ts4 contained dipeptidase 3 (DPEP3), a member of the membrane-bound dipeptidase family. A Western blot analysis using an anti-DPEP3 polyclonal antibody established in this study showed that this molecule was glycosylated and formed a disulfide-linked homodimer within the testis. Expression of DPEP3 protein was observed in the testicular germ cells, but not in the Sertoli or interstitial cells, or in any other major organs. Although Western blot analysis of testicular proteins separated by two-dimensional SDS-PAGE failed to demonstrate binding of Ts4 to DPEP3, we found that DPEP3 forms complexes with Ts4-immunoreactive molecules, such as TEX101, on the surfaces of spermatocytes, spermatids, and testicular spermatozoa. Based on data showing in the present study, further studies concerning DPEP3 on the testicular germ cells may help to clarify the molecular mechanisms of testicular germ-cell development.

Biosynthesis and metabolism of leukotrienes

Leukotrienes are metabolites of arachidonic acid derived from the action of 5-LO (5-lipoxygenase). The immediate product of 5-LO is LTA4 (leukotriene A4), which is enzymatically converted into either LTB4 (leukotriene B4) by LTA4 hydrolase or LTC4 (leukotriene C4) by LTC4 synthase. The regulation of leukotriene production occurs at various levels, including expression of 5-LO, translocation of 5-LO to the perinuclear region and phosphorylation to either enhance or inhibit the activity of 5-LO. Several other proteins, including cPLA2a (cytosolic phospholipase A2a) and FLAP (5-LO-activating protein) also assemble at the perinuclear region before production of LTA4. LTC4 synthase is an integral membrane protein that is present at the nuclear envelope; however, LTA4 hydrolase remains cytosolic. Biologically active LTB4 is metabolized by w-oxidation carried out by specific cytochrome P450s (CYP4F) followed by b-oxidation from the w-carboxy position and after CoA ester formation. Other specific pathways of leukotriene metabolism include the 12-hydroxydehydrogenase/15-oxo-prostaglandin-13-reductase that forms a series of conjugated diene metabolites that have been observed to be excreted into human urine. Metabolism of LTC4 occurs by sequential peptide cleavage reactions involving a g-glutamyl transpeptidase that forms LTD4 (leukotriene D4) and a membrane-bound dipeptidase that converts LTD4 into LTE4 (leukotriene E4) before w-oxidation. These metabolic transformations of the primary leukotrienes are critical for termination of their biological activity, and defects in expression of participating enzymes may be involved in specific genetic disease.

A patient with neurological symptoms and abnormal leukotriene metabolism: A new defect in leukotriene biosynthesis

A 15-year-old male patient presented with mental retardation, mild motor impairment, and partial deafness. Biochemical investigations showed an abnormal urinary profile of leukotrienes. Concentration of leukotriene D(4) (LTD(4)), which is usually not detectable, was highly increased, whereas LTE(4), the major urinary metabolite in humans, was completely absent. These data suggest membrane-bound dipeptidase deficiency, a new defect in leukotriene biosynthesis on the step of LTE(4) synthesis, as underlying defect.

New role for leucyl aminopeptidase in glutathione turnover.

A manganese-dependent cysteinyl-glycine hydrolysing activity has been purified to electrophoretic homogeneity from bovine lens. The characterization of the purified enzyme (molecular mass of the native protein, molecular mass of the subunit and extensive primary structure analysis) allowed the unequivocal attribution of the cysteinyl-glycine hydrolysing activity, which is usually associated with alanyl aminopeptidase (EC 3.4.11.2) or membrane-bound dipeptidase (EC 3.4.13.19), to LAP (leucyl aminopeptidase; EC 3.4.11.1). Analysis of the pH dependence of Cys-Gly hydrolysis catalysed by LAP, supported by a molecular modelling approach to the enzyme-substrate conformation, gave insights into the ability of the enzyme to recognize Cys-Gly as a substrate. Due to the effectiveness of LAP in hydrolysing Cys-Gly (K(m)=0.57 mM, kcat=6.0x10(3) min(-1) at pH 7.4 and 25 degrees C) with respect to other dipeptide substrates, a new role for this enzyme in glutathione turnover is proposed.

Cleavage of leukotriene D4 in mice with targeted disruption of a membrane-bound dipeptidase gene.

Leukotriene C4 (LTC4), leukotriene D4 (LTD4), and leukotriene E4 (LTE4), collectively known as cysteinyl leukotrienes, are members of the eicosanoid group of lipid mediators1. They have been implicated in a wide variety of acute and chronic inflammatory conditions including asthma, tissue injury, cardiac and liver diseases, and shock2,3. Production of LTC4 is initiated by the conjugation of the epoxide intermediate LTA4 with glutathione (GSH). LTC4 is further metabolized to the cysteinyl glycine conjugate of LTA4 known as LTD4 by the actions of two plasma-membrane-bound ectoenzymes, γ-glutamyl transpeptidase (GGT) and γ-glutamyl leukotrienase (GGL)4,5. LTD4 is believed to be converted to the less active cysteinyl glycine conjugate of LTA4 called LTE4 by a dipeptidase6. The rank order of molar potencies of cysteinyl leukotrienes is LTD4>LTC4 >LTE4. LTD4 is considered to be at least 10 to 100-fold more potent than LTE4 7. Consequently, conversion of LTD4 to LTE4 is a critical step in the cysteinyl leukotriene metabolism. LTC4, LTD4, and LTE4 are eliminated from the blood circulation with initial half-lives of 30–40 s. They are mainly taken up by kidney and liver and excreted into the urine and bile, respectively8. Thus, the liver seems to be the major site of their metabolic inactivation.

Leukotriene D4 and cystinyl-bis-glycine metabolism in membrane-bound dipeptidase-deficient mice.

We have developed mice deficient in membrane-bound dipeptidase (MBD, EC 3.4.13.19), the enzyme believed to be responsible for the conversion of leukotriene D4 (LTD4) to leukotriene E4 (LTE4). The MBD mutation generated by us was demonstrated to be a null mutation by Northern blot analysis and the absence of beta-lactamase activity in lung, kidney, small intestine, and heart. MBD gene deletion had no effect on viability or fertility. The mutant mice retain partial ability to convert LTD4 to LTE4, ranging from 80-90% of the wild-type values in small intestine and liver to 16% in kidney and 40% in lung, heart, and pancreas. MBD is also believed to function consecutively after gamma-glutamyl transpeptidase to cleave cystinyl-bis-glycine (cys-bis-gly) generated from glutathione cleavage. Our data indicate that kidney homogenates from MBD-deficient mice retain approximately 40% of their ability to cleave cys-bis-gly, consistent with only modest elevations (3-5-fold) of cys-bis-gly in urine from MBD-deficient mice. These observations demonstrate that the conversion of LTD4 to LTE4 and the degradation of cys-bis-gly are catalyzed by at least two alternative pathways (one of which is MBD) that complement each other to varying extents in different tissues.

Four Distinct Membrane-bound Dipeptidase RNAs Are Differentially Expressed and Show Discordant Regulation with γ-Glutamyl Transpeptidase

Membrane-bound dipeptidase (MBD) participates in the degradation of glutathione by cleaving the cysteinyl-glycine bond of cystinyl bisglycine (oxidized cysteinyl-glycine) following removal of a gamma-glutamyl group by gamma-glutamyl transpeptidase (GGT). In the mouse, MBD RNA is most abundant in small intestine, kidney, and lung and is represented by four distinct RNA species. These are generated by transcription from two promoters located 6 kilobases apart in the 5' flanking region of the gene and by the use of two different poly(A) addition sites. Promoter I is used primarily in small intestine and kidney, whereas promoter II is most active in lung and kidney. We found a discordance in the expected co-expression of MBD and GGT; as expected, MBD and GGT are both expressed at high levels in the kidney and small intestine. However, in the lung, MBD is expressed at high levels, whereas GGT is almost undetectable. The reverse is true in the seminal vesicles and fetal liver. Thus, although both enzymes may function in concert to metabolize glutathione in kidney and small intestine, in other tissues they appear to act independently, suggesting that they have independent roles in other biological processes.

Midgut dipeptidases from Rhynchosciara americana (diptera) larvae. Properties of soluble and membrane-bound forms

Dipeptidase activity in Rhynchosciara americana (Diptera: Sciaridae) is found mainly in midgut caeca cells. The caecal dipeptidase activity is partly soluble and partly membrane bound. Differential centrifugation of midgut caeca homogenates, followed by assays of enzyme markers and dipeptidase, suggest that soluble dipeptidase is cytosolic or weakly associated with the cell glycocalyx. Membranebound dipeptidase is likely a microvillar enzyme. Soluble dipeptidase activity is resolved by gel filtration and ion exchange chromatography into two enzymes (Mr 63,000 and 73,000), which hydrolyze both Gly-Leu and Pro-Gly, although with different efficiency. The two enzymes also differ in their stability in the presence of EDTA and degree of inhibition by phenanthroline and aminoacyl hydroxamates. Dipeptidase inhibition by phenanthroline is reversed by dialysis. Membrane-bound dipeptidase activity was solubilized by Triton X-100 and papain. Density-gradient ultracentrifugation, gel filtration, and ion-exchange chromatography suggest that there is only one detergent (M r 86,000) form of this enzyme, which is active on Gly-Leu and Pro-Gly. No activity upon Gly-Pro was found in R. americana midguts, whereas the weak activity observed upon carnosine is independent of the enzymes hydrolyzing Gly-Leu and Pro-Gly. Thus, R. americana midguts seem to have major soluble and membrane-bound dipeptide hydrolases (EC 3.4.13.11), which in contrast to the mammalian enzyme, are very active upon Pro-Gly. R. americana also has a minor carnosinase (EC 3.4.13.3).