Mammalian glutaminyl cyclases and their isoenzymes have identical enzymatic characteristics (original) (raw)
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Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006
Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Aβ(N3E-42) and Aβ(N3Q-42) was investigated. These amyloid-β peptides were expressed as fusion proteins with either the pre-pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Aβ(N3Q-42) after βand γ-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Aβ-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.
Mouse strain and brain region-specific expression of the glutaminyl cyclases QC and isoQC
International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience, 2014
Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) from glutamine precursors at the N-terminus of a number of peptide hormones, neuropeptides and chemokines. This post-translational modification stabilizes these peptides, protects them from proteolytical degradation or is important for their biological activity. However, QC is also involved in a pathogenic pGlu modification of peptides accumulating in protein aggregation disorders such as Alzheimer's disease and familial Danish and familial British dementia. Its isoenzyme (isoQC) was shown to contribute to aspects of inflammation by pGlumodifying and thereby stabilizing the monocyte chemoattractant protein CCL2. For the generation of respective animal models and for pharmacological treatment studies the characterization of the mouse strain and brain region-specific expression of QC and isoQC is indispensible.
Biological Chemistry, 2016
Secretory peptides and proteins are frequently modified by pyroglutamic acid (pE, pGlu) at their N-terminus. This modification is catalyzed by the glutaminyl cyclases QC and isoQC. Here, we decipher the roles of the isoenzymes by characterization of IsoQC-/- mice. These mice show a significant reduction of glutaminyl cyclase activity in brain and peripheral tissue, suggesting ubiquitous expression of the isoQC enzyme. An assay of substrate conversion in vivo reveals impaired generation of the pGlu-modified C-C chemokine ligand 2 (CCL2, MCP-1) in isoQC-/- mice. The pGlu-formation was also impaired in primary neurons, which express significant levels of QC. Interestingly, however, the formation of the neuropeptide hormone thyrotropin-releasing hormone (TRH), assessed by immunohistochemistry and hormonal analysis of hypothalamic-pituitary-thyroid axis, was not affected in isoQC-/-, which contrasts to QC-/-. Thus, the results reveal differential functions of isoQC and QC in the formatio...
Biochemistry, 2008
Compelling evidence suggests that N-terminally truncated and pyroglutamyl-modified amyloid-(A ) peptides play a major role in the development of Alzheimer's disease. Posttranslational formation of pyroglutamic acid (pGlu) at position 3 or 11 of A implies cyclization of an N-terminal glutamate residue rendering the modified peptide degradation resistant, more hydrophobic, and prone to aggregation. Previous studies using artificial peptide substrates suggested the potential involvement of the enzyme glutaminyl cyclase in generation of pGlu-A . Here we show that glutaminyl cyclase (QC) catalyzes the formation of A 3(pE)-40/42 after amyloidogenic processing of APP in two different cell lines, applying specific ELISAs and Western blotting based on urea-PAGE. Inhibition of QC by the imidazole derivative PBD150 led to a blockage of A 3(pE)-42 formation. Apparently, the QC-catalyzed formation of N-terminal pGlu is favored in the acidic environment of secretory compartments, which is also supported by doubleimmunofluorescence labeling of QC and APP revealing partial colocalization. Finally, initial investigations focusing on the molecular pathway leading to the generation of truncated A peptides imply an important role of the amino acid sequence near the -secretase cleavage site. Introduction of a single-point mutation, resulting in an amino acid substitution, APP(E599Q), i.e., at position 3 of A , resulted in significant formation of A 3(pE)-40/42 . Introduction of the APP KM595/596NL "Swedish" mutation causing overproduction of A , however, surprisingly diminished the concentration of A 3(pE)-40/42 . The study provides new cell-based assays for the profiling of small molecule inhibitors of QC and points to conspicuous differences in processing of APP depending on sequence at the -secretase cleavage site.
Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions
FEBS Letters, 2004
N-terminal pyroglutamate (pGlu) formation from glutaminyl precursors is a posttranslational event in the processing of bioactive neuropeptides such as thyrotropin-releasing hormone and neurotensin during their maturation in the secretory pathway. The reaction is facilitated by glutaminyl cyclase (QC), an enzyme highly abundant in mammalian brain. Here, we describe for the ¢rst time that human and papaya QC also catalyze N-terminal glutamate cyclization. Surprisingly, the enzymatic Glu 1 conversion is favored at pH 6.0 while Gln 1 conversion occurs with an optimum at pH 8.0. This unexpected ¢nding might be of importance for deciphering the events leading to deposition of highly toxic pyroglutamyl peptides in amyloidotic diseases. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
Biochemistry, 2002
Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues from glutamine at the N-terminus of peptides and proteins. In the current study, human QC was functionally expressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purification from the supernatant of a 5 L fermentation. Initial characterization studies of the recombinant QC using MALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at both potential sites with similar 2 kDa extensions. CD spectral analysis indicated a high alpha-helical content, which contrasts with plant QC from Carica papaya. The kinetic parameters for conversion of H-Gln-Tyr-Ala-OH by recombinant human QC were almost identical to those previously reported for purified bovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, and H-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularl...
Protein Expression and Purification, 2000
A full-length cDNA encoding Carica papaya glutamine cyclotransferase was cloned by RT-PCR on the basis of results from amino acid sequencing of tryptic fragments of the native enzyme. The cDNA of 1036 nucleotides encodes a typical 22-residue signal peptide and a mature protein of 266 residues with a calculated molecular mass of 30,923 Da. Five plant ESTs encoding putative QCs highly homologous to PQC were identified and the numbers and locations of cysteines and N-glycosylation sites are conserved. The plant QC amino acid sequences are very different from the known mammalian QC sequences and no clear homology was observed. The PQC cDNA was expressed in Escherichia coli as either His-tagged PQC, with three different signal peptides and in fusions with thioredoxin, glutathione S-transferase, and (pre-) maltose-binding protein. In all cases, the expressed protein was either undetectable or insoluble. Expression in Pichia pastoris of PQC fused to the ␣-factor leader resulted in low levels of PQC activity. Extracellular expression of PQC in the insect cell/baculovirus system was successful and 15-50 mg/liter of active PQCs with three different secretion signals was expressed and purified. Further, PQC N-terminally fused to a combined secretion signal/His-tag peptide was correctly processed by the host signal peptidase and the His-tag could subsequently be removed with dipeptidyl peptidase I. The expressed products were characterized by activity assays, SDS-PAGE, N-termi-nal amino acid sequencing, MALDI-TOF mass spectroscopy, and peptide mass fingerprint analysis.
Interaction between the catalytic and modifier subunits of glutamate-cysteine ligase
Biochemical Pharmacology, 2007
b i o c h e m i c a l p h a r m a c o l o g y 7 4 ( 2 0 0 7 ) 3 7 2 -3 8 1 Glutamate-cysteine ligase Protein-protein interactions Yeast two-hybrid system Histidine-tagged protein Nickel-NTA column Oxidative stress JEL classification: Protein interactions a b s t r a c t Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione (GSH)