Isolation and Characterization of Glutaminyl Cyclases from Drosophila:  Evidence for Enzyme Forms with Different Subcellular Localization

Abstract

Glutaminyl cyclases (QCs) present in plants and vertebrates catalyze the formation of pyroglutamic acid (pGlu) from N-terminal glutamine. Pyroglutamyl hormones also identified in invertebrates imply the involvement of QC activity during their posttranslational maturation. Database mining led to the identification of two genes in Drosophila, which putatively encode QCs, CG32412 (DromeQC) and CG5976 (isoDromeQC). Analysis of their primary structure suggests different subcellular localizations. While DromeQC appeared to be secreted due to an N-terminal signal peptide, isoDromeQC contains either an N-terminal mitochondrial targeting or a secretion signal due to generation of different transcripts from gene CG5976. According to the prediction, homologous expression of the corresponding cDNAs in S2 cells revealed either secreted protein in the medium or intracellular QC activity. Subcellular fractionation and immunochemistry support export of isoDromeQC into the mitochondrion. For enzymatic characterization, DromeQC and isoDromeQC were expressed heterologously in Pichia pastoris and Escherichia coli, respectively. Compared to mammalian QCs, the specificity constants were about 1 order of magnitude lower for most of the analyzed substrates. The pH dependence of the specificity constant was similar for both enzymes, indicating the necessity of an unprotonated substrate amino group and two protonated groups of the enzyme, resulting in an asymmetric bell-shaped characteristic. The determination of the metal content of DromeQC revealed equimolar protein-bound zinc. These results prove conserved enzymatic mechanisms between QCs from invertebrates and mammals. Drosophila is the first organism for which isoenzymes of glutaminyl cyclase have been isolated. The identification of a mitochondrial QC points toward yet undiscovered physiological functions of these enzymes.

Structures of Human Golgi-resident Glutaminyl Cyclase and Its Complexes with Inhibitors Reveal a Large Loop Movement upon Inhibitor Binding

“…This is likely due to different hydrophobic and charge interactions between this inhibitor and these two enzymes. Likewise, two distinct Drosophila QCs, named DromeQC and isoDromeQC, were previously identified and characterized in which DromeQC had an ϳ32-fold weaker sensitivity toward the PBD150 inhibition when compared with that of isoDromeQC (44). By aligning the sequences of Drosophila QCs with the gQC and sQC sequences (supplemental Fig.…”

Phosphate ions and glutaminyl cyclases catalyze the cyclization of glutaminyl residues by facilitating synchronized proton transfers

“…The QC used for enzymatic conversion of N-terminal glutamine was from Drosophila melanogaster [21]. Chemicals were purchased as follows: Glutamate dehydrogenase from Fluka, NADH/H+ and a-ketoglutaric acid from Sigma, deuterium oxide (99.8%) from Roth (Germany).…”

Isolation of an Isoenzyme of Human Glutaminyl Cyclase: Retention in the Golgi Complex Suggests Involvement in the Protein Maturation Machinery

“…In contrast to mammals, however, Drosophila apparently expresses an isoform that is translocated into mitochondria. 13 In human disease, compelling evidence suggests an involvement of QC in conditions such as rheumatoid arthritis, osteoporosis and Alzheimer's disease (AD). 14,15 Significant amounts of beta-amyloid (Aβ) peptides, deposited in amyloid plaques of AD, are N-terminally modified to possess a pGlu residue originating from glutamate cyclization.…”