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

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• 作者：Stephan Schilling ; Christiane Lindner ; Birgit Koch ; Michael Wermann ; Jens-Ulrich Rahfeld ; Alex von Bohlen ; Thomas Rudolph ; Gunter Reuter ; Hans-Ulrich Demuth
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：September 25, 2007
• 年：2007
• 卷：46
• 期：38
• 页码：10921 - 10930
• 全文大小：501K
• 年卷期：v.46,no.38(September 25, 2007)
• ISSN：1520-4995

文摘

Glutaminyl cyclases (QCs) present in plants and vertebrates catalyze the formation ofpyroglutamic acid (pGlu) from N-terminal glutamine. Pyroglutamyl hormones also identified in invertebratesimply the involvement of QC activity during their posttranslational maturation. Database mining led tothe identification of two genes in Drosophila, which putatively encode QCs, CG32412 (DromeQC) andCG5976 (isoDromeQC). Analysis of their primary structure suggests different subcellular localizations.While DromeQC appeared to be secreted due to an N-terminal signal peptide, isoDromeQC containseither an N-terminal mitochondrial targeting or a secretion signal due to generation of different transcriptsfrom gene CG5976. According to the prediction, homologous expression of the corresponding cDNAs inS2 cells revealed either secreted protein in the medium or intracellular QC activity. Subcellular fractionationand 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 magnitudelower for most of the analyzed substrates. The pH dependence of the specificity constant was similar forboth enzymes, indicating the necessity of an unprotonated substrate amino group and two protonatedgroups of the enzyme, resulting in an asymmetric bell-shaped characteristic. The determination of themetal content of DromeQC revealed equimolar protein-bound zinc. These results prove conserved enzymaticmechanisms between QCs from invertebrates and mammals. Drosophila is the first organism for whichisoenzymes of glutaminyl cyclase have been isolated. The identification of a mitochondrial QC pointstoward yet undiscovered physiological functions of these enzymes.