Journal of Molecular Biology
Isolation of an Isoenzyme of Human Glutaminyl Cyclase: Retention in the Golgi Complex Suggests Involvement in the Protein Maturation Machinery
Introduction
Posttranslational modifications, such as amidation, sulfatation and glycosylation are common events in the biosynthesis of a number of proteins. The formation of an N-terminal 5-oxoproline (pyroglutamate, pGlu) is a modification that is present at the N-terminus of a number of peptide hormones and secretory proteins, e.g., thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), neurotensin and fibronectin.1, 2 For TRH, the pGlu residue has been shown to be crucial for hormonal activity.3 Furthermore, the pGlu residue confers resistance against proteolysis by aminopeptidases.4
The enzymatic formation of the N-terminal pGlu from glutamine was first described in plants.5 The first mammalian glutaminyl cyclase (QC) cDNA was isolated from bovine pituitary, and further work by two different groups yielded in the isolation and characterization of human (hQC) and murine QC (mQC).6, 7, 8, 9 In contrast to the plant QCs, the mammalian counterparts represent zinc-dependent metalloenzymes.7, 10 The active-site-bound zinc ion, which presumably exerts a role in the polarization of the γ-amide group of the substrate, represents also the primary interaction of competitive inhibitors of QC.11 The tissue distribution of bovine QC revealed a marked abundance in different brain regions, with highest expression in the striatum and hippocampus.6 Within neurons, QC is co-localized with its products TRH and GnRH and is secreted from secretory granules into the extracellular space.12 The localization of soluble QCs in the regulated secretory pathway, which is primarily necessary for the processing of the hormonal precursors, was also corroborated for a QC of Drosophila that has been isolated recently. 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.16, 17. These pGlu-containing Aβ peptides are suggested to be potential targets in drug development due to their pronounced stability, neurotoxicity and aggregation propensity.18, 19, 20, 21, 22 With respect to the therapeutic intervention in those pathophysiological processes, the characterization of potential isoenzymes is important to reduce risk of cross-reactivity and, hence, side effects.23, 24
Here we describe the isolation and characterization of a mammalian isoenzyme of QC. The results provide evidence for a general role of glutaminyl cyclases in the protein-maturation machinery of the secretory pathway, revealing notable functional homology to glycosyltransferases. Furthermore, the results will have importance for the profiling of QC inhibitors for the treatment of QC-related disorders.
Section snippets
Identification of human and murine glutaminyl cyclase isoenzyme
On the basis of the primary structure of human and murine QC, similarity searches using BLAST at the National Institute for Biotechnology Information were performed. The putative glutaminyl cyclase isoenzyme (isoQC) sequences were selected from nucleotide entries NM_017659 (h-isoQC) and BC058181 (m-isoQC), respectively. On the basis of the identified nucleotide sequences, specific primers for the isolation of h-isoQC and m-isoQC were deduced. The coding sequences of h-isoQC and m-isoQC were
Discussion
Genes encoding glutaminyl cyclases (QPCT) have been recently described in numerous species. Examples are known from the lower phyla cnidaria, mollusks and arthropods,13, 28, 29, 30 lower vertebrates such as serpents31 and higher mammals such as mice,7 cattle6 and humans.9 In particular, the human enzyme is well characterized, since QC emerged as a potential drug target for treatment of pGlu-protein-mediated amyloidoses, such as AD.32 Although different QC activities in mammalian tissue have
Materials
E. coli strain DH5α was used for propagation of plasmids and E. coli strain BL21 was used for the expression of h-isoQC. E. coli strains were grown, transformed and analyzed according to the manufacturer's instructions [Qiagen (DH5α) Stratagene (BL21)].
Cultivation and transfection of mammalian cells
Human astrocytoma cell line LN405, human embryonal kidney cell line HEK293 and human hepatocellular carcinoma cell line Hep-G2 were cultured in appropriate cell culture media (Dulbecco's modified Eagle's medium, 10% fetal bovine serum for HEK293
Acknowledgements
The authors are grateful to Dr. Ingo Schulz for helpful discussion and Daniel Friedrich for providing the mouse organs. The authors also thank Dr. Steffen Rossner (Paul-Flechsig Institute for Brain Research, Leipzig) for the access to the confocal laser scanning microscope.
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2021, Journal of Molecular BiologyCitation Excerpt :As a consequence, a limited number of mammalian QCs and their orthologs have been identified, whereas a large number of M28 peptidases have been reported.16 In humans, the secreted (sQC) and Golgi-resident (gQC, originally named isoQC) isoforms of QC have been identified,17,18 sQC is reported as a drug target for treating Alzheimer’s disease and Huntington's disease, while gQC is a drug target for some inflammatory disorders and cancers.2,3,7,19,20 However, the majority of current QC inhibitors target the active-site zinc ion,2,3,7,19–23 so inhibitors targeting a more QC-specific site would be advantageous in avoiding possible off-target effects.