Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells
Introduction
The formation of N-terminal pyroglutamic acid (pGlu) is a posttranslational modification of several hormones such as gastrin, neurotensin and GnRH [1]. For some of these peptides, e.g. thyrotropin-releasing hormone (TRH), it has been shown that this pGlu-modification is crucial for hormonal activity [2]. Glutaminyl cyclase (QC; EC 2.3.2.5) is a zinc-dependent metalloenzyme catalyzing the cyclization of amino-terminal glutamine into pGlu under concomitant liberation of ammonia. QCs have been identified in a number of animals and plants [3], [4], [5], [6]. Because of its broad substrate specificity, QC has a key function in posttranslational pyroglutamyl formation of presumably all pGlu-containing peptides and hormones [7]. QC is expressed in various tissues of the body with a marked abundance in different brain regions. The highest expression was observed in striatum and anterior pituitary [8]. More detailed studies on the cellular and sub-cellular distribution in porcine and bovine hypothalamic and pituitary tissue (detection of QC-immunoreactivity on secretory granules of axonal nerve endings belonging to the tractus hypothalamo hypophysalis) revealed the striking evidence, that QC is transported via the same routes as its substrates, e.g. the hormone precursors of GnRH and TRH [9].
Recently, it has been shown that QC is also capable of converting amino-terminal glutamate to pyroglutamate [10]. Therefore, QC might also play a role in amyloidotic diseases, e.g. Alzheimer's Disease (AD), Familial British Dementia (FBD) or Familial Danish Dementia (FDD), because significant amounts of the deposited peptides (Aβ, ABri, ADan) are N-terminally modified by a pyroglutamyl residue resulting from glutamate cyclization [11], [12], [13]. These pGlu-containing peptide species have been suggested to be a potential target for the development of a treatment strategy due to their pronounced neurotoxicity, stability and aggregation propensity [12], [14], [15], [16]. After demonstrating QC-catalyzed N-glutamate peptide cyclization in vitro [10], the aim of our present study was to show that QC-inhibitors prevent the pyroglutamate formation in cultured mammalian cells. Hence, we have screened a number of established cell lines in culture for QC-activity using a HPLC method. Having successfully identified several QC-containing cell lines, we were interested in suppressing QC-activity using a recently characterized highly potent QC inhibitor P150/03 [17]. Because of the lack of specific antibodies to analyze the prevention of pGlu formation at the N-terminus of a variety of potential QC peptide substrates, we decided to test P150/03 using engineered Aβ(N3E-42) and Aβ(N3Q-42) because of the availability of specific sandwich ELISAs for Aβ(N3pGlu-42).
Section snippets
Reverse-transcription PCR
Total RNA was isolated from HEK293 and β-TC 3 cells using the Nucleospin Kit (Macherey-Nagel) and reversely transcribed by SuperScript II (Invitrogen). Subsequently, QC was amplified on a 1:12,5 dilution of generated cDNA product in a 25 μl reaction with Herculase Enhanced DNA-Polymerase (Stratagene). The primer sequences for amplification of QC were: β-TC 3, 5′-ATATGCATGCATGGCAGGCAGCGAAGACAAGC-3′ (mQC, sense) and 5′-ATATAAGCTTTTACAAGTGAAGATATTCCAACACAAAGAC-3′ (mQC, antisense); HEK293,
Determination of QC activity in cell lines
With emphasis on QC activity, we have analyzed mammalian cell lines representing different derivations in order to select an optimal line for expression of QC substrate precursors mTRH-Aβ(N3E-42), mTRH-Aβ(N3Q-42) or APP (NLQ). Murine QC was recently isolated from mouse insulinoma cell line β-TC 3 and detected in mouse monocyte/macrophage cell line RAW264.7 [19], [20]. In addition, the human cell lines HeLa, HEK293, U343 and L-363 as well as the murine cell line L929 were analyzed in the present
Discussion
For several bioactive peptides, a N-terminal pyroglutamyl residue is described, which is formed post-translationally from a glutamine precursor. This pGlu formation is catalyzed by QC and pGlu-containing peptides and proteins are conserved in invertebrates like the marine snail Aplysia californica and the Brazilian armed spider Phoneutria nigriventer as well as in vertebrates like Mus musculus, Bos taurus and Homo sapiens [4], [20], [25], [26], [27]. Two major functions are attributed to the
Acknowledgments
This work was supported by grants of the BMBF (grant #0313185). The authors gratefully thank Dr. Steffen Rossner (Paul Flechsig Institute for Brain Research, Leipzig, Germany) for providing cultures of murine primary cortical neurons. We thank Dr. Ingo Schulz and Mirko Buchholz for helpful discussions, Nadine Schreier for routine cytotoxicity tests and Anett Stephan for technical assistance. The help of Prof. Dr. Robert C. Bateman Jr. and Jan Eggert for critical reading of the manuscript is
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