Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells

https://doi.org/10.1016/j.bbapap.2006.08.003Get rights and content

Abstract

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 β- and 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.

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

References (34)

  • A.M. Cataldo et al.

    Abeta localization in abnormal endosomes: association with earliest Abeta elevations in AD and Down syndrome

    Neurobiol. Aging

    (2004)
  • A.C. Awade et al.

    Pyrrolidone carboxyl peptidase (Pcp): an enzyme that removes pyroglutamic acid (pGlu) from pGlu-peptides and pGlu-proteins

    Proteins

    (1994)
  • G.N. Abraham et al.

    Pyroglutamic acid. Non-metabolic formation, function in proteins and peptides, and characteristics of the enzymes effecting its removal

    Mol. Cell. Biochem.

    (1981)
  • W.H. Fischer et al.

    Identification of a mammalian glutaminyl cyclase converting glutaminyl into pyroglutamyl peptides

    Proc. Natl. Acad. Sci. U. S. A.

    (1987)
  • M. Messer

    Enzymatic cyclization of l-glutamine and l-glutaminyl peptides

    Nature

    (1963)
  • S. Schilling et al.

    Substrate specificity of glutaminyl cyclases from plants and animals

    Biol. Chem.

    (2003)
  • T. Pohl et al.

    Primary structure and functional expression of a glutaminyl cyclase

    Proc. Natl. Acad. Sci. U. S. A.

    (1991)
  • Cited by (0)

    View full text