|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Antimicrobial and immunostimulatory peptide, KLK, induces an increase in cytosolic Ca2+ concentration by mobilizing Ca2+ from intracellular stores
Julian Weghuber*1, Anna M Lipp†, Jacqueline Stadlbauer*, Michael C Aichinger‡, Verena Ruprecht*, Alois Sonnleitner†, Gerhard J Schütz* and Tamás Henics‡
*Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz, Austria, †Center for Advanced Bioanalysis, Scharitzerstrae 68, 4020 Linz, Austria, and ‡Department of Genetics, Max F. Perutz Laboratories, 1030 Vienna, Austria
The cationic antimicrobial immunomodulatory peptide, KLK (KLKL5KLK), exerts profound membrane interacting properties, impacting on ultrastructure and fluidity. KLK–membrane interactions that lead to these alterations require the ability of the peptide to move into an α-helical conformation. We show that KLK induces an increase of the intracellular Ca2+ concentration in human T24 cells. The effect of KLK is buffer-sensitive, as it is detected when HBSS buffer is used, but not with PBS. This, together with the lack of effect of the middle leucine-to-proline-substituted peptide derivative [KPK (KLKLLPLLKLK)], indicates that it is the conformational propensity rather than the net positive charge that contributes to the effect of KLK on intracellular Ca2+ level of T24 cells. We show that, although KLK slightly stimulates Ca2+ influx into the cell, the bulk increase of Ca2+ levels is due to KLK-induced depletion of intracellular Ca2+ stores. Finally, we demonstrate a KLK-induced switch of PS (phosphatidylserine) from the inner to the outer plasma membrane leaflet that contributes to the onset of early apoptotic changes in these cells.
Key words: apoptosis, Ca2+, influx, KLK, store depletion
Abbreviations: FBS, fetal bovine serum, KLK, KLKL5KLK, KPK, KLKLLPLLKLK, GFP, green fluorescent protein, HBSS, Hanks buffered saline solution, Lact-C2-GFP, lactadherin fused to GFP, PS, phosphatidylserine
1To whom correspondence should be addressed (email email@example.com).
Part of a series marking the 70th birthday of the Cell Biology International Editor-in-Chief Denys Wheatley
KLK (KLKL5KLK), the positively charged, amphipathic antimicrobial peptide has been studied in detail to uncover mechanisms about its interaction with membranes. We have demonstrated that, while KLK appears as a non-pore-forming peptide, it readily interacts with vesicle and cell membrane, profoundly altering their ultrastructure and fluidity (Aichinger et al., 2008). Depending on the composition of a membrane-mimicking environment, KLK can undergo multiple conformational transitions (Aichinger et al., 2008).
The second messenger Ca2+ influences a broad range of cellular and physiological processes. Changes in membrane fluidity and ultrastructure are often linked to perturbations of intracellular Ca2+ homoeostasis. For example, membrane injury caused by silica nanoparticles in macrophages is accompanied by increases of reactive oxygen species and intracellular Ca2+ concentration (Yang et al., 2009). Furthermore, increasing evidence points to the involvement of Ca2+ (namely, exocytotic insertion of vesicular Ca2+-conducting proteins into the plasma membrane) as a mechanism of coupling exocytotic activity to compensatory endocytosis, a fundamental process in maintaining cell surface area (Vogel, 2009; Usmani et al., 2010; Watanabe et al., 2010). Thus, we examined whether membrane-interacting effects of KLK were linked to changes in intracellular Ca2+ levels. We show that KLK induces an increase of the intracellular Ca2+ concentration in human T24 cells. This effect of KLK is buffer sensitive, since it is detected when HBSS (Hanks buffered saline solution) is used but is absent in PBS. An immunologically inert variant of KLK with a middle leucine to proline mutation (KLKLLPLLKLK, KPK) (Aichinger et al., 2008) lacks this effect, indicating that the increase of intracellular Ca2+ levels depends on the conformation of the peptide rather than its net positive charge. KLK slightly stimulates Ca2+ influx, but it also leads to a depletion of intracellular Ca2+ stores. Finally, we show that KLK induces a switch of PS (phosphatidylserine) from the inner to the outer plasma membrane leaflet, a characteristic feature of early apoptotic stages in eukaryotic cells.
2. Materials and methods
KLK and KPK were provided by Intercell AG. HBSS with/without Ca2+ and PBS with Ca2+ were from PAA Laboratories. BAPTA-AM was purchased from Invitrogen.
2.2. DNA constructs
pEGFP-C1-Lact-C2 [GFP (green fluorescent protein) fused with C2 domain of bovine lactadherin] was purchased from Haematologic Technologies Inc.).
2.3. Cell culture
Human T24 cells were from American Type Culture Collection. FBS (fetal bovine serum), media, antibiotics, trypsin and Geneticin (G418 sulfate) were purchased from PAA Laboratories GmbH. Culture plates were from Greiner Bio One International. A Gene Pulser electroporation unit (X-cell) and electroporation cuvettes were from Bio-Rad. T24 cells were cultured in RPMI medium supplemented with 10% FBS and grown at 37°C in a humidified incubator (≥95%) with 5% CO2 in air. For stable expression, 70% confluent cells were harvested and transfected with 10 μg of plasmid DNA using the X-cell electroporator with the following electroporation conditions: 240 V, 950 μF, unlimited resistance, 4 mm gap cuvettes and RPMI16 without FBS as the electroporation buffer. Cells were plated into 100 mm culture dishes and grown for 48 h. The medium was removed and replaced with fresh medium supplemented with 400 μg/ml G418. Medium was changed every 3 days, and 15–20 days later, individual neomycin-resistant colonies were selected for propagation and analysis. Transfected cells were grown on 3-cm glass coverslips and analysed 48 h after seeding.
2.4. Ca2+ measurements
T24 cells were loaded with Indo1-AM (3 μM, Invitrogen) in complete RPMI-1640 medium at room temperature for 30 min and washed twice with Ca2+-containing HBSS buffer. This cell permeable ratiometric dye allows for monitoring changes in free cytoplasmic Ca2+ levels (Grynkiewicz et al., 1985). All fluorescence measurements were performed with a self-developed microscope device (CytoScout®) (Paar et al., 2007). As a light source, a mercury lamp (HBO100, Zeiss) was used, and fluorescence was excited and collected via a ×10 Fluar objective (Zeiss). All bandpass filters and dichroic mirrors were from AHF Analysentechnik. Cells were illuminated at 333 nm (333/30), and fluorescence emission at 405 nm (405/20) and 485 nm (485/25) was detected simultaneously by 2 CoolSnap HQ™ CCD cameras (Photometrics). First, cells were acquired for 20 s without stimulation to monitor baseline activity, then KLK, or KPK, in their respective buffers, were added, and cells were acquired for another 3–4 min. Image processing and analysis was performed by ImageJ and MATLAB® (MathWorksTM, Inc.).
2.5. Annexin V staining
Human T24 cells were grown on 3-cm glass coverslips to 70% confluency and incubated with 10 nmol/ml KLK for 15 or 30 min, respectively, followed by fixation with 4% paraformaldehyde for 30 min at room temperature. After washing with HBSS buffer, Annexin V-Cy5 staining was performed following the manufacturers instructions (Apoptosis Detection Kit, BioVision).
2.6. Confocal microscopy
LSM images were taken by means of a LSM 510 Meta confocal laser scanning microscope using a ×40 1.2NA water immersion objective (Zeiss).
3.1. KLK but not KPK increases intracellular Ca2+ concentration
To study the impact of KLK on the intracellular Ca2+ concentration, we used the fluorescent Ca2+ indicator Indo1-AM. Human T24 cells were loaded with 3 μM Indo1-AM, and the ratio of fluorescence detected at 405 and 485 nm was determined for all cells in a selected area at given time-points (Figure 1A). Measurements were done in HBSS buffer containing Ca2+. After 20 s, cells were treated with 100 nmol/ml KLK, and Ca2+ levels were subsequently monitored for 4 min. A remarkable increase in intracellular Ca2+ concentration occurred 20 s after KLK addition (Figure 1B). The increase reached a plateau within 60 s and remained constant for 4 min (Figure 1B). When the middle leucine-to-proline-substituted and immunologically inert derivate of KLK, termed KPK (Aichinger et al., 2008), was tested in the same system, no increase in the intracellular Ca2+ concentration occurred (Figure 1C). Moreover, the presence of KPK in the incubation buffer did not prevent the effect of KLK on the Ca2+ level induced by subsequent administration of KLK to the cells (Figure 1C).
3.2. KLK-mediated changes of intracellular Ca2+ concentration are dependent on buffer conditions
To obtain more clues about the effects of KLK, the intracellular Ca2+ concentration of T24 cells in Ca2+-containing PBS buffer in the presence of 100 nmol/ml KLK was monitored. Interestingly, in contrast to our results obtained in HBSS including Ca2+, no effect of KLK on intracellular Ca2+ levels was observed under these conditions (Figure 2A). Since it was the α-helical conformation that could not be induced in sodium phosphate buffer in our previous measurements (Aichinger et al., 2008), it could be assumed that the presence of a high phosphate concentration prevented the correct folding of KLK into its α-helical form. This conformational hindrance might inhibit the interaction of KLK with the T24 cell membrane that is necessary for the peptide to induce ultrastructural and/or fluidity changes responsible for an increase in cytoplasmic Ca2+ levels.
3.3. KLK induces depletion of intracellular Ca2+ stores
Intracellular Ca2+ concentration was therefore measured after incubation of T24 cells with 100 nmol/ml KLK in nominally Ca2+-free HBSS buffer. The Ca2+ level was also increased under these conditions (Figure 2B), but with a lower magnitude compared with measurements performed in HBSS buffer containing Ca2+. Additionally, the Ca2+ concentration was slightly decreased within 4 min, a phenomenon not seen when extracellular Ca2+ was present (Figure 2B). We conclude that KLK induces a fast depletion of intracellular Ca2+ stores and also promotes the influx of Ca2+ from the extracellular buffer. To check this assumption, we used the membrane-permeable Ca2+ chelator, BAPTA-AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)]. T24 cells loaded with 3 μM Indo1-AM were incubated with 10 μM BAPTA-AM in Ca2+-free HBSS buffer for 20 min at 37°C in the dark. Addition of KLK did not result in an increase in the intracellular Ca2+ concentration (Figure 2C). These experiments clearly show that KLK addition leads to a fast elevation of the cytoplasmic Ca2+ level by depleting Ca2+ stores, probably in the endoplasmatic reticulum. This depletion may induce further entry of Ca2+ from extracellular sources via, for example, activation of SOCs (store-operated channels) (Csutora et al., 2006; Salido et al., 2009).
3.4. KLK mediates a switch of PS from the inner to the outer plasma membrane leaflet
Increased intracellular Ca2+ concentration can result in early apoptotic signalling (Mattson and Chan, 2003). The switch of PS from the inner to the outer plasma membrane leaflet is a characteristic event of cells undergoing the early stages of apoptosis (Li et al., 2003). Therefore, to investigate this possibility in KLK-treated T24 cells, two different methods were applied to follow the interleaflet switch of PS. First, fluorescently labelled Annexin V, which binds PS on the outer leaflet, was used. No fluorescent signal was observed in cells exposed to KLK for 15 min and subsequently stained with Annexin V (Figure 3A), but staining of some cells occurred 30 min post-KLK treatment (Figure 3B). Secondly, we used another fluorescent probe, which targets PS. The C2 domain of Lact-C2-GFP (lactadherin fused to GFP) specifically binds PS in the inner plasma membrane leaflet (Yeung et al., 2008). This probe in T24 cells was stably expressed (Figure 3C) and monitored a potential effect of KLK on its localization. Addition of 100 nmol/ml KLK resulted in a remarkable loss of the plasma membrane localization of Lact-C2-GFP and an increased staining of intracellular organelles by the probe within 30 min (Figure 3D). These data indicate that a PS switch occurs upon KLK treatment, and cells undergo early stages of apoptosis within 30 min of peptide addition.
Variations of intracellular Ca2+ concentrations are essential for numerous cellular processes transducing extracellular signals into living cells (Clapham, 1995). Once it enters the cytoplasm, Ca2+ exerts regulatory effects on different enzymes and proteins, playing a pivotal role in a wide range of cellular processes (Uhlen and Fritz, 2010).
We have described an easily detectable induction of intracellular Ca2+ levels by the cationic antimicrobial peptide, KLK, when human T24 cells are exposed to this peptide. This effect does not solely depend on the positive charge of the peptide and requires the α-helical conformation of KLK, no similar effect being detected by KPK and no KLK effect observed in PBS (Aichinger et al., 2008). KPK completely loses its propensity to aggregate into intermolecular β-sheeted structures as well as into α-helical structures upon appropriate environmental conditions (Aichinger et al., 2008). Circular dichroism spectra in water showed random coil conformation for both KLK and KPK. In Na-phosphate buffer, KLK, but not KPK, aggregated into β-sheeted complexes, while the lipid-mimicking environment rendered KLK into α-helical structure, a conformational transition, which was not observed with KPK (Aichinger et al., 2008). Because KLK and KPK carry the same net positive charge, we assume that the observed effect of KLK on the intracellular Ca2+ levels requires the onset of conformational transitions of the peptide. Although, KLK weakly promotes Ca2+ influx into T24 cells, its effect of increasing cytoplasmic Ca2+ levels is linked to depletion of intracellular Ca2+ stores, most likely the ER (endoplasmic reticulum). Finally, this phenomenon could be linked to early apoptotic signalling in T24 cells, demonstrated by a PS switch between the plasma membrane leaflets, a known sign of early apoptotic activity.
The mechanism(s) by which KLK elicits an increase of cytoplasmic Ca2+ concentration remain(s) elusive. We speculate that electrostatic interaction of KLK with certain surface receptors may contribute to the activation of Ca2+ signalling via different pathways, e.g. the phospholipase C-dependent pathway, which leads to the release of Ca2+ from the endoplasmic reticulum (Berridge, 2009).
Julian Weghuber designed and performed experiments and wrote the manuscript. Anna Lipp and Jacqueline Stadlbauer performed the Ca influx measurements. Michael Aichinger designed the experiments. Verena Ruprecht assisted in the digital data processing and evaluation. Alois Sonnleitner assisted in the Ca influx measurements. Gerhard Schütz designed the experiments. Tamás Henics wrote the manuscript.
This work was supported by the
Aichinger, MC, Ortbauer, M, Reipert, S, Zauner, W, Bogner, P, Froschauer, E, Nowikovsky, K, Lingnau, K, Von, GA, Schweyen, R and Henics, T (2008) Unique membrane-interacting properties of the immunostimulatory cationic peptide KLKL(5)KLK (KLK). Cell Biol Int 32, 1449-58
Csutora, P, Zarayskiy, V, Peter, K, Monje, F, Smani, T, Zakharov, SI, Litvinov, D and Bolotina, VM (2006) Activation mechanism for CRAC current and store-operated Ca2+ entry: calcium influx factor and Ca2+-independent phospholipase A2beta-mediated pathway. J Biol Chem 281, 34926-35
Paar, C, Wurm, S, Pfarr, W, Sonnleitner, A and Wechselberger, C (2007) Prion protein resides in membrane microclusters of the immunological synapse during lymphocyte activation. Eur J Cell Biol 86, 253-64
Usmani, SM, Fois, G, Albrecht, S, Von, As, Dietl, P and Wittekindt, OH (2010) 2-APB and capsazepine-induced Ca2+ influx stimulates clathrin-dependent endocytosis in alveolar epithelial cells. Cell Physiol Biochem 25, 91-102
Vogel, SS (2009) Channeling calcium: a shared mechanism for exocytosis–endocytosis coupling. Sci Signal 2, e80
Watanabe, H, Yamashita, T, Saitoh, N, Kiyonaka, S, Iwamatsu, A, Campbell, KP, Mori, Y and Takahashi, T (2010) Involvement of Ca2+ channel synprint site in synaptic vesicle endocytosis. J Neurosci 30, 655-60
Yeung, T, Gilbert, GE, Shi, J, Silvius, J, Kapus, A and Grinstein, S (2008) Membrane phosphatidylserine regulates surface charge and protein localization. Science 319, 210-3
Received 4 August 2010; accepted 9 August 2010
Published as Cell Biology International Immediate Publication 9 August 2010, doi:10.1042/CBI20100408
© 2010 The Author(s)
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)