Detection of phosphatidylserine with a modified polar head group in human keratinocytes exposed to the radical generator AAPH

https://doi.org/10.1016/j.abb.2014.02.002Get rights and content

Highlights

  • AAPH induce oxidation in fatty acyl chain and serine headgroup of PS.

  • Parent scan and neutral loss were used for analysis of oxPOPS.

  • Oxidation in the serine headgroup was observed in HaCaT cells.

Abstract

Phosphatidylserine (PS) is preferentially located in the inner leaflet of the cell membrane, and translocation of PS oxidized in fatty acyl chains to the outside of membrane has been reported as signaling to macrophage receptors to clear apoptotic cells. It was recently shown that PS can be oxidized in serine moiety of polar head-group. In the present work, a targeted lipidomic approach was applied to detecting OxPS modified at the polar head-group in keratinocytes that were exposed to the radical generator AAPH. Glycerophosphoacetic acid derivatives (GPAA) were found to be the major oxidation products of OxPS modified at the polar head-group during oxidation induced by AAPH-generated radicals, similarly to previous observations for the oxidation induced by OHradical dot radical. The neutral loss scan of 58 Da and a novel precursor ion scan of m/z 137.1 (HOPO3CH2COOH) allowed the recognition of GPAA derivatives in the total lipid extracts obtained from HaCaT cells treated with AAPH. The positive identification of serine head group oxidation products in cells under controlled oxidative conditions opens new perspectives and justifies further studies in other cellular environments in order to understand fully the role of PS polar head-group oxidation in cell homeostasis and disease.

Introduction

Phosphatidylserine (PS)1 is a phospholipid that has been identified to be a preferential target of in vivo oxidation. PS is located preferentially in the inner leaflet of cell membranes, but PS oxidation products are translocated to the outside of membrane. These products are considered to be markers of the early stages of apoptosis. It is known that one of the first steps of cellular apoptosis involves PS oxidization in the fatty acyl chains that are recognized by macrophages receptors for the clearance of apoptotic cells [1], [2], [3]. Several studies showed that oxidized PS is preferentially recognized by macrophage scavenger receptors over non-oxidized PS [4], [5], [6], [7]. The oxidation products of PS identified in vivo consisted of oxidative modifications in the fatty acyl chains, such as PS hydroxide and hydroperoxide derivatives and truncated sn-2 fatty acyl species [4], [8], [9]. These types of oxidation products retained an intact PS head-group and are usually identified by the typical fragmentation pathways under MS/MS conditions, which involved the loss of the serine group [8], [10], [11]. However, in a recent study of oxidation of PS standards using the Fenton reagent, it was observed that the PS head-group can also undergo oxidative modifications leading to the formation of modified polar head-groups [10], [12]. Among these oxidation products, the derivatives with a polar head-group containing an acetic acid linked to the phosphate group, called glycerophosphoacetic acid (GPAA), were found to be the most abundant [10]. These products showed a distinct fragmentation and neutral loss under MS/MS conditions and the typical loss of the serine group is absent. This behavior explains why this type of PS oxidation product has been overlooked. As yet, PS modified in the serine polar head-group has only been found in mitochondria from brain of rats treated with tacrine, which is associated with neurotoxicity and oxidative stress conditions [13]. Evaluation of pro-inflammatory activities of PS oxidation products with modifications in serine polar head-group was tested through cytokine production, and it was found that GPAA had no pro-inflammatory activity [12]. Until now, no other efforts have been made to detect these species with oxidative modifications on the polar head-group of PS. Nevertheless, it is likely that they can occur in vivo, in cells exposed to oxidative conditions.

To give new insight into this subject, the present work aimed to evaluate the formation of GPAA species in keratinocytes (HaCaT cells) after exposure to a radical generator. Keratinocytes were selected as a cell model since externalized PS and oxidation of fatty acyl chains in PS were identified in human keratinocytes during oxidative stress [14], [15]. These cells are frequently used in oxidation studies, because they are susceptible to modifications under oxidative stress conditions, such as UV, organic peroxides, or radical generators such as AAPH [16], [17], [18], [19]. The immortal human keratinocyte line HaCaT is frequently employed for studies of skin keratinocytes in vitro, since they retain their differentiation capacity [20]. Keratinocytes were incubated with the water-soluble azo-initiator (AAPH), which is frequently used in vitro for oxidation studies. The GPAA derivatives were detected in total lipid extracts using a targetted lipidomic approach involving neutral loss and precursor ion scanning modes, following optimization of this strategy with commercial 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) as a model system.

Section snippets

Materials

2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) was from Sigma Aldrich. Trypsin and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fetal calf serum, streptomycin and penicillin were purchased from Invitrogen (Paisley, UK). 1-Palmitoyl-2-oleoyl-phosphatidylserine (POPS) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AI, USA). Chloroform (Analytical reagent grade), and methanol (HPLC grade) were from Fisher (UK).

Oxidation of phosphatidylserine with AAPH

Vesicles of POPS

Results and discussion

To observe whether PS polar head-group oxidation occurred in HaCaT cells after AAPH incubation, as observed after radical oxidation of PS by hydroxyl radical [10], [25], cells were subjected to oxidation with AAPH (30 and 50 mM). The oxidative stimuli used were the same as reported in a previous study that mimicked oxidative stress injury in keratinocytes as a model of inflammation [18]. AAPH was chosen as it is a water-soluble azo-initiator frequently used for oxidation studies, using in in

Conclusions

This study highlights that oxidation of the serine polar head group, as well as fatty acyl chains, in phosphatidylserines in keratinocytes can occur after radical attack by the azo-initiator AAPH, and is likely to be missed by conventional techniques for identifying PS oxidation that depend on detection of the PS head-group. The GPAA derivatives formed due to serine head-group modification can be identified using an improved targetted lipidomic approach, based on the observation that GPAA

Acknowledgments

Thanks are due to Fundação para a Ciência e a Tecnologia (FCT, Portugal), European Union, QREN, FEDER, and COMPETE for funding the QOPNA research unit (project PEst-C/QUI/UI0062/2013; FCOMP-01-0124-FEDER-037296), RNEM (REDE/1504/REM/2005 regarding the Portuguese Mass Spectrometry Network) and to Grant number PTDC/SAL/OSM/099762/2008 and PTDC/QUI-BIQ/104968/2008. Ph.D. grants to Elisabete Maciel (SFRH/BD/73203/2010) funded POPH-advanced formation, subsidized by the European Social Fund and by

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