Placenta 35 (2014) 422e424

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Short communication

Arachidonic acid metabolism in the human placenta: Identification of a putative lipoxygenase A. Jadoon a, P. Cunningham b, L.C. McDermott a, * a

Diabetes and Nutritional Sciences Division, School of Medicine, Franklin Wilkins Building, Stamford Street, London SE1 9NH, UK Department of Biochemistry, School of Biomedical and Health Sciences, King’s College London, Franklin Wilkins Building, Stamford Street, London SE1 9NH, UK b

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 30 March 2014

Arachidonic acid (ARA) metabolites maintain pregnancy and control parturition. We generated a network of 77 proteins involved in placental ARA metabolism to identify novel proteins in this pathway. We identified a long pathway within this network which showed that secretory and cytosolic phospholipase A2 proteins act in concert. The functions of all network proteins expressed in the placental decidua were determined by database searches. Thus ARA metabolism was linked to carbohydrate metabolism. One protein, transmembrane protein 62 (TMEM62), expressed in decidua was previously uncharacterized, and was identified as a putative lipoxygenase. TMEM62 may play a role in pregnancy and/or parturition. Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

Keywords: Arachidonic acid Metabolism Proteineprotein interactions Leukotrienes Lipoxygenase

1. Introduction Arachidonic acid (ARA) metabolites play key roles in pregnancy maintenance and parturition. The mechanisms by which they do so remain elusive. Human term placenta is dominated by ARAcontaining membrane phosphoglycerides [1]. Of all the placental tissues, the decidua has the greatest turnover in metabolizing ARA [2]. Membrane stores of ARA are released by phospholipase A2 allowing for its transport, storage and/or metabolism. There are ten isozymes of secretory type II phospholipase A2 and four isozymes of cytosolic type IV phospholipase A2. Type IIA and type IVa proteins are the most studied. Both are expressed in amnion, decidua and trophoblast and function in human labour and delivery [3]. ARA is metabolized via a transcellular process using cyclooxygenases to produce prostaglandins, thromboxanes and prostacyclin; by cytochrome p450 to create epoxides and by lipoxygenases to yield leukotrienes and lipoxins. The lipoxygenase protein family includes 5-lipoxygenase, 12-lipoxygenase and 15-lipoxygenase. Only the expression of 5-lipoxygenase protein in the placenta has been reported. Its expression in decidua increases after labour compared with before labour, suggesting that it functions in the control of

Abbreviations: ARA, arachidonic acid; HPA, Human Protein Atlas. * Corresponding author. Tel.: þ44 (0) 207 848 4433; fax: þ44 (0) 207 848 4455. E-mail addresses: [email protected], dr.lindsay.mcdermott@gmail. com (L.C. McDermott). http://dx.doi.org/10.1016/j.placenta.2014.03.024 0143-4004/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

parturition at term [4]. The actions of 5-lipoxygenase produce leukotriene B4 (LTB4) [5]. The spontaneous onset of labour is associated with increased placental LTB4 output. As a hypothesis generating strategy, we employed a mathematical and computational approach to identify novel proteins that may participate in placental ARA metabolism. Analysis of a long pathway through a network of 77 proteins involved in placental ARA metabolism revealed that secretory and cytosolic phospholipase A2 proteins act in concert and ARA and carbohydrate metabolisms are linked. A previously uncharacterized protein, transmembrane protein 62, was identified as a putative lipoxygenase.

2. Methods 2.1. Network creation, pathway analysis, protein expression and function The 15 expressed enzymes participating in ARA metabolism in decidua were identified from the Human Protein Atlas (HPA) version 12 (http://www.proteinatlas. org/), a database of immunohistochemically-generated protein expression profiles from human tissues [6], and literature searches (cut-off date 3 January 2014). Results were recorded in table format. Protein partners of our 15 target proteins were identified using the Human Protein Reference Database release 9 [12]. Using a custom-written Cþþ code, interaction distances involved in longer pathways between proteins were calculated after penalizing shorter pathways. Distance measurements underwent average hierarchical clustering using the hclust function in the program “R” (http://www.R-project.org/). “R” was used to visualize the results as a dendrogram (Supplementary Methods). Expression of protein partners of our 15 targets in decidua was investigated using the HPA (cut-off date 13 January 2014). The Universal Protein Resource, the major repository for protein information (http://

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Fig. 1. Dendrogram of the longest proteineprotein path found within a placental ARA metabolism protein network. Proteins expressed in the decidua are coloured in red. Target proteins are highlighted in blue. The previously uncharacterized protein transmembrane protein 62 (gene name: TMEM62) is marked with a black arrow. Secretory (group V; gene name: PLA2G5) and cytosolic (group IV; gene name: PLA2G4A) phospholipase A2 proteins cluster at a distance of 2. Their close association is enclosed by a rectangle. The rainbow of coloured branches highlights that all other target protein cluster individually at a distance of 2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) www.uniprot.org/), was mined to determine the biological process for each protein partner (cut-off date 13 January 2014). Results were collated in table format. 2.2. Structural modelling A previously unidentified protein was located within our network. To visualize its three-dimensional structure, a structural model was created from its amino acid sequence using the fold prediction server I-TASSER on 21 October 2013 (http:// zhanglab.ccmb.med.umich.edu/I-TASSER/; [7]).

3. Results and discussion 3.1. Protein network of placental ARA metabolism Database and literature searches revealed 15 enzymes expressed in decidua (Supplementary Table 1). Sixty-two protein partners for these were identified in our protein network; 34 of

Fig. 2. Predicted structure of TMEM62. A) Sequence and domain coverage of structural model of TMEM62. B) Ribbon diagram of the lipoxygenase protein fold predicted for TMEM62 using I-TASSER software. The C-terminal domain is coloured blue and the catalytic domain green. Model parameters: C-score 1.63, template modelling score 0.52  0.15 and root mean square deviation 11.8  4.5 Å. The image was generated using PyMol. C) The ARA pathway in which TMEM62 may be involved in the conversion of LTB4 to 20-COOHLTB4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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which were expressed in decidua (Fig. 1). The dendrogram represents a long proteineprotein path through the network. A distance of 2 is biologically relevant. Secretory (group V) and cytosolic (group IVa) phospholipase A2 proteins appeared in the same cluster indicating that both function in concert. Other target proteins clustered individually suggesting that each operates independently. A distance of 1 denotes a direct proteineprotein interaction. The direct association of biglycan with secretory phospholipase A2 (group IB) links ARA metabolism to carbohydrate metabolism in decidua (Supplementary Table 2).

Conflict of interest There are no conflicts of interest that could inappropriately influence this work.

Acknowledgements We thank Prof. Anton Coolen for help with algorithms and the King’s Institute of Mathematical and Molecular Medicine for computational resources.

3.2. TMEM62, a putative lipoxygenase Our proteineprotein interaction network identified a previously uncharacterized protein, transmembrane protein 62, containing 643 amino acids. A standard protein BLAST search revealed that TMEM62 has no sequence similarities to other proteins of known function. To generate a molecular model covering the whole TMEM62 sequence, an iterative threading refinement programme I-TASSER was used to match its sequence with known protein structures. I-TASSER uses a C-score, typically in the range 5e2, to estimate the quality of the predicted model; a higher C-score indicating a model with higher confidence. The structure with the best C-score showed highest similarity to coral calcium-dependent 11-R lipoxygenase (Fig. 2B; [8]). The top 10 structural analogues and five enzyme homologues for the TMEM62 model in the Protein Data Bank were all lipoxygenases. Despite modest sequence identity, the identified lipoxygenases from different species showed similar three-dimensional structures, and accounted for the 643 residues of the TMEM62 sequence. All 15 related structures possess an N-terminal bebarrel domain, critical for membrane binding, and a predominantly a-helical catalytic domain [8]. Using an antibody raised against a recombinant 144 amino acid fragment of TMEM62, low staining in three decidua sections from different patients was recorded in the HPA. TMEM62 interacts directly with leukotriene A4 hydrolase (LTA4H; Fig. 1), an interaction that was identified using yeast-2-hybrid screening [9]. LTA4H catalyses the conversion of LTA4 into dihydroxy leukotriene, LTB4 (Fig. 2C [10],). LTB4 is further metabolized via u oxidation to inactive 20 COOH LTB4 [11]. We hypothesize therefore that TMEM62 may inactivate LTB4 in decidua. While the interaction of TMEM62 with LTA4H has yet to be experimentally confirmed, it will be interesting to analyse the contribution of TMEM62 in pregnancy and/or parturition. Funding support Ayesha Jadoon was supported by an Engineering and Physical Sciences Research Council doctoral teaching award (EP/P504961/1).

Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.placenta.2014.03.024.

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Arachidonic acid metabolism in the human placenta: identification of a putative lipoxygenase.

Arachidonic acid (ARA) metabolites maintain pregnancy and control parturition. We generated a network of 77 proteins involved in placental ARA metabol...
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