International Journal of Molecular Sciences Article Testis-Specific Isoform of Na/K-ATPase (ATP1A4) Interactome in Raft and Non-Raft Membrane Fractions from Capacitated Bovine Sperm Gayathri D. Rajamanickam 1, John P. Kastelic 2 and Jacob C. Thundathil 2,* 1 Department of Veterinary Clinical and Diagnostic Services, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada 2 Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada * Correspondence: [email protected]; Tel.: +1-403-220-8244 Received: 5 May 2019; Accepted: 16 May 2019; Published: 28 June 2019 Abstract: The plasma membrane of sperm contains highly dynamic lipid microdomains (rafts), which house signaling proteins with a role in regulating capacitation. We reported that ATP1A4, the testis-specific isoform of Na/K-ATPase, interacted with caveolin-1, Src, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinases 1/2 (ERK1/2) in raft and non-raft domains of the plasma membrane of bovine sperm during capacitation. The objective of the present study was to use a proteomic approach to characterize the ATP1A4 interactome in rafts and non-rafts from capacitated bovine sperm. The non-raft interactome included hexokinase 1, plakophilin 1, desmoglein 1, 14-3-3 protein ζ/δ, cathepsin D and heat shock protein beta1 proteins exclusively, whereas glutathione S-transferase and annexin A2 were unique to raft interactome. However, a disintegrin and metalloprotease 32 (ADAM 32), histone H4, actin, acrosin, serum albumin and plakoglobin were identified in both raft and non-raft fractions of capacitated sperm. Based on gene ontology studies, these differentially interacted proteins were implicated in cell–cell adhesion, signal transduction, fertilization, metabolism, proteolysis and DNA replication, in addition to acting as transport/carrier and cytoskeletal proteins. Overall, we identified proteins not previously reported to interact with ATP1A4; furthermore, we inferred that ATP1A4 may have a role in sperm capacitation. Keywords: bull; capacitation; proteomics; lipid rafts; Na/K-ATPase 1. Introduction Capacitation is a series of changes that sperm undergo in the female reproductive tract before they are capable of fertilization [1], including hyperactivated motility, tyrosine phosphorylation and other physiological events related to intracellular ions and initiation of signaling cascades. Although outcomes are well known, involvement of specific sperm proteins in regulating capacitation remain obscure. As an integral membrane protein in the plasma membrane, the heterodimeric Na/K-ATPase transports 3 Na+ and 2 K+ ions between intra- and extra-cellular environments. The α subunit of Na/K-ATPase binds to ouabain (cardiotonic ligand/receptor) and is responsible for catalytic activity, whereas the β subunit is an accessory subunit and helps the α subunit to remain stable in the plasma membrane. Four distinct α subunits (α1, α2, α3 and α4) and three β subunits (β1, β2 and β3) are expressed in various cells, depending on their physiological needs [2]. ATP1A4, the testis-specific (α4) isoform of Na/K-ATPase, is responsible for most Na/K-ATPase activity in sperm, whereas some activity is also attributed to the ubiquitous (α1) isoform (ATP1A1) [3]. In addition, it is noteworthy that bovine vaginal fluid contains ouabain [4]. Based on the existence of ATP1A4 in sperm and the presence of Int. J. Mol. Sci. 2019, 20, 3159; doi:10.3390/ijms20133159 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 3159 2 of 14 ouabain in the female reproductive tract, we inferred that inhibition of ATP1A4 by ouabain is involved in sperm physiology. ATP1A4 has two functions—classical and non-classical—namely, as an enzyme involved in ion transportation and a signaling molecule, respectively. Although Na/K-ATPase in raft microdomains has been regarded as being largely responsible for signaling (as it is close to other signaling molecules in the raft [5,6]), various other proteins outside raft domains (non-rafts) may have important roles in signal transduction [7]. In somatic cell studies, Na/K-ATPase signaling included both raft and non-raft domains and involved various signaling molecules, including Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt (a serine/threonine kinase) [8]. The plasma membrane of sperm has rafts and includes molecules with roles in sperm–oocyte interactions [9–11]. Although sperm have been reported to contain non-raft domains [12], their potential role in capacitation or other events prior to fertilization is not known. We reported two distinct pools of ATP1A4 (raft and non-raft) which trigger specific downstream signaling molecules under ouabain-induced capacitating conditions. Specifically, using candidate proteins, we identified that ATP1A4 interacted with caveolin-1 and EGFR in the raft fraction, whereas interaction of ATP1A4 with Src, EGFR and ERK1/2 occurred in the non-raft fraction of ouabain-capacitated sperm [13]. Sperm development is inherently complex, with sperm acquiring many unique proteins, including lactate dehydrogenase (LDH-C4), sperm adhesion molecule 1 (PH-20), testis-specific isoform of angiotensin-converting enzyme (t-ACE) and testis-specific isoform of Na/K-ATPase (ATP1A4) during spermatogenesis. Accordingly, raft and non-raft membrane fractions from ouabain-capacitated sperm were subjected to immunoprecipitation–mass spectrometry (IP-MS) to characterize the interactome profile of ATP1A4. In addition, a potential role for ATP1A4 on events leading to fertilization was also proposed. 2. Results 2.1. Mass Spectrometry to Identify Partners Interacting with ATP1A4 Raft and non-raft fractions were isolated from control and capacitated sperm, and LC-MS/MS was used to identify ATP1A4 interactomes. Protein identities, probability scores and peptide sequences (>95% confidence level) in raft and non-raft fractions are shown in Tables S1 and S2. We identified six (non-raft) and two (raft) differentially interacted proteins (upregulated or downregulated) unique to their respective fractions (Figure1a,b). Six proteins were common to both membrane fractions (Figure1c). Non-raft ATP1A4 interactome comprised hexokinase 1, plakophilin 1, desmoglein 1, 14-3-3 protein ζ/δ, cathepsin D and heat shock protein β1 (HSPβ1) proteins exclusively. Glutathione S-transferase (GST) and annexin A2 were unique to the raft interactome. However, ADAM32, histone H4, actin, acrosin, serum albumin and plakoglobin were common to both raft and non-raft fractions. There were significant differences (the log2fold change was >2 and p < 0.05) for hexokinase 1, actin, desmoglein 1, serum albumin and plakoglobin, which were significantly upregulated in their interactions with ATP1A4 in ouabain-capacitated sperm compared to their respective controls (Figure1a,c). Int. J. Mol. Sci. 2019, 20, 3159 3 of 14 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 15 Figure 1. Interactome profile of ATP1A4 in raft and non-raft fractions of control (control 4 h and controlFigure 0 h) 1. and Interactome ouabain-capacitated profile of sperm.ATP1A4 (a in) Fold raft changeand no ofn-raft upregulated fractions proteinsof control that (control interacted 4 h and withcontrol ATP1A4 0 h) in and non-raft ouabain-capacitated fraction. (b) Fold sperm. change (a) of Fold upregulated change of and upregulated downregulated proteins proteins that interacted that interactedwith ATP1A4 with ATP1A4 in non-raft in raft fraction. fraction. (b (c) )Fold Fold change change of of upregulated upregulated and and downregulated downregulated proteins proteins that identifiedinteracted in both with fractions. ATP1A4a inSignificant raft fraction. (p < (0.05)c) Fold diff changeerence of compared upregulated with and control downregulated 4 h group with proteins a identifiedb in both fractions. a Significant (p < 0.05) difference compared with control 4 h group with a log2 FC >2; Significant difference compared with control 0 h group with a log2FC >2. log2 FC >2; b Significant difference compared with control 0 h group with a log2FC >2. 2.2. Ontology and Pathway Analysis of ATP1A4 Interactome in Raft and Non-Raft Fractions 2.2. Ontology and Pathway Analysis of ATP1A4 Interactome in Raft and Non-Raft Fractions Based on PANTHER analysis, protein binding was a major classification in molecular function for bothBased raft (25%) on PANTHER and non-raft analysis, (33.33%) protein fractions. binding However, was a major among classification rafts, enzyme in molecular activity function and metalfor ion both binding raft (25%) had and equivalent non-raft contributions (33.33%) fractions. (25% However, each) to that among of protein rafts, enzyme binding activity (Figure and2a,b). metal Withinion non-rafts,binding had enzyme equivalent activity, contributions protease function (25% each) and to metal that of ion protein binding binding were the(Figure second 2a,b). major Within contributorsnon-rafts, (16% enzyme for eachactivity, category) protease to molecular function function.and metal Regarding ion binding biological were process,the second sperm major functionscontributors (motility, (16% fertilization) for each category) were a major to molecula categoryr function. in raft (50%) Regarding and non-raft biological (41%) process, fractions. sperm Proteinsfunctions involved (motility, in cell–cell fertilization) adhesion were contributed a major category 25% and in