Testis-Specific Isoform of Na/K-ATPase (ATP1A4) Interactome in Raft and Non-Raft Membrane Fractions from Capacitated Bovine Sperm.

Gayathri D Rajamanickam,John P Kastelic,Jacob C Thundathil

doi:10.3390/ijms20133159

Gayathri D Rajamanickam, John P Kastelic + Show 1 more

Open Access

https://doi.org/10.3390/ijms20133159

Copy DOI

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.

Highlights

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
Using candidate proteins, we identified that ATP1A4 interacted with caveolin-1 and epidermal growth factor receptor (EGFR) in the raft fraction, whereas interaction of ATP1A4 with Src, EGFR and extracellular signal-regulated kinases 1/2 (ERK1/2) occurred in the non-raft fraction of ouabain-capacitated sperm [13]
Raft and non-raft fractions were isolated from control and capacitated sperm, and Liquid chromatography (LC)-MS/MS was used to identify ATP1A4 interactomes

Summary

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. Outcomes are well known, involvement of specific sperm proteins in regulating capacitation remain obscure. 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. Based on the existence of ATP1A4 in sperm and the presence of ouabain in the female reproductive tract, we inferred that inhibition of ATP1A4 by ouabain is involved in sperm physiology

Objectives

Methods

Results

Conclusion