title: Heat Shock Protein member A2 forms a stable complex with angiotensin converting enzyme and protein disulfide isomerase A6 in human spermatozoa.
abstract: OBJECTIVE
  Given the importance of the chaperone Heat Shock Protein A2 (HSPA2) in the regulation of male fertility, this study aimed to identify and characterize additional proteins that may rely on the activity of this chaperone in human spermatozoa.
  
  
  RESULTS
  In view of the findings in this study we propose that angiotensin converting enzyme (ACE) and protein disulfide isomerase A6 (PDIA6) are novel interacting proteins of HSPA2 and that this multimeric complex may participate in key elements of the fertilization cascade.
  
  
  BACKGROUND
  The molecular chaperone HSPA2 plays a pivotal role in the remodelling of the sperm surface during capacitation. Indeed, human spermatozoa that are deficient in HSPA2 protein expression lack the ability to recognize human oocytes, resulting in repeated IVF failure in a clinical setting. Moreover, our recent work has shown that defective HSPA2 function induced by oxidative stress leads to the aberrant surface expression of one of its interacting proteins, arylsulfatase A, and thus contributes to a loss of sperm-zona pellucida adhesion.
  
  
  METHODS
  Human spermatozoa were collected from fertile donors, capacitated and prepared for Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) analysis. Protein complexes resolved via BN-PAGE were excised and their constituents were identified using mass spectrometry. The interactions between ACE, PDIA6 and HSPA2 were then confirmed using immunoprecipitation and proximity ligation assays and the localization of these proteins was assessed in isolated spermatozoa and commercially available human testis tissue sections. Finally, pharmacological inhibition of ACE was performed to assess the role of ACE in human sperm capacitation.
  
  
  RESULTS
  Herein we have identified ACE and PDIA6 as potential HSPA2-interacting proteins and shown that this assemblage resides in membrane raft microdomains located in the peri-acrosomal region of the sperm head. Additionally, the surface expression of PDIA6, but not ACE, was shown to be dynamically regulated during sperm capacitation and, like that of previously characterized HSPA2-interacting proteins, this surface expression proved vulnerable to oxidative stress. In terms of the functional significance of this protein complex, pharmacological inhibition of ACE significantly reduced the ability of human spermatozoa to undergo an agonist induced acrosome reaction (P < 0.01).
  
  
  CONCLUSIONS
  While these results provide a descriptive analysis of the PDIA6/ACE/HSPA2 complex, this study provides the impetus for further investigation into the role of PDIA6 and ACE in human sperm function.
  
  
  CONCLUSIONS
  As our research group, and others, have shown that HSPA2 is compromised in the spermatozoa of men with oocyte recognition defects, the characterization of these HSPA2-interacting proteins provides important insight into the complexity of the cellular pathways that may be affected in the spermatozoa of infertile individuals.
  
  
  METHODS
  Large scale proteomics data can be accessed through the Proteomics Identifications Database (PRIDE).
  
  
  BACKGROUND
  This work was supported by the National Health and Medical Research Council. Grant # APP1046346. The authors have no competing interests to declare.
authors.affiliation: Priority Research Centre for Reproductive Biology, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia elizabeth.bromfield@newcastle.edu.au.
authors.forename: Elizabeth G
authors.initials: EG
authors.lastname: Bromfield
authors.affiliation: Priority Research Centre for Reproductive Biology, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Eileen A
authors.initials: EA
authors.lastname: McLaughlin
authors.affiliation: Priority Research Centre for Reproductive Biology, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Robert John
authors.initials: RJ
authors.lastname: Aitken
authors.affiliation: Priority Research Centre for Reproductive Biology, School of Environmental and Life Sciences, Discipline of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Brett
authors.initials: B
authors.lastname: Nixon
mesh_terms: D020101:Acrosome Reaction / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D005298:Fertility / Q000235:genetics; D005786:Gene Expression Regulation; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007248:Infertility, Male / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D007703:Peptidyl-Dipeptidase A / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000235:genetics* / Q000378:metabolism; D055503:Protein Multimerization; D015398:Signal Transduction; D013075:Sperm Capacitation / Q000187:drug effects; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013737:Testis / Q000166:cytology / Q000378:metabolism
chemical_list: D018840:HSP70 Heat-Shock Proteins; C091879:HSPA2 protein, human; D000001:Calcimycin; D006861:Hydrogen Peroxide; C488034:ACE protein, human; D007703:Peptidyl-Dipeptidase A; C496931:PDIA6 protein, human; D019704:Protein Disulfide-Isomerases
keywords: acrosome reaction; angiotensin; infertility; spermatozoa; zona pellucida