Study hypothesis: 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. studyfinding: In viewof the findings in this studywe propose that angiotensin convertingenzyme (ACE) and protein disulfide isomeraseA6 (PDIA6) are novel interacting proteins of HSPA2 and that this multimeric complex may participate in key elements of the fertilization cascade. what is known already: 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. study design, samples/materials, 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. main results and the role of chance: 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). limitations, reasons for caution: 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. wider implications of the findings: 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. large scale data: Large scale proteomics data can be accessed through the Proteomics Identifications Database (PRIDE). study funding/competing interest(s): Thiswork wassupported by theNational Health and Medical ResearchCouncil. Grant # APP1046346. The authors have no competing interests to declare.