Boar spermadhesin AWN: novel insights in its binding behavior and localization on sperm.

As a major spermadhesin first found in the seminal plasma (SP) of boars, AWN is described to fulfil a variety of reproduction related tasks. Although being the best investigated boar spermadhesin, information about its interaction with membranes is inconsistent. In this regard, previous reports locate AWN either inside or on the surface of sperm cells and at different regions, depending on the method and antibody used. Here, we localize native AWN (natAWN) in/on epididymal, ejaculated, capacitated, and acrosome-reacted boar sperm using epifluorescence and electron microscopy as well as an analysis of potential lipid-binding partners of natAWN and recombinant AWN (recAWN). By applying a custom-made anti-AWN antibody, localization of AWN in the equatorial segment (EQS) of ejaculated, capacitated, and acrosome-reacted boar sperm was discovered. Electron microscopy showed that AWN is localized both on the sperm surface and on the cytoplasmic side of the plasma membrane and in close vicinity to the nuclear and both acrosomal membranes of sperm. Analysis of epididymal sperm indicated migration of AWN from the retral postacrosomal part to the EQS during the epididymal passage. In contrast to hypotheses claiming a specific association of AWN to phosphatidylethanolamine (PE) and in line with our previous study describing an interaction with phosphatidic acid (PA), the current results show a rather electrostatically driven binding mechanism of AWN to negative lipids. In conclusion, this work provides new insights into the arrangement of AWN in the EQS, which suggest a possible role in sperm-oocyte fusion.

Porcine spermadhesin AQN-3 binds to negatively charged phospholipids.

The spermadhesin AQN-3 is a major component of porcine seminal plasma. While various studies suggest that this protein binds to boar sperm cells, its attachment to the cells is poorly understood. Therefore, the capacity of AQN-3 to interact with lipids was investigated. For that purpose, AQN-3 was recombinantly expressed in E. coli and purified via the included His-tag. Characterizing the quaternary structure by size exclusion chromatography revealed that recombinant AQN-3 (recAQN-3) is largely present as multimer and/or aggregate. To determine the lipid specificity of recAQN-3, a lipid stripe method and a multilamellar vesicle (MLV)-based binding assay were used. Both assays show that recAQN-3 selectively interacts with negatively charged lipids, like phosphatidic acid, phosphatidylinositol phosphates, and cardiolipin. No interaction was observed with phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or cholesterol. The affinity to negatively charged lipids can be explained by electrostatic interactions because binding is partly reversed under high-salt condition. However, more factors have to be assumed like hydrogen bonds and/or hydrophobic forces because the majority of bound molecules was not released by high salt. To confirm the observed binding behavior for the native protein, porcine seminal plasma was incubated with MLVs comprising phosphatidic acid or phosphatidyl-4,5-bisphosphate. Attached proteins were isolated, digested, and analyzed by mass spectrometry. Native AQN-3 was detected in all samples analyzed and was - besides AWN - the most abundant protein. It remains to be investigated whether AQN-3, together with other sperm associated seminal plasma proteins, acts as decapacitation factor by targeting negative lipids with signaling or other functional roles in fertilization.