Contrasting effects of molecular crowding on the membrane-perturbing and chaperone-like activities of major bovine seminal plasma protein, PDC-109.

Crowded environments inside cells and biological fluids greatly affect protein stability and activity. PDC-109, a polydisperse oligomeric protein of the bovine seminal plasma selectively binds choline phospholipids on the sperm cell surface and causes membrane destabilization and lipid efflux, leading to acrosome reaction. PDC-109 also exhibits chaperone-like activity (CLA) and protects client proteins against various kinds of stress, such as high temperature and low pH. In the present work, we have investigated the effect of molecular crowding on these two different activities of PDC-109 employing Dextran 70 (D70) - a widely used polymeric dextran - as the crowding agent. The results obtained show that presence of D70 markedly increases membrane destabilization by PDC-109. Isothermal titration calorimetric studies revealed that under crowded condition the binding affinity of PDC-109 for choline phospholipids increases approximately 3-fold, which could in turn facilitate membrane destabilization. In contrast, under identical conditions, its CLA was reduced significantly. The decreased CLA could be correlated to reduced surface hydrophobicity, which was due to stabilization of the protein oligomers. These results establish that molecular crowding exhibits contrasting effects on two different functional activities of PDC-109 and highlight the importance of microenvironment of proteins in modulating their functional activities.

Semenogelin, a coagulum macromolecule monitoring factor involved in the first step of fertilization: A prospective review.

Human male infertility affects approximately 1/10 couples worldwide, and its prevalence is found more in developed countries. Along with sperm cells, the secretions of the prostate, seminal vesicle and epididymis plays a major role in proper fertilization. Many studies have proven the functions of seminal vesicle secretions, especially semenogelin protein, as an optimiser for fertilization. Semenogelin provides the structural components for coagulum formation after ejaculation. It binds with eppin and is found to have major functions like motility of sperm, transporting the sperm safely in the immune rich female reproductive tract until the sperm cells reach the egg intact. The capacitation process is essential for proper fertilization and semenogelin involved in mediating capacitation in time. Also, it has control of events towards the first step in the fertilization process. It is a Zn ions binding protein, and Zn ions act as a cofactor that helps in the proper motility of sperm cells. Therefore, any imbalance in protein that automatically affect sperm physiology and fertility status. This review sheds a comprehensive and critical view on the significant functions of semenogelin in fertilization. This review can open up advanced proteomics research on semenogelin towards unravelling molecular mechanisms in fertilization.

Supramolecular Mechanism of Viral Envelope Disruption by Molecular Tweezers.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.

Conserved core tryptophans of FnII domains are crucial for the membranolytic and chaperone-like activities of bovine seminal plasma protein PDC-109.

The fibronectin type II (FnII) domain, present in diverse vertebrate proteins, plays crucial roles in several fundamental biological processes. PDC-109, the major bovine seminal plasma protein, contains two FnII domains that bind to choline phospholipids on sperm plasma membrane and induce lipid efflux crucial for successful fertilization. PDC-109 also exhibits chaperone-like activity and protects other proteins against various types of stress. Here, we show that a core tryptophan residue is highly conserved across species in the FnII domains. Mutation of conserved tryptophan residues W47, W93, and W106 in the FnII domains of PDC-109 to alanine leads to drastic decrease or complete abolition of membrane-binding and chaperone-like activities. These observations suggest that conserved tryptophans are important for the function of FnII proteins.

Evidence for mouse sulfhydryl oxidase-assisted cross-linking of major seminal vesicle proteins.

Copulatory plug formation in animals is a general phenomenon by which competition is reduced among rival males. In mouse, the copulatory plug formation results from the coagulation of highly viscous seminal vesicle secretion (SVS) that is rich in proteins, such as dimers of SVS I, SVS I + II + III, and SVS II. These high-molecular-weight complexes (HMWCs) are also reported to be the bulk of proteins in the copulatory plug of the female mouse following copulation. In addition, mouse SVS contributes to the existence of sulfhydryl oxidase (Sox), which mediates the disulfide bond formation between cysteine residues. In this study, flavin adenine dinucleotide (FAD)-dependent Sox was purified from mouse SVS using ion exchange and high-performance liquid chromatography. The purified enzyme was identified to be Sox, based on western blot analysis with Sox antiserum and its capability of oxidizing dithiothreitol as substrate. The pH optima and thermal stability of the enzyme were determined. Among the metal ions tested, zinc showed an inhibitory effect on Sox activity. A prosthetic group of the enzyme was identified as FAD. The Km and Vmax of the enzyme was also determined. In addition to purification and biochemical characterization of seminal vesicle Sox, the major breakthrough of this study was proving its cross-linking activity among SVS I-III monomers to form HMWCs in SVS.

Identification and characterization of RSIY-11, a novel seminal peptide derived from semenogelin-1, which acts as a neutral endopeptidase inhibitor modulating sperm motility.

PURPOSE: Based on prior reports demonstrating that neutral endopeptidase (NEP) inhibitors increase sperm motility, the goal of our studies was to identify endogenous seminal peptides that inhibit NEP and investigate their potential effect on sperm motility. METHODS: Peptidomic analysis was performed on human seminal fluid, identifying 22 novel peptides. One peptide, named RSIY-11, derived from semenogelin-1, was predicted through sequence analysis to be a substrate and/or potential inhibitor of NEP. Enzymatic analysis was conducted to determine the inhibitory constant (Ki) of RSIY-11 as an inhibitor of NEP. Total and progressive sperm motility was determined at baseline and 30 and 60 min following addition of RSIY-11 to seminal fluid in 59 patients undergoing an infertility workup at an urban medical center. Additionally, the effects of RSIY-11 on sperm motility were evaluated in 15 of the 59 patients that met criteria for asthenospermia. RESULTS: RSIY-11 was shown to act as a competitive inhibitor of NEP with a Ki of 18.4 ± 1.6 µM. Addition of RSIY-11 at concentrations of 0.75 µM, 7.5 µM, and 75 µM significantly increased sperm motility at all time points investigated, with increases of 6.1%, 6.9%, and 9.2% at 60 min, respectively. Additionally, within the subgroup of patients with asthenospermia, RSIY-11 at concentrations of 0.75 µM, 7.5 µM, and 75 µM significantly increased sperm motility at all time points investigated, with increases of 7.6%, 8.8%, and 10.6% at 60 min, respectively. CONCLUSIONS: RSIY-11 is a newly identified semenogelin-1-derived peptide present in seminal fluid. RSIY-11 acts as a potent competitive inhibitor of NEP, which when added to seminal fluid significantly increases sperm motility. RSIY-11 could play a potential role in the treatment for male factor infertility related to asthenospermia and improve intrauterine insemination outcomes.

Glycosylation differentially modulates membranolytic and chaperone-like activities of PDC-109, the major protein of bovine seminal plasma.

The major bovine seminal plasma protein, PDC-109, is a mixture of glycosylated (BSP-A1) and non-glycosylated (BSP-A2) isoforms of a 109-residue long polypeptide. It binds to spermatozoa by specifically recognizing choline phospholipids on the plasma membrane and destabilizes it by penetrating the hydrophobic interior, resulting in lipid efflux, which is necessary for sperm capacitation and successful fertilization. PDC-109 also acts as a molecular chaperone and protects target proteins from denaturation and aggregation induced by various types of stress. In order to investigate the role of glycosylation in these activities, we have separated BSP-A1 and BSP-A2 from PDC-109, and also cloned and expressed BSP-A2 in E. coli and purified the recombinant BSP-A2 (rBSP-A2) to homogeneity. Employing biophysical and biochemical approaches we have investigated the membrane-perturbing and chaperone-like activities (CLA) of PDC-109, BSP-A1, BSP-A2 and recombinant BSP-A2 (rBSP-A2). The results obtained demonstrate that glycan-lacking wild-type BSP-A2 and rBSP-A2 exhibit higher membrane-perturbing activity but decreased CLA as compared to PDC-109. In contrast, BSP-A1 exhibits significantly higher CLA than PDC-109, but its ability to destabilize membranes is considerably lower. This differential modulation of the membrane-perturbing and chaperone-like activities has been explained on the basis of higher membrane-penetrating ability and lower solubility of glycan-lacking BSP-A2 as compared to the glycosylated BSP-A1.

Lectin-Binding Specificity of the Fertilization-Relevant Protein PDC-109 by Means of Surface Plasmon Resonance and Carbohydrate REcognition Domain EXcision-Mass Spectrometry.

Seminal plasma proteins are relevant for sperm functionality and some appear responsible for establishing sperm interactions with the various environments along the female genital tract towards the oocyte. In recent years, research has focused on characterizing the role of these proteins in the context of reproductive biology, fertility diagnostics and treatment of related problems. Herein, we focus on the main protein of bovine seminal plasma, PDC-109 (BSP-A1/-A2), which by virtue of its lectin properties is involved in fertilization. By means of surface plasmon resonance, the interaction of PDC-109 with a panel of the most relevant glycosidic epitopes of mammals has been qualitatively and quantitatively characterized, and a higher affinity for carbohydrates containing fucose has been observed, in line with previous studies. Additionally, using the orthogonal technique of Carbohydrate REcognition Domain EXcision-Mass Spectrometry (CREDEX-MS), the recognition domain of the interaction complexes between PDC-109 and all fucosylated disaccharides [(Fuc-α1,(3,4,6)-GlcNAc)] has been defined, revealing the specific glycotope and the peptide domain likely to act as the PDC-109 carbohydrate binding site.

Recombinant Human Semenogelin-1 (Sg1) and Sg1 (1-159) form Detergent Stable Amyloid like Aggregates in vitro.

Senile seminal vesicle amyloidosis (SSVA) is associated with deposition of semenogelin-1 (Sg1) protein aggregates in seminal vesicles that may manifest as hematospermia. Sg1 is the predominant protein that entraps spermatozoa which are freed upon fragmentation of Sg1 by the protease prostate specific antigen (PSA), post semen release. Certain small peptide fragments of Sg1 have been reported to form amyloid aggregates in vitro that can enhance HIV infectivity to cell cultures. However, the amyloid deposits in the seminal vesicles are expected to be that of the full length Sg1, as PSA is encountered downstream. So far, amyloid forming ability of full length Sg1 has not been established in vitro. Here, we examined the amyloidogenicity of full length Sg1 and a large fragment Sg1 (1-159), using recombinant proteins and tested if Zinc has any effect on their aggregation. Levels of Zinc, which is essential for health of male reproductive system, gradually decline with age. We succeeded in forming amyloid-like aggregates of Sg1 full length and Sg1 (1-159) fragment showing detergent stability and found that presence of Zn2+ substantially inhibits their amyloid aggregation in vitro. Possibly, high Zn2+ found in seminal plasma of young individuals may have preventive role against aggregation of Sg1 in seminal vesicles.

Evolution and function of mammalian binder of sperm proteins.

Binder of sperm (BSP) proteins are ubiquitous among mammals and have been extensively investigated over the last three decades. They were first characterized in bull seminal plasma and have now been identified in more than 15 different mammalian species where they represent a superfamily. In addition to sharing a common structure, BSP proteins share many characteristics. They are expressed by seminal vesicles and epididymides, interact with similar ligands and bind to the outer leaflet of sperm membranes via an interaction with choline phospholipids. In addition to playing a major role in sperm capacitation, they are implicated as molecular chaperones in sperm motility and viability, in the formation of the oviductal sperm reservoir, in the regulation of cell volume and possibly in the interaction between sperm and oocytes, making them crucial multifunctional proteins. Furthermore, BSP proteins can bind to egg yolk low-density lipoproteins and milk components, an interaction important for the protection of sperm during semen preservation in liquid or frozen state. Our current knowledge of BSP proteins strongly emphasizes their fundamental importance in male fertility and in the optimization of semen preservation techniques. Much work is still ahead in order to fully understand all the mysteries of BSP proteins.

New Insights into the Phylogeny and Gene Context Analysis of Binder of Sperm Proteins (BSPs).

Seminal plasma (SP) proteins support the survival of spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, resulting in higher fertilizing ability. Among SP proteins, BSP (binder of sperm) proteins are the most studied, since they may be useful for the improvement of semen diluents, storage and subsequent fertilization results. However, an updated and detailed phylogenetic analysis of the BSP protein superfamily has not been carried out with all the sequences described in the main databases. The update view shows for the first time an equally distributed number of sequences between the three families: BSP, and their homologs 1 (BSPH1) and 2 (BSPH2). The BSP family is divided in four subfamilies, BSP1 subfamily being the predominant, followed by subfamilies BSP3, BSP5 and BSP2. BSPH proteins were found among placental mammals (Eutheria) belonging to the orders Proboscidea, Primates, Lagomorpha, Rodentia, Chiroptera, Perissodactyla and Cetartiodactyla. However, BSPH2 proteins were also found in the Scandentia order and Metatheria clade. This phylogenetic analysis, when combined with a gene context analysis, showed a completely new evolutionary scenario for the BSP superfamily of proteins with three defined different gene patterns, one for BSPs, one for BSPH1/BSPH2/ELSPBP1 and another one for BSPH1/BSPH2 without ELSPBP1. In addition, the study has permitted to define concise conserved blocks for each family (BSP, BSPH1 and BSPH2), which could be used for a more reliable assignment for the incoming sequences, for data curation of current databases, and for cloning new BSPs, as the one described in this paper, ram seminal vesicle 20 kDa protein (RSVP20, Ovis aries BSP5b).

A peptide from human semenogelin I self-assembles into a pH-responsive hydrogel.

The peptide GSFSIQYTYHV derived from human semenogelin I forms a transparent hydrogel through spontaneous self-assembly in water at neutral pH. Linear rheology measurements demonstrate that the gel shows a dominating elastic response over a large frequency interval. CD, fluorescence and FTIR spectroscopy and cryo-TEM studies imply long fibrillar aggregates of extended ß-sheet. Dynamic light scattering data indicate that the fibril lengths are of the order of micrometers. Time-dependent thioflavin T fluorescence shows that fibril formation by GSFSIQYTYHV is a nucleated reaction. The peptide may serve as basis for development of smart biomaterials of low immunogenicity suitable for biomedical applications, including drug delivery and wound healing.

Structural characterization of semen coagulum-derived SEM1(86-107) amyloid fibrils that enhance HIV-1 infection.

SEM1(86-107) is a 22-residue peptide corresponding to residues 86-107 in the semenogelin I protein. SEM1(86-107) is an abundant component of freshly liquefied semen and forms amyloid fibrils capable of enhancing HIV infection. To probe the factors affecting fibril formation and gain a better understanding of how differences in pH between semen and vaginal fluid affect fibril stability, this study determined the effect of pH on SEM1(86-107) fibril formation and dissociation. The SEM1(86-107) fibril structure (i.e., residues that comprise the fibrillar core) was also probed using hydrogen-deuterium exchange mass spectrometry (HDXMS) and hydroxyl radical-mediated protein modification. The average percent exposure to hydroxyl radical-mediated modification in the SEM1(86-107) fibrils was determined without requiring tandem mass spectrometry spectral acquisition or complete separation of modified peptides. It was found that the residue exposures calculated from HDXMS and hydroxyl radical-mediated modification were similar. These techniques demonstrated that three regions of SEM1(86-107) comprise the amyloid fibril core and that positively charged residues are exposed, suggesting that electrostatic interactions between SEM1(86-107) and HIV or the cell surface may be responsible for mediating HIV infection enhancement by the SEM1(86-107) fibrils.

Murine Binder of SPerm homolog 2 (BSPH2): the black sheep of the BSP superfamily.

Proteins of the Binder of SPerm superfamily are known to bind choline phospholipids on sperm membrane and promote sperm capacitation. The current study focuses on the biochemical and functional characterization of the murine Binder of SPerm homolog 2 (BSPH2). A recombinant protein (rec-BSPH2) was expressed in Escherichia coli Rosetta-gami B (DE3)pLysS cells using pET32a vector. It was purified by immobilized metal ion affinity chromatography and refolded on column using a decreasing urea gradient. Rec-BSPH2 was found to share some binding characteristics with other BSP proteins, such as binding to gelatin, heparin, and epididymal sperm. Rec-BSPH2 as well as murine recombinant BSPH1 were found to have different immunofluorescence patterns when bound to uncapacitated versus capacitated sperm, indicating a rearrangement of these proteins on sperm surface during or following capacitation. Surprisingly, rec-BSPH2 was unable to bind phosphorylcholine liposomes or promote sperm capacitation. It is the first time that such results are reported for proteins of the BSP family. The results indicate that murine BSPH1 and BSPH2 might not have redundant functions, as is the case with bovine BSPs. This study could lead to a better understanding of the role of BSP proteins in sperm functions and the existence of redundant BSP proteins in the reproductive tract.

Correlation of epididymal protease inhibitor and fibronectin in human semen.

OBJECTIVE: Epididymal protease inhibitor (Eppin) was located on the surface of spermatozoa and modulates the liquefaction of human semen. Here, we identify the correlative protein partner of Eppin to explore the molecular mechanism of liquefaction of human semen. METHODS: (1) Human seminal vesicle proteins were transferred on the membrane by Western blotting and separated by 2-D electrophoresis and incubated in recombinant Eppin. The correlative protein was identified by Mass Spectrometry (MS) (2). Western blotting was used to determine the relation of rEppin and rFibronectin(Fn); (3) Co-localization in spermatozoa were detected using immunofluorescence; (4) Correalation of Eppin and Fn was proved by co-immunoprecipitation. RESULTS: Fn was identified as the binding partner of recombinant Eppin by MS. Recombinant of Eppin was made and demonstrated that the Eppin fragment binds the fn 607-1265 fragment. The Eppin-Fn complex presents on the sperm tail and particularly in the midpiece region of human ejaculated spermatozoa. Immunoprecipitation indicated that Eppin in the spermatozoa lysates was complexed with Fn. CONCLUSIONS: Our study demonstrates that Eppin and Fn bind to each other in human semen and on human ejaculated spermatozoa. Eppin-Fn complex may involve in semen coagulation, liquefaction and the survival and preparation of spermatozoa for fertility in the female reproductive tract.

Interacting proteins on human spermatozoa: adaptive evolution of the binding of semenogelin I to EPPIN.

Semenogelin I (SEMG1) is found in human semen coagulum and on the surface of spermatozoa bound to EPPIN. The physiological significance of the SEMG1/EPPIN interaction on the surface of spermatozoa is its capacity to modulate sperm progressive motility. The present study investigates the hypothesis that the interacting surface of SEMG1 and EPPIN co-evolved within the Hominoidea time scale, as a result of adaptive pressures applied by their roles in sperm protection and reproductive fitness. Our results indicate that some amino acid residues of SEMG1 and EPPIN possess a remarkable deficiency of variation among hominoid primates. We observe a distinct residue change unique to humans within the EPPIN sequence containing a SEMG1 interacting surface, namely His92. In addition, Bayes Empirical Bayes analysis for positive selection indicates that the SEMG1 Cys239 residue underwent positive selection in humans, probably as a consequence of its role in increasing the binding affinity of these interacting proteins. We confirm the critical role of Cys239 residue for SEMG1 binding to EPPIN and inhibition of sperm motility by showing that recombinant SEMG1 mutants in which Cys239 residue was changed to glycine, aspartic acid, histidine, serine or arginine have reduced capacity to interact to EPPIN and to inhibit human sperm motility in vitro. In conclusion, our results indicate that EPPIN and SEMG1 rapidly co-evolved in primates due to their critical role in the modulation of sperm motility in the semen coagulum, providing unique insights into the molecular co-evolution of sperm surface interacting proteins.

Cryopreservation increases coating of bull sperm by seminal plasma binder of sperm proteins BSP1, BSP3, and BSP5.

Artificial insemination with frozen semen allows affordable, worldwide dissemination of gametes with superior genetics. Nevertheless, sperm are damaged by the cryopreservation process. Elucidating the molecular effects of cryopreservation on sperm could suggest methods for improving fertility of frozen/thawed semen. This study was undertaken to examine the effect of cryopreservation on the coating of sperm by binder of sperm (BSP) proteins in seminal plasma. BSP proteins are secreted by the seminal vesicles and coat the surface of sperm by partially intercalating into the outer leaflet of the sperm plasma membrane. The BSP proteins are known to play roles in the formation of the oviductal sperm storage reservoir and in sperm capacitation. We investigated the effects of cryopreservation on the sperm BSP protein coat using Bovipure to separate live sperm from extended semen and then assaying the amounts of BSP proteins on sperm using quantitative western blotting with custom-made antibodies against unique sequences of each BSP protein. Greater amounts of all three BSP proteins (BSP1, BSP3, and BSP5) were detected on frozen/thawed sperm than on fresh sperm. Furthermore, the reduction of BSP3 from 15 to 13 kDa in mass, which occurs during incubation of sperm under mild capacitating conditions, was enhanced by cryopreservation. We concluded that freezing alters the BSP protein coating on sperm, which could account in part for reduced fertility of cryopreserved semen samples.

Bovine seminal PDC-109 protein: an overview of biochemical and functional properties.

Although long-term storage of bovine semen is desirable for wider use, successful cryopreservation depends on several factors, including various proteins present in seminal plasma. One such group of proteins, viz. bovine seminal plasma (BSP) proteins represents the major protein fraction in bovine seminal plasma. They constitute three major heparin-binding (HB-) acidic proteins secreted by seminal vesicles, viz. BSP-A1/-A2 (PDC-109), BSP-A3 and BSP-30-kDa. By purification studies it was deduced that PDC-109 is a polypeptide of 109 amino acids and contains two tandem repeating fibronectin type-II (Fn-II) domains, preceded by a 23 residue N-terminal domain. Though BSP-A1 and BSP-A2 are biochemically similar they differ only in glycosylation and their mixture is called PDC-109 or gonadostatins. PDC-109 exists as a polydisperse, multimeric self-associated molecule and possesses multifunctional properties, viz. binding to the surface of plasma membrane of spermatozoa causing conformational change in the sperm surface proteins and enhances motility. Besides binding, PDC-109 protein provokes cholesterol efflux from sperm membrane and promotes sperm reservoir by interacting with oviductal membrane. Interaction of sperm with PDC-109 protein induces sperm capacitation and acrosome reaction. However, prolonged exposure of spermatozoa with free floating PDC-109 protein as during processing for preservation, increases cholesterol efflux from spermatozoa. The efflux of sperm membrane cholesterol and disturbance in cholesterol:phospholipids ratio causes destabilization of plasma membrane thereby inducing cryoinjury to the sperm. In this review, the biochemical, functional properties of PDC-109 protein and its role during semen cryopreservation is summarized.

Chemical and physical requirements for lipid extraction by bovine binder of sperm BSP1.

The bovine seminal plasma contains phosphocholine-binding proteins, which associate to sperm membranes upon ejaculation. These binder-of-sperm (BSP) proteins then induce a phospholipid and cholesterol efflux from these membranes. In this work, we determined physical and chemical parameters controlling this efflux by characterizing the lipid extraction induced by BSP1, the most abundant of BSP protein in bull seminal plasma, from model membranes with different composition. The model membranes were formed from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) or cholesterol. The modulation of BSP1-induced lipid extraction from membranes by their chemical composition and their physical properties brings us to propose a 3-step extraction mechanism. First, the protein associates with membranes via specific binding to phosphocholine groups. Second, BSP1 penetrates in the membrane, essentially in the external lipid leaflet. Third, BSP1 molecules solubilize a lipid patch coming essentially from the outer lipid leaflet, without any lipid specificity, to ultimately form small lipid/protein auto-assemblies. The stoichiometry of these complexes corresponds to 10-15 lipids per protein. It is also shown that fluid-phase membranes are more prone to BSP1-induced lipid extraction than gel-phase ones. The inhibition of the lipid extraction in this case appears to be related to the inhibition of the protein penetration in the membrane (step 2) and not to the protein association with PC head groups (step 1). These findings contribute to our understanding of the mechanism by which BSP1 modify the lipid composition of sperm membranes, a key event in sperm capacitation.