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Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes
Biochemical and Biophysical Research Communications 399 (2010) 406–411
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes Michel Lafleur a,*, Lesley Courtemanche a, Göran Karlsson b, Katarina Edwards b, Jean-Louis Schwartz c, Puttaswamy Manjunath d a
Department of Chemistry, Center for Self-Assembled Chemical Systems, Université de Montréal, C.P. 6128, Succ. Centre Ville, Montréal, Québec, Canada H3C 3J7 Department of Physical and Analytical Chemistry, Uppsala University, Box 579, S-751 23 Uppsala, Sweden Department of Physiology, Groupe d’étude des Protéines Membranaires, Université de Montréal, C.P. 6128, Succ. Centre Ville, Montréal, Québec, Canada H3C 3J7 d Maisonneuve-Rosemont Hospital Research Center and Faculty of Medecine, Université de Montréal, 5415 L’Assomption Blvd, Montréal, Québec, Canada H1T 2M4 b c
a r t i c l e
i n f o
Article history: Received 20 July 2010 Available online 30 July 2010 Keywords: Binder-of-sperm (BSP) proteins Sperm Membranes Protrusions Nanotubes Fluorescence microscopy Cryo-electron microscopy Lipids Lipid curvature Lipid morphology
a b s t r a c t Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed with phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction With a concentration of about 1 mM in the bovine seminal plasma, binder-of-sperm (BSP) protein BSP1 [1], previously referred to as BSP-A1/-A2 or PDC-109, represents 25 (w/w)% of the total proteins [2,3]. It is known to be essential to sperm capacitation. The modification in the membrane lipid composition and the related reorganization of the lipid domains or rafts are key events in sperm maturation [4–8]. BSP1 was shown to extract selectively cholesterol and phosphatidylcholine (PC) upon its interaction with sperm membranes [5,6,9–11] and therefore, is likely involved in the sperm capacitation process. Its fibronectine-type II (Fn2) domains organized in tandem were proposed to act as binding sites for phosphocholine groups [12,13]. This relatively specific interaction could play a role in the lipid efflux induced by the proteins. Each Fn2 domain would correspond to one binding site and consequently, each BSP1 would bind two phosphocholines. Several studies of the BSP1 binding to PC model membranes indicated a saturation lipid/protein ratio of 10–12 [14–17], suggesting a more complex association. Despite the critical role of BSP1 in sperm * Corresponding author. Fax: +1 514 343 7586. E-mail address: michel.lafl[email protected] (M. Lafleur). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.07.088
capacitation and in reproduction, the details of the mechanism for the lipid extraction are not known. Recent results [18] indicate that homologue proteins can be ubiquitous in mammals, suggesting a more general importance than first thought. In this paper, we report drastic morphological changes of model membranes induced by BSP1, as studied by cryo-electron microscopy (cryo-EM) and fluorescence microscopy. The effect of the protein was examined on multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a phospholipid bearing the most abundant head group found in bovine sperm membranes [5,19]. Our findings provide new leads for the contribution of the BSP1 to the maturation process of sperm. 2. Material and methods BSP1 was extracted from bovine seminal plasma and purified according to an established procedure [20,21]. POPC was obtained from Avanti Polar Lipids (Birmingham, AL, USA) whereas b-BODIPYÒ500/510 C12-HPC (2-(4,4-difluoro-5-methyl-4-bora3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine) was purchased from Invitrogen (Eugene, OR, USA).
M. Lafleur et al. / Biochemical and Biophysical Research Communications 399 (2010) 406–411
For the cryo-electron microscopy (cryo-EM) experiments, POPC was hydrated in MOPS buffer (10 mM, pH 7.1). The lipid suspensions were submitted to six freeze-and-thaw cycles, from liquid nitrogen to room temperatures. Standard extrusion procedures were used to form large unilamellar vesicles (LUVs) using a hand-held extruder (Avestin, Ottawa, ON, Canada) and polycarbonate filters with pores of 100-nm diameter. For the samples including BSP1, an aliquot of the protein in solution (1 mM in MOPS buffer) was added to preformed LUVs or MLVs to obtain the desired lipid/protein incubation ratio (Ri). The samples were then incubated at 37 °C for, at least two minutes. Longer incubation periods did not appear to have an influence on the resulting structures. This is consistent with the association kinetics data obtained by Isothermal Titration Calorimetry [17] and Surface Plasmon Resonance [22]. An aliquot of the samples was deposited on a copper grid covered with a perforated polymer film. The sample was equilibrated at 37 °C, and 99% relative humidity in an environmental chamber. Excess of water was blotted with a filter paper and the sample was then vitrified by plunging it into liquid ethane held at a temperature just above its freezing point. The frozen sample was transferred to the microscope, keeping it at 165 °C and protected from the atmospheric conditions. Cryo-EM pictures were obtained with a Zeiss EM 902A transmission electron microscope (Carl Zeiss NTS, Oberkochem, Germany). The instrument was operating at 80 kV, in zero loss bright-field mode. Digital images were recorded under low dose conditions with a BioVision Pro-SM Slow Scan CCD camera (ProScan, GmbH, Scheuring, Germany) and analySIS software (Soft Imaging System, GmbH, Münster, Germany). For the fluorescence experiments, POPC, and b-BODIPYÒ500/ 510 C12-HPC were respectively solubilized in benzene/methanol (90/10 (v/v)) and methanol. Aliquots of appropriate volumes were mixed in order to obtain 0.01 (mol/mol)% of fluorescent probe. The lipid organic solution was then freeze-dried. The resulting powder was hydrated in MOPS buffer (10 mM, pH 7.4), to obtain a final lipid concentration of about 1 mM. The sample was submitted to five freeze-and-thaw cycles (from liquid nitrogen to 35 °C) to ensure proper hydration of the lipid bilayers. The exact lipid concentration was determined using the Fiske-Subbarow assay [23]. For pure lipid MLVs, an aliquot of this suspension was deposited on a thin coverslip located in a custom-made holder and covered with a glass slide to prevent water evaporation. In order to observe the effect of BSP1, the appropriate volume of protein solution (0.2 mM in the MOPS buffer) was added to an aliquot of lipid MLVs to obtain an Ri of 24. This sample was mounted as described above. Fluorescence imaging was performed with a Molecular Dynamics Multiprobe 2001 Inverted confocal laser-scanning microscope (Multiprobe, Santa Barbara, CA, USA), with the excitation laser set at 488 nm and the sample fluorescent images recorded through a 510-nm bandpass filter. Alternatively, epifluorescence images and time-lapse sequences were obtained with an Olympus IX81 microscope (Olympus, Melville, NY, USA). 3. Results A typical cryo-EM micrograph of freshly extruded POPC LUVs is presented in Fig. 1A. As expected from the preparation procedure, most LUVs were unilamellar with a mean diameter of about 100 nm. The addition of BSP1, at a Ri of 48, modified the morphology of several LUVs and gave rise to various self-assembly morphologies (Fig. 1B). This representative micrograph shows elongated LUVs, some of them displaying protrusions. Analogous budding and the formation of small vesicles have been previously reported in transmission electron microscopy experiments on phosphatidylcholine LUVs in the presence of BSP1, stained with
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Fig. 1. Modification of the POPC LUV morphology induced by BSP1 (Ri = 48), as observed by cryo-EM. (A) Typical micrograph of POPC LUVs. (B) Morphologies observed in the presence of BSP1, including (a) protrusion, (b) bead necklace selfassemblies, (c) small vesicles and (d) long spiral-like threads. Bar = 100 nm.
phosphotungstic acid [15]. Series of small vesicles connected to each other were observed in our unstained samples, as well as small, isolated vesicles, with diameter as small as 15 nm. The formation of zigzag structures, resembling long spiral-like threads, was also observed. The pitch of these spiral-like structures appeared to be rather constant (about 15 nm). When the Ri was decreased to 12, this type of structure was considerably more abundant. Similar cryo-EM experiments were performed on POPC MLVs. Lipid self-assemblies with modified morphologies were induced by the addition of BSP1 at an Ri of 48 (Fig. 2). One striking feature was the formation of long tubes of various diameters. As assessed by an estimate of the wall thickness, these nanotubes appeared to be formed by one or several lipid bilayer cylinders. Their diameter was typically between 40 and 50 nm. Some tubes appeared to contain a series of small unilamellar vesicles and were then wider. The lipid nanotubes were long; at lower magnification, their length could be estimated to reach several micrometers, corresponding to an aspect ratio of 2000 or more. These nanotubes appeared to originate from MLVs, which represent a large pool of lipids. Several long spiral-like threads, similar to those obtained from the LUVs, were also frequently observed. BODYPY-labeled MLVs were observed by fluorescence confocal microscopy (Fig. 3). As expected, their size was rather heterogeneous, with diameters varying from less than 1 lm up to 10 lm. Self-assemblies with distinct morphologies could be observed from samples of labeled POPC MLVs in the presence of BSP1, at an Ri of
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0.25 lm. During the time-resolved investigations, the transformation of a necklace into a thin tube was observed (Fig. 4). This observation gives a clear indication of the sequence of structural transformations involved in the evolution of the new lipid morphologies. This time sequence of images also illustrates the dynamic nature and the considerable flexibility of these tubular structures. The video is available on the journal site. 4. Discussion
Fig. 2. Modification of the POPC MLV morphology induced by BSP1 (Ri = 48), as observed by cryo-EM. (A) Lipid nanotubes with walls of varying thickness and in some cases containing a series of LUVs. The presence of spiral-like threads is also evident. (B) Lipid nanotubes at a lower magnification. (C) A nanotube that appeared to originate from a MLV that acted as a lipid reservoir. Unless otherwise stated the size bar equals 100 nm.
24. Bead necklace-like structures were regularly found; the diameter of the small vesicles was in the order of 1 to 3 lm. These could include up to ten ‘‘beads”. Short cylindrical vesicles with a diameter of about 1 lm were also observed. Their length was generally not greater than 20 lm. Protrusions appeared on some liposomes. The walls of some structures seemed brighter than most others, suggesting that they were made of several bilayers. Long and thin lipid nanotubes were also observed. Their length could reach 0.1 mm. Their diameter was difficult to evaluate because of the resolution of the microscope but it roughly corresponded to
The addition of BSP1 to liposomes leads to changes in the morphology of vesicles. Cryo-EM and fluorescence microscopy provides a consistent view of the evolution of the shapes. The observation of these morphologies by fluorescence microscopy confirms their lipidic nature because of the lipidic nature of the fluorescent probe. Upon the addition of the protein, the vesicles become elongated and evolve towards bead-necklace structures. The latter appear to transform into series of isolated small vesicles, as series of aligned but distinct small vesicles were often seen in the cryo-EM pictures. In other cases, the bead necklace-like structures seem to rearrange and form thinner and longer lipid nanotubes, as illustrated by the fluorescence time-lapse imaging sequences. Theoretical models have been developed to rationalize the formation of different morphologies by lipid fluid bilayers. In the elastic model of vesicle shape, these morphologies are associated with equilibrium shapes that minimize the bending energy. As a parameter playing a crucial role in the bending energy, the spontaneous curvature of lipid bilayers is directly affected by the difference between the areas of the inner and the outer leaflets. The elastic model [24–26] predicted the evolution of the lipid self-assembly morphologies taking into account the leaflet area imbalance. This work led to a vesicle shape phase diagram that illustrates the various vesicle morphologies that may be adopted for different effective differential areas and volume-to-area ratios. Interestingly, this model predicts for increasing relative area of the external leaflet an evolution from a sphere, to elongated ellipsoid, bead-necklace structures and elongated thinner tubes. The protrusions and tube formation observed in the presence of BSP1 are therefore totally compatible with an increase in the external leaflet area relative to that of the internal one. The presence of very long nanotubes in BSP1-exposed MLVs indicates that these can only be formed when a large amount of lipid material is present; these tubes result from the reorganization of the MLVs and not from the fusion of smaller lipid objects. The correspondence of the lipid self-assembly morphology sequence observed in the presence of BSP1 and that proposed by the theoretical models reinforces the hypothesis suggesting that an increase of external leaflet area relative to that of the inner leaflet is the likely origin of the observed supramolecular changes. Similar morphologies, i.e. the formation of long tubular structures and protrusions, were observed when dioleoylphosphatidylglycerol (DOPG) was transported from the inner to the outer leaflet of bilayers [24] or when lyso-phosphatidylcholine was added to preformed liposomes, partitioning exclusively into the external leaflet of the membrane [27]. The increase of external leaflet area can be caused by an increased molecular area of the lipids of the external leaflet, by a contribution of the proteins interacting with the bilayers, or both. Previous electron spin resonance (ESR) spectroscopy studies showed that the presence of BSP1 has actually an ordering effect on the lipid chains for model membranes [14,28], as well as for plasma membranes of intact bovine epididymal sperm cells [29]. These results discount the increase of lipid molecular area of the external leaflet caused by interaction with the protein. The penetration of BSP1 in the external leaflet is therefore the likely origin of the putative area augmentation. The Fn2 motifs were proposed
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Fig. 3. Morphology modification of the labeled POPC MLVs induced by BSP1 (Ri = 24), as observed by fluorescence microscopy. (A) Typical micrograph of POPC MLVs. (B) Morphologies observed in the presence of BSP1: (B) protrusions; (C) bead-necklace assembly; (D) thin nanotube. (A), (C) and (D) were obtained with an oil-immersion objective (100) in confocal mode, and (B) with an oil-immersion objective (60), measuring the epifluorescence.
to act as binding sites for BSP1 [12] and these might account for the initial anchoring of the proteins. BSP1 has hydrophobic cavities that give the protein an amphiphilic character [30,31]. ESR spectroscopy studies showed that BSP1 reduces the mobility of the lipid chains up to the 14th carbon atom [14], a finding consistent with the protein penetration in membranes. Furthermore, spectrofluorescence data showing a change in intrinsic tryptophan fluorescence of BSP1 upon membrane binding supports the concept of the protein penetrating into the hydrophobic core of bilayers [16,32]. Moreover, it was concluded, comparing the interactions of BSP1 with uniformly labeled vesicles and vesicles labeled only on the inner leaflet, that the interaction of BSP1 with lipid membranes is confined to the outer leaflet of bilayers [33]. Therefore, the partial penetration of BSP1 into the external leaflet of the lipid bilayer should induce an increase of its relative area, which in turn would result in the formation of morphologies with altered and various curvatures. Based on the present results, it is unlikely that the protein added to preformed vesicles can translocate the membrane because this would result in a similar effect on both leaflets. A similar rational was used to explain the formation of protrusions induced by the phospholipase A2 adsorption on vesicles [34]. It has been shown that there is a significant rearrangement of small lipid domains in sperm membranes during the capacitation process [7,8,35]. The specific recognition of the phosphocholine group likely initiates the association of BSP1 to the lipid
membranes. The present work may imply that domain formation is also related to local morphologies induced by BSP1 because different partitioning coefficients of the various lipids in regions with different local membrane curvatures may lead to domain formation. Such phase separation induced by local bilayer curvature has been observed in bilayers supported on rippled matrices [36] as well as in tubes pulled from giant unilamellar vesicles [37]. The interaction of BSP1 with sperm membranes ultimately leads to the extraction of lipids from the membrane. This process has been observed in biological membranes of fibroblasts, red blood cells, sperm cells [10,11,29,33] as well as pure lipid vesicles [33]. Independent determinations estimated that one BSP1 protein extracts 10 [33] or 20 PC molecules [10]. Up to now, the structure of the particles resulting from the lipid extraction is unknown. It is proposed that the thread-like spiral structures observed in the present work are POPC/BSP1 complexes obtained in the presence of high concentration of the protein. These structures were more commonly observed when LUVs were incubated with a low Ri. For the MLV/BSP1 system, numerous thread-like spiral structures were observed; it must be noted that the effective lipid/protein incubation ratio in this case was much greater than 48 if one considers that the protein did not translocate bilayers and that only a small fraction of the lipids (those of the external leaflet of the outer bilayer) were exposed to the protein. The frequent observation of thread-like spiral structures in this system reinforces the
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Fig. 4. Transition from a bead-necklace to a long tube structure, recorded from a sample with a POPC/BSP1 Ri of 24. The micrographs were recorded from epifluorescence with an oil-immersion objective (60). The delay between successive pictures is about 10 s. The arrow highlights the location of the transition.
hypothesis that these structures correspond to lipid/protein complexes obtained in the presence of large amounts of BSP1. These structures would involve several lipids and proteins as they often reach several hundreds of micrometers in length. It has been shown that that the addition of BSP1 to liposomes leads to the formation of smaller self-assemblies as inferred from a drastic decrease in turbidity [14]. The supramolecular structures proposed here are compatible with this observation. It has been proposed
that lipid extraction and its specificity are associated with phosphocholine-containing lipids mainly located on the external leaflet of sperm membranes [33]. This finding fits well with our conclusion indicating that BSP1 interacts essentially with the external membrane leaflet, a process leading to the reported morphology changes. The insertion of BSP1 in the external lipid leaflet of the membrane induces local changes in bilayer curvature, and these may favor phase separations that could subsequently be involved
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in the specificity of the lipid extraction process. This original hypothesis is currently being checked in our laboratories. Acknowledgments The authors thank the Natural Sciences and Engineering Council of Canada, the Fonds Québécois de la Recherche sur la Nature et les Technologies of Québec, and the Swedish Research Council for their financial support. M.L. is grateful to the Royal Academy of Arts and Sciences of Uppsala for financial support during his stay at Uppsala University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.07.088. References [1] P. Manjunath, J. Lefebvre, P.S. Jois, J. Fan, M.W. Wright, New nomenclature for mammalian BSP genes, Biol. Reprod. 80 (2009) 394–397. [2] V. Nauc, P. Manjunath, Radioimmunoassays for bull seminal plasma proteins (BSP-A1/-A2, BSP-A3, and BSP-30-kilodaltons), and their quantification in seminal plasma and sperm, Biol. Reprod. 63 (2000) 1058–1066. [3] J.J. Calvete, M. Raida, L. Sanz, F. Wempe, K.H. Scheit, A. Romero, E. TöpferPetersen, Localization and structural characterization of an oligosaccharide Olinked to bovine PDC-109/quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa, FEBS Lett. 350 (1994) 203–206. [4] B.K. Davis, Timing of fertilization in mammals: sperm cholesterol/phospholipid ratio as a determinant of the capacitation interval, Proc. Natl. Acad. Sci. USA 78 (1981) 7560–7564. [5] J.P. Nolan, R.H. Hammerstedt, Regulation of membrane stability and the acrosome reaction in mammalian sperm, FASEB J. 11 (1997) 670–682. [6] P. Manjunath, I. Thérien, Role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation, J. Reprod. Immunol. 53 (2002) 109–119. [7] C.D. Thaler, M. Thomas, J.R. Ramalie, Reorganization of mouse sperm lipid rafts by capacitation, Mol. Reprod. Dev. 73 (2006) 1541–1549. [8] V. Selvaraj, A. Asano, D.E. Buttke, J.L. McRlwee, J.L. Nelson, C.A. Wolff, T. Merdiushev, M.W. Fornés, A.W. Cohen, M.P. Lisanti, G.H. Rothblat, G. Kopf, A.J. Travis, Segregation of micron-scale membrane sub-domains in live murine sperm, J. Cell. Physiol. 206 (2006) 636–646. [9] R. Moreau, P.G. Frank, C. Perreault, Y.L. Marcel, P. Manjunath, Seminal plasma choline phospholipid-binding proteins stimulate cellular cholesterol and phosphpolipid efflux, Biochim. Biophys. Acta 1438 (1999) 38–46. [10] R. Moreau, P. Manjunath, Characterization of lipid efflux particles generated by seminal phospholipid-binding proteins, Biochim. Biophys. Acta 1438 (1999) 175–184. [11] I. Thérien, R. Moreau, P. Manjunath, Bovine seminal plasma phospholipidbinding proteins stimulate phospholipid efflux from epididymal sperm, Biol. Reprod. 61 (1999) 590–598. [12] D.A. Wah, C. Fernandez-Tornero, L. Sanz, A. Romero, J.J. Calvete, Sperm coating mechanism from the 1.8 Å crystal structure of PDC-109-phosphorylcholine complex, Structure 10 (2002) 505–514. [13] H. Sticht, A.R. Pickford, J.R. Potts, I.D. Campbell, Solution structure of the glycosilated second type 2 module of fibronectin, J. Mol. Biol. 276 (1998) 177– 187. [14] M. Ramakrishnan, V. Anbazhagan, T.V. Pratap, D. Marsh, M.J. Swamy, Membrane insertion and lipid–protein interactions of bovine seminal plasma protein PDC-109 investigated by spin-label electron spin resonance spectroscopy, Biophys. J. 81 (2001) 2215–2225.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes Michel Lafleur a,*, Lesley Courtemanche a, Göran Karlsson b, Katarina Edwards b, Jean-Louis Schwartz c, Puttaswamy Manjunath d a
Department of Chemistry, Center for Self-Assembled Chemical Systems, Université de Montréal, C.P. 6128, Succ. Centre Ville, Montréal, Québec, Canada H3C 3J7 Department of Physical and Analytical Chemistry, Uppsala University, Box 579, S-751 23 Uppsala, Sweden Department of Physiology, Groupe d’étude des Protéines Membranaires, Université de Montréal, C.P. 6128, Succ. Centre Ville, Montréal, Québec, Canada H3C 3J7 d Maisonneuve-Rosemont Hospital Research Center and Faculty of Medecine, Université de Montréal, 5415 L’Assomption Blvd, Montréal, Québec, Canada H1T 2M4 b c
a r t i c l e
i n f o
Article history: Received 20 July 2010 Available online 30 July 2010 Keywords: Binder-of-sperm (BSP) proteins Sperm Membranes Protrusions Nanotubes Fluorescence microscopy Cryo-electron microscopy Lipids Lipid curvature Lipid morphology
a b s t r a c t Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed with phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction With a concentration of about 1 mM in the bovine seminal plasma, binder-of-sperm (BSP) protein BSP1 [1], previously referred to as BSP-A1/-A2 or PDC-109, represents 25 (w/w)% of the total proteins [2,3]. It is known to be essential to sperm capacitation. The modification in the membrane lipid composition and the related reorganization of the lipid domains or rafts are key events in sperm maturation [4–8]. BSP1 was shown to extract selectively cholesterol and phosphatidylcholine (PC) upon its interaction with sperm membranes [5,6,9–11] and therefore, is likely involved in the sperm capacitation process. Its fibronectine-type II (Fn2) domains organized in tandem were proposed to act as binding sites for phosphocholine groups [12,13]. This relatively specific interaction could play a role in the lipid efflux induced by the proteins. Each Fn2 domain would correspond to one binding site and consequently, each BSP1 would bind two phosphocholines. Several studies of the BSP1 binding to PC model membranes indicated a saturation lipid/protein ratio of 10–12 [14–17], suggesting a more complex association. Despite the critical role of BSP1 in sperm * Corresponding author. Fax: +1 514 343 7586. E-mail address: michel.lafl[email protected] (M. Lafleur). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.07.088
capacitation and in reproduction, the details of the mechanism for the lipid extraction are not known. Recent results [18] indicate that homologue proteins can be ubiquitous in mammals, suggesting a more general importance than first thought. In this paper, we report drastic morphological changes of model membranes induced by BSP1, as studied by cryo-electron microscopy (cryo-EM) and fluorescence microscopy. The effect of the protein was examined on multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a phospholipid bearing the most abundant head group found in bovine sperm membranes [5,19]. Our findings provide new leads for the contribution of the BSP1 to the maturation process of sperm. 2. Material and methods BSP1 was extracted from bovine seminal plasma and purified according to an established procedure [20,21]. POPC was obtained from Avanti Polar Lipids (Birmingham, AL, USA) whereas b-BODIPYÒ500/510 C12-HPC (2-(4,4-difluoro-5-methyl-4-bora3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine) was purchased from Invitrogen (Eugene, OR, USA).
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For the cryo-electron microscopy (cryo-EM) experiments, POPC was hydrated in MOPS buffer (10 mM, pH 7.1). The lipid suspensions were submitted to six freeze-and-thaw cycles, from liquid nitrogen to room temperatures. Standard extrusion procedures were used to form large unilamellar vesicles (LUVs) using a hand-held extruder (Avestin, Ottawa, ON, Canada) and polycarbonate filters with pores of 100-nm diameter. For the samples including BSP1, an aliquot of the protein in solution (1 mM in MOPS buffer) was added to preformed LUVs or MLVs to obtain the desired lipid/protein incubation ratio (Ri). The samples were then incubated at 37 °C for, at least two minutes. Longer incubation periods did not appear to have an influence on the resulting structures. This is consistent with the association kinetics data obtained by Isothermal Titration Calorimetry [17] and Surface Plasmon Resonance [22]. An aliquot of the samples was deposited on a copper grid covered with a perforated polymer film. The sample was equilibrated at 37 °C, and 99% relative humidity in an environmental chamber. Excess of water was blotted with a filter paper and the sample was then vitrified by plunging it into liquid ethane held at a temperature just above its freezing point. The frozen sample was transferred to the microscope, keeping it at 165 °C and protected from the atmospheric conditions. Cryo-EM pictures were obtained with a Zeiss EM 902A transmission electron microscope (Carl Zeiss NTS, Oberkochem, Germany). The instrument was operating at 80 kV, in zero loss bright-field mode. Digital images were recorded under low dose conditions with a BioVision Pro-SM Slow Scan CCD camera (ProScan, GmbH, Scheuring, Germany) and analySIS software (Soft Imaging System, GmbH, Münster, Germany). For the fluorescence experiments, POPC, and b-BODIPYÒ500/ 510 C12-HPC were respectively solubilized in benzene/methanol (90/10 (v/v)) and methanol. Aliquots of appropriate volumes were mixed in order to obtain 0.01 (mol/mol)% of fluorescent probe. The lipid organic solution was then freeze-dried. The resulting powder was hydrated in MOPS buffer (10 mM, pH 7.4), to obtain a final lipid concentration of about 1 mM. The sample was submitted to five freeze-and-thaw cycles (from liquid nitrogen to 35 °C) to ensure proper hydration of the lipid bilayers. The exact lipid concentration was determined using the Fiske-Subbarow assay [23]. For pure lipid MLVs, an aliquot of this suspension was deposited on a thin coverslip located in a custom-made holder and covered with a glass slide to prevent water evaporation. In order to observe the effect of BSP1, the appropriate volume of protein solution (0.2 mM in the MOPS buffer) was added to an aliquot of lipid MLVs to obtain an Ri of 24. This sample was mounted as described above. Fluorescence imaging was performed with a Molecular Dynamics Multiprobe 2001 Inverted confocal laser-scanning microscope (Multiprobe, Santa Barbara, CA, USA), with the excitation laser set at 488 nm and the sample fluorescent images recorded through a 510-nm bandpass filter. Alternatively, epifluorescence images and time-lapse sequences were obtained with an Olympus IX81 microscope (Olympus, Melville, NY, USA). 3. Results A typical cryo-EM micrograph of freshly extruded POPC LUVs is presented in Fig. 1A. As expected from the preparation procedure, most LUVs were unilamellar with a mean diameter of about 100 nm. The addition of BSP1, at a Ri of 48, modified the morphology of several LUVs and gave rise to various self-assembly morphologies (Fig. 1B). This representative micrograph shows elongated LUVs, some of them displaying protrusions. Analogous budding and the formation of small vesicles have been previously reported in transmission electron microscopy experiments on phosphatidylcholine LUVs in the presence of BSP1, stained with
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Fig. 1. Modification of the POPC LUV morphology induced by BSP1 (Ri = 48), as observed by cryo-EM. (A) Typical micrograph of POPC LUVs. (B) Morphologies observed in the presence of BSP1, including (a) protrusion, (b) bead necklace selfassemblies, (c) small vesicles and (d) long spiral-like threads. Bar = 100 nm.
phosphotungstic acid [15]. Series of small vesicles connected to each other were observed in our unstained samples, as well as small, isolated vesicles, with diameter as small as 15 nm. The formation of zigzag structures, resembling long spiral-like threads, was also observed. The pitch of these spiral-like structures appeared to be rather constant (about 15 nm). When the Ri was decreased to 12, this type of structure was considerably more abundant. Similar cryo-EM experiments were performed on POPC MLVs. Lipid self-assemblies with modified morphologies were induced by the addition of BSP1 at an Ri of 48 (Fig. 2). One striking feature was the formation of long tubes of various diameters. As assessed by an estimate of the wall thickness, these nanotubes appeared to be formed by one or several lipid bilayer cylinders. Their diameter was typically between 40 and 50 nm. Some tubes appeared to contain a series of small unilamellar vesicles and were then wider. The lipid nanotubes were long; at lower magnification, their length could be estimated to reach several micrometers, corresponding to an aspect ratio of 2000 or more. These nanotubes appeared to originate from MLVs, which represent a large pool of lipids. Several long spiral-like threads, similar to those obtained from the LUVs, were also frequently observed. BODYPY-labeled MLVs were observed by fluorescence confocal microscopy (Fig. 3). As expected, their size was rather heterogeneous, with diameters varying from less than 1 lm up to 10 lm. Self-assemblies with distinct morphologies could be observed from samples of labeled POPC MLVs in the presence of BSP1, at an Ri of
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0.25 lm. During the time-resolved investigations, the transformation of a necklace into a thin tube was observed (Fig. 4). This observation gives a clear indication of the sequence of structural transformations involved in the evolution of the new lipid morphologies. This time sequence of images also illustrates the dynamic nature and the considerable flexibility of these tubular structures. The video is available on the journal site. 4. Discussion
Fig. 2. Modification of the POPC MLV morphology induced by BSP1 (Ri = 48), as observed by cryo-EM. (A) Lipid nanotubes with walls of varying thickness and in some cases containing a series of LUVs. The presence of spiral-like threads is also evident. (B) Lipid nanotubes at a lower magnification. (C) A nanotube that appeared to originate from a MLV that acted as a lipid reservoir. Unless otherwise stated the size bar equals 100 nm.
24. Bead necklace-like structures were regularly found; the diameter of the small vesicles was in the order of 1 to 3 lm. These could include up to ten ‘‘beads”. Short cylindrical vesicles with a diameter of about 1 lm were also observed. Their length was generally not greater than 20 lm. Protrusions appeared on some liposomes. The walls of some structures seemed brighter than most others, suggesting that they were made of several bilayers. Long and thin lipid nanotubes were also observed. Their length could reach 0.1 mm. Their diameter was difficult to evaluate because of the resolution of the microscope but it roughly corresponded to
The addition of BSP1 to liposomes leads to changes in the morphology of vesicles. Cryo-EM and fluorescence microscopy provides a consistent view of the evolution of the shapes. The observation of these morphologies by fluorescence microscopy confirms their lipidic nature because of the lipidic nature of the fluorescent probe. Upon the addition of the protein, the vesicles become elongated and evolve towards bead-necklace structures. The latter appear to transform into series of isolated small vesicles, as series of aligned but distinct small vesicles were often seen in the cryo-EM pictures. In other cases, the bead necklace-like structures seem to rearrange and form thinner and longer lipid nanotubes, as illustrated by the fluorescence time-lapse imaging sequences. Theoretical models have been developed to rationalize the formation of different morphologies by lipid fluid bilayers. In the elastic model of vesicle shape, these morphologies are associated with equilibrium shapes that minimize the bending energy. As a parameter playing a crucial role in the bending energy, the spontaneous curvature of lipid bilayers is directly affected by the difference between the areas of the inner and the outer leaflets. The elastic model [24–26] predicted the evolution of the lipid self-assembly morphologies taking into account the leaflet area imbalance. This work led to a vesicle shape phase diagram that illustrates the various vesicle morphologies that may be adopted for different effective differential areas and volume-to-area ratios. Interestingly, this model predicts for increasing relative area of the external leaflet an evolution from a sphere, to elongated ellipsoid, bead-necklace structures and elongated thinner tubes. The protrusions and tube formation observed in the presence of BSP1 are therefore totally compatible with an increase in the external leaflet area relative to that of the internal one. The presence of very long nanotubes in BSP1-exposed MLVs indicates that these can only be formed when a large amount of lipid material is present; these tubes result from the reorganization of the MLVs and not from the fusion of smaller lipid objects. The correspondence of the lipid self-assembly morphology sequence observed in the presence of BSP1 and that proposed by the theoretical models reinforces the hypothesis suggesting that an increase of external leaflet area relative to that of the inner leaflet is the likely origin of the observed supramolecular changes. Similar morphologies, i.e. the formation of long tubular structures and protrusions, were observed when dioleoylphosphatidylglycerol (DOPG) was transported from the inner to the outer leaflet of bilayers [24] or when lyso-phosphatidylcholine was added to preformed liposomes, partitioning exclusively into the external leaflet of the membrane [27]. The increase of external leaflet area can be caused by an increased molecular area of the lipids of the external leaflet, by a contribution of the proteins interacting with the bilayers, or both. Previous electron spin resonance (ESR) spectroscopy studies showed that the presence of BSP1 has actually an ordering effect on the lipid chains for model membranes [14,28], as well as for plasma membranes of intact bovine epididymal sperm cells [29]. These results discount the increase of lipid molecular area of the external leaflet caused by interaction with the protein. The penetration of BSP1 in the external leaflet is therefore the likely origin of the putative area augmentation. The Fn2 motifs were proposed
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Fig. 3. Morphology modification of the labeled POPC MLVs induced by BSP1 (Ri = 24), as observed by fluorescence microscopy. (A) Typical micrograph of POPC MLVs. (B) Morphologies observed in the presence of BSP1: (B) protrusions; (C) bead-necklace assembly; (D) thin nanotube. (A), (C) and (D) were obtained with an oil-immersion objective (100) in confocal mode, and (B) with an oil-immersion objective (60), measuring the epifluorescence.
to act as binding sites for BSP1 [12] and these might account for the initial anchoring of the proteins. BSP1 has hydrophobic cavities that give the protein an amphiphilic character [30,31]. ESR spectroscopy studies showed that BSP1 reduces the mobility of the lipid chains up to the 14th carbon atom [14], a finding consistent with the protein penetration in membranes. Furthermore, spectrofluorescence data showing a change in intrinsic tryptophan fluorescence of BSP1 upon membrane binding supports the concept of the protein penetrating into the hydrophobic core of bilayers [16,32]. Moreover, it was concluded, comparing the interactions of BSP1 with uniformly labeled vesicles and vesicles labeled only on the inner leaflet, that the interaction of BSP1 with lipid membranes is confined to the outer leaflet of bilayers [33]. Therefore, the partial penetration of BSP1 into the external leaflet of the lipid bilayer should induce an increase of its relative area, which in turn would result in the formation of morphologies with altered and various curvatures. Based on the present results, it is unlikely that the protein added to preformed vesicles can translocate the membrane because this would result in a similar effect on both leaflets. A similar rational was used to explain the formation of protrusions induced by the phospholipase A2 adsorption on vesicles [34]. It has been shown that there is a significant rearrangement of small lipid domains in sperm membranes during the capacitation process [7,8,35]. The specific recognition of the phosphocholine group likely initiates the association of BSP1 to the lipid
membranes. The present work may imply that domain formation is also related to local morphologies induced by BSP1 because different partitioning coefficients of the various lipids in regions with different local membrane curvatures may lead to domain formation. Such phase separation induced by local bilayer curvature has been observed in bilayers supported on rippled matrices [36] as well as in tubes pulled from giant unilamellar vesicles [37]. The interaction of BSP1 with sperm membranes ultimately leads to the extraction of lipids from the membrane. This process has been observed in biological membranes of fibroblasts, red blood cells, sperm cells [10,11,29,33] as well as pure lipid vesicles [33]. Independent determinations estimated that one BSP1 protein extracts 10 [33] or 20 PC molecules [10]. Up to now, the structure of the particles resulting from the lipid extraction is unknown. It is proposed that the thread-like spiral structures observed in the present work are POPC/BSP1 complexes obtained in the presence of high concentration of the protein. These structures were more commonly observed when LUVs were incubated with a low Ri. For the MLV/BSP1 system, numerous thread-like spiral structures were observed; it must be noted that the effective lipid/protein incubation ratio in this case was much greater than 48 if one considers that the protein did not translocate bilayers and that only a small fraction of the lipids (those of the external leaflet of the outer bilayer) were exposed to the protein. The frequent observation of thread-like spiral structures in this system reinforces the
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Fig. 4. Transition from a bead-necklace to a long tube structure, recorded from a sample with a POPC/BSP1 Ri of 24. The micrographs were recorded from epifluorescence with an oil-immersion objective (60). The delay between successive pictures is about 10 s. The arrow highlights the location of the transition.
hypothesis that these structures correspond to lipid/protein complexes obtained in the presence of large amounts of BSP1. These structures would involve several lipids and proteins as they often reach several hundreds of micrometers in length. It has been shown that that the addition of BSP1 to liposomes leads to the formation of smaller self-assemblies as inferred from a drastic decrease in turbidity [14]. The supramolecular structures proposed here are compatible with this observation. It has been proposed
that lipid extraction and its specificity are associated with phosphocholine-containing lipids mainly located on the external leaflet of sperm membranes [33]. This finding fits well with our conclusion indicating that BSP1 interacts essentially with the external membrane leaflet, a process leading to the reported morphology changes. The insertion of BSP1 in the external lipid leaflet of the membrane induces local changes in bilayer curvature, and these may favor phase separations that could subsequently be involved
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in the specificity of the lipid extraction process. This original hypothesis is currently being checked in our laboratories. Acknowledgments The authors thank the Natural Sciences and Engineering Council of Canada, the Fonds Québécois de la Recherche sur la Nature et les Technologies of Québec, and the Swedish Research Council for their financial support. M.L. is grateful to the Royal Academy of Arts and Sciences of Uppsala for financial support during his stay at Uppsala University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.07.088. References [1] P. Manjunath, J. Lefebvre, P.S. Jois, J. Fan, M.W. Wright, New nomenclature for mammalian BSP genes, Biol. Reprod. 80 (2009) 394–397. [2] V. Nauc, P. Manjunath, Radioimmunoassays for bull seminal plasma proteins (BSP-A1/-A2, BSP-A3, and BSP-30-kilodaltons), and their quantification in seminal plasma and sperm, Biol. Reprod. 63 (2000) 1058–1066. [3] J.J. Calvete, M. Raida, L. Sanz, F. Wempe, K.H. Scheit, A. Romero, E. TöpferPetersen, Localization and structural characterization of an oligosaccharide Olinked to bovine PDC-109/quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa, FEBS Lett. 350 (1994) 203–206. [4] B.K. Davis, Timing of fertilization in mammals: sperm cholesterol/phospholipid ratio as a determinant of the capacitation interval, Proc. Natl. Acad. Sci. USA 78 (1981) 7560–7564. [5] J.P. Nolan, R.H. Hammerstedt, Regulation of membrane stability and the acrosome reaction in mammalian sperm, FASEB J. 11 (1997) 670–682. [6] P. Manjunath, I. Thérien, Role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation, J. Reprod. Immunol. 53 (2002) 109–119. [7] C.D. Thaler, M. Thomas, J.R. Ramalie, Reorganization of mouse sperm lipid rafts by capacitation, Mol. Reprod. Dev. 73 (2006) 1541–1549. [8] V. Selvaraj, A. Asano, D.E. Buttke, J.L. McRlwee, J.L. Nelson, C.A. Wolff, T. Merdiushev, M.W. Fornés, A.W. Cohen, M.P. Lisanti, G.H. Rothblat, G. Kopf, A.J. Travis, Segregation of micron-scale membrane sub-domains in live murine sperm, J. Cell. Physiol. 206 (2006) 636–646. [9] R. Moreau, P.G. Frank, C. Perreault, Y.L. Marcel, P. Manjunath, Seminal plasma choline phospholipid-binding proteins stimulate cellular cholesterol and phosphpolipid efflux, Biochim. Biophys. Acta 1438 (1999) 38–46. [10] R. Moreau, P. Manjunath, Characterization of lipid efflux particles generated by seminal phospholipid-binding proteins, Biochim. Biophys. Acta 1438 (1999) 175–184. [11] I. Thérien, R. Moreau, P. Manjunath, Bovine seminal plasma phospholipidbinding proteins stimulate phospholipid efflux from epididymal sperm, Biol. Reprod. 61 (1999) 590–598. [12] D.A. Wah, C. Fernandez-Tornero, L. Sanz, A. Romero, J.J. Calvete, Sperm coating mechanism from the 1.8 Å crystal structure of PDC-109-phosphorylcholine complex, Structure 10 (2002) 505–514. [13] H. Sticht, A.R. Pickford, J.R. Potts, I.D. Campbell, Solution structure of the glycosilated second type 2 module of fibronectin, J. Mol. Biol. 276 (1998) 177– 187. [14] M. Ramakrishnan, V. Anbazhagan, T.V. Pratap, D. Marsh, M.J. Swamy, Membrane insertion and lipid–protein interactions of bovine seminal plasma protein PDC-109 investigated by spin-label electron spin resonance spectroscopy, Biophys. J. 81 (2001) 2215–2225.
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