Reorganization of mouse sperm lipid rafts by capacitation

One of the hallmarks of mammalian sperm capacitation is the loss of cholesterol from the plasma membrane. Cholesterol has been associated with the formation of detergent insoluble membrane microdomains in many cell types, and sperm from several mammalian species have been shown to contain detergent-resistant membranes (DRMs). The change in cholesterol composition of the sperm plasma membrane during capacitation raises the question of whether the contents of DRMs are altered during this process. In this study, we investigated changes in protein composition of DRMs isolated from uncapacitated or capacitated mouse sperm. TX-100 insoluble membranes were fractionated by sucrose flotation gradient centrifugation and analyzed by Western and lectin blotting, and capacitation-related differences in protein composition were identified. Following capacitation, the detergent insoluble fractions moved to lighter positions on the sucrose gradients, reflecting a global change in density or composition. We identified several individual proteins that either became enriched or depleted in DRM fractions following capacitation. These data suggest that the physiological changes in sperm motility, ability to penetrate the zona pellucida (ZP), ZP responsiveness, and other capacitation-dependent changes, may be due in part to a functional reorganization of plasma membrane microdomains.     

Characterization of MDGA1, a novel human glycosylphosphatidylinositol-anchored protein localized in lipid rafts

We report the characterization of the novel human protein MDGA1 encoded by MDGA1 (MAM domain containing glycosylphosphatidylinositol anchor-1) gene, firstly termed as GPIM. MDGA1 has been mapped to 6p21 and it is expressed in human tissues and tumors. The deduced polypeptide consists of 955 amino acids and exhibits structural features found in different types of cell adhesion molecules (CAMs), such as the presence of both immunoglobulin domains and a MAM domain or the capacity to anchor to the cell membrane by a GPI (glycosylphosphatidylinositol) motif. Our results demonstrate that human MDGA1 (hMDGA1) is localized in the membrane of eukaryotic cells. The protein follows the secretion pathway and finally it is retained in the cell membrane by a GPI anchor, susceptible to be cleavaged by phospholipase C (PI-PLC). Moreover, our results reveal that hMDGA1 is localized specifically into membrane microdomains known as lipid rafts. Finally, as other proteins of the secretory pathway, hMDGA1 undergoes other post-translational modification consisting of N-glycosylation.     

N-terminal processing is essential for release of epithin, a mouse type II membrane serine protease

Epithin was originally identified as a mouse type II membrane serine protease. Its human orthologue membrane type-serine protease 1 (MT-SP1)/matriptase has been reported to be localized on the plasma membrane. In addition, soluble forms of matriptase were isolated from human breast milk and breast cancer cell-conditioned medium. In this paper, we report a processing mechanism that appears to be required for the release of epithin. CHO-K1 or COS7 cells transfected with single full-length epithin cDNA generated two different-sized proteins in cell lysates, 110 and 92 kDa. The 92-kDa epithin was found to be an N-terminally truncated form of the 110-kDa epithin, and it was the only form detected in the culture medium. The 92-kDa epithin was also found on the cell surface, where it was anchored by the N-terminal fragment. The results of in vivo cell labeling experiments indicate that the 110-kDa epithin is rapidly processed to the 92-kDa epithin. Using site-directed mutagenesis experiments, we identified Gly(149) of the GSVIA sequence in epithin as required for the processing and release of the protein. These results suggest that N-terminal processing of epithin at Gly(149) is a necessary prerequisite step for release of the protein.     

Bluetongue virus outer capsid protein VP5 interacts with membrane lipid rafts via a SNARE domain

Bluetongue virus (BTV) is a nonenveloped double-stranded RNA virus belonging to the family Reoviridae. The two outer capsid proteins, VP2 and VP5, are responsible for virus entry. However, little is known about the roles of these two proteins, particularly VP5, in virus trafficking and assembly. In this study, we used density gradient fractionation and methyl beta cyclodextrin, a cholesterol-sequestering drug, to demonstrate not only that VP5 copurifies with lipid raft domains in both transfected and infected cells, but also that raft domain integrity is required for BTV assembly. Previously, we showed that BTV nonstructural protein 3 (NS3) interacts with VP2 and also with cellular exocytosis and ESCRT pathway proteins, indicating its involvement in virus egress (A. R. Beaton, J. Rodriguez, Y. K. Reddy, and P. Roy, Proc. Natl. Acad. Sci. USA 99:13154-13159, 2002; C. Wirblich, B. Bhattacharya, and P. Roy J. Virol. 80:460-473, 2006). Here, we show by pull-down and confocal analysis that NS3 also interacts with VP5. Further, a conserved membrane-docking domain similar to the motif in synaptotagmin, a protein belonging to the SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptor) family was identified in the VP5 sequence. By site-directed mutagenesis, followed by flotation and confocal analyses, we demonstrated that raft association of VP5 depends on this domain. Together, these results indicate that VP5 possesses an autonomous signal for its membrane targeting and that the interaction of VP5 with membrane-associated NS3 might play an important role in virus assembly.     

Isolation and proteomic analysis of mouse sperm detergent-resistant membrane fractions: evidence for dissociation of lipid rafts during capacitation

Mammalian sperm acquire fertilization capacity after residing in the female tract during a process known as capacitation. The present study examined whether cholesterol efflux during capacitation alters the biophysical properties of the sperm plasma membrane by potentially reducing the extent of lipid raft domains as analyzed by the isolation of detergent-resistant membrane fractions using sucrose gradients. In addition, this work investigated whether dissociation of the detergent-resistant membrane fraction during capacitation alters resident sperm raft proteins. Mouse sperm proteins associated with such fractions were studied by silver staining, tandem mass spectrometry, and Western blot analysis. Caveolin 1 was identified in sperm lipid rafts in multimeric states, including a high-molecular-weight oligomer that is sensitive to degradation under reducing conditions at high pH. Capacitation resulted in reduction of the light buoyant-density, detergent-resistant membrane fraction and decreased the array of proteins isolated within this fraction, including loss of the high-molecular-weight caveolin 1 oligomers. Proteomic analysis of sperm proteins isolated in the light buoyant-density fraction identified several proteins, including hexokinase 1, testis serine proteases 1 and 2, TEX101, hyaluronidase (PH20, SPAM1), facilitated glucose transporter 3, lactate dehydrogenase A, carbonic anhydrase IV, IZUMO, pantophysin, basigin, and cysteine-rich inhibitory secretory protein 1. Capacitation also resulted in a significant reduction of sperm labeling by the fluorescent lipid-analog DiIC16, indicating that capacitation alters the liquid-ordered domains in the sperm plasma membrane. The observations that capacitation alters the protein composition of the detergent-resistant membrane fractions is consistent with the hypothesis that cholesterol efflux during capacitation dissociates lipid raft constituents, initiating signaling events that lead to sperm capacitation.     

Identification of mouse trp homologs and lipid rafts from spermatogenic cells and sperm.

Intracellular Ca(2+) has an important regulatory role in the control of sperm motility, capacitation, and the acrosome reaction (AR). However, little is known about the molecular identity of the membrane systems that regulate Ca(2+) in sperm. In this report, we provide evidence for the expression of seven Drosophila transient receptor potential homolog genes (trp1-7) and three of their protein products (Trp1, Trp3 and Trp6) in mouse sperm. Allegedly some trps encode capacitative Ca(2+) channels. Immunoconfocal images showed that while Trp6 was present in the postacrosomal region and could be involved in sperm AR, expression of Trp1 and Trp3 was confined to the flagellum, suggesting that they may serve sperm to regulate important Ca(2+)-dependent events in addition to the AR. Likewise, one of these proteins (Trp1) co-immunolocalized with caveolin-1, a major component of caveolae, a subset of lipid rafts potentially important for signaling events and Ca(2+) flux. Furthermore, by using fluorescein-coupled cholera toxin B subunit, which specifically binds to the raft component ganglioside GM1, we identified caveolin- and Trp-independent lipid rafts residing in the plasma membrane of mature sperm. Notably, the distribution of GM1 changes drastically upon completion of the AR.     

Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts

A number of recent studies have demonstrated the significance of detergent-insoluble, glycolipid-enriched membrane domains or lipid rafts, especially in regard to activation and signaling in T lymphocytes. These domains can be viewed as floating rafts composed of sphingolipids and cholesterol which sequester glycosylphosphatidylinositol (GPI)-linked proteins, such as Thy-1 and CD59. CD45, a 200-kDa transmembrane phosphatase protein, is excluded from these domains. We have found that human immunodeficiency virus type 1 (HIV-1) particles produced by infected T-cell lines acquire the GPI-linked proteins Thy-1 and CD59, as well as the ganglioside GM1, which is known to partition preferentially into lipid rafts. In contrast, despite its high expression on the cell surface, CD45 was poorly incorporated into virus particles. Confocal fluorescence microscopy revealed that HIV-1 proteins colocalized with Thy-1, CD59, GM1, and a lipid raft-specific fluorescent lipid, DiIC(16)(3), in uropods of infected Jurkat cells. CD45 did not colocalize with HIV-1 proteins and was excluded from uropods. Dot immunoassay of Triton X-100-extracted membrane fractions revealed that HIV-1 p17 matrix protein and gp41 were present in the detergent-resistant fractions and that [(3)H]myristic acid-labeled HIV Gag showed a nine-to-one enrichment in lipid rafts. We propose a model for the budding of HIV virions through lipid rafts whereby host cell cholesterol, sphingolipids, and GPI-linked proteins within these domains are incorporated into the viral envelope, perhaps as a result of preferential sorting of HIV Gag to lipid rafts.     

Identification of heat shock protein 5, calnexin and integral membrane protein 2B as Adam7-interacting membrane proteins in mouse sperm

In mammals, sperm acquire their motility and ability to fertilize eggs in the epididymis. This maturation process involves the acquisition of particular proteins from the epididymis. One such secretory protein specifically expressed in the epididymis is Adam7 (a disintegrin and metalloprotease 7). Previous studies have shown that Adam7 that resides in an intracellular compartment of epididymal cells is transferred to sperm membranes, where its levels are dependent on the expression of Adam2 and Adam3, which have critical roles in fertilization. Here, using a proteomics approach based on mass spectrometry, we identified proteins that interact with Adam7 in sperm membranes. This analysis revealed that Adam7 forms complexes with calnexin (Canx), heat shock protein 5 (Hspa5), and integral membrane protein 2B (Itm2b). Canx and Hspa5 are molecular chaperones, and Itm2b is a type II integral membrane protein implicated in neurodegeneration. The interaction of Adam7 with these proteins was confirmed by immunoprecipitation-Western blot analysis. We found that Adam7 and Itm2b are located in detergent-resistant regions known to be highly correlated with membrane lipid rafts. We further found that the association of Adam7 with Itm2b is remarkably promoted during sperm capacitation owing to a conformational change of Adam7 that occurs in concert with the capacitation process. Thus, our results suggest that Adam7 functions in fertilization through the formation of a chaperone complex and enhanced association with Itm2b during capacitation in sperm.     

Spatio-temporal localization of membrane lipid rafts in mouse oocytes and cleaving preimplantation embryos

We report for the first time the detection of membrane lipid rafts in mouse oocytes and cleaving preimplantation embryos. Cholera toxin β (CTβ), which binds to the raft-enriched ganglioside GM1, was selected to label rafts. In a novel application a Qdot reagent was used to detect CTβ labeling. This is the first reported use of nanocrystals in mammalian embryo imaging. Comparative membrane labeling with CTβ and lipophilic membrane dyes containing saturated or unsaturated aliphatic tails showed that the detection of GM1 in mouse oocytes and embryo membranes was consistent with the identification of cholesterol- and sphingolipid-enriched rafts in the cell membrane. Distribution of the GM1 was compared with the known distribution of non-raft membrane components, and disruption of membrane rafts with detergents confirmed the cholesterol dependence of GM1 on lipid raft labeling. Complementary functional studies showed that cholesterol depletion using methyl-β-cyclodextrin inhibited preimplantation development in culture. Our results show that the membranes of the mouse oocyte and zygote are rich in lipid rafts, with heterogeneous and stage-dependent distribution. In dividing embryos, the rafts were clearly associated with the cleavage furrow. At the morula stage, rafts were also apically enriched in each blastomere. In blastocysts, rafts were detectable in the trophectoderm layer, but could not be detected in the inner cell mass without prior fixation and permeabilization of the embryo. Lipid rafts and their associated proteins are, therefore, spatio-temporally positioned to a play a critical role in preimplantation developmental events.     

Further evidence that paroxysmal nocturnal haemoglobinuria is a disorder of defective cell membrane lipid rafts

The glycolipid glycosylphosphatidylinositol anchor (GPI-A) plays an important role in lipid raft formation, which is required for proper expression on the cell surface of two inhibitors of the complement cascade, CD55 and CD59. The absence of these markers from the surface of blood cells, including erythrocytes, makes the cells susceptible to complement lysis, as seen in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). However, the explanation for why PNH-affected hematopoietic stem/progenitor cells (HSPCs) expand over time in BM is still unclear. Here, we propose an explanation for this phenomenon and provide evidence that a defect in lipid raft formation in HSPCs leads to defective CXCR4- and VLA-4-mediated retention of these cells in BM. In support of this possibility, BM-isolated CD34⁺ cells from PNH patients show a defect in the incorporation of CXCR4 and VLA-4 into membrane lipid rafts, respond weakly to SDF-1 stimulation, and show defective adhesion to fibronectin. Similar data were obtained with the GPI-A⁻ Jurkat cell line. Moreover, we also report that chimeric mice transplanted with CD55^−/− CD59^−/− BM cells but with proper GPI-A expression do not expand over time in transplanted hosts. On the basis of these findings, we propose that a defect in lipid raft formation in PNH-mutated HSPCs makes these cells more mobile, so that they expand and out-compete normal HSPCs from their BM niches over time.     

The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors

Serine proteases are involved in many processes in the nervous system and specific inhibitors tightly control their proteolytic activity. Thrombin is thought to play a role in tissue development and homeostasis. To date, protease nexin-1 is the only known endogenous protease inhibitor that specifically interferes with thrombotic activity and is expressed in the brain. In this study, we report the detection of a novel thrombin inhibitory activity in the brain of protease nexin-1(-/-) mice. Purification and subsequent analysis by tandem mass spectrometry identified this protein as the phosphatidylethanolamine-binding protein (PEBP). We demonstrate that PEBP exerts inhibitory activity against several serine proteases including thrombin, neuropsin, and chymotrypsin, whereas trypsin, tissue type plasminogen activator, and elastase are not affected. Since PEBP does not share significant homology with other serine protease inhibitors, our results define it as the prototype of a novel class of serine protease inhibitors. PEBP immunoreactivity is found on the surface of Rat-1 fibroblast cells and although its sequence contains no secretion signal, PEBP-H(6) can be purified from the conditioned medium upon recombinant expression.     

Glucocorticoids alter the lipid and protein composition of membrane rafts of a murine T cell hybridoma

Glucocorticoids (GC) are widely used anti-inflammatory agents known to suppress T cell activation by interfering with the TCR activation cascade. The attenuation of early TCR signaling events by these compounds has been recently attributed to a selective displacement of key signaling proteins from membrane lipid rafts. In this study, we demonstrate that GC displace the acyl-bound adaptor proteins linker for activation of T cells and phosphoprotein associated with glycosphingolipid-enriched microdomains from lipid rafts of murine T cell hybridomas, possibly by inhibiting their palmitoylation status. Analysis of the lipid content of the membrane rafts revealed that GC treatment led to a significant decrease in palmitic acid content. Moreover, we found an overall decrease in the proportion of raft-associated saturated fatty acids. These changes were consistent with a decrease in fluorescence anisotropy of isolated lipid rafts, indicating an increase in their fluidity. These findings identify the mechanisms underlying the complex inhibitory effects of glucocorticoids on early TCR signaling and suggest that some of the inhibitory properties of GC on T cell responses may be related to their ability to affect the membrane lipid composition and the palmitoylation status of important signaling molecules.     

SERPINE2, a serine protease inhibitor extensively expressed in adult male mouse reproductive tissues, may serve as a murine sperm decapacitation factor

SERPINE2, one of the potent serine protease inhibitors that modulates the activity of plasminogen activator and thrombin, is implicated in many biological processes. In the present study, we purified SERPINE2 from mouse seminal vesicle secretion (SVS), using liquid chromatography and identified it by liquid chromatography/tandem mass spectrometry, and it showed potent inhibitory activity against the urokinase-type plasminogen activator. SERPINE2 was expressed predominantly in seminal vesicles among murine male reproductive tissues. It was immunolocalized to the SVS and mucosal epithelium of the seminal vesicle, epididymis, coagulating gland, and vas deferens. In the testes, SERPINE2 was immunostained in spermatogonia, spermatocytes, spermatids, Leydig cells, and spermatozoa. SERPINE2 was also detected on the acrosomal cap of testicular and epididymal sperm and was suggested to be an intrinsic sperm surface protein. The purified SERPINE2 protein could bind to epididymal sperm. A prominent amount of SERPINE2 was detected on ejaculated and oviductal spermatozoa. Nevertheless, SERPINE2 was detected predominantly on uncapacitated sperm, indicating that SERPINE2 is lost before initiation of the capacitation process. Moreover, SERPINE2 could inhibit in vitro bovine serum albumin-induced sperm capacitation and prevent sperm binding to the egg, thus blocking fertilization. It acts through preventing cholesterol efflux, one of the initiation events of capacitation, from the sperm. These findings suggest that the SERPINE2 protein may play a role as a sperm decapacitation factor.     

Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.     

Uroplakin III, a novel Src substrate in Xenopus egg rafts, is a target for sperm protease essential for fertilization

In a previous study, we identified Xenopus egg uroplakin III (xUPIII), a single-transmembrane protein that localized to lipid/membrane rafts and was tyrosine-phosphorylated upon fertilization. An antibody against the xUPIII extracellular domain abolishes fertilization, suggesting that xUPIII acts not only as tyrosine kinase substrate but also as a receptor for sperm. Previously, it has been shown that the protease cathepsin B can promote a transient Ca2+ release and egg activation as seen in fertilized eggs (Mizote, A., Okamoto, S., Iwao, Y., 1999. Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization. Dev. Biol. 208, 79-92). Here, we show that activation of Xenopus eggs by cathepsin B is accompanied by tyrosine phosphorylation of egg-raft-associated Src, phospholipase Cgamma, and xUPIII. Cathepsin B also promotes a partial digestion of xUPIII both in vitro and in vivo. A synthetic xUPIII-GRR peptide, which contains a potential proteolytic site, inhibits the cathepsin-B-mediated proteolysis and tyrosine phosphorylation of xUPIII and egg activation. Importantly, this peptide also inhibits sperm-induced tyrosine phosphorylation of xUPIII and egg activation. Protease activity that digests xUPIII in an xUPIII-GRR peptide-sensitive manner is present in Xenopus sperm. Several protease inhibitors, which have been identified to be inhibitory toward Xenopus fertilization, are shown to inhibit sperm-induced tyrosine phosphorylation of xUPIII. Uroplakin Ib, a tetraspanin UP member, is found to be associated with xUPIII in egg rafts. Our results highlight novel mechanisms of fertilization signaling by which xUPIII serves as a potential target for sperm protease essential for fertilization.     

Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5

We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression.     

Annexin A6 is recruited into lipid rafts of Niemann–Pick type C disease fibroblasts in a Ca-dependent manner

Niemann–Pick type C (NPC) disease is characterized by excessive accumulation of cholesterol in the late endosome/lysosome compartment. Some members of the annexin family of proteins such as annexin A2 (AnxA2) and annexin A6 (AnxA6) follow the same route as cholesterol during the endocytic pathway and are found, as AnxA6, attached to the membranes of the cholesterol storage compartment in NPC disease fibroblasts. Therefore, the purpose of this work was to test the hypothesis that AnxA6 participates in the NPC-induced changes in the organization of membrane microdomains resistant to solubilization by a nonionic detergent, Triton X-100, i.e., detergent-resistant microdomains (DRMs). Using cellular fractionation, fluorescence microscopy and specific antibodies we observed that in the absence of calcium AnxA6 was found in the DRM-depleted membrane fractions isolated from NPC and control fibroblasts. In the presence of calcium, AnxA6 re-located to the fractions enriched in DRMs only in the NPC cells, suggestive of AnxA6 participation in organization of these microdomains.     

Annexin A6 is recruited into lipid rafts of Niemann-Pick type C disease fibroblasts in a Ca2+-dependent manner

Niemann-Pick type C (NPC) disease is characterized by excessive accumulation of cholesterol in the late endosome/lysosome compartment. Some members of the annexin family of proteins such as annexin A2 (AnxA2) and annexin A6 (AnxA6) follow the same route as cholesterol during the endocytic pathway and are found, as AnxA6, attached to the membranes of the cholesterol storage compartment in NPC disease fibroblasts. Therefore, the purpose of this work was to test the hypothesis that AnxA6 participates in the NPC-induced changes in the organization of membrane microdomains resistant to solubilization by a nonionic detergent, Triton X-100, i.e., detergent-resistant microdomains (DRMs). Using cellular fractionation, fluorescence microscopy and specific antibodies we observed that in the absence of calcium AnxA6 was found in the DRM-depleted membrane fractions isolated from NPC and control fibroblasts. In the presence of calcium, AnxA6 re-located to the fractions enriched in DRMs only in the NPC cells, suggestive of AnxA6 participation in organization of these microdomains.