PS-binding Site Research Articles

Genome-regulated Assembly of a ssRNA Virus May Also Prepare It for Infection

Many single-stranded, positive-sense RNA viruses regulate assembly of their infectious virions by forming multiple, cognate coat protein (CP)-genome contacts at sites termed Packaging Signals (PSs). We have determined the secondary structures of the bacteriophage MS2 ssRNA genome (gRNA) frozen in defined states using constraints from X-ray synchrotron footprinting (XRF). Comparison of the footprints from phage and transcript confirms the presence of multiple PSs in contact with CP dimers in the former. This is also true for a virus-like particle (VLP) assembled around the gRNA in vitro in the absence of the single-copy Maturation Protein (MP) found in phage. Since PS folds are present at many sites across gRNA transcripts, it appears that this genome has evolved to facilitate this mechanism of assembly regulation. There are striking differences between the gRNA-CP contacts seen in phage and the VLP, suggesting that the latter are inappropriate surrogates for aspects of phage structure/function. Roughly 50% of potential PS sites in the gRNA are not in contact with the protein shell of phage. However, many of these sit adjacent to, albeit not in contact with, PS-binding sites on CP dimers. We hypothesize that these act as PSs transiently during assembly but subsequently dissociate. Combining the XRF data with PS locations from an asymmetric cryo-EM reconstruction suggests that the genome positions of such dissociations are non-random and may facilitate infection. The loss of many PS-CP interactions towards the 3′ end of the gRNA would allow this part of the genome to transit more easily through the narrow basal body of the pilus extruding machinery. This is the known first step in phage infection. In addition, each PS-CP dissociation event leaves the protein partner trapped in a non-lowest free-energy conformation. This destabilizes the protein shell which must disassemble during infection, further facilitating this stage of the life-cycle.

Phosphatidylserine binding directly regulates TIM-3 function.

Co-signaling receptors for the T cell receptor (TCR) are important therapeutic targets, with blockade of co-inhibitory receptors such as PD-1 now central in immuno-oncology. Advancing additional therapeutic immune modulation approaches requires understanding ligand regulation of other co-signaling receptors. One poorly understood potential therapeutic target is TIM-3 (T cell immunoglobulin and mucin domain containing-3). Which of TIM-3's several proposed regulatory ligands is/are relevant for signaling is unclear, and different studies have reported TIM-3 as a co-inhibitory or co-stimulatory receptor in T cells. Here, we show that TIM-3 promotes NF-κB signaling and IL-2 secretion following TCR stimulation in Jurkat cells, and that this activity is regulated by binding to phosphatidylserine (PS). TIM-3 signaling is stimulated by PS exposed constitutively in cultured Jurkat cells, and can be blocked by mutating the PS-binding site or by occluding this site with an antibody. We also find that TIM-3 signaling alters CD28 phosphorylation. Our findings clarify the importance of PS as a functional TIM-3 ligand, and may inform the future exploitation of TIM-3 as a therapeutic target.

Dendron-Functionalized Surface: Efficient Strategy for Enhancing the Capture of Microvesicles.

SummaryMicrovesicles (MVs) are used by various types of cells in the human body for intercellular communication, making them biomarkers of great potential for the early and non-evasive diagnosis of a spectrum of diseases. An integrated analysis including morphological, quantitative, and compositional studies is most desirable for the clinical application of MV detection; however, such integration is limited by the currently available analysis techniques. In this context, exploiting the phosphatidylserine (PS) exposure of MVs, we synthesized a series of dendritic molecules with PS-binding sites at the periphery. PS-dendron binding was studied at the molecular level using NMR approaches, whereas PS-containing membrane-dendron interaction was investigated in an aqueous environment using plasmon waveguide resonance spectroscopy. As a proof of concept, polyethylene terephthalate surface was functionalized with the synthetic dendrons, forming devices that can capture MVs to facilitate their subsequent analyses.

Phosphatidylserine-exposing blood and endothelial cells contribute to the hypercoagulable state in essential thrombocythemia patients.

The mechanisms of thrombogenicity in essential thrombocythemia (ET) are complex and not well defined. Our objective was to explore whether phosphatidylserine (PS) exposure on blood cells and endothelial cells (ECs) can account for the increased thrombosis and distinct thrombotic risks among mutational subtypes in ET. Using flow cytometry and confocal microscopy, we found that the levels of PS-exposing erythrocytes, platelets, leukocytes, and serum-cultured ECs were significantly higher in each ET group [JAK2, CALR, and triple-negative (TN) (all P < 0.001)] than those in controls. Among ET patients, those with JAK2 mutations showed higher levels of PS-positive erythrocytes, platelets, neutrophils, and serum-cultured ECs than TN patients or those with CALR mutations, which show similar levels. Coagulation function assays showed that higher levels of PS-positive blood cells and serum-cultured ECs led to markedly shortened coagulation time and dramatically increased levels of FXa, thrombin, and fibrin production. This procoagulant activity could be largely blocked by addition of lactadherin (approx. 70% inhibition). Confocal microscopy showed that the FVa/FXa complex and fibrin fibrils colocalized with PS on ET serum-cultured ECs. Additionally, we found a relationship between D-dimer, prothrombin fragment F1 + 2, and PS exposure. Our study reveals a previously unrecognized link between hypercoagulability and exposed PS on cells, which might also be associated with distinct thrombotic risks among mutational subtypes in ET. Thus, blocking PS-binding sites may represent a new therapeutic target for preventing thrombosis in ET.

The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation.

Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases.

Effects of phospholipids on the functional regulation of tBID in membranes

The functional interplay between tBID and phospholipids was investigated in this study. The binding of tBID to model membranes was increased by an incorporation of phosphatidylserine (PS) into the liposomes. Using limited proteolysis and mass spectrometry, two peptide regions, which correspond to Ser(100)-Arg(114) and His(89)-Arg(114) in BID, revealed the specific PS-binding site. tBID also decreased the light scattering values of PS-containing liposomes and increased the leakage of fluorescent dye encapsulated in vesicles, which suggest that tBID reduces membrane integrity by fragmentation. The membrane fragmentation by tBID was also observed using confocal and transmission electron microscopy. The activity of tBID paralleled results that were obtained with cardiolipin (CL)-containing membranes. However, other anionic phospholipids had little effect. CL- and PS-induced conformational changes of tBID were observed by circular dichroism and intrinsic fluorescence. CL and PS also stimulated the insertion of BID into lipid monolayers. tBID stimulated the leakage of Ca(2+) from purified microsomes and mitochondria in a protein concentration-dependent manner. In contrast, BID showed significantly reduced effects when compared to tBID in all of the experiments performed. These results suggest that tBID specifically interacts with PS as well as CL and decreases membrane integrity without the aid of other pro-apoptotic proteins.

Snake Cytotoxins Bind to Membranes via Interactions with Phosphatidylserine Head Groups of Lipids

The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of cells.

Phosphatidylserine-positive erythrocytes bind to immobilized and soluble thrombospondin-1 via its heparin-binding domain

Phosphatidylserine (PS)-dependent erythrocyte adhesion to endothelium and subendothelial matrix components is mediated in part via thrombospondin (TSP). Although TSP exhibits multiple cell-binding domains, the PS-binding site on TSP is unknown. Because a cell-binding domain for anionic heparin is located at the amino-terminus, we hypothesized that PS-positive red blood cells (PS(+ve)-RBCs) bind to this domain. We demonstrate that both heparin and its low-molecular-weight derivative enoxaparin (0.5-50 u/mL) inhibited PS(+ve)-RBC adhesion to immobilized TSP in a concentration-dependent manner (21% to 77% inhibition, P < 0.05). Preincubation of immobilized TSP with an antibody against the heparin-binding domain blocked PS(+ve)-RBC adhesion to TSP. Antibodies that recognize the collagen- and the carboxy-terminal CD47-binding domain on TSP had no effect on this process. Although preincubation of PS(+ve)-RBCs with TSP peptides from the heparin-binding domain that contained the specific heparin-binding motif KKTRG inhibited PS(+ve)-erythrocyte adhesion to matrix TSP (P < 0.001), these peptides in the immobilized form supported PS-mediated erythrocyte adhesion. A TSP-peptide that lacks the binding motif neither inhibited nor supported PS(+ve)-RBC adhesion. Additional experiments show that soluble TSP also interacted with PS(+ve)-RBCs via its heparin-binding domain. Our results demonstrate that PS-positive erythrocytes bind to both immobilized and soluble TSP via its heparin-binding domain and that both heparin and enoxaparin, at clinically relevant concentrations, block this interaction. Other studies have shown that heparin inhibited P-selectin- and soluble-TSP-mediated sickle erythrocyte adhesion to endothelial cells. Our results, taken together with the previously documented findings, provide a rational basis for clinical use of heparin or its low-molecular-weight derivatives as therapeutic agents in treating vaso-occlusive pain in patients with sickle cell disease.

Role of Gamma-Carboxyglutamic Acid (GLA) Domain in Phosphatidylserine (PS)-Regulated Activity of Factor IXa.

Both factors Xa and IXa are vitamin-K-dependent serine proteases that consist of disulfide-linked heavy and light chains. The heavy chain contains the serine protease active site, while the light chain contains the GLA (gamma-carboxyglutamic acid) domain, EGF-I (epidermal growth factor-like region) and EGF-II domains. Effect of PS on FXa: We have extensively studied the interaction of factor Xa with 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS). C6PS is a soluble analogue of phosphatidylserine, which is present in platelet membranes; it serves as a model for the effect of membrane-bound PS on factor Xa activity and structure. We located three C6PS binding sites to different domains of factor Xa using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements, showing that :i.the Gla domain binds C6PS only in the absence of calcium (kd ∼ 1 mM);ii.a calcium-dependent, regulatory, PS-binding site exists in the EGFN domain when linked by calcium to the Gla domain; andiii.a second calcium-requiring site exists in the EGFC-catalytic domains and shares residues with the substrate recognition site.We have shown that full functional response to C6PS requires linkage of the Gla, EGFNC, and catalytic domains in the presence of calcium. Recently we have observed that the proteolytic activity of des EGFN factor Xa is not affected in the presence of C6PS, locating more precisely a significant component of the PS-triggered regulatory site to the EGFN domain. Efect of PS on FIXa: We have previously reported that C6PS induces a calcium-dependent conformational change in factor IXa that regulates the amidolytic and proteolytic activities. We have also shown that factor IXa binds 2 molecules of C6PS. Here we have examined the role of the GLA domain in this C6PS regulation of factor IXa using a GLA-domainless variant of factor IXa (GD-IXa). In the absence of the GLA domain, binding to C6PS has no effect on the rate of proteolytic or amidolytic activity of GD-IXa toward both factor X and synthetic substrates. The binding of C6PS to GD-IXa studied using intrinsic tryptophan fluorescence (Kd = ∼50μM) was different from that for native factor IXa (Kd = ∼2μM). The critical micelle concentration (CMC) of C6PS under the conditions of these experiments (∼300μM) was much greater than the C6PS concentrations used in our experiments. We conclude that:1.C6PS does bind to GD-IXa, but at a reduced affinity compared to factor IXa;2.C6PS does not regulate the amidolytic or proteolytic activities of factor IXa in the absence of GLA domain.This could be due to two C6PS sites existing in FIXa, with the regulatory site requiring the Gla domain (as observed for FXa), or to a requirement for the Gla domain for the regulatory activity of a single site. Future studies looking at the stoichiometry of binding of C6PS with GD-IXa will help distinguish between these possibilities.Supported by Supported by NHLBI (HL 072827).

Phosphatidylserine (PS)-Positive Erythrocytes Bind to Both Immobilized and Soluble Thrombospondin-1 (TSP-1) Via Its Heparin Binding Domain.

Phosphatidylserine (PS)-dependent erythrocyte adhesion to both cultured endothelial cells and the components of sub-endothelial matrix appears to be mediated in part via thrombospondin-1 (TSP). While TSP exhibits multiple cell-binding domains, the PS-binding site on TSP has not been identified. Since a cell-binding domain for anionic heparin is located at the amino-terminal domain of TSP, we hypothesized that anionic PS-positive (PS+ve) red cells bind to this domain. In a recent preliminary study, using a flow adhesion system and PS+ve erythrocytes (prepared by treating control AA red cells with A23187), we have demonstrated that heparin inhibited PS+ve erythrocyte adhesion to immobilized TSP in a concentration-dependent manner with 58 to 77% inhibition noted at concentrations between 1 and 50 U/ml (n=9, P<0.001). Other anionic polysaccharides including high molecular weight dextran sulfate and chondroitin sulfate A also inhibited PS+ve erythrocyte adhesion to immobilized TSP with the magnitude of the inhibitory effects comparable to heparin. These results suggested that the heparin-binding domain on TSP may be involved in PS-mediated red cell adhesion to immobilized TSP. We have extended these studies to characterize the PS-binding site on TSP using monoclonal antibodies directed against specific cell-binding domains on the molecule and also using specific TSP peptides. We demonstrate that pre-incubation of immobilized TSP with an antibody directed against the heparin-binding domain on TSP (TSP-Ab9, Lab Vision) blocked PS-mediated red cell adhesion to immobilized TSP (80% inhibition compared to an isotype-matched negative control antibody, n=7, P<0.001), whereas an antibody that recognizes the collagen-binding domain on TSP (TSP-Ab4) did not affect this process. In addition, incubation of PS+ve erythrocytes with a TSP peptide containing the specific heparin-binding motif, KKTRG, inhibited PS-mediated red cell adhesion by 55% (P<0.001, n=6), whereas a peptide lacking the binding motif had no effect. Since protein confirmation of immobilized TSP appears to be different from that of soluble TSP, we next investigated whether soluble TSP, like immobilized TSP, also interacted with PS+ve erythrocytes. Erythrocytes containing 8 to 10% PS+ve cells were incubated in the absence or the presence of increasing concentrations of soluble TSP (0.1 to 10 μg/ml), and then analyzed by flow cytometry for surface bound TSP using both adhesion blocking (TSP-Ab9) and non-blocking (TSP-Ab4) anti-TSP antibodies. We demonstrate that soluble TSP binds to PS+ve erythrocytes in a concentration-dependent manner with 3 to 11% TSP-positive (TSP+ve) red cells measured at soluble TSP concentrations between 1 to 10 μg/ml (n=4). In addition, TSP binding could be detected only with the non-adhesion blocking antibody TSP-Ab4, which recognizes the collagen-binding domain on TSP. The adhesion blocking antibody TSP-Ab9 that interacts with the heparin binding domain, failed to detect any TSP+ve red cells. No TSP+ve erythrocytes were detected when PS-negative control red cells were evaluated in binding assays. In parallel adhesion experiments, soluble TSP inhibited PS+ve erythrocyte adhesion to immobilized TSP at concentrations at which significant TSP binding to erythrocytes occurred (43% and 44% inhibition at 5 and 10 μg of soluble TSP per ml, n=4). These results conclusively demonstrate that PS-positive erythrocytes interact with both immobilized and fluid phase TSP through the heparin-binding domain, and that heparin blocks this interaction.

Cell Migration and Signaling Specificity Is Determined by the Phosphatidylserine Recognition Motif of Rac1

The Rho guanosine triphosphatases (GTPases) control cell shape and motility and are frequently overexpressed during malignant growth. These proteins act as molecular switches cycling between active GTP- and inactive GDP-bound forms. Despite being membrane anchored via their isoprenylated C termini, Rho GTPases rapidly translocate between membrane and cytosolic compartments. Here, we show that the Rho GTPase Rac1 preferentially interacts with phosphatidylserine (PS)-containing bilayers through its polybasic motif (PBM). Rac1 isoprenylation contributes to membrane avidity but is not critical for PS recognition. The similar protein Cdc42 (cell division cycle 42), however, only associates with PS when prenylated. Conversely, other Rho GTPases such as Rac2, Rac3, and RhoA do not bind to PS even when they are prenylated. Cell stimulation with PS induces translocation of Rac1 toward the plasma membrane and stimulates GTP loading, membrane ruffling, and filopodia formation. This stimulation also promotes Cdc42 activation and phosphorylation of mitogen-activated protein kinase through Rac1/PS signaling. Consequently, the PBM specifically directs Rac1 to effect cytoskeletal rearrangement and cell migration by selective membrane phospholipid targeting.

A Phosphatidylserine-binding Site in the Cytosolic Fragment of Clostridium sordellii Lethal Toxin Facilitates Glucosylation of Membrane-bound Rac and Is Required for Cytotoxicity

Large clostridial toxins glucosylate some small G proteins on a threonine residue, thereby preventing their interactions with effector molecules and regulators. We show that the glucosyltransferase domain of lethal toxin from Clostridium sordellii (LT(cyt); amino acids 1-546), which is released into the cytosol during cell infection, binds preferentially to liposomes containing phosphatidylserine as compared with other anionic lipids. The binding of LT(cyt) to phosphatidylserine increases by two orders of magnitude the rate of glucosylation of liposome-bound geranyl-geranylated Rac-GDP. Limited proteolysis and deletion studies show that the binding site for phosphatidylserine lies within the first 18 N-terminal residues of LT(cyt). Deletion of these residues abolishes the effect of phosphatidylserine on the activity of LT(cyt) on liposome-bound geranyl-geranylated Rac-GDP and prevents the morphological effects induced by LT(cyt) microinjection into various cells, but it does not affect the intrinsic activity of LT(cyt) on non-geranyl-geranylated Rac-GDP in solution. We conclude that the avidity of LT(cyt) for phosphatidylserine facilitates its targeting to the cytosolic leaflet of cell membranes and, notably, the plasma membrane, where this anionic lipid is abundant and where several targets of lethal toxin reside.

Phosphatidylserine binding sites in red cell spectrin

Spectrin has been shown to interact with phosphatidylserine (PS), however, the precise binding sites for PS in spectrin have not been defined. In the present study, we have identified specific PS binding sites in spectrin using recombinant spectrin fragments encompassing the entire sequences of both spectrin chains. We show that sites of high affinity are located within eight of the 38 triple-helical structural repeats which make up the bulk of both chains: these are: α8 and α9-10, and β2, β3, β4, β12, β13 and β14, and PS affinity was also found in the non-homologous N-terminal domain of the β-chain. It is noteworthy that the PS-binding sites in β-spectrin are clustered in close proximity to the sites of attachment both of ankyrin and of 4.1R, the proteins engaged in attachment of spectrin to the membrane. We conjecture that direct interaction of spectrin with PS in the membrane complements modulates its interactions with the proteins, and that (considering also the known affinity of 4.1R for PS) the formation of PS-rich lipid domains, which have been observed in the red cell membrane, may be a result.

Roles of Ionic Residues of the C1 Domain in Protein Kinase C-α Activation and the Origin of Phosphatidylserine Specificity

On the basis of extensive structure-function studies of protein kinase C-alpha (PKC-alpha), we have proposed an activation mechanism for conventional PKCs in which the C2 domain and the C1 domain interact sequentially with membranes (Medkova, M., and Cho, W. (1999) J. Biol. Chem. 274, 19852-19861). To further elucidate the interactions between the C1 and C2 domains during PKC activation and the origin of phosphatidylserine specificity, we mutated several charged residues in two C1 domains (C1a and C1b) of PKC-alpha. We then measured the membrane binding affinities, activities, and monolayer penetration of these mutants. Results indicate that cationic residues of the C1a domain, most notably Arg(77), interact nonspecifically with anionic phospholipids prior to the membrane penetration of hydrophobic residues. The mutation of a single aspartate (Asp(55)) in the C1a domain to Ala or Lys resulted in dramatically reduced phosphatidylserine specificity in vesicle binding, activity, and monolayer penetration. In particular, D55A showed much higher vesicle affinity, activity, and monolayer penetration power than wild type under nonactivating conditions, i.e. with phosphatidylglycerol and in the absence of Ca(2+), indicating that Asp(55) is involved in the tethering of the C1a domain to another part of PKC-alpha, which keeps it in an inactive conformation at the resting state. Based on these results, we propose a refined model for the activation of conventional PKC, in which phosphatidylserine specifically disrupts the C1a domain tethering by competing with Asp(55), which then leads to membrane penetration and diacylglycerol binding of the C1a domain and PKC activation.

Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis.

We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg(2074), Asp(2098), Arg(2171), Arg(2174), and Glu(2189) are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg(2074), Asp(2098), Arg(2171), and Arg(2174)) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys(2060) through Glu(2069) in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His(2128) through Lys(2137). The PS-binding site in the factor V C2-domain includes amino acid residues Trp(2063) and Trp(2064). This site overlaps with the epitope for monoclonal antibody HV-1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.

Differential membrane-binding and activation mechanisms of protein kinase C-alpha and -epsilon.

To elucidate the mechanisms of membrane binding and activation of conventional and novel protein kinase C (PKC), we measured the interactions of rat PKC-alpha and -epsilon with phospholipid monolayers and vesicles of various compositions. Besides the established difference in calcium requirement, the two isoforms showed major differences in their membrane-binding and activation mechanisms. For PKC-alpha, diacylglycerol (DG) specifically enhanced the binding of PKC-alpha to phosphatidylserine (PS)-containing vesicles by 2 orders of magnitude, allowing PKC-alpha high specificity for PS. Also, PKC-alpha could penetrate into the phospholipid monolayer with a packing density comparable to that of the cell membrane only in the presence of Ca2+ and PS. When compared to PKC-alpha, PKC-epsilon had lower binding affinity for PS-containing vesicles both in the presence and in the absence of DG. As a result, PKC-epsilon did not show pronounced specificity for PS. Also, PKC-epsilon showed reduced penetration into PS-containing monolayers, which was comparable to the Ca2+-independent penetration of PKC-alpha into the same monolayers. Taken together, these results suggest the following: (1) The role of Ca2+ in the membrane binding of PKC-alpha is to expose a specific PS-binding site. (2) Once bound to membrane surfaces, PS specifically induces the partial membrane penetration of PKC-alpha that allows its optimal interactions with DG, hence the enhanced membrane binding and activation. (3) PKC-epsilon, due to the lack of Ca2+ binding, cannot specifically interact with PS and DG, which implies the presence of other physiological activator(s) for this isoform.

Localization of the phosphatidylserine-binding site of glyceraldehyde-3-phosphate dehydrogenase responsible for membrane fusion.

In this study, we demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phosphatidylserine (PS)-binding protein and localized the putative PS-binding site involved in the membrane fusion induced by this enzyme. In an attempt to identify the PS-binding proteins, we raised polyclonal antibodies against a 15-amino-acid synthetic peptide (amino acid residues 390-403 of phosphatidylserine decarboxylase), which was shown to bind specifically to PS. One polyclonal antibody, designated aPSD-2, crossreacted with GAPDH, and its binding to GAPDH was inhibited by PS but not by other phospholipids such as phosphatidylethanolamine and phosphatidylinositol. Kinetic analysis of GAPDH binding to phospholipid membranes by measuring surface plasmon resonance showed that GAPDH associated with the phospholipid membrane containing PS rapidly (k[on] =2.8 X 10(4) M(-1) X s[-1]) and dissociated extremely slowly (k[off]=5.9 X 10(-5) s[-1]), giving a low dissociation constant (KD=2.6nM). GAPDH bound less effectively to membranes without PS with a dissociation constants of 0.2 microM. GAPDH-induced vesicle fusion was also inhibited by aPSD-2, suggesting that this antibody recognizes the putative PS-binding site on GAPDH involved in the enzyme-induced membrane fusion. Chemical fragmentation of GAPDH with cyanogen bromide followed by separation and sequence analysis of the reactive peptide resulted in the identification of a single reactive peptide with the sequence of amino acid residues 45-103 of GAPDH. Analysis of aPSD-2 binding to synthetic peptides derived from the corresponding region localized the antibody-binding site to amino acid residues 70-94 of GAPDH. Both the 25-amino-acid synthetic peptide (amino acid residues 70-94 of GAPDH) and polyclonal antibody raised against this peptide inhibited GAPDH-induced membrane fusion, suggesting that these amino acid residues play a crucial role in this membrane fusion process.

Phosphatidylserine liposomes can be tethered by caldesmon to actin filaments

Rotary shadowing electron microscopy revealed that attachment of caldesmon to phosphatidylserine (PS) liposomes was mainly through its C-terminal end. To determine the PS-binding sites of caldesmon, we have made use of synthetic peptides covering the two C-terminal calmodulin binding sites and a recombinant fragment corresponding to the N-terminal end of the C-terminal domain that contains an amphipathic helix. Interactions of these peptides with the PS liposomes were studied by nondenaturing gel electrophoresis and fluorescence spectroscopy. The results showed that both calmodulin-binding sites of caldesmon were able to interact with PS. The affinity (Kd) of PS for these sites was in the range of 1.8-14.3 x 10(-5) M, compared to 0.69 x 10(-5) M for the whole caldesmon molecule. Fragments located outside of calmodulin-binding sites bound PS weakly (3.85 x 10(-4) M) and thus may contain a second class of lipid-binding sites. Binding of PS induced conformational changes in regions other than the C-terminal PS-binding sites, as evidenced by the changes in the susceptibility to proteolytic cleavages. Most significantly, the presence of caldesmon greatly increased binding of PS to F-actin, suggesting that caldesmon may tether PS liposomes to actin filaments. These results raise the possibility that caldesmon-lipid interactions could play a functionally important role in the assembly of contractile filaments near the membranes.

Soluble phospholipids enhance factor Xa-catalyzed prothrombin activation in solution.

Acidic phospholipids play an important but incompletely understood role in prothrombin activation. Here we report the effect of short-chain phosphatidylserine (dicaproylphosphatidylserine, C6PS) and the corresponding phosphatidylglycerol (C6PG) and phosphatidylcholine (C6PC) derivatives on the rate of prothrombin activation by factor Xa. The critical micellar concentrations of these short-chained phospholipids have been determined under a variety of conditions that we used for kinetic and structural studies. Under conditions for which these lipids exist in a soluble form, the results demonstrate that: (i) the rate of human prothrombin activation by human factor Xa was enhanced in a calcium-dependent fashion up to 60-fold by addition of C6PS, roughly 20% of the optimal enhancement seen with bovine phosphatidylserine/palmitoyloleoylphosphatidylcholine (25/75 PS/POPC) membranes; (ii) C6PS inhibited the rate of hydrolysis of synthetic factor Xa substrate (S-2765), an effect that was mimicked, but at much lower lipid concentrations, by PS/POPC membranes; (iii) there was no enhancement of prothrombin activation and much less inhibition of hydrolysis of S-2765 by factor Xa in the presence of C6PG or C6PC; and (iv) the thermal denaturation of prothrombin was altered in a calcium-independent but dose-dependent fashion by either C6PS or C6PG. These results have been interpreted in terms of the existence of (a) specific PS binding site(s) on factor Xa (Kd approximately 73 microM) that regulate(s) the activity of this serine protease. Our results do not rule out the possibility that the rate of prothrombin activation is also influenced by a weaker, calcium-independent, and less specific acidic lipid binding site on prothrombin, the occupancy of which results in conformational changes in this protein. The results clearly suggest that PS binding regulates the rate of prothrombin activation.

Some Factor VIII Inhibitor Antibodies Recognize a Common Epitope Corresponding to C2 Domain Amino Acids 2248 Through 2312, Which Overlap a Phospholipid-Binding Site

The finding that human factor VIII (fVIII) inhibitor antibodies with C2 domain epitopes interfere with the binding of fVIII to phosphatidylserine (PS) suggested that this is the mechanism by which they inactivate fVIII. We constructed a recombinant C2 domain polypeptide and demonstrated that it bound to all six human inhibitors with fVIII light chain specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Recombinant C2 also partially or completely neutralized the inhibitor titer of these plasmas, demonstrating that anti-C2 antibodies inhibit fVIII activity. Immunoblotting of a series of C2 deletion polypeptides, expressed in Escherichia coli, with inhibitor plasmas showed that the epitopes for human inhibitors consist of a common core of amino acid residues 2248 through 2312 with differing extensions for individual inhibitors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was localized to residues 2248 through 2285. Three human antibodies and anti-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS-binding site, but MoAb ESH8 did not. These antibodies also inhibited the binding of fVIII to synthetic phospholipid membranes of PS and phosphatidylcholine, confirming that the blocked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal function of activated fVIII in the intrinsic factor Xase complex requires its binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes with the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-containing membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies.