New sensitive assay for phosphatidylserine decarboxylase based on the detection of CO2 from nonradiolabeled phosphatidylserine

A new rapid assay for phosphatidylserine decarboxylase, which is sensitive in the nanomolar range, is described. Synthesis of radiolabeled phosphatidylserine for use as a substrate is not required, since the assay, unlike previous ones, is based on the detection of CO(2) liberated from unlabeled phosphatidylserine. The assay employs a gas chromatographic procedure for the analysis of methane formed by catalytic conversion of the CO(2) produced as a product of the enzymatic reaction.     

Dimeric galectin-1 induces surface exposure of phosphatidylserine and phagocytic recognition of leukocytes without inducing apoptosis

We report that human galectin-1 (dGal-1), a small dimeric beta-galactoside-binding protein, induces phosphatidylserine (PS) exposure, measured by Annexin V staining, on human promyelocytic HL-60 cells, T leukemic MOLT-4 cells, and fMet-Leu-Phe-activated, but not resting, human neutrophils. This effect of dGal-1 on HL-60 and MOLT-4 cells is enhanced by pretreatment of the cells with neuraminidase, but treatment of resting neutrophils with neuraminidase does not enhance their sensitivity to dGal-1. Although the induction of staining with Annexin V is often associated with apoptosis, the dGal-1-treated HL-60 cells, MOLT-4 cells, and activated neutrophils do not undergo apoptosis, and there is no detectable DNA fragmentation. HL-60 and MOLT-4 cells treated with dGal-1 continue to grow normally. By contrast, camptothecin-treated HL-60 cells, etoposide-treated MOLT-4 cells, and anti-Fas-treated neutrophils exhibit extensive DNA fragmentation and/or cell death. Lactose inhibits the dGal-1-induced effects, indicating that dGal-1-induced signaling requires binding to cell surface beta-galactosides. The dimeric form of Gal-1 is required for signaling, because a monomeric mutant form of Gal-1, termed mGal-1, binds to cells but does not cause these effects. Importantly, dGal-1, but not mGal-1, treatment of HL-60 cells and activated human neutrophils significantly promotes their phagocytosis by activated mouse macrophages. These dGal-1-induced effects are distinguishable from apoptosis, but like apoptotic agents, prepare cells for phagocytic removal. Such effects of dGal-1 may contribute to leukocyte homeostasis.     

A new consensus sequence for phosphatidylserine recognition by annexins

Annexins are abundant and ubiquitous proteins that bind, by their four structurally identical domain cores, to phosphatidylserine-containing membranes in the presence of Ca2+. Using molecular simulation and mutagenesis, we have identified a new phosphatidylserine-binding site in annexin V domain 1 and established its structure. The residues involved in this site constitute a consensus sequence highly conserved in all annexins. Remarkably, this consensus sequence is exclusively found in domains 1 or 2, sometimes in both, but never in domains 3 and 4. Such a pattern actually delineates three classes of annexins, shedding new light on the role played by the four-domain core of annexins that could encode specific information discriminating the different annexins that compete within a given cell for membrane binding. Our findings thus provide new strategies for understanding the regulation of the cellular functions of annexins.     

Externalization of phosphatidylserine during apoptosis does not specifically require either isoform of phosphatidylserine synthase

Phosphatidylserine (PtdSer) is made in mammalian cells by two PtdSer synthases, PSS1 and PSS2. In the plasma membrane PtdSer is normally localized on the inner leaflet but undergoes transbilayer movement during apoptosis and becomes exposed on the cell surface. We induced apoptosis with staurosporine in four Chinese hamster ovary (CHO) cell lines that are deficient in PSS1 and/or PSS2 to determine if PtdSer generated by either of these enzymes is required for externalization on the cell surface during apoptosis. The onset of apoptosis was confirmed by the appearance of morphological changes and DNA fragmentation while the plasma membrane remained largely intact. In all cell lines, regardless of their content of PSS1 and/or PSS2, apoptosis occurred to approximately the same extent, and within approximately the same time frame, as in parental CHO-K1 cells. The exposure of PtdSer on the cell surface was assessed by annexin V labeling and flow cytometry. Cells that were deficient in either PSS1 or PSS2, as well as cells that were deficient in both PSS1 and PSS2, externalized normal amounts of PtdSer. Our study demonstrates, that reduction of in vitro serine-exchange activity, even by 97%, does not restrict the externalization of PtdSer during apoptosis. Moreover, a normal level of expression of PSS1 and/or PSS2 is not required for generating the pool of PtdSer externalized during apoptosis.     

Effective production of monoclonal antibodies against phosphatidylserine: stereo-specific recognition of phosphatidylserine by monoclonal antibody

A system based on the direct immunization of phospholipid Ag into mouse spleen has been used to produce mAb against phosphatidylserine (PS). mAb that bind to PS but not to phosphatidylcholine were selected. Remarkable frequency of the production of mAb against PS was observed with the immunization protocol. The mAb exhibited three distinct reactivity profiles ranging from highly specific to broadly cross-reactive. Among 61 hybridomas, 15 mAb were established for further analysis. The reactivities of three typical mAb, designated PS4A7, PS3A, and PSC8, are described. PS4A7 is highly specific to PS and no cross-reaction with other acidic phospholipids was observed. In the experiments using PS derivatives with a modified polar head group, PS4A7 was shown to bind to 1,2-diacyl-sn-glycero-3-phospho-L-serine (PS) but not to 1,2-diacyl-sn-glycero-3-phospho-D-serine or 1,2-diacyl-sn-glycero-3-phospho-L-homoserine, indicating that the antibody recognizes the stereo-specific configuration of serine residue in PS. PS3A binds to both PS and phosphatidylethanolamine, whereas no cross-reaction with other acidic phospholipids was observed. The analysis using the derivatives of PS and phosphatidylethanolamine shows that the antibody recognizes the amino group of the phospholipid Ag and cannot distinguish the conformational structure of serine residue in PS. PSC8 represents the family of mAb that cross-react considerably with other acidic phospholipids.     

Oxidative signaling pathway for externalization of plasma membrane phosphatidylserine during apoptosis

Active maintenance of membrane phospholipid asymmetry is universal in normal cell membranes and its disruption with subsequent externalization of phosphatidylserine is a hallmark of apoptosis. Externalized phosphatidylserine appears to serve as an important signal for targeting recognition and elimination of apoptotic cells by macrophages, however, the molecular mechanisms responsible for phosphatidylserine translocation during apoptosis remain unresolved. Studies have focused on the function of aminophospholipid translocase and phospholipid scramblase as mediators of this process. Here we present evidence that unique oxidative events, represented by selective oxidation of phosphatidylserine, occur during apoptosis that could promote phosphatidylserine externalization. We speculate that selective phosphatidylserine oxidation could affect phosphatidylserine recognition by aminophospholipid translocase and/or directly result in enzyme inhibition. The potential interactions between the anionic phospholipid phosphatidylserine and the redox-active cationic protein effector of apoptosis, cytochrome c, are presented as a potential mechanism to account for selective oxidation of phosphatidylserine during apoptosis. Thus, cytochrome c-mediated phosphatidylserine oxidation may represent an important component of the apoptotic pathway.     

The role of phosphatidylserine in recognition and removal of erythrocytes.

During the time that erythrocytes (RBC) spend in the circulation, a series of progressive events take place that lead to their removal and determine their apparent aging and limited survival. In addition, a fraction of RBC precursors will be removed during erythropoiesis by apoptotic processes, often described as "ineffective erythropoiesis". Both will determine the survival of erythroid cells and play an important role in red cell pathology, including hemoglobinopathies and red cell membrane disorders. The loss of phospholipid asymmetry, and the exposure of phosphatidylserine (PS) on the surface of plasma membranes may be a general trigger by which cells, including aging RBC and apoptotic cells, are removed. Oxidant stress and inactivation of the system that maintains phospholipid asymmetry play a central role in the events that will lead to PS exposure, death and removal.     

Polyacrylic polyhydrazides as reagents for detection of glycoproteins

Glycoproteins immobilized on membranes can be detected with high selectivity and sensitivity by the four-step procedure described in this work. The glycoproteins are first oxidized by sodium periodate and then polyacrylic polyhydrazides are coupled to the aldehyde groups generated in the sugar part of the glycoproteins. In the third step, a glycoenzyme, such as horseradish peroxidase, is coupled to the remaining hydrazide groups on the polymer through the aldehydes formed in its glycan chains. In the last step, the visualization of glycoproteins is achieved through the reaction product of the bound glycoenzyme. The sensitivity of the glycoprotein detection is most critically dependent on the hydrazide reagent. Thus, dihydrazides were not satisfactory, a trihydrazide was better, and polyhydrazides were the best. Two different polyhydrazides were used. One was based on acrylamide and the other on N-acryloyl-tris(hydroxymethyl)aminomethane. The second one proved to be superior because it gave higher sensitivity with no detectable background staining. We have also investigated the influence of various reaction conditions on staining of glycoproteins having oligomannose and N-acetyllactosamine type glycan chains. Some of them, invertase and fetuin, could be detected with sensitivity similar to that of silver staining in gels and colloidal gold staining on the membranes. The detection of small quantities of Endo H-deglycosylated glycoproteins was possible under standard conditions only if several N-acetylglucosamine residues remained bound to the protein.     

Regulated externalization of phosphatidylserine at the cell surface: implications for apoptosis

The regulated loss of plasma membrane phosphatidylserine (PS) asymmetry is critical to many biological processes. In particular, the appearance of PS at the cell surface, a hallmark of apoptosis, prepares the dying cell for engulfment and elimination by phagocytes. While it is well established that PS externalization is regulated by activation of a calcium-dependent phospholipid scramblase activity in concert with inactivation of the aminophospholipid translocase, there is no evidence indicating that these processes are triggered and regulated by apoptotic regulatory mechanisms. Using a novel model system, we show that PS externalization is inducible, reversible, and independent of cytochrome c release, caspase activation, and DNA fragmentation. Additional evidence is presented indicating that the outward movement of plasma membrane PS requires sustained elevation in cytosolic Ca2+ in concert with inactivation of the aminophospholipid translocase and is inhibited by calcium channel blockers.     

Caspase-independent phosphatidylserine exposure during apoptosis of primary T lymphocytes

Exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane is a key feature of apoptosis. As the signals underlying these phenomena are unknown, it is generally assumed that PS exposure is a consequence of caspase activation, another hallmark of apoptosis. In this study we investigated the role of caspases in PS externalization during apoptosis of activated PBL triggered by drugs (etoposide, staurosporine), CD95 engagement, or IL-2 withdrawal. Anti-CD95 mAb induces a rapid activation of caspases, followed by PS exposure and mitochondrial transmembrane potential (DeltaPsim) disruption. In contrast, etoposide (ETO), staurosporine (STS), or IL-2 withdrawal triggers concomitant caspase activation, PS exposure, and DeltaPsim disruption. Such kinetics suggest that PS exposure could be independent of caspase activation. As expected, in activated PBL treated by anti-CD95 mAb, the pan-caspase inhibitor Cbz-Val-Ala-Asp(OMe)-fluoromethylketone and the caspase-8 inhibitor Cbz-Leu-Glu-Thr-Asp(OMe)-fluoromethylketone, but not the caspase-9 inhibitor Cbz-Leu-Glu-His-Asp(OMe)-fluoromethylketone, inhibit PS externalization and DeltaPsim disruption. Surprisingly, during apoptosis induced by ETO, STS, or IL-2 withdrawal, none of those caspase inhibitors prevents PS externalization or DeltaPsim disruption, whereas they all inhibit DNA fragmentation as well as the morphological features of nuclear apoptosis. In Jurkat and H9 T cell lines, as opposed to activated PBL, PS exposure is inhibited by Cbz-Val-Ala-Asp(OMe)-fluoromethylketone during apoptosis induced by CD95 engagement, ETO, or STS. Thus, caspase-independent PS exposure occurs in primary T cells during apoptosis induced by stimuli that do not trigger death receptors.     

Visual methods for the nanomolar detection of electrophilic reagents

A series of highly colored nitrophenolates and nitrothiophenolates has been tested as spray reagents for the detection of electrophilic species of the types commonly used in peptide and protein chemistry. Sensitive TLC detection of agents for alkylation, acylation, sulfonylation and phosphorylation was demonstrated. In addition, the thiophenolate sprays were sensitive for oxidizing agents in nanomolar quantities. Selective TLC detection of acylating and phosphorylating agents was accomplished by subsequent alkali treatment resulting in the restoration of color.     

Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved

Exposure of the aminophospholipid phosphatidylserine at the outer leaflet of the plasma membrane by apoptotic cells can trigger phagocytic removal of these dying cells. This functionality of phosphatidylserine exposure in the process of phagocytosis is indicated by in vitro studies of mammalian and insect phagocytes. We have studied the in vivo distribution of cell-surface exposed phosphatidylserine by injecting biotinylated Annexin V, a Ca 2+ -dependent phosphatidyl-serine binding protein, into viable mouse and chick embryos and Drosophila pupae. The apparent binding of Annexin V to cells with a morphology which is characteristicof apoptosis and which was present in regions of developmental cell death indicates that phosphatidylserine exposure by apoptotic cells is a phylogenetically conserved mechanism.     

Appetizing rancidity of apoptotic cells for macrophages: oxidation,externalization, and recognition of phosphatidylserine

Programmed cell death (apoptosis) functions as a mechanism to eliminate unwanted or irreparably damaged cells ultimately leading to their orderly phagocytosis in the absence of calamitous inflammatory responses. Recent studies have demonstrated that the generation of free radical intermediates and subsequent oxidative stress are implicated as part of the apoptotic execution process. Oxidative stress may simply be an unavoidable yet trivial byproduct of the apoptotic machinery; alternatively, intermediates or products of oxidative stress may act as essential signals for the execution of the apoptotic program. This review is focused on the specific role of oxidative stress in apoptotic signaling, which is realized via phosphatidylserine-dependent pathways leading to recognition of apoptotic cells and their effective clearance. In particular, the mechanisms involved in selective phosphatidylserine oxidation in the plasma membrane during apoptosis and its association with disturbances of phospholipid asymmetry leading to phosphatidylserine externalization and recognition by macrophage receptors are at the center of our discussion. The putative importance of this oxidative phosphatidylserine signaling in lung physiology and disease are also discussed.     

The role of phosphatidylserine in recognition of apoptotic cells by phagocytes

Exposure of phosphatidylserine on the outer leaflet of the plasma membrane is a surface change common to many apoptotic cells. Normally restricted to the inner leaflet, phosphatidylserine appears as a result of decreased aminophospholipid translocase activity and activation of a calcium-dependent scramblase. Phosphatidylserine exposure has several potential biological consequences, one of which is recognition and removal of the apoptotic cell by phagocytes. It is still not clear which receptors mediate PS recognition on apoptotic cells; however, several interesting candidates have been proposed. These include the Class B scavenger and thrombospondin receptor CD36, an oxLDL receptor (CD68), CD14, annexins, beta2 glycoprotein I, gas-6 and a novel activity expressed on macrophages stimulated with digestible particles such as beta-glucan. Whether PS is the sole ligand recognized by phagocytes or whether it associated with other molecules to form a complex ligand remains to be determined.     

New reagents and solid support for automated oligonucleotide synthesis

The optimal system for the rapid, efficient, convenient, and economical synthesis and purification of synthetic oligonucleotides has been advancing. By recognizing the very rapid reaction kinetics and taking advantage of an efficient, low volume delivery system, cycle times have decreased to about 5.5 minutes, without compromising synthesis performance. A new set of base protecting groups for cyanoethylphosphoramidite nucleoside monomers have been developed, which decreases the post-synthesis time requirements. A particular form of polystyrene has also been developed as a solid support for automated oligonucleotide synthesis. Typical sequencing or PCR primers (20mers) now require less than 2 hours for synthesis and 2 hours for cleavage and deprotection.     

A role for oxidative stress in apoptosis: oxidation and externalization of phosphatidylserine is required for macrophage clearance of cells undergoing Fas-mediated apoptosis

Exposure of phosphatidylserine (PS) on the surface of apoptotic cells has been suggested to serve as an important recognition signal for macrophages. In this work we show that triggering of the death receptor Fas on Jurkat cells results in the generation of reactive oxygen species with oxidation and externalization of PS but not of the other major aminophospholipid, phosphatidylethanolamine. These cells were readily ingested by several classes of macrophages, whereas Raji cells, which are defective for Fas-induced PS exposure, remained unengulfed. However, when Raji cells were incubated with the thiol-reactive agent N-ethylmaleimide to induce PS exposure in the absence of other features of apoptosis, these cells were also engulfed by macrophages. Phagocytosis of Fas-triggered Jurkat cells was inhibited by superoxide dismutase and catalase, which prevent oxidation of PS while allowing PS to remain externalized on these cells. Moreover, liposomes containing oxidized PS (PS-OX) were more potent inhibitors of phagocytosis than those containing its nonoxidized counterpart. Finally, enrichment of the plasma membrane of Jurkat or Raji cells, or myeloid leukemic HL-60 cells, with exogenous PS resulted in phagocytic cell clearance, and this process was further enhanced when PS was substituted for by PS-OX. Taken together, our data suggest that the presence of PS-OX in conjunction with nonoxidized PS on the cell surface is an important signal for macrophage clearance of apoptotic cells.