Surface proteins of Plasmodium falciparum merozoites binding to the erythrocyte receptor, glycophorin

Invasion of erythrocytes by the malarial parasite is a receptor- mediated process. P. falciparum merozoites recognize and bind to erythrocyte surface sialoglycoproteins, glycophorins A and B, and the glycophorins bind to saturable sites on the merozoite surface. The purpose of the present work was to identify a receptor or ligand molecule on the merozoite surface that mediates binding to the erythrocyte. A fraction containing the sialoglycoproteins was coupled to an acrylamide matrix and incubated with metabolically labeled merozoites. A merozoite protein of 155 kD that labeled prominently with [3H]glycine bound to glycophorin. A minor protein of 130 kD also bound. Both proteins are rich in proline and glycine, poor in methionine, and may be related. The proteins are also stable to heating to 100 degrees C for 10 min. Immunoelectron microscopy demonstrated that the 155 kD and 130 kD proteins are located on the merozoite surface coat. The antibodies significantly inhibited merozoite invasion into erythrocytes and also binding of the proteins to the glycophorin-matrix. The specific binding of the 155-kD and 130-kD proteins to the erythrocyte receptor and the demonstration that they are located on the merozoite surface suggest they could be receptor proteins that mediate binding of the merozoite to the erythrocyte surface.     

Coated-platelets: an emerging component of the procoagulant response.

Coated-platelets, formerly known as COAT-platelets, represent a subpopulation of cells observed after dual agonist stimulation of platelets with collagen and thrombin. This class of platelets retains on its surface high levels of several procoagulant proteins, including fibrinogen, von Willebrand factor, fibronectin, factor V and thrombospondin. Coated-platelets also express surface phosphatidylserine and strongly support prothrombinase activity. Retention of alpha-granule proteins on the surface of coated-platelets involves an unexpected derivatization of these proteins with serotonin and an interaction of serotonin-conjugated proteins with serotonin binding sites on fibrinogen and thrombospondin. This review will also detail experimental systems where coated-platelets are generated as a result of other agonist(s). Finally, the putative physiological consequences of coated-platelet formation will be discussed.     

Role of surface proteins in staphylococcal adherence to fibers in vitro.

To study the role of surface proteins in the adherence of Staphylococcus aureus to fibers that are used in tampon and surgical gauze pad manufacture, we have developed an adherence assay with S. aureus cells and cotton and rayon fibers. Results suggest that staphylococcal adherence is dependent on both the substrate and the material used to coat these fibers. Scanning electron micrographs supported the adherence results and revealed more cells on the surface of cotton than rayon fibers. Treatment of staphylococcal cells with proteolytic enzymes significantly reduced binding to pure cotton and detergent-treated cotton fibers. Immunoblot analysis of cell wall proteins suggested that surface proteins in the mol wt range of 120-220 kD were involved in the adherence of S. aureus to cotton fibers. Although the adherence of S. aureus to cotton fibers alone appeared to be mediated through surface charge or hydrophobic interactions, bacterial binding to fibers which have been pretreated with defibrinated blood appeared to be more specific and independent of the surface constituents of the fibers. The results of these studies implicate staphylococcal surface proteins in the adherence of S. aureus to commercially available tampon fibers and surgical gauze pads.     

Membrane proteins as diagnostic biomarkers and targets for new therapies

Membrane proteins, especially plasma membrane proteins, form one of the most interesting classes of proteins among disease biomarker candidates. Because of their localization on the surface of cells and organelles, membrane proteins also represent potential drug targets. In this review, developments in the characterization of membrane proteins and their role in the treatment of disease, in particular cancer treatment, are presented.     

LYMPHOCYTE MEMBRANE DYNAMICS : METABOLIC RELEASE OF CELL SURFACE PROTEINS

Cell surface proteins of normal and neoplastic lymphocytes were labeled with iodide-¹²⁵I by lactoperoxidase-catalyzed iodination. Incubation of ¹²⁵I-labeled iodide cells in vitro resulted in the release of iodinated surface proteins at a rapid rate which was dependent on cellular respiration and protein synthesis. Comparisons by disc electrophoresis showed a marked similarity between urea-soluble surface proteins extracted from iodinated cells and iodinated material released by the cells during in vitro incubation. The rate of release of cell surface proteins from thymus cells was three times faster than that of spleen cells or bone marrow-derived thoracic duct lymphocytes. In addition, different proteins were released at different rates as evidenced by the rate of release of ¹²⁵I of rabbit anti-mouse immunoglobulin specifically bound to mouse spleen cells and comparisons by disc electrophoresis of urea-soluble iodinated surface proteins extracted from cells before and after incubation. The results suggest that a dynamic state exists at the cell surface. The possible role of the release of cell surface proteins in cell regulation and communication is discussed.     

Antibacterial activity of cationic proteins from human granulocytes.

Human granulocytes contain several cationic proteins with a molecular weight of approximately 25,000, almost identical amino acid composition, and complete immunologic identity. These proteins possess a chymotrypsin-like protease activity at a neutral pH. The antibacterial activity of the cationic proteins has been studied. Bactericidal activities are found against both Gram-positive (Streptococcus faecalis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) organisms. Gram-positive bacteria are, however, the most sensitive. The pH-optimum is near neutrality, and the microbicidal activity shows an inverse relationship to the ionic strength, indicating an ionic interaction between the cationic proteins and the bacterial surface. The microbicidal effect of the cationic proteins is generally independent of the chymotrypsin-like activity of the same proteins since the activity against several bacterial species is heat stable while the chymotrypsin-like activity is heat labile. The surface properties of S. aureus that are determined by protein A do not seem to influence the susceptibility to cationic proteins. The properties of the Gram-negative envelope of E. coli that determine the susceptibility to the lytic action of serum do not influence the sensitivity to the action of cationic proteins. The present study shows that cationic proteins of human granulocytes represent one potential microbicidal mechanism that is independent of hydrogen peroxide and myeloperoxidase.     

Cell surface organization of stress-inducible proteins ULBP and MICA that stimulate human NK cells and T cells via NKG2D

Cell surface proteins major histocompatibility complex (MHC) class I-related chain A (MICA) and UL16-binding proteins (ULBP) 1, 2, and 3 are up-regulated upon infection or tumor transformation and can activate human natural killer (NK) cells. Patches of cross-linked raft resident ganglioside GM1 colocalized with ULBP1, 2, 3, or MICA, but not CD45. Thus, ULBPs and MICA are expressed in lipid rafts at the cell surface. Western blotting revealed that glycosylphosphatidylinositol (GPI)-anchored ULBP3 but not transmembrane MICA, MHC class I protein, or transferrin receptor, accumulated in detergent-resistant membranes containing GM1. Thus, MICA may have a weaker association with lipid rafts than ULBP3, yet both proteins accumulate at an activating human NK cell immune synapse. Target cell lipid rafts marked by green fluorescent protein-tagged GPI also accumulate with ULBP3 at some synapses. Electron microscopy reveals constitutive clusters of ULBP at the cell surface. Regarding a specific molecular basis for the organization of these proteins, ULBP1, 2, and 3 and MICA are lipid modified. ULBP1, 2, and 3 are GPI anchored, and we demonstrate here that MICA is S-acylated. Finally, expression of a truncated form of MICA that lacks the putative site for S-acylation and the cytoplasmic tail can be expressed at the cell surface, but is unable to activate NK cells.     

Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages

It has long been known from the results of ultrastructural studies that complement- and immunoglobulin G (IgG)-opsonized particles are phagocytosed differently by macrophages (Kaplan. G. 1977. Scand. J. Immunol. 6:797-807). Complement-opsonized particles sink into the cell, whereas IgG-coated particles are engulfed by lamellipodia, which project from the cell surface. The molecular basis for these differences is unknown. We used indirect immunofluorescence and confocal microscopy to examine how cytoskeletal proteins associate with phagosomes containing complement-opsonized zymosan (COZ) particles or IgG beads in phorbol-myristateacetate-treated peritoneal macrophages. During ingestion of COZ, punctate structures rich in F-actin, vinculin, alpha-actinin, paxillin, and phosphotyrosine-containing proteins are distributed over the phagosome surface. These foci are detected beneath bound COZ within 30 s of warming the cells to 37 degrees C, and their formation requires active protein kinase C. By contrast, during Fc receptor-mediated phagocytosis, all proteins examined were uniformly distributed on or near the phagosome surface. Moreover, ingestion of IgG beads was blocked by tyrosine kinase inhibitors, whereas phagocytosis of COZ was not. Thus, the signals required for particle ingestion, and the arrangement of cytoskeletal proteins on the phagosome surface, vary depending upon which phagocytic receptor is engaged. Moreover, complement receptor (CR)-mediated internalization required intact microtubules and was accompanied by the accumulation of vesicles beneath the forming phagosome, suggesting that membrane trafficking plays a key role in CR-mediated phagocytosis.     

A century of human blood groups.

During the course of the twentieth century, at least 270 authenticated alloantigens have been recognised on the red cell surface. Most of these have been classified into 26 blood group systems, each of which represents a single gene or a cluster of two or three closely-linked homologous genes. Most blood group polymorphisms result from single nucleotide changes encoding amino acid substitutions in cell surface proteins. Many other mechanisms, including recombination between homologous genes, are also involved, especially in the complex Rh and MNS systems. There are at least three common molecular backgrounds to the RhD-negative phenotype. Some blood group antigens are carbohydrates, the polymorphisms resulting from mutations within genes encoding glycosyltransferases. Red cell surface proteins perform a variety of functions. For some the functions are well understood, but for most they can only be surmised from the structure of the protein. Putative functions include, membrane transport, cell adhesion, complement inactivation, binding chemokines, and anchoring the plasma membrane to the cytoskeleton. Some erythroid cell surface antigens may serve their primary purpose during erythropoiesis. Analysis of the development of these proteins on erythroid cells during erythropoiesis, ex vivo, has provided clues to their functions and a useful set of markers for the study of erythropoiesis.     

Immune response to stage-specific surface antigens of the parasitic nematode Trichinella spiralis

Rats were infected with the nematode Trichinella spiralis and the primary serum antibody response to antigenic surface proteins of infective larvae, intestinal worms, and newborn larvae was studies. 1 wk after infection, the sera contained antibodies to surface antigens of both infective larvae and intestinal worms. These early sera, however, failed to react with newborn larvae surface antigens. In addition, adsorption of sera with living intestinal worms or infective larvae removed antibodies to surface antigens of the homologous stage only. Finally, the time-course of appearance of antibodies that mediate eosinophil adherence to the surface of each stage of the parasite. We concluded that in a primary infection in rats, the surface proteins of T. spiralis used in this study are antigenically stage specific. Furthermore, they could be targets for the stage-specific, antibody- dependent eosinophil-mediated destruction of this parasite, known to occur in vitro.     

Outer membrane proteins of Neisseria meningitidis: structure and importance in meningococcal disease

Meningococcal meningitis is a serious disease with a high incidence in children. Since meningococcal surface structures play a role in invasion of the host and initiation of disease, host defense mechanisms are directed against these meningococcal structures. All virulent meningococci are encapsulated, but other factors are also involved. Only certain serotypes of group B and group C meningococci are strongly associated with disease. Our studies have been directed toward a better understanding of the structure of the meningococcal cell surface. There are five classes of major outer membrane proteins exposed on the cell surface, designated class 1 through 5, based in part upon the protein's molecular weight. Using monoclonal antibodies we have shown that antibodies to the class 1, 2 and 5 proteins of serotype 2 are bactericidal and therefore probably protective. Convalescent sera of meningococcal disease patients have elevated antibody levels to each of these classes of proteins. Outer membrane protein vaccines have been prepared, and clinically evaluated in adults and children as young as 6 mo of age. The vaccines induce serotype specific bactericidal antibodies in all age groups, but they induced lower levels in young children. We are now working to improve the immune response of children through the use of aluminium adjuvants.     

Studies on platelet plasma membranes. I. Characterization of surface proteins of human platelets labeled with diazotized (125i)-diiodosulfanilic acid.

A polar, nonpenetrating compound of high specific activity, diazotized (125I)-diiodosulfanilic acid (DD125ISA), has been developed as a label for exposed proteins of the human platelet plasma membrane, and platelet proteins and the pattern of labeling have been studied with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). That DD125ISA binds specifically to membrane proteins was demonstrated by: (1) the specific activity of isolated membrane protein was five to seven times that of whole platelet protein and (2) no proteins of intact platelets were labeled which were not represented in the isolated plasma membrane. That the DD125ISA-labeled membrane proteins were exposed on the cell surface was demonstrated by: (1) DD125ISA-labeled proteins were altered by trypsin treatment of intact, labeled platelets and (2) the pattern of labeling produced by reaction of isolated membranes with DD125ISA was quite different from that produced by the labeling of intact platelets. Analysis of platelet membrane proteins by SDS-PAGE demonstrated the glycoproteins previously described at 150,000 daltons (termed glycoprotein I) and 92,000 daltons (glycoprotein III) but we could discriminate two apparently distinct glycoproteins in the intermediate region (IIa: 125,000 daltons, and II: 118,000 daltons). Glycoproteins I and III were constant whereas IIa was clearly visible only in unreduced samples and II was predominant in reduced samples. Reaction of DD125ISA with intact platelets resulted in equal labeling of three of these four membrane glycoproteins (IIa, II, and III). The pattern of exposed proteins on the platelet surface labeled by DD125ISA was different from lactoperoxidase-131I, which labeled predominantly the 92,000 dalton glycoprotein, as demonstrated by simultaneous SDS-PAGE analysis. Therefore three glycoproteins of the human platelet plasma membrane are exposed to a radioisotope probe on the platelet surface and are accessible for contact interactions.     

Developmentally regulated expression of CD3 components independent of clonotypic T cell antigen receptor complexes on immature thymocytes

CD3 signal transducing proteins are thought to be expressed on the surface of T cells only as part of clonotypic T cell receptor (TCR) complexes. Contrary to this paradigm, the present study describes surface expression of CD3 proteins independently of clonotypic TCR complexes, but only on immature thymocytes. Such novel clonotype- independent CD3 (CIC) complexes are composed primarily of CD3 gamma epsilon and secondarily of CD3 delta epsilon heterodimers that are independent of one another and are expressed on the cell surface in association with an unknown 90-100 kD protein termed CD3-associated protein (CD3AP). CIC complexes are expressed in normal mice on early thymocytes through the CD4+CD8+ stage of development, but not on mature peripheral T cells. Furthermore, CIC complexes are expressed by both TCR- severe combined immunodeficiency (SCID) thymocytes and thymoma cell lines, in the absence of any clonotypic chains. The isolation and biochemical characterization of surface CIC complexes provides a structural basis for the signaling effects of anti-CD3 epsilon antibody treatment in early thymocyte development.     

Restricted or absent immune responses in human populations to Plasmodium falciparum gamete antigens that are targets of malaria transmission-blocking antibodies

We have studied the antibodies to sexual stage antigens of Plasmodium falciparum in human sera from Papua New Guinea where intense transmission of P. falciparum occurs as well as the less prevalent P. malariae and P. vivax. In extracts of gametes of P. falciparum we have studied the reactivity of serum antibodies with antigens labeled with 125I on the surface of the gametes as well as intracellular gamete antigens. A prominent 27-kD sexual stage-specific intracellular protein was recognized more or less in proportion to the general antibody response to gamete proteins. The response to the gamete surface proteins, however, was quite unrepresentative of the general antibody response to the intracellular gamete proteins. No antibodies were detected against Pfs25, a 21-kD protein expressed on zygotes and ookinetes of P. falciparum and known to be a sensitive target of malaria transmission-blocking antibodies. The antibody response to two other target antigens of transmission-blocking antibodies on the surface of gametes of P. falciparum, a 230- and a 48- and 45-kD protein doublet, was very variable and independent of the response to the internal protein antigens. Several possibilities are discussed that may account for the variable response to these gamete surface antigens in individuals with otherwise good antibody responses to internal sexual stage proteins. Among these is the possibility that there is MHC restriction of the immune response to the gamete surface antigens in the human population. This interpretation accords well with evidence for MHC-restricted immune response to the same P. falciparum gamete surface antigens in studies with H-2 congenic mice (24).     

Sequential expression of antigens on sexual stages of Plasmodium falciparum accessible to transmission-blocking antibodies in the mosquito

Plasmodium falciparum gametocytes contain specific antigens, some of which (Mr 230,000, 48,000, 45,000) are expressed on the surface of the newly emerged macrogamete. A different antigen (Mr 25,000) surrounds the surface of the ookinete and, although present to some extent in the developing gametocyte, is synthesized in high quantities by the macrogamete/zygote and expressed progressively on the transforming zygote surface. These antigens are targets of transmission blocking antibodies that are effective at two distinct points after gametogenesis: fertilization of the macrogamete and ookinete to oocyst development. The antigens involved in the fertilization blockade are the Mr 48 and 45 proteins, which are expressed on the macrogamete surface. The Mr 230 K coprecipitating protein probably plays no part in transmission block. mAb directed against the Mr 25 K ookinete surface protein blocked transmission without inhibiting ookinete formation, indicating that this protein has an important role in the transformation of ookinete into oocyst. A combination of mAb recognizing different epitopes on the same protein molecule acted synergistically in inhibiting oocyst formation. Using a mixture of two blocking mAb reacting against the Mr 48/45 and 25 K proteins, respectively, an additive blocking effect could be demonstrated.     

Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (mean survival: 34.2% +/- 9.0% of albumin, P less than 0.0001) despite similar efficient ingestion of extracellular bacteria. Enhancement of killing was observed when surfaces were coated with purified constituents of extracellular matrix, i.e., fibronectin, fibrinogen, laminin, vitronectin, or type IV collagen. In addition to matrix proteins, the tetrapeptide RGDS (the sequence recognized by integrins) crosslinked to surface bound albumin was also active (survival: 74.5% +/- 5.5% of albumin, P less than 0.02), and fibronectin-increased killing was inhibited by soluble RGDS. Chemiluminescence measurements and experiments with CGD neutrophils revealed that both oxygen-dependent and -independent bactericidal mechanisms are involved. In conclusion, matrix proteins enhance intracellular bactericidal activity of adherent neutrophils, presumably by integrin recognition of RGDS-containing ligands. These results indicate a role for extracellular matrix proteins in the enhancement of the host defense against pyogenic infections.     

Platelet structure and function: immunocytochemical localizations of membrane glycoprotein and alpha-granule protein]

The platelet membrane glycoproteins (GPs) are receptors or binding sites for adhesive proteins. GPIb and GPIIb/IIIa complex are major glycoproteins and have important roles, functionally. GPIb plays an essential role in primary hemostasis as receptor for the von Willebrand factor. The GPIIb/IIIa complex acts as the binding site for adhesive proteins on activated platelets and, as such, is essential for platelet aggregation. On the other hand, four adhesive proteins (fibrinogen, fibronectin, thrombospondin and von Willebrand factor), which are present not only in plasma but also in alpha-granules, mediate or modulate the platelet adhesive response. The interaction between these adhesive proteins and platelet membrane GPs are essential for platelet adhesion and aggregation. The present report will focus on the localization of GPIb and GPIIb/IIIa on the platelet surface and that of adhesive proteins in alpha-granules in both resting and activated human platelets.     

Membrane-tethered proteins for basic research, imaging, and therapy

Secreted and intracellular proteins including antibodies, cytokines, major histocompatibility complex molecules, antigens, and enzymes can be redirected to and anchored on the surface of mammalian cells to reveal novel functions and properties such as reducing systemic toxicity, altering the in vivo distribution of drugs and extending the range of useful drugs, creating novel, specific signaling receptors and reshaping protein immunogenicity. The present review highlights progress in designing vectors to target and retain chimeric proteins on the surface of mammalian cells. Comparison of chimeric proteins indicates that selection of the proper cytoplasmic domain and introduction of oligiosaccharides near the cell surface can dramatically enhance surface expression, especially for single-chain antibodies. We also describe progress and limitations of employing surface-tethered proteins for preferential activation of prodrugs at cancer cells, imaging gene expression in living animals, performing high-throughput screening, selectively activating immune cells in tumors, producing new adhesion molecules, creating local immune privileged sites, limiting the distribution of soluble factors such as cytokines, and enhancing polypeptide immunogenicity. Surface-anchored chimeric proteins represent a rich source for developing new techniques and creating novel therapeutics.     

The effect of ambient storage on platelet membrane structure and response to thrombin

Blood platelets rapidly lose hemostatic function during in vitro storage at room temperature. In view of recent studies implicating platelet surface proteins in some aspects of platelet function, we have examined the effect of platelet storage on membrane structure and function. Platelets obtained from a single donor by double plateletpheresis were stored for seven days at 22 C with constant agitation. On succeeding days, platelets were removed and analyzed for membrane surface structure determined by iodination of membrane surface proteins, membrane function determined by serotonin uptake and equilibrium binding of thrombin to surface receptors, and platelet (unction determined by serotonin release and platelet aggregation in response to thrombin. The earliest changes in membrane surface structure were observed within 48 hours and consisted of decreased iodination of membrane surface proteins, and increased iodination of membrane proteins with estimated molecular weights of 260,000, 240,000, and 84,000. No changes were observed in the overall protein composition of the platelet, but changes in membrane function were observed. Relative uptake of serotonin decreased from 89 to 37 per cent and appeared to occur in parallel with the changes in membrane surface proteins. Changes in thrombin binding, on the other hand, were progressive throughout the storage period. The number of molecules of thrombin bound with high affinity decrease from 1,040 to 520 per platelet while the number of molecules bound with low affinity decreased from 103,600 to 4,500. No persistent alterations were observed in the affinity of thrombin for its receptors. Thrombin-induced platelet aggregation and release of serotonin decreased in parallel with the changes in thrombin binding.