Dimer dissociation of the pore-forming toxin aerolysin precedes receptor binding

The pore-forming toxin aerolysin is secreted by Aeromonas hydrophila as an inactive precursor. Based on chemical cross-linking and gel filtration, we show here that proaerolysin exists as a monomer at low concentrations but is dimeric above 0.1 mg/ml. At intermediate concentrations, monomers and dimers appeared to be in rapid equilibrium. All together our data indicate that, at low concentrations, the toxin is a monomer and that this species is competent for receptor binding. In contrast, a mutant toxin that forms a covalent dimer was unable to bind to target cells.     

Cytometrically detected specific binding of interleukin 2 to cells.

We have developed a new technique for detecting binding of interleukin 2 (IL-2) to cells. This technique involves incubating the cells with IL-2 and then analysing the cell surface with specific anti-IL-2 antibodies and flow cytometry. This binding was only detected on tumor cells that possessed the p55 subunit of the IL-2 receptor. The role of p55 was ascertained by inhibition of the binding with a monoclonal antibody to p55. Although p55 is necessary for cytometrically detected IL-2 binding, further studies demonstrated that p55 is not sufficient. Thus, cytometrically-detected binding is likely to involved the contribution of other IL-2 surface receptors. Interleukin-2 binding to peripheral blood T lymphocytes and to a non-transformed T-cell clone was also detected cytometrically and it was shown that this binding is regulated by the activation status of the cells. Whereas IL-2 binding to quiescent T cells could not be detected, upon activation abundant binding was seen. The functional consequences of this type of cellular binding were studied. Interleukin-2 binding to cells during a short pulse was shown to have significant long-term consequences both for T-cell proliferation and for the enhancement of major histocompatibility complex (MHC)-non-restricted cytotoxicity.     

The erythrocyte receptor for the channel-forming toxin aerolysin is a novel glycosylphosphatidylinositol-anchored protein

The plasma membrane of rat erythrocytes contains a 47-kDa glycoprotein that binds the channel-forming toxin aerolysin with high affinity and accounts for the sensitivity of these cells to the toxin. The receptor was purified so that its N-terminal sequence could be determined after Western blotting. The sequence did not match any sequences in the databases, indicating that the receptor is a novel erythrocyte surface protein. However, it exhibited considerable homology to the N-termini of a group of membrane proteins that are thought to be involved in ADP-ribosyl transfer reactions. A common property of these proteins is that they are attached to plasma membranes by C-terminal glycosylphosphatidylinositol (GPI) anchors. The aerolysin receptor was shown to be anchored in the same way by treating rat erythrocytes with phosphatidylinositol-specific phospholipase C. This caused the selective release of the receptor and a reduction in the rodent cells' sensitivity to aerolysin. Human and bovine erythrocytes were shown to contain an aerolysin-binding protein with similar properties to the rat erythrocyte receptor. Proteins with GPI anchors are thought to have unusually high lateral mobility, and this may be an advantage for a toxin, such as aerolysin, which must oligomerize after binding to become insertion competent.     

The interleukin 1 receptor. Dynamics of interleukin 1 binding and internalization in T cells and fibroblasts

In this study, we have demonstrated that a murine T cell lymphoma, EL 4, and a murine fibroblast cell line, Swiss 3T3, possess a single class of high affinity interleukin 1 (IL 1) receptors that exist in a dynamic state of equilibrium that is influenced by IL 1. In the absence of IL 1, the IL 1 receptor appears to turnover with a t1/2 of approximately 11 hr. However, when cells are incubated in the presence of IL 1, the IL 1 receptor undergoes extensive ligand-induced down-regulation. IL 1 itself is internalized at 37 degrees C; 50% of the surface-bound IL 1 is internalized in 60 to 120 min. IL 1 does not undergo degradation for at least 6 hr after internalization. By using electron microscopy and autoradiography, we observed several important features of the internalization process. When cells having bound 125I-IL 1 at 4 degrees C were shifted to 37 degrees C, IL 1 moved from the cell membrane to the cytoplasm where it was found in proximity to nuclei or within lysosomes. IL 1 appeared to progressively accumulate in nuclei. Six hours after shifting cells to 37 degrees C, 30 to 35% of the internalized 125I-IL 1 is associated with the cell nucleus. The accumulation of relatively high levels of IL 1 in the nucleus raises the interesting possibility that IL 1 may not only interact in a highly specific manner with cell surface receptors, but also with potentially important nuclear receptors.     

NMR solution structure of a peptide from the mdm-2 binding domain of the p53 protein that is selectively cytotoxic to cancer cells

We have recently found that a peptide from the mdm-2 binding domain of the p53 protein induced rapid membranolytic necrosis of a variety of different human cancer cell lines. To determine the role of solution structure in this peptide's selective and rapid tumor membrane disruptive behavior, we have performed two-dimensional NMR on a 32-residue sequence called PNC-27, in both an aqueous cytosolic-like and a mixed organic membrane-mimetic solution environment. In an aqueous milieu, PNC-27 contains three alpha-helical domains connected by loop structures, forming an S shape, and another similar structure with less helical structure. In a solution environment simulating a membrane, the helical domains found in water increase in length, forming three classes of structures, all of which form a U-shaped helix-coil-helix ensemble. In both solvent systems, this peptide forms amphipathic structures such that its hydrophobic residues coalesce on one face while the polar residues aggregate on the opposite face. The ability to form these unique structures in these two solution environments may allow the PNC-27 peptide to selectively and rapidly disrupt cancer cell membranes.     

Interleukin 2 (IL2) PE40 is cytotoxic to cells displaying either the p55 or p70 subunit of the IL2 receptor

IL2-PE40 is a chimeric protein composed of human interleukin 2 (IL2) genetically fused to the amino terminus of a modified form of pseudomonas exotoxin (PE). Internalization of IL2 via the individual p55 and p70 subunits of the IL2 receptor was studied using IL2-PE40 on several mouse and human cell lines expressing either the p55, the p70, or both IL2 receptor subunits. Internalization was assessed by measuring inhibition of protein synthesis caused by the toxin moiety of IL2-PE40. The results demonstrate that IL2 internalization is mediated by either the p55 receptor subunit or by the p70 subunit but is much more efficient when high affinity receptors composed of both subunits are present. IL2-PE40 is a powerful reagent for studying IL2 receptor interactions and for analyzing pathways of the immune response and its regulation.     

Expression of interleukin 2 and the interleukin 2 receptor in aging rats

Lymphocytes of aged animals exhibit a marked decrease in proliferative capacity in response to mitogen stimulation when compared to those of younger animals. In humans and mice the decreased proliferation is due at least in part (i) to the inability of lymphocytes to synthesize sufficient interleukin 2 (IL-2) and (ii) to decreased expression of IL-2 receptors (IL-2R) on the surface of aged lymphocytes. We compared proliferative abilities, IL-2 production, and IL-2R expression in splenocyte cultures of 4- to 5- and 22- to 24-month-old Fischer 344 rats stimulated with either concanavalin A (Con A) or A23187 and phorbol myristate acetate (PMA). Proliferation was significantly decreased in aged lymphocytes (30-50%) with both treatment protocols. However, unlike mice and humans we observed no difference in IL-2 activity, IL-2 mRNA levels, or IL-2R cell surface expression of lymphocytes from young and aged rats stimulated with either Con A or A23187 and PMA. These results indicate that factors other than decreased expression of IL-2 and IL-2R are responsible for the diminished proliferative capacity of aged rat lymphocytes following mitogen stimulation.     

Interleukin 1-dependent induction of both interleukin 2 secretion and interleukin 2 receptor expression by thymoma cells

The macrophage-derived product, interleukin 1 (IL 1) is thought to play an important regulatory role in the proliferation of T lymphocytes; however, its mechanism of action is unknown. We describe in this report a variant subline of EL4 thymoma cells (EL4-6.1) that displays a high degree of responsiveness to IL 1. We show that recombinant IL 1 can induce both the secretion of interleukin 2 (IL 2) and the expression of IL 2 receptors (IL 2-R) by these cells. EL4-6.1 cells do not constitutively secrete IL 2, nor do they express IL 2-R; but when cultured in the presence of recombinant IL 1, they secrete detectable amounts of IL 2 (5 to 15 U/ml). In the presence of either suboptimal levels of phorbol ester (PMA) or Ionomycin, the addition of IL 1 resulted in up to an 80-fold enhancement in the amount of IL 2 secreted. Stimulation with IL 1 alone or in combination with Ionomycin was unable to induce detectable IL 2-R expression by EL4-6.1 cells. However, in the presence of suboptimal concentrations of PMA, IL 1 induced expression of about 3000 high affinity (dissociation constant, Kd of 31 pM) and 50,000 low affinity (Kd of 2800 pM) IL 2-R. These IL 2-R were functional, based on their ability to rapidly internalize IL 2. This model system will allow a detailed analysis of the mechanisms involved in the regulation of the immune response by IL 1 and IL 2.     

Analysis of receptor binding by the channel-forming toxin aerolysin using surface plasmon resonance.

Aerolysin is a channel-forming bacterial toxin that binds to glycosylphosphatidylinositol (GPI) anchors on host cell-surface structures. The nature of the receptors and the location of the receptor-binding sites on the toxin molecule were investigated using surface plasmon resonance. Aerolysin bound to the GPI-anchored proteins Thy-1, variant surface glycoprotein, and contactin with similar rate constants and affinities. Enzymatic removal of N-linked sugars from Thy-1 did not affect toxin binding, indicating that these sugars are not involved in the high affinity interaction with aerolysin. Aerolysin is a bilobal protein, and both lobes were shown to be required for optimal binding. The large lobe by itself bound Thy-1 with an affinity that was at least 10-fold weaker than that of the whole toxin, whereas the small lobe bound the GPI-anchored protein at least 1000-fold more weakly than the intact toxin. Mutation analyses provided further evidence that both lobes were involved in GPI anchor binding, with certain single amino acid substitutions in either domain leading to reductions in affinity of as much as 100-fold. A variant with single amino acid substitutions in both lobes of the protein was completely unable to bind the receptor. The membrane protein glycophorin, which is heavily glycosylated but not GPI-anchored, bound weakly to immobilized proaerolysin, suggesting that interactions with cell-surface carbohydrate structures other than GPI anchors may partially mediate toxin binding to host cells.     

A soluble binding protein specific for interleukin 1 beta is produced by activated mononuclear cells

Soluble interleukin 1 (IL 1) binding proteins were identified by gel filtration and covalent cross-linking of 125I IL 1 in normal human serum and inflammatory exudate. High molecular weight 125I IL 1 protein complexes occurred with both IL 1 alpha and IL 1 beta, however, high molecular weight binding appeared to be non-specific. One specific IL 1 beta binding protein was observed to elute at approximately 100 kDa on gel filtration when bound to 125I IL 1 beta. This complex migrated as a broad band at 60 kDa when covalently cross-linked and analyzed by SDS-PAGE. The protein did not bind 125I IL 1 alpha and 125I IL 1 beta binding was only displaceable by excess cold IL-1 beta. The production of the specific IL 1 beta binding protein was assessed in a number of cell populations. Unstimulated peripheral blood mononuclear cells (PBMNC) did not produce the binding protein, but stimulation with phytohemagglutinin (PHA) caused production within 24 hr and binding protein levels remained elevated for up to 7 days. Stimulation with lipopolysaccharide (LPS) and IL 1 alpha did not consistently induce synthesis of the binding protein. Ligand-binding studies were performed to compare solubilized EL 4 NOB.1 cell membrane IL 1 receptor (sIL 1R) with semi-purified IL 1 beta binding protein from pooled synovial fluid. The sIL 1R preparation bound ligand with an affinity of 168 pM while the IL 1 beta binding protein bound 125I IL 1 beta with an affinity of 370 pM. This protein may function as an important carrier molecule for IL 1 beta and determine its distribution and kinetics in vivo.     

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which - due to its severity - is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149-amino acid protein belongs to the ribotoxin family, whose best characterized member is alpha-sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Delta(7-22) have been produced as recombinant proteins; the deleted region corresponds to an exposed beta-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Delta(7-22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted beta-hairpin is involved in ribosome recognition and is a significant allergenic region.     

The murine interleukin 2 receptor. Irreversible cross-linking of radiolabeled interleukin 2 to high affinity interleukin 2 receptors reveals a noncovalently associated subunit

The murine interleukin 2 (IL-2) receptor is a 55- to 60-kDa glycoprotein (p58) that binds IL-2 at a high and low affinity. In this investigation, we have identified sublines of EL4 that vary in their capacity to express high affinity IL-2 receptors after transfection of the IL-2 receptor cDNA. These and other cell populations were used to determine whether unique membrane molecules were specifically associated with the high affinity IL-2 receptor. Irreversible chemical cross-linking of [125I]IL-2 to only high affinity IL-2 receptors resulted in detection of IL-2 cross-linked to p58 as a 70- to 75-kDa band and other complexes of 90 to 95 kDa, 115 kDa, 150 kDa, 170 to 190 kDa, and 245 kDa. Antibodies specific for p58 resulted in precipitation of each of these complexes. However, disruption of noncovalent interactions prior to immunoprecipitation resulted in an inability to detect the material at 90 to 95 kDa. Therefore, we conclude that this complex most likely represented IL-2 cross-linked to a 75- to 80-kDa subunit that was noncovalently associated with p58. The other complexes greater than 150 kDa may represent these subunits cross-linked to each other. The detection of all the cross-linked complexes larger than 75 kDa appeared to be directly related to formation of high affinity IL-2 receptors because IL-2 was cross-linked only to p58 for three cell lines that exclusively expressed low affinity IL-2 receptors. Thus, high affinity murine IL-2 receptors are comprised of at least one alpha (p58)- and beta (p75)-subunit. Our data also raise the possibility of a more complex subunit structure.     

1,25-Dihydroxyvitamin D3 modulates the effects of interleukin 2 independent of IL-2 receptor binding

Previous studies have shown that 1,25-dihydroxyvitamin D3 (calcitriol) is a macrophage-derived cytokine and a potent inhibitor of IL-2 and interferon-gamma (IFN-gamma) production and T lymphocyte proliferation. The growth inhibitory effect of calcitriol is only partially reversed by IL-2 addition, suggesting IL-2 independent effects. In this report we characterize the IL-2-independent effects of calcitriol on lymphocyte activation. Calcitriol inhibited cellular transition from early to late G1 (G1A-G1B transition) in both the absence and presence of IL-2. Exogenous IL-2 did not increase either IFN-gamma production or transferrin receptor (TfR) expression in the presence of calcitriol despite increases in cell entry into late G1 and proliferation. Calcitriol treatment reduced TfR expression by activated T lymphocytes independent of their location in the cell cycle, further suggesting its independence from IL-2-mediated events. Combinations of rIL-2 and rIL-4 did not reverse calcitriol-dependent inhibition of proliferation and TfR expression to any greater degree than rIL-2 alone. Northern blot analysis demonstrated the decrease in IFN-gamma and TfR mRNA accumulation with calcitriol treatment was unaffected by exogenous IL-2. In contrast, IL-2R mRNA and protein were increased by IL-2, with superinduction in the presence of calcitriol, demonstrating that the lack of effect on IFN-gamma and TfR was not due to IL-2 insensitivity. Moreover, equivalent numbers of high-affinity IL-2R were expressed by both control and calcitriol-treated T lymphoblasts. Thus, lectin-activated T lymphocyte responsiveness to IL-2, as measured by IL-2R expression and proliferation, can be partly to completely dissociated from IFN-gamma production and TfR expression in the presence of calcitriol. Finally, IL-2-induced proliferation of unstimulated mononuclear cells and purified T lymphocytes was inhibited by calcitriol. These data indicate that local production of calcitriol by activated macrophages is capable of regulating T lymphocyte activation not only through suppression of IL-2 production, but also through additional mechanism(s), that are mediated at a post-IL-2R level.     

Modulation of interleukin 2 high-affinity binding by lymphocyte-derived tetrahydrobiopterin: pterins as potential participants in the control of interleukin 2 receptor assembly

In this report, we have examined whether (6R)-tetrahydrobiopterin (H4biopterin) modulates the binding of interleukin 2 to high-affinity sites of the cloned mouse cytotoxic T-lymphocyte clone CTLL-2. Scatchard plot analysis of the equilibrium binding data reveals increased affinity when the cells are exposed simultaneously to interleukin 2 and to the pterin. The Kd values are statistically significantly reduced from 1.4 x 10(-11) M to 0.78 x 10(-11) M interleukin 2. The dissociation kinetics of the ligand were followed at 4 degrees C after equilibrium binding under high-affinity conditions (1.2 x 10(-10) M interleukin 2). In the presence of H4 biopterin, the dissociation rate constant (k-1) decreases from 6.2 x 10(-3) min-1 to 3.0 x 10(-3) min-1 and the half-time for dissociation increases from 106.8 min to 218.0 min. As a third approach interleukin 2 was bound to the surface of cells under high-affinity conditions by incubation in the cold and the internalization kinetics upon warming were determined. Sigmoidal-shaped kinetics of endocytosis in control cells indicate that the internalization rates increase only gradually. The presence of H4 biopterin causes an apparent immediate transition from higher-order kinetics to a linear response so that maximum internalization rates are reached immediately upon warming. The data show that lymphocyte-derived H4 biopterin in vitro at concentrations ranging from 2-8 x 10(-7) M modulates interleukin 2 high-affinity binding and that H4 biopterin potentially participates in the control of interleukin 2 receptor assembly.     

Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system

This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple-affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.     

High-affinity interleukin 2 receptor alpha and beta chains are internalized and remain associated inside the cells after interleukin 2 endocytosis.

High-affinity interleukin 2 (IL2) receptors on human T lymphocytes are multimeric complexes containing two IL2-binding polypeptides, alpha and beta chains of 50-55 and 70-75 kDa, respectively, associated by noncovalent bonds. IL2 binds to high-affinity IL2 receptors on the surface of T lymphocytes, mediates cell growth, and is internalized. In this paper, we used a biochemical method to directly identify the receptors components internalized together with the ligand. 125I-IL2-receptor complexes were solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, and IL2-binding polypeptides were identified by cross-linking with disuccinimidyl suberate. Under such conditions, the noncovalent association between alpha and beta is maintained. After IL2 internalization, two complexes of about 70 and 90 kDa, IL2 crosslinked to alpha and beta, respectively, were found inside the cells. Both components were immunoprecipitated with either anti-alpha or anti-beta monoclonal antibodies. This shows that the alpha and beta chains are found in an intracellular compartment after IL2 endocytosis, and remain associated as a ternary complex with IL2.