A1 Peptide Research Articles

Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains.

The mechanism by which cholera toxin (CT) is internalized from the plasma membrane before its intracellular reduction and subsequent activation of adenylyl cyclase is not well understood. Ganglioside GM1, the receptor for CT, is predominantly clustered in detergent-insoluble glycolipid rafts and in caveolae, noncoated, cholesterol-rich invaginations on the plasma membrane. In this study, we used filipin, a sterol-binding agent that disrupts caveolae and caveolae-like structures, to explore their role in the internalization and activation of CT in CaCo-2 human intestinal epithelial cells. When toxin internalization was quantified, only 33% of surface-bound toxin was internalized by filipin-treated cells within 1 h compared with 79% in untreated cells. However, CT activation as determined by its reduction to form the A1 peptide and CT activity as measured by cyclic AMP accumulation were inhibited in filipin-treated cells. Another sterol-binding agent, 2-hydroxy-beta-cyclodextrin, gave comparable results. The cationic amphiphilic drug chlorpromazine, an inhibitor of clathrin-dependent, receptor-mediated endocytosis, however, affected neither CT internalization, activation, nor activity in contrast to its inhibitory effects on diphtheria toxin cytotoxicity. As filipin did not inhibit the latter, the two drugs appeared to distinguish between caveolae- and coated pit-mediated processes. In addition to its effects in CaCo-2 cells that express low levels of caveolin, filipin also inhibited CT activity in human epidermoid carcinoma A431 and Jurkat T lymphoma cells that are, respectively, rich in or lack caveolin. Thus, filipin inhibition correlated more closely with alterations in the biochemical characteristics of CT-bound membranes due to the interactions of filipin with cholesterol rather than with the expressed levels of caveolin and caveolar structure. Our results indicated that the internalization and activation of CT was dependent on and mediated through cholesterol- and glycolipid-rich microdomains at the plasma membrane rather than through a specific morphological structure and that these glycolipid microdomains have the necessary components required to mediate endocytosis.

Intralesional selection of T cell clonotypes in the immune response to melanoma antigens occurring during vaccination.

T cells infiltrating pre- and postvaccine metastases obtained from melanoma patients vaccinated with either dinitrophenyl (DNP)-modified autologous tumor or with the MAGE-3.A1 peptide display selective T cell receptor (TCR) beta chain variable region (BV) repertoire changes at the tumor site as a consequence of vaccination. Restricted sets of BV families expand in all postvaccine lesions when compared with prevaccine specimens and often contain dominant clones. A protocol devised to obtain T cell lines highly enriched for expression of a given BV region through the use of anti-BV monoclonal antibodies was used to understand whether responses to specific antigen(s) accounted for these clonal expansions. In one of the patients vaccinated with DNP-modified tumor cells, BV-driven selection of the T lymphocytes expanded in two infiltrated postvaccine metastases resulted in T cell lines able to exert HLA class I-restricted lysis of the autologous tumor. These results indicate that TCR repertoire analysis at the tumor site facilitates the detection of T cell responses elicited by a vaccine and potentially cytotoxic for the autologous tumor.

Mini-proinsulin and mini-IGF-I: homologous protein sequences encoding non-homologous structures

Protein minimization highlights essential determinants of structure and function. Minimal models of proinsulin and insulin-like growth factor I contain homologous A and B domains as single-chain analogues. Such models (designated mini-proinsulin and mini-IGF-I) have attracted wide interest due to their native foldability but complete absence of biological activity. The crystal structure of mini-proinsulin, determined as a T 3R 3hexamer, is similar to that of the native insulin hexamer. Here, we describe the solution structure of a monomeric mini-proinsulin under physiologic conditions and compare this structure to that of the corresponding two-chain analogue. The two proteins each contain substitutions in the B-chain (His B10→Asp and Pro B28→Asp) designed to destabilize self-association by electrostatic repulsion; the proteins differ by the presence or absence of a peptide bond between Lys B29 and Gly A1. The structures are essentially identical, resembling in each case the T-state crystallographic protomer. Differences are observed near the site of cross-linking: the adjoining A1-A8 α-helix (variable among crystal structures) is less well-ordered in mini-proinsulin than in the two-chain variant. The single-chain analogue is not completely inactive: its affinity for the insulin receptor is 1500-fold lower than that of the two-chain analogue. Moreover, at saturating concentrations mini-proinsulin retains the ability to stimulate lipogenesis in adipocytes (native biological potency). These results suggest that a change in the conformation of insulin, as tethered by the B29–A1 peptide bond, optimizes affinity but is not integral to the mechanism of transmembrane signaling. Surprisingly, the tertiary structure of mini-proinsulin differs from that of mini-IGF-I (main-chain rms deviation 4.5 Å) despite strict conservation of non-polar residues in their respective hydrophobic cores (side-chain rms deviation 4.9 Å). Three-dimensional profile scores suggest that the two structures each provide acceptable templates for threading of insulin-like sequences. Mini-proinsulin and mini-IGF-I thus provide examples of homologous protein sequences encoding non-homologous structures.

Interacting Regions in the A1 and A2 Subunits of Factor VIIIa Identified by Zero-Length Cross-Linking

AbstractFactor VIIIa is a heterotrimer of A1, A2, and A3-C1-C2 subunits, the activity of which is labile due to a weak affinity interaction of the A2 subunit with the A1/A3-C1-C2 dimer. We have used the zero-length cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), to localize regions of interaction within the A1 and A2 subunits. Reaction of factor VIIIa with EDC resulted in the formation of a cross-linked product of approximately 90 kD consisting of the A1 and A2 subunits as judged by Western blotting. Alkaline resistance of this product indicated an amide rather than ester linkage. Factor VIIIa activity decreased as the concentration of cross-linked product increased, suggesting that flexibility in the inter-subunit interaction may be required for proper cofactor function. This product was not formed in the contiguous A1-A2 domains of factor VIII, suggesting that, upon cofactor activation, a conformational change occurs that leads to the formation of a new interdomainal salt bridge(s). Reaction of the EDC-treated factor VIIIa with activated protein C (APC), which cleaves the A1 subunit at Arg336 and bisects the A2 subunit at Arg562, resulted in the formation of an approximately 30 kD product that contains the C-terminus region of A1 covalently linked to the N-terminal half of the A2. The approximately 90 kD cross-linked product was generated after reaction of A2 subunit with A1/A3-C1-C2 dimer but not with A1336/A3-C1-C2, a form of the dimer produced by APC cleavage and lacking the C-terminal acidic region of A1. A synthetic peptide corresponding to this acidic region (Met337-Arg372) was found to covalently cross-link to the isolated A2 subunit in 1:1 stoichiometry, suggesting that this region is both necessary and sufficient for the interaction of the A1 and A2 subunits. Sequence analysis of this product suggested that Glu344 in the A1 peptide may contribute to the cross-linkage. These results indicate that activation of factor VIII results in formation of a new ionic linkage(s) localized to the acidic C-terminal region of A1 and the N-terminal half of A2.

Protein-disulfide Isomerase-mediated Reduction of the A Subunit of Cholera Toxin in a Human Intestinal Cell Line

A key step in the action of cholera toxin (CT) is the reduction of its A subunit to the A1 peptide. The latter is an ADP-ribosyltransferase, which activates the alpha-subunit of the stimulatory G protein of adenylyl cyclase. In this study, the enzymatic reduction of membrane-bound CT in CaCo-2 human intestinal epithelial cells was characterized. Whereas diphtheria toxin was found to be reduced by a cell surface population of protein-disulfide isomerase (PDI) and its cytotoxicity was inhibited by p-chloromercuribenzenesulfonic acid, bacitracin, or anti-PDI antibodies, these inhibitors had no effect on CT reduction or activity in intact cells. In contrast, the reduction of CT in vitro by either postnuclear supernatants (PNS) or microsomal membranes in the presence of Triton X-100 was significantly inhibited by p-chloromercuribenzenesulfonic acid and bacitracin. Anti-PDI monoclonal antibodies likewise inhibited the in vitro reduction of CT and also were effective in depleting reductase activity from PNS. Since inhibition and depletion were not observed in the absence of detergent, these results suggested that the reductase activity was a soluble component localized to the lumen of microsomal vesicles and correlated with the presence of protein-disulfide isomerase. This was further confirmed by showing a corresponding depletion of reductase activity and PDI in alkali-treated microsomes. This activity was restored when purified bovine PDI was added back to alkali-treated microsomes in a redox buffer that reflected conditions found in the lumen of the endoplasmic reticulum (ER). When the CT-related reductase activity was assayed in subcellular fractions of PNS-derived membranes isolated on a 9-30% Iodixanol gradient, the activity, as measured by CT-A1 peptide formation localized to those fractions containing PDI. Likewise CT-A1 peptide formed in intact cells co-localized to those membrane fractions containing the majority of cellular PDI. Furthermore, the banding density corresponded to a region of the gradient containing ER-derived membranes. These results indicated that CT was a substrate for PDI-catalyzed reduction in intact cells and supported the hypothesis that CT reduction and activation occurs in the ER.

Determination of HLA-B7 T-cell epitopes

Abstract A series of peptides de rived from the αl and α2 regions of the HLA-B7 and HLA-A2 mole cules was synthesized with an auto matic peptide synthesizer using the FMOC (9–fluorenylmethoxycarbon-yl) technique. Peptides were ana lyzed and purified by reversed-phase high pressure liquid chroma-tography (HPLC). Peptide purity was > 96%. The effect of B7 pep tides on mixed lymphocyte cultures was tested in vivo. The alloresponse in the cultures with B7 peptides was strongly enhanced. The peptides that were most effective inhibited cytotoxic T-lymphocyte (CTL) cy-tolysis. The data show that the pep tides are immunogenic and that they are recognised by both the direct and indirect pathways. Further, the mechanism of peptide recognition was studied. We coupled peptide B7 (residues 62–70 in HLA-B7) and peptide A2 (residues 62–70 in HLA-B2) covalently to fluorescein isothiocyanate (FITC). B7–specific CTLs were incubated with these peptides at 37 °C for 90 min and cell fluorescence measured by flow cy-tometry. The B7 peptide was bound by 60 % of the CTLs whereas the A2 peptide, as a negative control, bound only 10 %. The molecular size of the ligands to which the peptides bind are being characterized by im-munoprecipitation.

Determination of HLA-B7 T-cell epitopes.

A series of peptides derived from the alpha 1 and alpha 2 regions of the HLA-B7 and HLA-A2 molecules was synthesized with an automatic peptide synthesizer using the FMOC (9-fluorenylmethoxycarbonyl) technique. Peptides were analyzed and purified by reversed-phase high pressure liquid chromatography (HPLC). Peptide purity was > 96%. The effect of B7 peptides on mixed lymphocyte cultures was tested in vivo. The alloresponse in the cultures with B7 peptides was strongly enhanced. The peptides that were most effective inhibited cytotoxic T-lymphocyte (CTL) cytolysis. The data show that the peptides are immunogenic and that they are recognised by both the direct and indirect pathways. Further, the mechanism of peptide recognition was studied. We coupled peptide B7 (residues 62-70 in HLA-B7) and peptide A2 (residues 62-70 in HLA-B2) covalently to fluorescein isothiocyanate (FITC). B7-specific CTLs were incubated with these peptides at 37 degrees C for 90 min and cell fluorescence measured by flow cytometry. The B7 peptide was bound by 60% of the CTLs whereas the A2 peptide, as a negative control, bound only 10%. The molecular size of the ligands to which the peptides bind are being characterized by immunoprecipitation.

Alterations of adenylyl cyclase-linked G proteins in rat liver during aging.

beta-Adrenergic stimulation of adenylyl cyclase in rat liver increases during aging. We examined whether this increase is related to alterations in the stimulatory and inhibitory G proteins (Gs and Gi) linked to adenylyl cyclase. Levels of immunoreactive alpha- and beta-subunits of Ga and Gi in liver plasma membranes from 6-, 12-, 18-, and 24-mo-old rats were unchanged with age, as was pertussis toxin-catalyzed [32P]ADP ribosylation of Gi alpha. Cholera toxin-catalyzed [32P]ADP ribosylation of Ga alpha and Gs bioactivity, assessed as reconstitution of adenylyl cyclase activity in S49 cyc- cell membranes, increased two- to threefold between 6 and 12-18 mo, and declined by 24 mo. Recombinant ADP ribosylation factor (ARF) enhanced cholera toxin labeling of Gs alpha at all ages, yet abolished the increase in toxin labeling at 12-18 mo. Auto-ADP ribosylation of the cholera toxin A1 peptide also increased transiently with age. Alteration of Gs alpha, as reflected by increased cholera toxin labeling and Gs bioactivity, may be involved in the regulation of beta-adrenergic-responsive adenylyl cyclase in rat liver during aging. Moreover, changes in endogenous ARF levels could contribute to age differences in cholera toxin labeling of Gs alpha.

Insertion and orientation of a synthetic peptide representing the C-terminus of the A1 domain of Shiga toxin into phospholipid membranes.

Shiga toxin is a bacterial protein composed of one A and five B subunits. Its A chain possesses a protease sensitive loop (Cys-242-Cys-261) that is cleaved to produce an enzymatically active A1 domain and an A2 fragment associated with its B subunit pentamer. The proposed mode of action of the toxin is linked to its retrograde transport to the ER lumen followed by the translocation of its catalytic A1 chain to the cytoplasmic side of the ER membrane. A signal sequence-like domain (residues 220-246) which constitutes the C-terminus of the A1 chain precedes a region within the protease sensitive loop (residues 247-258) that contains known and putative cleavage sites. Two peptides corresponding to this C-terminus (residues 220-246) were chemically synthesized to investigate if this signal sequence-like domain can interact with membranes. Such a property may provide a clue to the mechanism of translocation of the A1 domain across the ER membrane. The first peptide represented the native sequence, which includes a naturally occurring cysteine at position 242 and provided a thiol moiety for the attachment of a spinlabel. A second peptide was designed to contain a single tryptophan residue (Ile232Trp) located within the hydrophobic core of the sequence which served as an intrinsic fluorescence probe. The interactions of both peptides with lipid vesicles were analyzed by circular dichroism, fluorescence, and EPR spectroscopy. The peptides lack structure in aqueous buffers and adopted an alpha-helical geometry when bound to negatively charged lipid vesicles. The addition of lipid vesicles to a solution of the tryptophan-containing peptide results in a blue shift in the wavelength of its fluorescence maxima as well as an increase in fluorescence intensity at 335 nm, suggesting that the hydrophobic core of this A1 peptide relocated to a nonpolar environment. EPR measurements of a proxyl-labeled analog of the peptide (introduced at Cys-242) indicated a decreased mobility of a fraction of the proxyl probe in the presence of lipid vesicles. At pH 7, the membrane-bound probe was completely reduced by ascorbate trapped inside vesicles but only partially reduced by ascorbate added outside the vesicles, suggesting that the C-terminal region of the peptide traversed the membrane bilayer or relocated close to the surface of its inner lipid leaflet. Finally, the peptide was shown to insert into lipid vesicles, causing the release of calcein at a high peptide:lipid ratio. These results suggest that the C-terminal tail of the A1 chain may anchor this domain into the ER membrane.

Pheromones trigger filamentous growth in Ustilago maydis.

Cell recognition and mating in the smut fungus Ustilago maydis have been proposed to involve specific pheromones and pheromone receptors. The respective structural genes are located in the a mating type locus that exists in the alleles a1 and a2. We demonstrate that binding of pheromone to the receptor can induce a morphological switch from yeast-like to filamentous growth in certain strains. Using this as biological assay we were able to purify both the a1 and a2 pheromone. The structure of the secreted pheromones was determined to be 13 amino acids for a1 and nine amino acids for a2. Both pheromones are post-translationally modified by farnesylation and carboxyl methyl esterification of the C-terminal cysteine. An unmodified a1 peptide exhibits dramatically reduced activity. The pheromone alone is able to induce characteristic conjugation tubes in cells of opposite mating type and confers mating competence; even cells of the same mating type undergo fusion. We discuss the role of pheromones in initiating filamentous growth and pathogenic development.

Evidence that proteolytic separation of Shiga-like toxin type IIv A subunit into A1 and A2 subunits is not required for toxin activity.

The role for proteolytic activation of Shiga-like toxin type II variant (SLT-IIv) A subunit was examined using site-directed mutagenesis. Processing of the enzymatically active A subunit by trypsin results in cleavage at an arginine residue(s) (Arg247 and/or Arg250) located between two cysteines. After reduction of the disulfide bond, the processed A subunit separates into an enzymatically active A1 and an A2 peptide. Substitution mutations were created in SLT-IIv that replaced each or both of the two arginines with either glutamic acid (R247E, R250E, or R247E/R250E) or histidine (R247H, R250H, or R247H/R250H). The products of all glutamic acid substitution mutations were immunoreactive but were not cytotoxic due to an inability to assemble into holotoxin. The products of all histidine substitution mutations had cytotoxic activities, enzymatic activities, and a lethal dose for mice similar to that of native toxin. R247H and R250H were susceptible to proteolytic cleavage while R247H/R250H was resistant to processing by exogenously added trypsin. Native toxin incubated with Vero cells was completely cleaved while only a fraction of R247H/R250H was cleaved. These results demonstrate that cleavage of SLT-IIv can be mediated by proteases with different specificities and suggest efficient cleavage is not required for toxicity.

Orientation of cholera toxin bound to target cells

Cholera toxin (CT) consists of a pentameric B subunit that binds to specific cell surface receptors identified as ganglioside GM1 and an A subunit that activates adenylylcyclase. The A subunit consists of A1 and A2 peptides linked by a disulfide bond; A2 acts to connect A to B, whereas A1 is an ADP-ribosyltransferase that modifies the alpha subunit of the stimulatory G protein (Gs). How the toxin is oriented when it binds to the cell surface and the related issue of the mechanism by which A1 gains access to Gs alpha are not known. In the present study, we used subunit-specific antibodies and their corresponding Fab fragments to assess their affects on holotoxin binding to target cells and their immunoreactivity to cell-bound toxin. Our results suggest that CT binds with A1 facing away from the membrane. Our hypothesis is further supported by the ability to assemble active CT on the cell surface of cultured human intestinal and neurotumor cells by the sequential addition of purified B and A subunits. We also observed that when cells containing bound CT were incubated at 37 degrees C, both subunits rapidly became inaccessible to their respective antibodies. We propose that the holotoxin binds with its A subunit facing away from the membrane and must enter the cell in order for A1 to be released, gain access to Gs alpha, and activate adenylylcyclase.

Brefeldin A blocks the response of cultured cells to cholera toxin. Implications for intracellular trafficking in toxin action

Cholera toxin (CT) consists of a pentameric B subunit which binds to ganglioside GM1 on the cell surface and an A subunit which activates adenylylcyclase. The latter process involves the reduction of A to the A1 peptide which ADP-ribosylates the stimulatory G protein, Gs of adenylylcyclase. There is a distinct lag phase between toxin binding and activation of adenylylcyclase. Little is known about the events during this lag including where A1 is generated and how it gains access to Gs on the cytoplasmic side of the plasma membrane. We explored the effects of several inhibitors of intracellular trafficking on the response of human SK-N-MC neurotumor and Caco-2 intestinal tumor cells to CT. Whereas chloroquine or monensin had little or no effect on CT stimulation of cyclic AMP accumulation, brefeldin A (BFA) totally inhibited the response to CT in a time- and dose-dependent and reversible manner. BFA was effective when added at the same time as CT and had an IC50 of 30 ng/ml. BFA did not alter cell surface GM1 as cells treated with BFA for 30 min bound as much 125I-CT as control cells. Furthermore, BFA inhibited CT stimulation of GM1-treated rat glioma C6 cells. BFA treatment did not affect beta-adrenergic agonist stimulation of cyclic AMP. In addition, adenylylcyclase was activated by A1 peptide and NAD+ to the same extent in membranes from control and BFA-treated cells, or when BFA was added directly to the assay. Whereas control cells generated small amounts of A1 from bound CT with time, no A1 was detected in BFA-treated cells. BFA treatment did not prevent the internalization of CT but did inhibit its degradation. BFA is known to disrupt the organization of the Golgi complex, resulting in inhibition of protein transport from the endoplasmic reticulum and redistribution of Golgi enzymes to the endoplasmic reticulum. BFA also prevents the formation of non-clathrin-coated vesicles from Golgi membranes and thus vesicular transport between Golgi cisternae. We confirmed that BFA caused the morphological disruption of the Golgi apparatus in Caco-2 cells. The data support a role for a functional Golgi apparatus with its associated vesicular routing in CT action.

Selective Activation of Th1‐ and Th2‐like Cells in vivo– Response to Human Collagen IV

Mice immunized with human collagen IV develop either antibody responses or T-cell proliferative responses as a function of the MHC genotype of the immunized mice. CD4+ T cells, similar to Th1 and Th2 cells, participate in these two types of responses, with CD4+ T-cell proliferative responses associating with IL-2 and IFN gamma release, and antibody production associating with CD4+ T-cell IL-4 and IL-5 release. Thus it would appear that the same antigen can induce responses consistent with either cell-mediated or humoral immunity depending on MHC class II genotype. In attempting to understand how MHC genotype controls the class of immunity observed several models are discussed. It was proposed, based on results obtained upon priming with human collagen IV, that the activation of Th1 and Th2 responses may be regulated at several levels (presentation by different APCs, presentation of different densities of the T-cell receptor ligand and presentation of different T-cell epitopes). With the identification of the peptide recognized by the CD4+ T cells, it was clear that the inability to induce Th1 responses in H-2b and the inability to induce Th2 responses in H-2s could not be accounted for by the failure to generate an immunodominant peptide during processing or the failure of the peptides to bind to the MHC class II molecules. Furthermore, the difference in the type of response generated could not be explained by the use of different peptides of the human collagen IV molecule in the two mouse strains, as a single peptide will induce both types of CD4+ T-cell response. However, it cannot be ruled out that the a2 peptide-class II interaction forms different T-cell ligands in the two strains either because the two class II MHC molecules are different or that the peptide is processed and reveals a different antigenic activity (Fox et al. 1988). Perhaps the most important finding from the peptide studies is that the lack of proliferative response in H-2b mice is not absolute, but can be overcome either by priming with high doses of the a2 peptide or by increasing the amount of peptide needed to elicit a recall response. It seems reasonable to speculate that changes in the dose required for priming Th1 or Th2 responses may reflect differences in the activation requirements of the two types of cells with Th1 cells requiring a high ligand density and Th2 cells a low ligand density.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of rat liver adenylate cyclase by cholera toxin requires toxin internalization and processing in endosomes

Involvement of acidic cell compartments in processing and action of cholera toxin (CT) in rat liver has been examined using subcellular fractionation. Liver cell fractions prepared various times after CT injection display, after a lag phase, a progressive increase in adenylate cyclase activity, detectable earlier in Golgi-endosomal fractions (20 min) than in plasma membrane fractions (30 min), with a maximum (3-fold basal activity) achieved by 60-90 min. Endosomes containing in vivo internalized CT display a time-dependent increase in their ability to bind anti-A-subunit antibodies and to stimulate exogenous adenylate cyclase, which kinetically parallels the generation of A1 peptide, suggesting a translocation of A-subunit (or A1 peptide) across the endosomal membrane. In vivo chloroquine treatment inhibits endocytosis of CT taken up into the liver, lengthens the lag phase for adenylate cyclase activation by CT, and reduces by 3- to 10-fold the apparent affinity of the toxin for the enzyme. Incubation of endosomes containing internalized toxin at 37 degrees C under isotonic conditions results in a pH-dependent increase in generation of A1 peptide, membrane translocation of A-subunit (or A1 peptide), and degradation of toxin, with a maximum at pH 5. Addition of ATP, by decreasing the internal endosomal pH, stimulates both generation of the A1 peptide and degradation of toxin at pH 6-8. It is concluded that activation of adenylate cyclase by CT in intact liver requires association and subsequent processing of toxin in an acidic cell compartment, presumably endosomal.

Interactions of serum proteins with small unilamellar liposomes composed of dioleoylphosphatidylethanolamine and oleic acid: high-density lipoprotein, apolipoprotein A1, and amphipathic peptides stabilize liposomes

Small unilamellar liposomes composed to dioleoylphosphatidylethanolamine (DOPE) and oleic acid (OA) are stabilized by incubation with normal human serum or plasma [Liu, D., & Huang, L. (1989) Biochemistry 28, 7700-7707]. The present report describes a systematic study of interactions of purified serum proteins and lipoproteins with these liposomes. Albumin destabilized liposomes by extracting OA from the liposomes, whereas immunoglobulins and lipoproteins (HDL, LDL, and VLDL) had no effect. However, HDL and, to some extent, VLDL showed a rapid stabilization activity against the lytic effect of albumin. HDL added together with or shortly after the addition of albumin completely abolished the liposome leakage and aggregation effects induced by albumin. SDS-PAGE analysis of the HDL-stabilized liposomes revealed that apolipoprotein A1 was associated with liposomes. Purified apolipoprotein A1, but not a lipid mixture resembling the lipid composition of HDL, showed comparable liposome stabilization activity as HDL. Furthermore, synthetic peptides resembling the amphipathic helices found in apolipoprotein A1 also showed strong liposome stabilization activity. Peptides which were able to form amphipathic helixes of a wedge shape were more effective stabilizers than those which could not. These data indicate that HDL plays a major role in human serum or plasma for the liposome stabilization activity. HDL exerts its activity probably by the interactions of the amphipathic helices of apolipoprotein A1 with the hydrophobic voids found on the outer surface of the highly curved, small liposomes.

Stimulation of thromboxane release by extracellular UTP and ATP from perfused rat liver. Role of icosanoids in mediating the nucleotide responses.

1. In isolated perfused rat liver, infusion of UTP (20 microM) led to a transient, about sevenfold stimulation of thromboxane release (determined as thromboxane B2), which did not parallel the time course of the UTP-induced stimulation of glucose release. An increased thromboxane release was also observed after infusion of ATP (20 microM). Although the maximal increase of portal pressure following ATP was much smaller than with UTP (4.2 vs 11.5 cm H2O), the peak thromboxane release was similar with both nucleotides. 2. Indomethacin (10 microM) inhibited the UTP-induced stimulation of thromboxane release and decreased the UTP-induced maximal increase of glucose output and of portal pressure by about 30%. The thromboxane A2 receptor antagonist BM 13.177 (20 microM) completely blocked the pressure and glucose response to the thromboxane A2 analogue U-46619 (200 nM) and decreased the ATP- and UTP-induced stimulation of glucose output by about 25%, whereas the maximal increase of portal pressure was inhibited by about 50% and 30%, respectively. BM 13.177 and indomethacin inhibited the initial nucleotide-induced overshoot of portal pressure increase, but had no effect on the steady-state pressure increase which is obtained about 5 min after addition of ATP or UTP. 3. The leukotriene D4/E4 receptor antagonist LY 171883 (50 microM) inhibited not only the glucose and pressure response of perfused rat liver to leukotriene D4, but also to leukotriene C4 by about 90%. This suggests that leukotriene D4 (not C4) is the active metabolite in perfused liver and the effects of leukotriene C4 are probably due to its rapid conversion to leukotriene D4. LY 171883 also inhibited the response to the thromboxane A2 analogue U-46619 by 75-80%, whereas the response of perfused liver to leukotriene C4 was not affected by the thromboxane receptor antagonist BM 13.177 (20 microM). The glucose and pressure responses of the liver to extracellular UTP were inhibited by LY 171883 and by BM 13.177 by about 30%. This suggests that the inhibitory action of LY 171883 was due to a thromboxane receptor antagonistic side-effect and that peptide leukotrienes do not play a major role in mediating the UTP response. 4. In isolated rat hepatocytes extracellular UTP (20 microM), ATP (20 microM), cyclic AMP (50 microM) and prostaglandin F2 alpha (3 microM) increased glycogen phosphorylase a activity by more than 100%.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of nicking on Shiga-like toxin I of enterohaemorrhagic Escherichia coli

The Shiga-like toxin I (SLT-I), which had an unnicked A subunit, was purified from Escherichia coli O157: H7. After incubation of the unnicked SLT-I with trypsin, the A subunit of the toxin was nicked and converted to A1 and A2 peptides, which were linked by the disulfide bond. The effects of nicking of SLT-I on its heat stability and cytotoxic activity against Vero cells were examined. The trypsin treatment of SLT-I neither reduced nor increased the ability of cytotoxic activity or the heat stability of the toxin. The time course of binding of the toxin to Vero cells also was not affected by nicking of the toxin. These results indicate the absence of a difference in biological or physical properties of SLTs between nicked and unnicked conditions.

Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action.

The major steps in cholera-toxin action, i.e. binding, internalization, generation of A1 peptide and activation of adenylate cyclase, were examined in isolated hepatocytes. The binding of toxin involves a single class of high-affinity sites (KD congruent to 0.1 nM; Bmax. congruent to 10(7) sites/cell). At 37 degrees C, cell-associated toxin is progressively internalized, as judged by the loss of its accessibility to antibodies against whole toxin, A and B subunits (about 50, 75 and 30% of initially bound toxin after 40 min respectively). Two distinct pathways are involved in this process: endocytosis of the whole toxin, and selective penetration of the A subunit into the plasma membrane. Exposure of hepatocytes to an acidic medium (pH 5) results in a rapid and marked disappearance of the A subunit from the cell surface. Generation of A1 peptide and activation of adenylate cyclase by the toxin occur after a lag phase (10 min at 37 degrees C), and increase with time in a parallel manner up to 2-3% A1 peptide generated; they are unaffected by exposure of cells to an acidic medium. Chloroquine and monensin, which elevate the pH in acidic organelles, inhibit by 2-4-fold both the generation of A1 peptide and the activation of adenylate cyclase. Unexpectedly, these drugs also inhibit the internalization of the toxin. These results suggest that an acidic pH facilitates the penetration of A subunit into the plasma membrane and presumably the endosomal membrane as well, and that endocytosis of cholera toxin is required for generation of A1 peptide and activation of adenylate cyclase.

Lecithin:cholesterol acyltransferase activation by synthetic amphipathic peptides.

The amphipathic helical theory of Segrest and colleagues (FEBS Lett.:38:247-253, 1974) proposes that the lipid-binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30-residue peptide, GALA, which in the alpha helical conformation has a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26:2964-2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphosphatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC-GALA:19/1 (molar ratio) complex can be isolated by gel-permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44-A edge thickness and a 170- to 350-A diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100-fold larger than into egg phosphatidylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1-like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.