A conserved residue in the tip proteins of CS1 and CFA/I pili of enterotoxigenic Escherichia coli that is essential for adherence

Enterotoxigenic Escherichia coli associated with human diarrheal disease utilize any of a limited group of serologically distinguishable pili for attachment to intestinal cells. These include CS1 and CFA/I pili. We show here that chemical modification of arginyl residues in CS1 pili abolishes CS1-mediated agglutination of bovine erythrocytes, which serves as a model system for attachment. Alanine substitution of the single arginyl residue in CooA, the major pilin, had no effect on the assembly of pili or on hemagglutination. In contrast, substitution of alanine for R181 in CooD, the minor pilin associated with the pilus tip, abolished hemagglutination, and substitution of R20 reduced hemagglutination. Neither of these substitutions affected CS1 pilus assembly. This shows that CooD is essential for CS1-mediated attachment and identifies specific residues that are involved in receptor binding but not in pilus assembly. In addition to mediating agglutination of bovine erythrocytes, CFA/I also mediates agglutination of human erythrocytes. Substitution of R181 by alanine in the CooD homolog, CfaE, abolished both of these reactions. We conclude that the same region of the pilus tip protein is involved in adherence of CS1 and CFA/I pili, although their receptor specificities differ. This suggests that the region of the pilus tip adhesin protein that includes R181 might be an appropriate target for therapeutic intervention or for a vaccine to protect against human diarrhea caused by enterotoxigenic E. coli strains that have serologically different pili.

Search for the pathogenesis of the differing phenotype in two compound heterozygote Hungarian brothers with the same genotypic triosephosphate isomerase deficiency

In a Hungarian family with triosephosphate isomerase (TPI) deficiency, two compound heterozygote brothers were found with the same severe decrease in TPI activity, but only one of them had the classical symptoms. In search for the pathogenesis of the differing phenotype of the same genotypic TPI deficiency, an increase in red cell membrane fluidity was found. There were roughly 100% and 30% more 16:0/20:4 and 18:0/20:4 diacyl-phosphatidylcholine species in erythrocytes from the two TPI-deficient brothers than in the probes from healthy controls. The activities of acethylcholinesterase and calmodulin induced Ca2+ ATPase were significantly enhanced in erythrocytes from the propositus as compared with those of the neurologically symptom-free brother and other members of the TPI-deficient family as well as to those from healthy controls. Both enzymes are crucially involved in the function of nerve cells. The observed differences in membrane fluidity and enzyme activities between the erythrocytes from the phenotypically differing TPI-deficient brothers underline the importance of investigations into the effect of biophysical changes in the lipid environment of the membrane proteins on the development of disseminated focal neurological disorders of unknown pathogenic origin.

Transmembrane β-barrel of staphylococcal α-toxin forms in sensitive but not in resistant cells

Staphylococcal α-toxin is a 293-residue, single-chain polypeptide that spontaneously assembles into a heptameric pore in target cell membranes. To identify the pore-forming domain, substitution mutants have been produced in which single cysteine residues were introduced throughout the toxin molecule. By attaching the environmentally sensitive dye acrylodan to the sulfhydryl groups, the environment of individual amino acid side chains could be probed. In liposomes, a single 23-amino acid sequence (residues 118–140) was found to move from a polar to a nonpolar environment, indicating that this sequence forms the walls of the pore. However, periodicity in side chain environmental polarity could not be detected in the liposomal system. In the present study, the fluorimetric analyses were extended to physiological target cells. With susceptible cells such as rabbit erythrocytes and human lymphocytes, the 23 central amino acids 118–140 were again found to insert into the membrane; in contrast to the previous study with liposomes, the expected periodicity was now detected. Thus, every other residue in the sequence 126–140 entered a nonpolar environment in a striking display of an amphipathic transmembrane β-barrel. In contrast, human granulocytes were found to bind α-toxin to a similar extent as lymphocytes, but the heptamers forming on these cells failed to insert their pore-forming domain into the membrane. As a consequence, nonfunctional heptamers assembled and the cells remained viable. The data resolve the molecular organization of a pore-forming toxin domain in living cells and reveal that resistant cells can prevent insertion of the functional domain into the bilayer.

Temporal mapping of gene expression levels during the differentiation of individual primary hematopoietic cells

A hierarchical order of gene expression has been proposed to control developmental events in hematopoiesis, but direct demonstration of the temporal relationships between regulatory gene expression and differentiation has been difficult to achieve. We modified a single-cell PCR method to detect 2-fold changes in mRNA copies per cell (dynamic range, 250–250,000 copies/cell) and used it to sequentially quantitate gene expression levels as single primitive (CD34+,CD38−) progenitor cells underwent differentiation to become erythrocytes, granulocytes, or monocyte/macrophages. Markers of differentiation such as CD34 or cytokine receptor mRNAs and transcription factors associated with their regulation were assessed. All transcription factors tested were expressed in multipotent progenitors. During lineage-specific differentiation, however, distinct patterns of expression emerged. SCL, GATA-2, and GATA-1 expression sequentially extinguished during erythroid differentiation. PU.1, AML1B, and C/EBPα expression profiles and their relationship to cytokine receptor expression in maturing granulocytes could be distinguished from similar profiles in monocytic cells. These data characterize the dynamics of gene expression accompanying blood cell development and define a signature gene expression pattern for specific stages of hematopoietic differentiation.

Mapping regions containing binding residues within functional domains of Plasmodium vivax and Plasmodium knowlesi erythrocyte-binding proteins

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Whereas Plasmodium vivax and Plasmodium knowlesi use the Duffy blood group antigen, Plasmodium falciparum uses sialic acid residues of glycophorin A as receptors to invade human erythrocytes. P. knowlesi uses the Duffy antigen as well as other receptors to invade rhesus erythrocytes by multiple pathways. Parasite ligands that bind these receptors belong to a family of erythrocyte-binding proteins (EBP). The EBP family includes the P. vivax and P. knowlesi Duffy-binding proteins, P. knowlesi β and γ proteins, which bind alternate receptors on rhesus erythrocytes, and P. falciparum erythrocyte-binding antigen (EBA-175), which binds sialic acid residues of human glycophorin A. Binding domains of each EBP lie in a conserved N-terminal cysteine-rich region, region II, which contains around 330 amino acids with 12 to 14 conserved cysteines. Regions containing binding residues have now been mapped within P. vivax and P. knowlesi β region II. Chimeric domains containing P. vivax region II sequences fused to P. knowlesi β region II sequences were expressed on the surface of COS cells and tested for binding to erythrocytes. Binding residues of P. vivax region II lie in a 170-aa stretch between cysteines 4 and 7, and binding residues of P. knowlesi β region II lie in a 53-aa stretch between cysteines 4 and 5. Mapping regions responsible for receptor recognition is an important step toward understanding the structural basis for the interaction of these parasite ligands with host receptors.

Cell membrane fluctuations are regulated by medium macroviscosity: Evidence for a metabolic driving force

Extracellular fluid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body fluids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical fluctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase fluid viscosity. The parameters of cell membrane fluctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane fluctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATP-depleted red blood cells elevation of EMF did not affect cell membrane fluctuations. This study demonstrates that (i) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (ii) cell membrane fluctuations are driven by a metabolic driving force in addition to the thermal one.

v-K-ras leads to preferential farnesylation of p21ras in FRTL-5 cells: Multiple interference with the isoprenoid pathway

The isoprenoid pathway in FRTL-5 thyroid cells was found to be deeply altered on transformation with v-K-ras. A dramatic overall reduction of protein prenylation was found in v-K-ras-transformed cells in comparison with the parent FRTL-5 cells, as shown by labeling cells with [3H]mevalonic acid. This phenomenon was accompanied by a relative increase of p21ras farnesylation and by a decrease of the ratio between the amounts of geranylgeraniol and farnesol bound to prenylated proteins. Analysis of protein prenylation in FRTL-5 cells transformed by a temperature-sensitive mutant of the v-K-ras oncogene indicated that these variations represent an early and specific marker of active K-ras. Conversely, FRTL-5 cells transformed with Harvey-ras showed a pattern of [3H]-mevalonate (MVA)-labeled proteins similar to that of nontransformed cells. The K-ras oncogene activation also resulted in an overall decrease of [3H]-MVA incorporation into isopentenyl-tRNA together with an increase of unprocessed [3H]-MVA and no alteration in [3H]-MVA uptake. The effects of v-K-ras on protein prenylation could be mimicked in FRTL-5 cells by lowering the concentration of exogenous [3H]-MVA whereas increasing the [3H]-MVA concentration did not revert the alterations observed in transformed cells. Accordingly, v-K-ras expression was found to: (i) down-regulate mevalonate kinase; (ii) induce farnesyl-pyrophosphate synthase expression; and (iii) augment protein farnesyltransferase but not protein geranylgeranyl-transferase-I activity. Among these events, mevalonate kinase down-regulation appeared to be related strictly to differential protein prenylation. This study represents an example of how expression of the v-K-ras oncogene, through multiple interferences with the isoprenoid metabolic pathway, may result in the preferential farnesylation of the ras oncogene product p21ras.

DNA Replication in Quiescent Cell Nuclei: Regulation by the Nuclear Envelope and Chromatin Structure

Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H10 are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.

Identification of a Plasmodium falciparum intercellular adhesion molecule-1 binding domain: A parasite adhesion trait implicated in cerebral malaria

Binding of infected erythrocytes to brain venules is a central pathogenic event in the lethal malaria disease complication, cerebral malaria. The only parasite adhesion trait linked to cerebral sequestration is binding to intercellular adhesion molecule-1 (ICAM-1). In this report, we show that Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) binds ICAM-1. We have cloned and expressed PfEMP1 recombinant proteins from the A4tres parasite. Using heterologous expression in mammalian cells, the minimal ICAM-1 binding domain was a complex domain consisting of the second Duffy binding-like (DBL) domain and the C2 domain. Constructs that contained either domain alone did not bind ICAM-1. Based on phylogenetic criteria, there are five distinct PfEMP1 DBL types designated α, β, γ, δ, and ɛ. The DBL domain from the A4tres that binds ICAM-1 is DBLβ type. A PfEMP1 cloned from a distinct ICAM-1 binding variant, the A4 parasite, contains a DBLβ domain and a C2 domain in tandem arrangement similar to the A4tres PfEMP1. Anti-PfEMP1 antisera implicate the DBLβ domain from A4var PfEMP1 in ICAM-1 adhesion. The identification of a P. falciparum ICAM-1 binding domain may clarify mechanisms responsible for the pathogenesis of cerebral malaria and lead to interventions or vaccines that reduce malarial disease.

Efficient IgG-mediated suppression of primary antibody responses in Fcγ receptor-deficient mice

IgG antibodies can suppress more than 99% of the antibody response against the antigen to which they bind. This is used clinically to prevent rhesus-negative (Rh−) women from becoming immunized against Rh+ erythrocytes from their fetuses. The suppressive mechanism is poorly understood, but it has been proposed that IgG/erythrocyte complexes bind to the inhibitory Fc receptor for IgG (FcγRIIB) on the B cell surface, thereby triggering negative signals that turn off the B cell. We show that IgG induces the same degree of suppression of the response to sheep erythrocytes in animals lacking the known IgG-binding receptors FcγRIIB, FcγRI + III, FcγRI + IIB + III, and FcRn (the neonatal Fc receptor) as in wild-type animals. Reinvestigation of the ability of F(ab′)2 fragments to suppress antibody responses demonstrated that they were nearly as efficient as intact IgG. In addition, monoclonal IgE also was shown to be suppressive. These findings suggest that IgG inhibits antibody responses through Fc-independent mechanisms, most likely by masking of antigenic epitopes, thereby preventing B cells from binding and responding to antigen. In agreement with this, we show that T cell priming is not abolished by passively administered IgG. The results have implications for the understanding of in vivo regulation of antibody responses and Rh prophylaxis.

A host–guest system to study structure–function relationships of membrane fusion peptides

We designed a host–guest fusion peptide system, which is completely soluble in water and has a high affinity for biological and lipid model membranes. The guest sequences are those of the fusion peptides of influenza hemagglutinin, which are solubilized by a highly charged unstructured C-terminal host sequence. These peptides partition to the surface of negatively charged liposomes or erythrocytes and elicit membrane fusion or hemolysis. They undergo a conformational change from random coil to an obliquely inserted (≈33° from the surface) α-helix on binding to model membranes. Partition coefficients for membrane insertion were measured for influenza fusion peptides of increasing lengths (n = 8, 13, 16, and 20). The hydrophobic contribution to the free energy of binding of the 20-residue fusion peptide at pH 5.0 is −7.6 kcal/mol (1 cal = 4.18 J). This energy is sufficient to stabilize a “stalk” intermediate if a typical number of fusion peptides assemble at the site of membrane fusion. The fusion activity of the fusion peptides increases with each increment in length, and this increase strictly correlates with the hydrophobic binding energy and the angle of insertion.

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inflection point energy for cysteine and cystine are observed. For methionine sulfoxide the inflection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfur-containing components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.

Mutated plant lectin library useful to identify different cells

The 24 nucleotides comprising the carbohydrate-recognition domain of Maackia amurensis hemagglutinin (MAH) cDNA were randomly mutated. The mutant lectins were expressed as glutathione-S-transferase fusion proteins in Escherichia coli and 16 clones were randomly chosen. Although all of 16 recombinant lectins reacted strongly with anti-MAH polyclonal antibody, the carbohydrate-recognition domain of each was unique. As shown by agglutination studies, each mutant MAH lectin was able to bind to erythrocytes from one or more of five animal species in very distinct patterns. Thus, novel plant lectin libraries can be used to discriminate in a highly specific manner among a variety of cell types. This technology may prove to be very useful in a number of different applications requiring a high level of specificity in cell identification.

The surface variant antigens of Plasmodium falciparum contain cross-reactive epitopes

Plasmodium falciparum parasites evade the host immune system by clonal expression of the variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Antibodies to PfEMP1 correlate with development of clinical immunity but are predominantly variant-specific. To overcome this major limitation for vaccine development, we set out to identify cross-reactive epitopes on the surface of parasitized erythrocytes (PEs). We prepared mAbs to the cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 that is functionally conserved for binding to CD36. Two mAbs, targeting different regions of CIDR1, reacted with multiple P. falciparum strains expressing variant PfEMP1s. One of these mAbs, mAb 6A2-B1, recognized nine of 10 strains tested, failing to react with only one strain that does not bind CD36. Flow cytometry with Chinese hamster ovary cells expressing variant CIDR1s demonstrated that both mAbs recognized the CIDR1 of various CD36-binding PfEMP1s and are truly cross-reactive. The demonstration of cross-reactive epitopes on the PE surface provides further credence for development of effective vaccines against the variant antigen on the surface of P. falciparum-infected erythrocytes.

Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175

A member of a Plasmodium receptor family for erythrocyte invasion was identified on chromosome 13 from the Plasmodium falciparum genome sequence of the Sanger Centre (Cambridge, U.K.). The protein (named BAEBL) has homology to EBA-175, a P. falciparum receptor that binds specifically to sialic acid and the peptide backbone of glycophorin A on erythrocytes. Both EBA-175 and BAEBL localize to the micronemes, organelles at the invasive ends of the parasites that contain other members of the family. Like EBA-175, the erythrocyte receptor for BAEBL is destroyed by neuraminidase and trypsin, indicating that the erythrocyte receptor is a sialoglycoprotein. Its specificity, however, differs from that of EBA-175 in that BAEBL can bind to erythrocytes that lack glycophorin A, the receptor for EBA-175. It has reduced binding to erythrocytes with the Gerbich mutation found in another erythrocyte, sialoglycoprotein (glycophorin C/D). The interest in BAEBL's reduced binding to Gerbich erythrocytes derives from the high frequency of the Gerbich phenotype in some regions of Papua New Guinea where P. falciparum is hyperendemic.