Interaction of human erythrocyte multicatalytic proteinase with polycations

The multicatalytic proteinase from human erythrocytes (macropain, proteasome) is a large enzyme composed of at least six distinct subunits ranging in molecular masses from 20 to 30 kDa. As its name implies, this proteinase appears to contain multiple catalytic sites with differing specificities toward peptide substrates. Several polycationic substances, including polylysines, polyarginine, protamine and histone H1 markedly stimulated caseinolytic activity of the proteinase. Activation was instantaneous, and involved increasing the Vmax of the proteinase for casein. Prolonged preincubation with polylysine at 37 degrees C resulted in autolytic inactivation of the proteinase. The polylysine concentrations required for half-maximal activation or autolytic inactivation were the same. A 23 kDa subunit of the proteinase disappeared at the same rate as loss of catalytic activity, and with the same pH dependence and polylysine concentration dependence. These results suggest that polylysine perturbs the structure of the multicatalytic proteinase, resulting in increased catalytic activity toward substrates; and, with prolonged exposure, allowing autoproteolytic inactivation to occur. The 23 kDa subunit appeared to be required for expression of caseinolytic activity, and may therefore be a catalytic subunit of the complex having activity against casein.     

Turnover of cell-surface macromolecules in cultured dog tracheal epithelial cells

We studied the metabolism of sulfated cell-surface macromolecules in dog tracheal epithelial cells in primary culture. To examine the time-course and rate of appearance of sulfated macromolecules at the cell surface, the cells were pulsed with 35SO4 for short periods (5-15 min), and the incubation medium was sampled for spontaneously released macromolecules (basal secretions) and for release induced by trypsin (trypsin-accessible secretions). Trypsin-accessible 35S-labeled macromolecules appeared on the cell surface within 5-10 min, increased linearly, and plateaued by 40 min; the median transit time for 35S-labeled macromolecules to reach the cell surface was 21 min. 35S-labeled macromolecules in basal secretions increased with a similar time-course, reaching a plateau by 40 min. Incorporation of [3H]serine into the protein moiety of trypsin-accessible macromolecules occurred more slowly; trypsin-accessible 3H-labeled macromolecules were barely detectable at 1 h and increased to a maximum after 2 h, suggesting the presence of a preformed pool of nonsulfated core protein. Pretreatment with cycloheximide, an inhibitor of protein synthesis, decreased trypsin-accessible 35S-labeled macromolecules log-linearly depending on the duration of pretreatment providing an estimate of the rate of depletion of the core protein pool (t1/2 = 32 min). During continuous exposure to 35SO4, 35S-labeled macromolecules accumulated on the cell surface (trypsin-accessible compartment) for 16 h, at which point the cell-surface pool was saturated (t1/2 = 7.5 h). After pulse-labeling the cells with 35SO4 for 15 min, the 35S-labeled macromolecules disappeared continuously from the cell surface (t1/2 = 4.6 h), and 79% of the radioactivity was recovered in the medium as nondialyzable macromolecules. Release of the 35S-labeled macromolecules from the cell surface was abolished at 4 degrees C, indicative of an energy-dependent process, but multiple proteinase inhibitors did not affect the release. We conclude that sulfate is metabolized rapidly into epithelial cell-surface macromolecules, which accumulate continuously into a relatively large cell-surface pool, before they are released by an undefined energy-dependent mechanism.     

Interaction of enteropathogenicEscherichia coli with host epithelial cells

EnteropathogenicEscherichia coli (EPEC) causes severe diarrhea in young children. Upon infection, EPEC induces the assembly of highly organized pedestal-like actin structures in host epithelial cells. All the EPEC genes that are involved in inducing formation of actin pedestals are located in a unique 35 kbp chromosomal pathogenicity island, termed LEE. These genes include thesep genes that encode components of type III protein secretion system, and genes that encode proteins secreted by this system, theesp genes. This protein secretion system is activated upon contact with the host cell, resulting in increased secretion of Esp proteins. Some of these Esp proteins form the translocation apparatus while others are translocated into the cytoplasm of the host cell. Concerted activity of the LEE genes including theeae, esp and thesep genes is needed to trigger signal transduction in the host cell which results in formation of an actin pedestal. Presented at the1st International Minisymposium on Cellular Microbiology: Cell Biology and Signalization in Host-Pathogen Interactions, Prague, October 6, 1997.     

Interaction of Arcobacter spp. with human and porcine intestinal epithelial cells

Little is known about the pathogenic mechanisms or potential virulence factors of Arcobacter spp. The aim of the study described here was to obtain more insights in the pathogenicity mechanisms of Arcobacter spp. by testing their ability to adhere to, invade and induce interleukin-8 expression in human Caco-2 and porcine IPI-2I cell lines. Eight Arcobacter strains were tested. Four strains were obtained from a culture collection, and represent the four Arcobacter spp. known to be associated with animals and humans. The other four strains were field isolates from the amniotic fluid of sows and from newborn piglets. All eight Arcobacter strains were able to adhere to both cell lines, and induced interleukin-8 production as early as 2 h after a 1h incubation period. This production was still increased 6 h postinfection. Differences in the cell association of the eight strains were obvious, with A. cibarius showing the highest adhesion ability. Invasion of intestinal epithelial cells was only observed for A. cryaerophilus strains. No correlation between invasiveness or strong adhesion of the tested strains and the level of interleukin-8 induction was observed.     

Interaction of bacterial lipopolysaccharides with host soluble proteins and polycations

Lipopolysaccharides (LPS) are unique cell wall components of gram-negative bacteria. They represent amphiphilic biopolymeric compounds combining in a single molecule hydrophilic (O-specific chains, core oligosaccharide, etc.) and hydrophobic (lipid A) entities. LPS play a crucial role in various interactions between micro- and macroorganisms and display a broad range of biological activities including toxic activity and ability to activate immune cells. Biological activities of LPS are based on their ability to bind with high affinity to mammalian proteins, e.g., lipoproteins, bactericidal permeability-increasing proteins, lysozyme, etc., and thus to neutralize toxic effects of endotoxins. LPS are specific targets for antimicrobial polycationic compounds used in the therapy of bacterial infections. Studies of mechanisms of toxic effects of LPS culminated in the development of novel approaches to LPS neutralization. One of them is based on the use of compounds able to neutralize LPS toxicity at the expense of formation of macromolecular complexes with them. This approach is highly specific and has no effect on functional activity of antipathogenic defense mechanisms of the host. Interaction of LPS with various classes of cationic amphiphilic molecules including proteins, peptides, and polyamines was the subject of intensive studies in the past decade. Binding of cationic polymers is provided by electrostatic interactions between LPS and negatively charged phosphate and carboxylic groups of LPS localized in lipid A core. The present study is an overview of recently published data on different mechanisms of interactions of LPS with soluble proteins and polycations and modification of physiological activity of LPS.     

Aggregation of sponge cells: 23. Interaction of Geodia lectin with Tethya cell-surface glycoprotein

A macromolecule has been isolated from the cell membranes of the sponge Tethyalyncurium. This macromolecule was purified and found to bind to a d-galactose specific lectin from the sponge Geodia cydonium. The lectin receptor was characterized as a glycoprotein with a molecular weight in the region of 155 000. Evidence is presented indicating that the binding of lectin with the lectin receptor is caused by hydrophobic interactions.     

Cell-density-dependent changes in cell-surface glycopeptides and in adhesion of cultured intestinal epithelial cells

We studied mannose-containing glycopeptides and glycoproteins of subconfluent and confluent intestinal epithelial cells in culture. Cells were labelled with d-[2-³H]mannose for 24h and treated with Pronase or trypsin to release cell-surface components. The cell-surface and cell-residue fractions were then exhaustively digested with Pronase and the resulting glycopeptides were fractionated on Bio-Gel P-6, before and after treatment with endo-β-N-acetylglucosaminidase H to distinguish between high-mannose and complex oligosaccharides. The cell-surface glycopeptides were enriched in complex oligosaccharides as compared with residue glycopeptides, which contained predominantly high-mannose oligosaccharides. Cell-surface glycopeptides of confluent cells contained a much higher proportion of complex oligosaccharides than did glycopeptides from subconfluent cells. The ability of the cells to bind [³H]concanavalin A decreased linearly with increasing cell density up to 5 days in culture and then remained constant. When growth of the cells was completely inhibited by either retinoic acid or cortisol, no significant difference was observed in the ratio of complex to high-mannose oligosaccharides in the cell-surface glycopeptides of subconfluent cells. Only minor differences were found in total mannose-labelled glycoproteins between subconfluent and confluent cells by two-dimensional gel analysis. The adhesion of the cells to the substratum was measured at different stages of growth and cell density. Subconfluent cells displayed a relatively weak adhesion, which markedly increased with increased cell density up to 6 days in culture. It is suggested that alterations in the structure of the carbohydrates of the cell-surface glycoproteins are dependent on cell density rather than on cell growth. These changes in the glycopeptides are correlated with the changes in adhesion of the cells to the substratum.     

Correlation between cell-surface hydrophobicity of Candida albicans and adhesion to buccal epithelial cells

Adhesion of four isolates of Candida albicans to buccal epithelial cells was determined after growth of the yeasts in defined medium containing 50 mM glucose or 500 mM galactose as the carbon source. With each isolate, adhesion of galactose-grown yeasts was significantly higher than that of glucose-grown organisms. Yeast cell-surface hydrophobicity was assessed by two methods, a modified hydrocarbon adhesion assay and a more sensitive polystyrene microsphere assay. All four isolates were significantly more hydrophobic after growth on 500 mM galactose than after growth on 50 mM glucose. Overall, a strong positive correlation between adhesion and surface hydrophobicity was observed (r = 0.965). These results are discussed in relation to the role of yeast-surface hydrophobicity in pathogenesis.     

Interaction between Erbin and a Catenin-related protein in epithelial cells.

Integrity of epithelial tissues relies on the proper apical-basolateral polarity of epithelial cells. Members of the LAP (LRR and PDZ) protein family such as LET-413 and Scribble are involved in maintaining epithelial cell polarity in Caenorhabditis elegans and Drosophila melanogaster, respectively. We previously described Erbin as a mammalian LET-413 homologue interacting with ERBB2/HER2, an epidermal growth factor receptor family member. Erbin and ERBB2/HER2 are located in the basolateral membranes of epithelial cells. We show here that Erbin interacts with p0071 (also called plakophilin-4), an armadillo repeat protein linked to the cytoskeleton. Erbin binds to p0071 in vitro and in vivo in a PDZ domain-dependent manner, and both proteins colocalized in desmosomes of epithelial cells. Using a dominant negative approach, we found that integrity of epithelial cell monolayer is impaired when interaction between Erbin and p0071 is disrupted. We propose that Erbin is connected by p0071 to cytoskeletal networks in an interaction crucial for epithelial homeostasis.     

Interaction of streptavidin-based peptide-MHC oligomers (tetramers) with cell-surface TCRs

The binding of oligomeric peptide-MHC (pMHC) complexes to cell surface TCR can be considered to approximate TCR-pMHC interactions at cell-cell interfaces. In this study, we analyzed the equilibrium binding of streptavidin-based pMHC oligomers (tetramers) and their dissociation kinetics from CD8(pos) T cells from 2C-TCR transgenic mice and from T cell hybridomas that expressed the 2C TCR or a high-affinity mutant (m33) of this TCR. Our results show that the tetramers did not come close to saturating cell-surface TCR (binding only 10-30% of cell-surface receptors), as is generally assumed in deriving affinity values (K(D)), in part because of dissociative losses from tetramer-stained cells. Guided by a kinetic model, the oligomer dissociation rate and equilibrium constants were seen to depend not only on monovalent association and dissociation rates (k(off) and k(on)), but also on a multivalent association rate (μ) and TCR cell-surface density. Our results suggest that dissociation rates could account for the recently described surprisingly high frequency of tetramer-negative, functionally competent T cells in some T cell responses.     

DNA complexes with polycations used for delivery of DNA into cells

The formation and physicochemical properties of high-molecular thymus and plasmid DNA complexes with synthetic polymers based on (dimethyl-amino)ethyl methacrylate (DMAEM), (diethyl-amino)ethyl methacrylate (DEAEM), and polyvinyl amine (PVA) were investigated in solutions of different ionic strength by low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The toxicity of complexes in T98G cells was studied. It was shown that, when the ratio of polycations to DNA charged groups concentration (N+/P) reaches values > 1, DNA condensation occurs. It is accompanied by increasing optical density of solutions. Changes in DNA size after condensation were estimated. Phase diagrams of systems DNA/polycation in the presence of NaCl were obtained. It was shown by MTT-analysis that DNA complexes with polycations in the range of concentrations used have low toxicity.     

Interaction of syntaxins with the amiloride-sensitive epithelial sodium channel.

Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na(+) entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (alpha, beta, gamma subunits) with syntaxin 1A or 3 cRNAs in Xenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving protein-protein interactions.     

Changes in cell-surface fucose-containing glycopeptides and adhesion of cultured intestinal epithelial cells as a function of cell density.

Confluent cultured intestinal epithelial cells displayed greater adhesion to the substratum than did subconfluent cells. Subconfluent and confluent cells were labelled with [3H]fucose for 24h and the cell-surface components were released by mild Pronase treatment. After extensive Pronase digestion, cell-surface and cell-residue glycopeptides were fractionated on Bio-Gel P-6. The cell surface contained a higher proportion of lower-molecular-weight glycopeptides than the residue. No significant difference in elution pattern was found between total cell-surface glycopeptides of subconfluent and confluent cells. However, confluent cells contained almost twice as much [3H]-fucose-labelled glycopeptides that were bound to concanavalin A-Sepharose and were subsequently eluted with 20mM-methyl alpha-D-glucopyranoside as subconfluent cells. When the bound glycopeptides were chromatographed on Bio-Gel P-6, it was found that confluent cells contained a larger proportion of lower-molecular-weight glycopeptides than subconfluent cells. This difference in size was eliminated after treatment of glycopeptides with sialidase. When growth of subconfluent cells was inhibited with a non-toxic concentration of retinoic acid, no significant effect on the elution pattern of [3H]fucose-labelled glycopeptides was observed on either Bio-Gel P-6 or concanavalin A-Sepharose. No significant difference was found in the total [3H]fucose-labelled glycoproteins from subconfluent and confluent cells by two-dimensional gel electrophoresis. It is suggested that the differences in [3H]fucose-labelled glycopeptides between subconfluent and confluent cells are cell-density-dependent rather than growth-dependent, and that these differences are likely to result from some changes in glycosylation mechanism(s). Furthermore, the differences in cell-surface glycopeptides may be related to the changes in the adhesion of the cells to the substratum.     

Interaction of epithelial cells with the edges of fluid and solid lipid films

Capping of Concanavalin A (Con A) on the surface of epithelial cells near the cell-cell contacts has been compared with that in the regions of cell contacts with the edges of lipid films. If the lipids are in "fluid" state, Con A is capped likely as on the free edges of epithelial sheets, while contacts with the edge of solid lipid film inhibit capping of Con A as do cell-cell contacts. The same is true for capping of liposomes adsorbed on the surface of epithelial cells. We suppose that solid rather than fluid domains in plasma membranes may play a significant role in establishing cell-cell contacts.     

DNA complexes with polycations useful for delivery of DNA into cells

Generation and physicochemical properties of complexes formed by high-molecular thymus DNA and plasmid DNA with synthetic polymers of (dimethyl amino)ethyl methacrylate, (diethyl amino)ethyl methacrylate, and poly(vinyl amine) were studied in solutions of different ionic strength using low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The complexes were tested for toxicity with T98G cell cultures. Condensation of DNA was shown to occur when the ratio of charged groups in the polycations and DNA exceeded unity. This condensation manifested itself as an increase in the optical density of DNA solutions. Condensation-associated changes in the dimensions of DNA molecules were determined, and phase diagrams of DNA-polycation systems were analyzed in the presence of NaCl. MTT analysis revealed no toxicity of these complexes.     

Interaction of tumour promoters with epithelial cells in culture : An immunofluorescence study

12-O-tetradecanoyl phorbol-13-acetate (TPA) has a profound and rapid influence on the cytoskeleton of Madin-Darby Canine Kidney (MDCK) cells. Within 10 min, TPA induces a rapid change in morphology, from a flat, cuboidal state to a rounded or elongated morphology in which the cell membranes become convoluted. Concomitant with this morphological change is a rapid dissolution of stress fibres and a redistribution of F-actin from microfilament bundles to a membrane or sub-membranous location. The rearrangement of actin is paralleled by a rearrangement of alpha-actinin and a reduction in the number of vinculin-containing adhesion plaques. Unusual F-actin configurations are often found emanating from a perinuclear location, usually containing alpha-actinin and terminating in a vinculin-containing adhesion plaque. The cytoskeletal rearrangements occur in the presence of inhibitors of protein synthesis or oxidative phosphorylation, but do not occur if glycolysis is also inhibited. The rearrangements are partly abrogated by the presence of cytochalasin B (CB). Despite these dramatic changes in microfilaments the polymerization state of actin remained unaltered after TPA treatment. Furthermore, although changes in the movement of membrane lipids have been reported, no obvious differences in the ability of glycoproteins to redistribute in the plane of the membrane were found as judged by FITC-concanavalin A (conA) induced patching. The rapidity of the morphological response of MDCK cells to TPA indicates that the cytoskeleton is one of the primary targets of TPA, but that tumour promoters differ from RNA tumour viruses in their effect on the state of actin polymerization.     

Interaction of alveolar epithelial cells with CFP21, a mycobacterial cutinase-like enzyme

Mycobacterium tuberculosis (M. tb), an intracellular pathogen, has the ability to infect alveolar epithelial cells (AEC) also in addition to alveolar macrophages. The virulence of M. tb is attributed to proteins encoded by genomic regions of deletion (RD) and till date 16 such regions (RD1-RD16) have been identified. Culture filtrate protein 21 (CFP21), a RD2 secretory protein, is a cutinase-like enzyme that possesses esterase and lipolytic activity. It is hypothesized that CFP21 could be playing a role in M. tb virulence by disrupting the host cell integrity. In this study, recombinant CFP21 was expressed and purified. The in vitro exposure of type I (WI26) and type II (A549) AEC to CFP21 resulted in a significant decline in their cellular viability by inducing cell apoptosis. However, the cytotoxic effects were more pronounced in WI26 cells than in A549 cells. The analysis of immune responses in CFP21-treated AEC exhibited significant production of reactive oxygen species and anti-inflammatory cytokine TGF-β which indicated oxidative stress-mediated cell death. These results show that CFP21 could play an important role in M. tb pathogenesis by disrupting the host alveolar barrier and thereby facilitating mycobacterial dissemination.     

Interaction of plasmin with endothelial cells.

Interaction of human plasmin with a monolayer culture of mini-pig aortic endothelial cells was studied by using the 125I-labelled enzyme. The binding of plasmin was time- and concentration-dependent. Equilibrium between bound and free enzyme was obtained within 90s, and Scatchard analysis indicated a high- and a low-affinity population of binding sites of approx. 1.24 X 10(4) sites/cell having a Kd of 1.4 X 10(-9) M and 7.2 X 10(4) sites/cell with a Kd of 2 X 10(-8) M respectively. Plasmin, bound to cell, was spontaneously released within 2 min, suggesting a rapid equilibrium. Chemical modification of the enzyme with phenylmethanesulphonyl fluoride or pyridoxal 5'-phosphate revealed that neither the active centre nor the heparin-binding site of plasmin was involved in the interaction with the endothelial cell. In terms of endothelial-cell receptors, the binding sites of cells for plasmin and thrombin were different: the two enzymes did not compete with each other, and the pretreatment of cells with neuraminidase or chondroitin ABC lyase resulted in a 50% decrease of thrombin or plasmin binding respectively. Arachidonic acid incorporated into phospholipids of the cell was released by plasmin, but a change in the rate of prostacyclin formation was not measurable. The interaction of plasmin with endothelial cells seems to be specific in the fibrinolytic system, since plasminogen did not bind to these cells under similar conditions.