Catharine B. Saelinger

Receptor-mediated internalization of pseudomonas toxin by mouse fibroblasts

Pseudomonas exotoxin (PE) was used as a probe to study the mechanism by which protein ligands are internalized by mammalian cells. Both biochemical and electron microscopic methods were used to look at the internalization of PE by mouse LM cell fibroblasts. Our data suggest that PE enters cells by receptor-mediated endocytosis, a process previously thought to be restricted to the entry of biologically significant molecules such as lysosomal enzymes and peptide hormones. Biochemical studies showed that methylamine (20 mM) and chloroquine (10 microM) protected LM cells from the action of PE. Full protection was observed if methylamine or chloroquine was added to the monolayers simultaneously with toxin or if they were added up to 10 min after toxin binding. Later addition of amine or chloroquine afforded partial protection to the monolayers. With immunoelectron microscopy we observed that in the cold toxin bound diffusely to the cell surface but was rapidly internalized when cells were warmed to 37 degrees C. In the presence of methylamine, chloroquine or ammonium chloride, internalization did not occur. We propose that PE enters mouse fibroblasts by receptor-mediated endocytosis and that chloroquine and methylamine, agents which are known to block this process, prevent expression of toxicity.

PIG3V, an immortalized human vitiligo melanocyte cell line, expresses dilated endoplasmic reticulum.

SummaryVitiligo is an enigmatic pigmentary disorder of the skin. Factors potentially involved in the progressive loss of melanocytes from the basal layer of the epidermis include genetically determined aberrancies of the vitiligo melanocyte. It follows that analysis of melanocytes cultured from vitiligo donors can contribute to a further understanding of the etiopathomechanism. A setback for vitiligo research has been the limited availability of vitiligo-derived melanocytes. To overcome this limitation, we have generated a vitiligo melanocyte cell line according to a protocol established previously for the immortalization of normal human melanocytes. Vitiligo melanocytes Ma9308P4 were transfected with HPV16 E6 and E7 genes using the retroviral construct LXSN16E6E7. Successful transformants were selected using geneticin and subsequently cloned to ensure genetic homogeneity. The resulting cell line PIG3V has undergone more than 100 cell population doublings ince its establishment as a confluent primary culture, whereas untransfected melanocytes derived from adult skin senesce after a maximum of 50 population doublings. Cells immortalized by this transfection procedure retain lineage-specific characteristics and proliferate significantly faster than parental cells. In this study, the phenotype of PIG3V resembled melanocytes rather than melanoma cells in culture. Tyrosinase was processed properly and melanosomes remained pigmented. Importantly, ultrastructural characterization of PIG3V cells revealed dilated endoplasmic reticulum profiles characteristic of vitiligo melanocytes. An explanation for this dilation may be found in the retention of proteins with molecular weight of 37.5, 47.5, and 56.5 kDa, as determined by gel electrophoresis of microsomal proteins isolated from radiolabeled cells.

Visualization of intracellular trafficking: use of biotinylated ligands in conjunction with avidin-gold colloids.

A simple, sensitive method to visualize the binding and internalization of protein ligands by cells in culture is described. A biotinylated toxin was used as ligand, and succinoylated avidin adsorbed onto 5.2 nm gold sols was the electron-dense marker. This method affords direct localization of proteins that are on the cell surface or intracellular without need for techniques that alter membrane integrity.

Validation of the biotinyl ligand-avidin-gold technique.

We demonstrate here that the intracellular routing of biotinylated ligands was not affected by the attachment of streptavidin gold colloids so long as the electron-dense marker was added after the biotinyl ligand-receptor interaction had occurred. The binding, internalization, and intracellular routing of three different biotinyl ligands were followed in mouse LM fibroblasts. The biotinyl (B) ligands included B-choleragenoid (B-CTd), B-wheat germ agglutinin (B-WGA), and B-Pseudomonas exotoxin A (B-PE). All three ligands showed distinct intracellular trafficking patterns. B-WGA and B-PE entered via clathrin-coated pits, whereas B-CTd did not. After entry, B-CTd was routed to the lysosomal compartment without involvement of the Golgi. Although B-PE and B-WGA were also routed to the lysosomal compartment, a significant portion of these two ligands was observed in association with the Golgi. B-WGA, however, remained in the endosomal and Golgi compartments longer than did B-PE. We also monitored the internalization and routing of native PE by an indirect immunoperoxidase technique done in conjunction with saponin solubilization. The results corroborated the observations with the biotinyl-PE-streptavidin-gold method. In contrast, biotinyl-PE added to streptavidin-gold before addition to LM cells was poorly internalized and routed aberrantly. From these observations we conclude that the biotinyl ligand-avidin-gold technique is a valid method for following the binding, internalization, and intracellular routing of ligands.

Toxicity of pseudomonas toxin for mouse LM cell fibroblasts

Pseudomonas toxin inhibited protein synthesis in mouse LM fibroblast monolayers. Incubation of toxin with LM cell monolayers resulted in a depletion of functional elongation factor 2. The initial interaction of pseudomonas toxin with mouse LM cells was rapid; within 2.5 min, toxin was rendered inaccessible to neutralization with specific pseudomonas antitoxin. At 4°C toxin adsorbed to the cell surface, but remained at a site where it could be neutralized with antitoxin. Ammonium chloride (20 mM) rendered LM cells insensitive to the action of toxin. The ammonium salt did not prevent adsorption of toxin to the cell membrane; rather, it appeared to maintain toxin at a site amenable to antitoxin neutralization.

Problems in the production and use of 5 nm avidin-gold colloids

Over the past 5 years we have encountered several problems in the production and use of 5 nm avidin‐gold colloids for markers in electron microscopy. These problems include flocculation of colloids during reduction of chloroauric acid, insoluble gold pellets following ultracentrifugation, and non‐specific binding of avidin‐gold colloids to biological membranes. We are able to avoid these problems by: (1) avoiding the use of crystalline chloroauric acid; (2) succinoylating egg white avidin prior to adsorption on the gold sols; (3) resuspending the pellets following ultracentrifugation in 5 mM phosphate buffer, pH 7.5; and (4) using the avidin‐gold colloids within 4 weeks of production.

Visualization of intracellular trafficking of proteins.

Publisher Summary This chapter describes a method in which the intracellular routing of protein ligands can be followed by conventional electron microscopy. The technique described permits the intracellular visualization of biotinylated proteins when used in conjunction with avidin–gold colloids. The biotinylation of proteins is a mild procedure involving the nucleophilic attack on proteins by the N -hydroxysuccinimide ester group of biotinyl- N -hydroxysuccinimide ester. The biotinylation is done at a 5:1 molar ratio (BNHS/protein) and a 1:50 volume ratio. The method involves placing the dialyzed protein solution in a glass test tube and clarifying by centrifugation if necessary. The interaction between egg-white avidin and biotin is among the most avid interactions known. The binding of avidin to biotin is several orders of magnitude greater than that found for most antigen–antibody interactions and can be considered essentially an irreversible noncovalent interaction. Gold colloids of varying sizes can be easily prepared by the reduction of gold chloride. Colloids with average diameters ranging from five to 20 nm are routinely used for electron microscopy.

Gold enhancement of gold-labeled probes: gold-intensified staining technique (GIST).

In this report we present a staining method in which gold chloride is used to enhance the size of gold colloids. We show the utility of this technique when used in conjunction with small gold colloids, i.e., 5 nm, 4 nm, and 2.6 nm. Post-embedding staining of epoxy-embedded, gold-labeled mouse LM fibroblasts showed that staining with 0.1% gold chloride facilitated the visualization of the smallest gold colloids.

[32] Use of exotoxin A to inhibit protein synthesis

Publisher Summary This chapter describes the usage of exotoxin A to inhibit protein synthesis. The importance of Pseudomonas exotoxin A (PE) as one of the prime virulence factors of Pseudomonas aeruginosa is well established. The toxin is characterized on both the structural and biochemical level. Pseudomonas exotoxin A has a molecular weight of 66,583. PE exerts its toxic effect by specifically stopping protein synthesis both in vitro and in vivo . At the biochemical level, it acts in a manner identical to fragment A of diphtheria toxin—for example, by the adenosine diphosphate (ADP) ribosylation of cytoplasmic elongation factor 2 (EF-2). PE is produced by P. aeruginosa as a proenzyme and must be activated to express enzyme activity. A wide range of cells are susceptible to the cytotoxic action of PE, with mouse fibroblasts (L929, LM, 3T3) being the most sensitive cell lines. One simple assay to measure PE activity is to assess inhibition of protein synthesis.

[21] Preparation of Pseudomonas exotoxin A

Publisher Summary This chapter describes the preparation of Pseudornonas exotoxin A (PE). PE is one of the major virulence factors of Pseudomonas aeruginosa . A low protease-producing strain PAl03 is usually used for exotoxin production. The undefined growth medium for PE production is the dialysate of trypticase soy broth (TSB). The medium routinely used for the culture of P. aeruginosa and the production of PE is the dialyzable portion of trypticase soy broth (DTSB) originally described by Liu. The dialysate (DTSB) is used for PE production. The concentration of iron in the dialysate must be monitored carefully because excess iron depresses toxin production; the optimum iron concentration is 0.05 μg/ml. Dialyzed trypticase soy agar (DTSA) for initial colony growth is made by the addition of agar to a final concentration of 1.5% (w/v) to the DTSB, sterilized, then supplemented with glycerol and monosodium glutamate to the concentrations that is also described in the chapter.