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

Vitiligo 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 since 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.

Level of receptor-associated protein moderates cellular susceptibility to pseudomonas exotoxin A.

Pseudomonas exotoxin A (PE) enters mammalian cells via a receptor-mediated endocytic pathway. The initial step in this pathway is binding to the multiligand receptor termed the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (LRP). Binding of toxin, and of the many other ligands that bind to LRP, is blocked by the addition of a 39-kDa receptor-associated protein (RAP). Here we show that approximately 40% of the cell-associated LRP is on the surface of toxin-sensitive mouse LM fibroblasts and thus accessible for toxin internalization. The remainder is located intracellularly, primarily in the Golgi region. Mammalian cells exhibit a wide range of sensitivity to PE. To investigate possible reasons for this, we examined the expression levels of both LRP and RAP. Results from a variety of cell lines indicated that there was a positive correlation between LRP expression and toxin sensitivity. In the absence of LRP, cells were as much as 200-fold more resistant to PE compared with sensitive cells. A second group of resistant cells expressed LRP but had a high level of RAP. Thus, a toxin-resistant phenotype would be expected when cells expressed either low levels of LRP or high levels of LRP in the presence of high levels of RAP. We hypothesize that RAP has a pivotal role in moderating cellular susceptibility to PE.

Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein receptor-related protein.

The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, protease-inhibitor complexes, and various ligands associated with lipid metabolism. While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associated protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands. Previously, we (Kounnas, M.Z., R.E. Morris, M.R. Thompson, D.J. FitzGerald, D.K. Strickland, and C.B. Saelinger. 1992. J. Biol. Chem. 267:12420-12423) [corrected] showed that Pseudomonas exotoxin (PE) could bind immobilized LRP. Also, the addition of RAP blocked toxin-mediated cell killing. These findings suggested that PE might use LRP to gain entry into toxin-sensitive cells. Here we report on a strategy to select PE-resistant lines of Chinese hamster ovary cells that express altered amounts of LRP. An important part of this strategy is to screen PE-resistant clones for those that retain sensitivity to both diphtheria toxin and to a fusion protein composed of lethal factor (from anthrax toxin) fused to the adenosine diphosphate-ribosylating domain of PE. Two lines, with obvious changes in their expression of LRP, were characterized in detail. The 14-2-1 line had significant amounts of LRP, but in contrast to wild-type cells, little or no receptor was displayed on the cell surface. Instead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state. The 14-2-1 line showed no uptake of chymotrypsin-alpha 2M and was 10-fold resistant to PE compared with wild-type cells. A second line, 13-5-1, had no detectable LRP mRNA or protein, did not internalize alpha 2M-chymotrypsin, and exhibited a 100-fold resistance to PE. Resistance to PE appeared to be due to receptor-specific defects, since these mutant lines showed no resistance to a PE chimeric toxin that was internalized via the transferrin receptor. The results of this investigation confirm that LRP mediates the internalization of PE.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is a large cell-surface glycoprotein consisting of a 515-kDa and an 85-kDa polypeptide; this receptor is thought to be responsible for the binding and endocytosis of activated alpha 2-macroglobulin and apoE-enriched beta-very low density lipoprotein. A similar high molecular weight glycoprotein has been identified as a potential receptor for Pseudomonas exotoxin A (PE). We demonstrate that the alpha 2 MR/LRP and the PE-binding glycoprotein have a similar mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are immunologically indistinguishable. Furthermore, affinity-purified alpha 2 MR/LRP binds specifically to PE but not to a mutant toxin defective in its ability to bind cells. The 39-kDa receptor-associated protein, which blocks binding of ligands to alpha 2 MR/LRP, also prevents binding and subsequent toxicity of PE for mouse fibroblasts. The concentration of receptor-associated protein that was required to reduce binding and toxicity to 50% was approximately 14 nM, a value virtually identical to the KD measured for the interaction of receptor-associated protein with the purified receptor. Overall, the studies strongly suggest that the alpha 2 MR/LRP is responsible for internalizing PE.

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.

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.

Mouse liver contains a Pseudomonas aeruginosa exotoxin A-binding protein.

The opportunistic pathogen Pseudomonas aeruginosa produces several potential virulence factors, including the ADP-ribosylating toxin, exotoxin A (PE). Studies using a burned mouse model have shown that PE consistently inhibits protein synthesis and depletes elongation factor 2 in mouse liver and variably in other organs. One reason for toxin sensitivity could be the presence of a PE receptor on the surface of cells. Therefore we examined detergent extracts of mouse tissues for the presence of toxin-binding proteins. Proteins which specifically bind PE were present in extracts from liver, kidney, lung, spleen, and heart. Because liver appears to be a prominent target for the toxin in a burned animal, we choose to isolate the PE-binding protein from mouse liver and compare this protein to the recently characterized toxin-binding protein from toxin-sensitive mouse LM fibroblasts. The toxin-binding proteins from both sources have a molecular mass of approximately 350 kDa, share similar protease digestion profiles, and are glycosylated. However the glycosylation patterns for the two species are quite different. Both glycoproteins bind toxin with high avidity. The toxin-binding moiety is located, at least in part, on the plasma membrane and thus could represent the receptor involved in internalization of toxin molecules responsible for cell death.

Isolation and characterization of Pseudomonas aeruginosa exotoxin A binding glycoprotein from mouse LM cells.

A Pseudomonas aeruginosa exotoxin A (PE) binding glycoprotein was affinity purified from toxin sensitive mouse LM cells. The binding protein was solubilized with Triton X-100 or Nonidet P-40 and purified on a PE-Sepharose affinity column. Polyacrylamide gel electrophoresis yielded a single band with an estimated molecular mass of greater than 300,000 Da. N-Linked carbohydrate was present, accounting for approximately 10% of the total mass of the molecule. The purified protein specifically bound PE. Incubation of purified protein specifically bound PE. Incubation of purified PE binding protein with toxin reduced toxicity to LM cells. We speculate on the role of this toxin binding glycoprotein in the intoxication process.

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: avoiding the use of crystalline chloroauric acid; succinoylating egg white avidin prior to adsorption on the gold sols; resuspending the pellets following ultracentrifugation in 5 mM phosphate buffer, pH 7.5; and using the avidin-gold colloids within 4 weeks of production.

Reduced temperature alters Pseudomonas exotoxin A entry into the mouse LM cell.

The movement of Pseudomonas exotoxin A (PE) into the cytoplasm of mouse LM fibroblasts was followed by using inhibition of protein synthesis as a biochemical index of toxin activity; biotinyl-PE and avidin-gold colloids were used for electron microscopy. At 37 degrees C both specific antitoxin and pronase-trypsin protected cells against PE toxicity when added within seconds of warming cells, whereas methylamine was protective when added during the first 7 min of endocytosis. Lowering the temperature to 19 degrees C afforded protection when the temperature transition was accomplished within 15 min of the original endocytic event. These data suggest that PE enters an acidic compartment before reaching a step blocked by shifting cells from 37 to 19 degrees C. PE expressed toxicity for LM cells at 19 degrees C, but at a concentration 1 order of magnitude higher than that required at 37 degrees C. At 19 degrees C, antitoxin or trypsin-pronase protection was rapidly ablated. In contrast cells were fully protected by methylamine for 90 min. Using electron microscopy we demonstrated that toxin moved normally (30 s) to coated areas at 19 degrees C, but remained at this site for up to 20 min before being internalized. The majority of the toxin internalized at 19 degrees C remained in endosomes or in Golgi-associated vesicles and was not delivered to lysosomes. The results suggest that, under physiological conditions (37 degrees C), PE rapidly enters cells through coated areas, moves to an acidic compartment (i.e., the endosome), and then probably to the Golgi region en route to lysosomes. The evidence suggests that movement of toxin from endosomes or Golgi vesicles to lysosomes is blocked at 19 degrees C. We hypothesize that the active form of PE enters the cytosol, where it expresses its toxicity during fusion of Golgi-derived, toxin-laden vesicles with lysosomes.

Receptor-mediated entry of diphtheria toxin into monkey kidney (Vero) cells: electron microscopic evaluation.

To express toxicity in living cells, diphtheria toxin (DT) must cross a membrane barrier and reach its target in the cytosol. Here we examine the entry of DT into the toxin-sensitive monkey kidney (Vero) cells. Using electron microscopy we directly demonstrated for the first time that DT is internalized by receptor-mediated endocytosis, i.e., via clathrin-coated pits, and enters the endosomal system. Methylamine, which is known to protect cells from DT, stopped the movement of toxin to coated areas of the cell membrane. In the presence of amine, prebound biotinyl-DT was internalized, but toxicity was inhibited. Biochemical evidence revealed that methylamine maintained toxin molecules at a site accessible to neutralization by antitoxin. The data suggest that DT entering Vero cells in the presence of methylamine is sequestered within the cell and does not express toxicity.

Trafficking of Pseudomonas exotoxin A in mammalian cells.

Experiments designed to elucidate cellular internalization of Pseudomonas aeruginosa exotoxin A are described. Inhibition of protein synthesis was used as an index of the biological activity of exotoxin A, and a biotinyl-toxin: avidin-gold system to follow its movement on the ultrastructural level. Addition of amantadine, methylamine and dansylcadaverine to cells enhanced the toxicity of exotoxin A at lower concentrations, while protecting cells at higher concentrations. In general, both sensitive and resistant cell lines responded similarly. Exposure of LM or Vero cells to an acidic extracellular pH did not overcome the protection afforded by ammonium chloride against exotoxin A cytotoxicity. This and other data suggest that sensitive and resistant cells may internalize exotoxin A in a similar manner, the toxin entering the cytosol from a prelysosomal acidic vacuole.

Evidence for pseudomonas exotoxin A receptors on plasma membrane of toxin-sensitive lm fibroblasts.

Pseudomonas exotoxin A enters mouse LM fibroblasts by receptor-mediated endocytosis and ultimately causes cell death. Here we present evidence for the existence of a specific receptor for the toxin. Toxin association with LM cells at 18 and 37 degrees C, but not at 4 degrees C, was highly specific. At 37 degrees C, the association increased with time, reaching a steady state by 5 h. Binding to paraformaldehyde-fixed cells at 37 degrees C was saturable (Kd = 5.4 nM), was reversible, and indicated ca. 100,000 binding sites per cell. It is believed that receptor-bound toxin is responsible for cell death. Once the kinetics of toxin entry were described, we examined the effect of reduced temperatures on the intracellular processing of toxin and thus its expression. Toxin-induced inhibition of protein synthesis was minimal at temperatures below 20 degrees C. This was seen even though at 20 degrees C sufficient toxin was internalized to kill cells, and toxin enzyme activity was maximal. Internalization of 125I-labeled toxin, but not of 125I-labeled horseradish peroxidase (marker of fluid-phase endocytosis), became rate limiting at 20 degrees C or below. These data suggest that reduced temperatures block a step in the receptor-mediated endocytic pathway essential for the expression of Pseudomonas toxin activity.