Acyl-Protein Thioesterase 2 Catalizes the Deacylation of Peripheral Membrane-Associated GAP-43

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution

Targeted delivery of immunotoxin by antibody to ganglioside GD3: a novel drug delivery route for tumor cells.

Gangliosides are sialic acid-containing glycolipids expressed on plasma membranes from nearly all vertebrate cells. The expression of ganglioside GD3, which plays essential roles in normal brain development, decreases in adults but is up regulated in neuroectodermal and epithelial derived cancers. R24 antibody, directed against ganglioside GD3, is a validated tumor target which is specifically endocytosed and accumulated in endosomes. Here, we exploit the internalization feature of the R24 antibody for the selective delivery of saporin, a ribosome-inactivating protein, to GD3-expressing cells [human (SK-Mel-28) and mouse (B16) melanoma cells and Chinese hamster ovary (CHO)-K1 cells]. This immunotoxin showed a specific cytotoxicity on tumor cells grew on 2D monolayers, which was further evident by the lack of any effect on GD3-negative cells. To estimate the potential antitumor activity of R24-saporin complex, we also evaluated the effect of the immunotoxin on the clonogenic growth of SK-Mel-28 and CHO-K1(GD3+) cells cultured in attachment-free conditions. A drastic growth inhibition (>80-90%) of the cell colonies was reached after 3 days of immunotoxin treatment. By the contrary, colonies continue to growth at the same concentration of the immuntoxin, but in the absence of R24 antibody, or in the absence of both immunotoxin and R24, undoubtedly indicating the specificity of the effect observed. Thus, the ganglioside GD3 emerge as a novel and attractive class of cell surface molecule for targeted delivery of cytotoxic agents and, therefore, provides a rationale for future therapeutic intervention in cancer

Golgi Phosphoprotein 3 Regulates the Physical Association of Glycolipid Glycosyltransferases

Glycolipid glycosylation is an intricate process that mainly takes place in the Golgi by the complex interplay between glycosyltransferases. Several features such as the organization, stoichiometry and composition of these complexes may modify their sorting properties, sub-Golgi localization, enzymatic activity and in consequence, the pattern of glycosylation at the plasma membrane. In spite of the advance in our comprehension about physiological and pathological cellular states of glycosylation, the molecular basis underlying the metabolism of glycolipids and the players involved in this process remain not fully understood. In the present work, using biochemical and fluorescence microscopy approaches, we demonstrate the existence of a physical association between two ganglioside glycosyltransferases, namely, ST3Gal-II (GD1a synthase) and β3GalT-IV (GM1 synthase) with Golgi phosphoprotein 3 (GOLPH3) in mammalian cultured cells. After GOLPH3 knockdown, the localization of both enzymes was not affected, but the fomation of ST3Gal-II/β3GalT-IV complex was compromised and glycolipid expression pattern changed. Our results suggest a novel control mechanism of glycolipid expression through the regulation of the physical association between glycolipid glycosyltransferases mediated by GOLPH3

Selective cytotoxicity of R24-targeted immunotoxin on mouse B16^GD3+ melanoma cells. A

<p>) Wild-type B16 cells (B16^wt) and B16 cells genetically modified to express GD3 (B16^GD3+) grown on coverslips were incubated at 4°C to inhibit intracellular transport, then with R24 antibody for 45 min at 4°C, washed and fixed. R24 antibody was detected by using anti-mouse IgG conjugated with Alexa Fluor⁴⁸⁸ (45 min, 4°C; left panels). Cells were incubated with R24 for 45 min at 4°C and after washing temperature was shifted to 37°C for 30 min to allow the endocytosis of the complex GD3-R24. Then, cells were fixed and R24 antibody detection was carried out as indicated above (30 min, 37°C; right panels). <b>B</b>) B16^GD3+ cells transiently expressing Rab5-GFP or Lamp1-GFP were incubated with R24 for 45 min at 4°C. After washing, temperature was shifted to 37°C for 30 min to allow the endocytosis of the complex GD3-R24. R24 antibody was detected by using anti-mouse IgG conjugated with Alexa Fluor⁵⁴³. Expression of Rab5 and Lamp1 was detected by the intrinsic fluorescence of GFP. In another set of experiments, uptake of Alexa Fluor⁶⁴⁷-transferrin (Tf) was monitored simultaneously with R24 endocytosis. In this case, R24 antibody was detected by using anti-mouse IgG conjugated with Alexa Fluor⁴⁸⁸. Insets in merge panels (right column) show details at higher magnifications. In all experimental conditions, single confocal sections were taken every 0.8 µm parallel to the coverslip. The fluorescence micrographs shown are representative of three independent experiments. Scale bar: 10 µm. <b>C</b>) B16^wt and B16^GD3+ cells were cultured at 37°C for 72 h in 96-well plates at the indicated concentration of monoclonal antibody to GD3 (R24 antibody) in combination with goat antibody to mouse IgG (squares, black lines) or saporin conjugated goat antibody anti mouse IgG secondary antibody (circles, grey lines). The concentration of the secondary antibodies was 0.95 nM. As negative control (100% viability), B16^GD3+ cells were incubated only with culture medium. Cell viability was determined using the colorimetric MTT metabolic activity assay. Absorbance was measured at 595 nm using a multiplate reader. Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. Note that R24-targeted saporin selectively kills B16^GD3+ melanoma cells (** p<0.001, respect to control condition).</p

R24 antibody is specifically internalized in GD3-expressing cells. A

<p>) CHO-K1^GD3+, CHO-K1^WT (GD3-) and SK-Mel-28 cells grown on coverslips were incubated at 4°C to inhibit intracellular transport, then with R24 antibody for 45 min at 4°C, washed and fixed. R24 antibody was detected by using goat anti-mouse IgG conjugated with Alexa Fluor⁴⁸⁸ (45 min, 4°C; left panels). <b>B</b>) Cells were incubated with R24 for 45 min at 4°C and after washing temperature was shifted to 37°C for 30 min to allow the endocytosis of the complex GD3-R24. Then, cells were fixed and R24 antibody detection was carried out as indicated in <b>A</b> (30 min, 37°C; right panel). Single confocal sections were taken every 0.8 µm parallel to the coverslip. The fluorescence micrographs shown are representative of three independent experiments. Scale bar: 10 µm.</p