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

Immunotoxin inhibits CHO-K1^GD3+ and SK-Mel-28 cells colony formation. A

<p>) A schematical representation of the experimental procedure used in <b>B</b> and <b>C</b>. CHO-K1^GD3+(<b>B</b>) and SK-Mel-28 (<b>C</b>) cells (50–80 cells) were seed in 24-well plates previously coated with 0.5% agar in DMEM supplemented with 20% FBS. Cultures were supplemented with 0.95 nM Saporin-Ab or 30 nM R24/0.95 nM Saporin-Ab and maintained at 37°C in a hummed atmosphere. Quantification of the colony area was performed every day, but only indicated at 7 and 10 days. Cells maintained only with medium were used as negative control (control). Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. Note the drastic inhibition of colony formation only in presence of R24/Saporin-Ab.</p

Targeted delivery of immunotoxin by R24 antibody inhibit the clonogenic growth of CHO-K1^GD3+ cells. A

<p>) A schematical representation of the experimental procedure used in (<b>B</b> and <b>C</b>). <b>B</b>) CHO-K1^GD3+ cells (50–80 cells) were grown in 24-well plates previously coated with 0.5% agar in DMEM supplemented with 20% FBS. Cells were maintained at 37°C in a hummed atmosphere until cell colonies appeared (7 days, upper row. Colonies indicated with arrows). Then, cells were treated for 3 days (7+3 days, lower row) with 0.95 nM Saporin-Ab (Saporin-Ab, middle panel) or 30 nM R24/0.95 nM Saporin-Ab (R24/Saporin-Ab, right panel). CHO-K1^GD3+ cells maintained only with medium were used as negative control (control, left panel). The micrographs are representative of three independent experiments. <b>C</b>) Quantification of the colony area at 7 and 7+3 days at the different conditions indicated in <b>B</b>. Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. Note that the clonogenic growth of CHO-K1^GD3+ cells was severely affected only in presence of R24/Saporin-Ab (*** p<0.0001, respect to control condition at 7+3 days).</p

Selective delivery of saporin via R24 antibody drastically reduces the clonogenic growth of human SK-Mel-28 melanoma cells. A)

<p>SK-Mel-28 cells (50–80 cells) were grown in 24-well plates previously coated with 0.5% agar in DMEM supplemented with 20% FBS. Cells were maintained at 37°C in a hummed atmosphere until cell colonies appeared (7 days). Then, cells were exposed for 3 days (7+3 days) to 0.95 nM Saporin-Ab or 30 nM R24/0.95 nM Saporin-Ab. Colonies are indicated with arrows. <b>B)</b> Quantification of the colony area was performed at 7 and 7+3 days. SK-Mel-28 cells maintained only with medium were used as negative control (control). Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means±S.E. Note that the clonogenic growth of SK-Mel-28 cells was severely affected only in presence of R24/Saporin-Ab (***p<0.0001, respect to control condition at 7+3 days).</p

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

Deacylation kinetic and membrane association of GAP-43 at different doses of 2-BP.

<p>CHO-K1 cells coexpressing diacylated GAP-43-YFP and GalNAc-T-CFP were treated with 25, 50 or 150 µM 2-BP or vehicle (DMSO, Control), and the GAP-43 subcellular distribution was analyzed by live cell confocal microscopy at the indicated times. CHX and protein degradation inhibitors were added and maintained in the culture media until the end of each experiment. Representative images show the effect of different doses of 2-BP on the membrane association of GAP-43. The fluorescent signals from YFP and CFP were pseudocoloured green and red, respectively. Scale bars: 5 µm.</p

Deacylation kinetic of ^N13GAP-43(C3S) at different doses of 2-BP.

<p><b>A</b>) Schematic representation of the experimental procedure used in <b>B</b>, <b>C</b> and <b>E</b>. The CHO-K1 cells expressing ^N13GAP-43(C3S)-YFP, 72 h after transfection, were treated at 20°C with 25, 50 or 150 µM 2-BP or DMSO (Control) in the presence of CHX and protein degradation inhibitors which were added 1 h before imaging and during all the experiments. The ^N13GAP-43(C3S) subcellular distribution was analyzed by live cell confocal microscopy. <b>B</b>) Representative images showing the effect of different doses of 2-BP or DMSO (vehicle, Control) on the TGN-membrane association of ^N13GAP-43(C3S)-YFP. The fluorescent signal from YFP (pseudocoloured gray) at 0, 5, 15 and 30 min after 2-BP or vehicle addition is shown. The insets show the expression of the TGN marker GalNAc-T-CFP (pseudocolored gray). Cell boundaries (white lines) are indicated. <b>C</b>) Quantification of the images shown in <b>B</b> (for details see Materials and methods). Curves were fitted to the exponential decay function for each data set, and data are expressed as means±SEM for a representative experiment from nine independent ones. <b>D</b>) The half-life for deacylation at each 2-BP dose calculated from the <b>C</b> data (n = 6). (*p<0.05; ***p<0.0001; compared to 25 µM). <b>E</b>) Representative images showing the effect of 50 µM 2-BP on the TGN-membrane association of GalNAc-T-YFP over time. The fluorescent signal from YFP (pseudocoloured gray) at 0, 5, 15 and 30 min after 2-BP addition is shown. Scale bars: 5 µm.</p

2-BP inhibits acylation and membrane association of newly synthesized ^N13GAP-43(C3S).

<p><b>A</b>) Schematic representation of the experimental procedure used in <b>B</b> and <b>C</b>. The CHO-K1 cells were transfected at −8 h with the plasmid encoding ^N13GAP-43(C3S)-YFP. At 30 min before CHX withdrawal (-CHX), cells were incubated with 25, 50 or 150 µM 2-BP or DMSO (vehicle, Control). At 0 h, CHX was removed and cells were further incubated with 2-BP at the concentrations indicated above, or with DMSO, at 37°C for 9 h. Finally, cells were analyzed by confocal fluorescent microscopy or subjected to biochemical assays. <b>B</b>) Representative images showing the effect of 2-BP or DMSO (Control) on the TGN association of ^N13GAP-43(C3S). The fluorescent signal from YFP was pseudocoloured gray. The inset shows details of the boxed area at a higher magnification. Scale bars: 5 µm. <b>C</b>) After treatment with 2-BP, CHO-K1 cells transiently expressing ^N13GAP-43(C3S)-YFP were lysed, ultracentrifuged and the supernatant (S) and pellet (P) fractions were recovered. Proteins from these fractions were western blotted with an antibody to GFP (α-GFP) and α-tubulin (α-tub).</p

Dose-response curve for APT activities following different concentrations of palmitoyl-CoA and the structural characterization of the substrate by SAXS analysis.

<p>The initial rate of palmitoyl-CoA hydrolase activity was measured with 0.5 µg of recombinant APT1 (A) or APT2 (B), both in control conditions (-CHAPS -Mg²⁺, left bars) or in the presence of 7.5 mM CHAPS and 2 mM MgCl₂ (+CHAPS +Mg²⁺, right bars) at 150, 300, 600 and 1500 µM. Data show the initial rate of the reaction (V, µM/min) at different concentrations of palmitoyl-CoA (P-CoA), which are from representative experiments performed in triplicate. C) SAXS analysis. Palmitoyl-CoA was resuspended in buffer (50 mM Hepes, pH 8.0) at 50, 300, 600 and 1775 µM, and measurement were carried out as indicated in Materials and methods. The figure shows the SAXS raw data (after subtraction of the buffer background and the concentration normalization) for increasing concentrations of palmitoyl-CoA. As can be seen, no noticeable diffraction peak (due to any strong correlation) is observed in any of the curves. The curve for 50 µM palmitoyl-CoA does not display any obvious tendencies. The curves for 300 and 600 µM show increasing intensity at a very low angle, adopting similar slopes and absolute values. The curve at 1775 shows a different behavior with an increment at a low angle, which reached a plateau below 0.3 nm⁻¹ with a prominent bump centered at 1.6–1.7 nm⁻¹ (very common in bilayers and micelles). In agreement with the wedge-shaped molecular structure, this molecule did not display the global form factor of bilayers, but rather one of the micelles. This is evident from the non-quadratic decay of the intensity as a function of q. The saturation value at low q (Guinieŕs approximation) for the 1775 µM may indicate globular micelles. The clear differences present between the curves at 300–600 µM and the one at 1775 µM is probably due to the fact that the micelles have a different geometry, with the decay at low q values (q<0.5 nm⁻¹) having a finite slope closer to an inverse (first power) behavior, suggesting rod-like structures.</p