Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations

Abstract

Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. The TGs identified to date do not have a typical endoplasmic reticulum (ER)-signal peptide, and the molecular mechanisms of their secretion under physiologic conditions are unclear. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. We also found that TG-A, but not TG-B, was modified concomitantly by N-myristoylation and S-palmitoylation, and N-myristoylation was a pre-requisite for S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca²⁺ ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. Ultracentrifugation, electron microscopy analyses, and treatments with inhibitors of multivesicular body formation revealed that TG-A was secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes.

Keywords: Drosophila; exosome (vesicle); protein myristoylation; protein palmitoylation; protein secretion; transglutaminase.

Figure 1.

TG-A is a target for N-myristoylation. A, the N-terminal sequences of TG-A and TG-B. The N-myristoyl consensus sequence is underlined, and the N-myristoylated glycine residue and the S-palmitoylation sites are shown in red and green, respectively. B, the N-myristoylated proteins were detected by streptavidin blotting (left) and the expressed proteins were checked by Western blotting using the anti-FLAG antibody (right). C, third instar larvae (w¹¹¹⁸) ingested myristic acid-azide and the resulting N-myristoylated proteins were labeled with biotin. Proteins purified on avidin-immobilized agarose were analyzed by Western blotting using the anti-TG-A/B antibody. D, hemocytes of third instar larvae were analyzed by immunocytochemistry using the anti-TG-A-specific antibody (green). PI, propidium iodide (red). Arrowheads indicate puncta structures. The scale bar in white is 10 μm. E, Drosophila S2 cells expressing TG-A, TG-B, or G2A tagged with the C-terminal V5-His₆ tag were analyzed by immunocytochemistry using the anti-His₆ tag antibody. The percentages of cells with the plasma membrane-localized signal are shown (n = 300). Arrowheads indicate puncta structures. The scale bar in white is 10 μm. F, S2 cells were co-transfected with the C-terminal mCherry (red)-tagged TG-A and the N-terminal EGFP (green)-tagged Rab7 and analyzed by fluorescence microscopy. G, S2 cells expressing AN⁴⁶-EGFP and BN³⁸-EGFP were incubated with myristic acid-azide (8 or 80 μm) and analog-incorporated proteins were labeled with biotin alkyne using click chemistry. The resulting proteins were purified using anti-GFP-agarose, and detected using NeutrAvidin-horseradish peroxidase. Myr-Az, myristic acid-azide.

Figure 2.

TG-A is a target for S-palmitoylation. A, the biotin-switch assay for lysates of adult w¹¹¹⁸ flies. Proteins that precipitated on avidin-immobilized agarose after the biotin-switch assay were detected by Western blotting using the anti-TG-A/B antibody. B, the biotin-switch assay for lysates of cells expressing the C-terminal V5-His₆-tagged TG-A and TG-B (S2 cells) and the C-terminal FLAG-tagged TG-A and TG-B (Sf-9 cells). Proteins that precipitated on avidin-immobilized agarose after the biotin-switch assay were detected by Western blotting using the anti-His₆ tag and anti-FLAG-tagged antibodies, respectively. C, S2 cells expressing AN⁴⁶-EGFP and BN³⁸-EGFP were incubated with palmitic acid/azide (8 or 80 μm) and analog-incorporated proteins were labeled with biotin alkyne using click chemistry. The resulting proteins were purified using anti-GFP-agarose, and detected using NeutrAvidin-horseradish peroxidase. Pal-Az, palmitic acid/azide. D, the biotin-switch assay for lysates of Sf-9 cells expressing the C-terminal V5-His₆-tagged C7A, C8A, and C7A/C8A. Proteins that precipitated on avidin-immobilized agarose after the biotin-switch assay were detected by Western blotting using the anti-TG-A/B antibody. E and F, the C-terminal FLAG-tagged TG-expressing Sf-9 cells (E) and the C-terminal V5-His₆-tagged TG-expressing S2 cells (F) were analyzed by immunocytochemistry using the anti-FLAG antibody and anti-V5 antibody, respectively. The percentage of cells with the MVB-localized signal are shown (E, n = 50; F, n = 100). The scale bar in white is 10 μm. G, The biotin-switch assay for lysates of S2 cells expressing the C-terminal V5-His₆-tagged TG-A and G2A. Proteins that precipitated on avidin-immobilized agarose after the biotin-switch assay were detected by Western blotting using the anti-His₆ tag antibody.

Figure 3.

TG is secreted in response to stimulation by Ecc15. A, TG-A or TG-B antigen secreted from hemocytes after treatment with Ecc15 was detected by Western blotting using the anti-TG-A/B antibody. The band intensity at 90 min after infection was defined as 100%. Line graph shows the band intensity analyzed by ImageJ software. B-D, Ecc15 was added to the C-terminal V5-His₆-tagged TG-expressing S2 cells (B and D) or FLAG-tagged TG-A-expressing S2 cells (C), and cell lysates or the conditioned media were analyzed by Western blotting using the anti-TG-A/B (B and D), anti-FLAG (C), and anti-His₆ tag (D) antibodies, respectively. Bar graph shows the band intensity analyzed by ImageJ software (B). E, lipopolysaccharides or peptidoglycans were added to the C-terminal FLAG-tagged TG-A-expressing S2 cells and cultured supernatants were analyzed by Western blotting using the anti-FLAG antibody. LPS, lipopolysaccharide; DAP-PGN, diaminopimelic acid-type peptidoglycan; Lys-PGN, lysine-type peptidoglycan. One and 10 μg/ml of bacteria cell components were used. F, the supernatant from Ecc15-stimulated hemocytes was separated into the soluble (S100) and precipitated (P100) fractions by ultracentrifugation at 100,000 × g and analyzed by Western blotting using the anti-TG-A/B antibody. Numbers show the band intensity analyzed by ImageJ software, and the intensity of the P100 fraction was set to 1.0. G, A23187, ionomycin (1 μm each), or 20 nm uracil was added to the C-terminal FLAG-tagged TG-A-expressing S2 cells for 1 h, and the resulting P100 fraction was analyzed by Western blotting using the anti-FLAG antibody. H, C-terminal mCherry-tagged TG-A or G2A-expressing S2 cells were treated with A23187 and analyzed by Western blotting using the anti-TG-A/B antibody.

Figure 4.

TG-A is secreted in exosomes via an unconventional secretion pathway. A and D, brefeldin A (1 μg/ml, B) or monensin (1 μm, M) were added to the C-terminal FLAG-tagged TG-A, TG-B, AN⁴⁶-EGFP, or BN³⁸-EGFP-expressing S2 cells, and subsequently treated with A23187 or ionomycin (1 μm each). Cell lysates and the P100 fraction were analyzed by Western blotting using the anti-TG-A/B (A) or anti-GFP tag antibody (D). B, GFP fused with the BiP secretion signal sequence at the N terminus was expressed in S2 cells and treated with brefeldin A (1 μg/ml) or monensin (1 μm). The resulting cultured medium was analyzed by Western blotting using the anti-GFP tag antibody. C, A23187 or ionomycin (1 μm each) were added to the C-terminal EGFP-tagged N-terminal fragment of TG-A and TG-B (AN⁴⁶-EGFP, BN³⁸-EGFP)-expressing S2 cells for 1 h, and analyzed by Western blotting using the anti-GFP tag antibody. E, the C-terminal EGFP-tagged TG-A-expressing S2 cells were treated with or without 10 μm GW4869 for 40 h, and then with or without 1 μm A23187 for 1 h. Bar graph shows the band intensity analyzed by ImageJ software, and error bars indicate ± S.E. (n = 3). F, the P100 fraction from dsRab27 or dsEGFP (negative control)-treated C-terminal V5-His₆-tagged TG-A-expressing S2 cells were analyzed by Western blotting using the anti-His₆ tag antibody. Bar graph shows the band intensity analyzed by ImageJ software, and error bars indicate ± S.E. (n = 6). G, the P100 fraction from the C-terminal FLAG-tagged TG-A and/or the C-terminal EGFP-tagged human CD63-expressing S2 cells were analyzed by Western blotting using the anti-FLAG, CD63, or GFP antibody.

Figure 5.

Profiles of TG-A containing exosomes. A, the P100 fraction prepared from A23187-stimulated the C-terminal FLAG tagged-TG-A- and the N-terminal EGFP-tagged-CD63-expressing S2 cells was analyzed by density gradient centrifugation using OptiPrep. B and C, the P100 fraction from A23187-stimulated the C-terminal FLAG tagged-TG-A-expressing S2 cells was analyzed by the biotin-switch assay with or without HA (B), or treated with or without 10 μm proteinase K in the presence or absence of 1% Triton X-100 for 1 h at 37 °C (C). D, immunotransmission electron microscopic analysis of S2 cells expressing the C-terminal FLAG-tagged TG-A. The TG-A-specific antibody was used as a primary antibody and was detected by the anti-rabbit colloidal-gold conjugated secondary antibody. a′ and b′ represent magnified photographs of square area of a and b, respectively. Arrowheads indicate 10-nm colloidal gold signals. The scale bars in white in a and b, or a′ and b′ are 500 and 100 nm, respectively. E, intracellular gold particles from 10 cells in the immune transmission electron microscopic analysis were counted and analyzed by Student's t test. ***, p < 0.001. Error bars indicate ± S.E. (n = 10). F, the P100 fraction prepared from A23187-treated (1 or 2 μm) the C-terminal V5-His₆-tagged TG-A-expressing S2 cells were incubated with Ecc15 for 1 h at room temperature. After incubation, bacteria were collected and washed three times, and then bacteria-bound proteins were analyzed by Western blotting using the anti-His₆ tag antibody. Numbers show the band intensity of the bacteria-bound fraction analyzed by ImageJ software, and the intensity at the 2 μm A23187 was set to 1.0. ND, not detectable. G, S2 cells expressing the C-terminal EGFP tagged-TG-A were labeled with the exosomal marker BODIPY TR ceramide, and stimulated with A23187, and then the resulting P100 fraction was collected and added to untransfected S2 cells for 1 h. The exosome-treated S2 cells were analyzed under a fluorescence microscope. The scale bars in white are 10 μm.

Figure 6.

A schematic model of the two types of fatty acid modifications and secretion of TG-A. TG-A is co-translationally N-myristoylated and subsequently S-palmitoylated probably on the cytoplasmic face of the ER/Golgi membrane. The dually fatty acid-modified TG-A is finally transported to the MVBs by unknown mechanisms and secreted as exosomes following activation by external stimuli, such as bacteria-derived uracil. The secreted TG-A retains the fatty acid modification and locates in the inner leaflet of the exosomes.