Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression

Abstract

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5-ZNT6 heterodimers [ZNT5-6] and ZNT7-ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.

Keywords: ER quality control; ZNT; cell surface; early secretory pathway; ectoenzyme; glycosylphosphatidylinositol (GPI anchor); phosphatidylinositol glycan anchor biosynthesis (PIG); transporter; zinc.

Figure 1

Properties of transiently expressed GPI-anchored and single membrane–spanning polypeptide-anchored zinc ectoenzymes in Z5Z7-DKO cells.A, the activity and protein expression of GPI-anchored zinc ectoenzymes (TNAP, PLAP, and CD73). B, immunofluorescence staining of TNAP, PLAP, and CD73, detected in red fluorescence. C, the activity and protein expression of single membrane–spanning polypeptide-anchored zinc ectoenzymes (sACE, gACE, and ACE2). gACE, detected in the lower left panel for the blotting of β-gal, is indicated with an asterisk (∗). D, immunofluorescence staining of sACE, gACE, and ACE2. In (A) and (C), expression plasmids were transiently transfected in WT A549 cells and A549-Z5Z7-DKO cells. β-galactosidase (β-gal) was used as the internal control. All activities are expressed as the mean ± SD of triplicate experiments (upper graphs). Ten micrograms of total cellular lysate prepared from transiently transfected cells was subjected to immunoblot analysis (lower panels). In (B) and (D), WT SK-MEL-2 and SK-Z5Z7-DKO cells were transiently transfected with expression plasmid harboring each cDNA in IRES-GFP plasmid, and transfected cells were discriminated by GFP fluorescence. Merged images with GFP and DAPI are shown. Each experiment was performed at least three times and representative results from independent experiments are shown. ACE, angiotensin-converting enzyme; cDNA, complementary DNA; DAPI, 4,6-diamino-2-phenylindole; DKO, double KO; gACE, germline specific ACE; GPI, glycosylphosphatidylinositol; IRES, internal ribosome entry site; PLAP, placental ALP; sACE, somatic ACE; TNAP, tissue nonspecific ALP.

Figure 2

Properties of chimeric zinc ectoenzyme expression in Z5Z7-DKO cells.A, activity and protein expression of chimeric PLAP mutants (PLAP-TM(H) or PLAP-TM(G)) in which the GPI anchor was substituted with a single membrane–spanning polypeptide anchor. B, immunofluorescence staining of transiently expressed PLAP-TM(H) or PLAP-TM(G). C, activity and protein expression of chimeric mutants of sACE and ACE2 (sACE-GPI or ACE2-GPI), in which the single membrane–spanning polypeptide-anchor was substituted with the GPI-anchor. In the left graph, sACE activity is shown on the left-hand axis of ordinate, while sACE-GPI activity is shown on the right-hand axis. ACE2, detected in the lower right panel for the blotting of β-gal, is indicated by an asterisk (∗). D, immunofluorescence staining of transiently expressed ACE-GPI or ACE2-GPI. In (A) and (C), expression plasmids were transiently transfected in WT A549 cells and A549-Z5Z7-DKO cells. In (B) and (D), WT SK-MEL-2 and SK-Z5Z7-DKO cells were transiently transfected with expression plasmid harboring each cDNA in IRES-GFP plasmid. Each experiment was performed at least three times, and representative results from independent experiments are shown. ACE, angiotensin-converting enzyme; cDNA, complementary DNA; DKO, double KO; gACE, germline specific ACE; GPI, glycosylphosphatidylinositol; IRES, internal ribosome entry site; PLAP, placental ALP; sACE, somatic ACE.

Figure 3

Properties of GPI-anchored proteins transiently expressed in Z5Z7-DKO cells.A, protein expression of CD55, CD59, and CP in A549-Z5Z7-DKO cells and WT A549 cells. B, immunofluorescence staining of CD55, CD59, and CP in SK-Z5Z7-DKO cells and WT SK-MEL-2 cells. C, restoration of CD55 and CD59 expression on the cell surface in SK-Z5Z7-DKO cells by simultaneous expression of HA-ZNT5 or HA-ZNT7 but not by zinc transport–incompetent HA-ZNT5_H451A or HA-ZNT7_H70A. Cells transfected with zinc transport incompetent ZNT5 or ZNT7 expression plasmids were cultured in the presence of 75 μM ZnSO₄. D and E, expression of chimeric CD55 and CP proteins in which the GPI anchor was substituted with a single membrane–spanning polypeptide anchor was examined using immunofluorescence staining (D) and immunoblotting (E). Immunofluorescence staining and immunoblotting were performed in the same method as described in Figure 1. In (E), arrowheads indicate the position of CD-55-TM or CP-TM. Nonreducing SDS-PAGE was performed to detect CD55 in (A) and (E). A nonspecific band is indicated by an asterisk (∗). Each experiment was performed at least three times and representative results from independent experiments are shown. CP, ceruloplasmin; DKO, double KO; GPI, glycosylphosphatidylinositol; HA, hemagglutinin.

Figure 4

Endogenous GPI-anchored proteins substantially decreased in Z5Z7-DKO cells.A and B, immunoblotting of endogenous CD55, CD59, BST2, and TNAP in WT HAP1 cells and HAP-Z5Z7-DKO cells (A) and WT SK-MEL-2 and SK-Z5Z7-DKO cells (B). C and D, CD55 and CD59 expression in HAP-Z5Z7-DKO cells following re-expression of FLAG-tagged ZNT5. Immunofluorescence staining was performed as in Figure 1. E, immunoblotting of CD55 and CD59 expression in HAP-Z5Z7-DKO cells following re-expression of FLAG-tagged ZNT5. In (A), (B), and (E), 20 μg of membrane protein prepared from the cells was used for immunoblotting analysis. Calnexin (CANX) or Calreticulin (CALR) is shown as a loading control. CNX was detected as the higher molecular weight band in the higher percentage gels. Each experiment was performed at least three times and representative results from independent experiments are shown. DKO, double KO; GPI, glycosylphosphatidylinositol; TNAP, tissue nonspecific ALP; ZNT, Zn transporter.

Figure 5

GPI-anchored protein expression in Z5Z7-DKO cells with impaired PIG protein.A, CD55, CD59, TNAP, and BST2 expression in HAP-PIGN-KO, HAP-PIGO-KO, and HAP-PIGG-KO cells. B, CD55, CD59, TNAP, and BST2 expression in HAP-Z5Z7PIGG-TKO cells or HAP-Z5Z7PIGN-TKO cells. C, CD55, CD59, TNAP, and BST2 expression in HAP-Z5Z7PIGA-TKO cells, HAP-Z5Z7PIGT-TKO, HAP-PIGA-KO, and HAP-PIGT-KO cells. Twenty micrograms of membrane protein prepared from the cells was used for immunoblotting analysis. Calreticulin (CALR) or CANX is shown as a loading control. CANX was detected as the higher molecular weight band in the higher percentage gels. Each experiment was performed at least three times and representative results from independent experiments are shown. CANX, calnexin; DKO, HAP-Z5Z7-DKO cells; GPI, glycosylphosphatidylinositol; PIG, phosphatidylinositol glycan anchor biosynthesis; TNAP, tissue nonspecific ALP.

Figure 6

Disruption of both Znt5 and Znt7 genes resulted in touch-insensitive phenotype in medaka fish (Oryzias latipes).A–E, representative lateral or dorsal, views of whole larvae of each genotype, 7 to 8 days postfertilization (dpf) in WT (A), Znt5^+/−;Znt7^+/− (B), Znt5^+/−;Znt7^−/− (C), Znt5^−/−;Znt7^+/− (D), Znt5^−/−;Znt7^−/− (E) medaka. Note that Znt5^−/−;Znt7^+/−, and Znt5^−/−;Znt7^−/− larvae failed to show a dorsal photo because they did not move. F–I, mechanosensory stimulation induced swimming away in WT (F), Znt5^+/−;Znt7^+/− (G), and Znt5^+/−;Znt7^−/− (H), but not in Znt5^−/−;Znt7^+/− (I) medaka. Znt5^−/−;Znt7^+/− medaka did not respond to touch.