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Zinc supply mediated by ZNT5-ZNT6 and ZNT7 is critical to glycosylphosphatidylinositol-anchored protein expression

ABSTRACT: Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. ZNT type zinc transporter proteins mobilize cytosolic zinc to the extracellular space and intracellular compartments. Here, we report that the early secretory pathway ZNTs [ZNT5-ZNT6 heterodimers (ZNT5-6) and ZNT7 homodimers (ZNT7)], which supply zinc into the lumen of organelles, are essential for GPI-anchored protein expression on the cell surface. Loss of ZNT5-6 and ZNT7 zinc transport functions results in a significant reduction of GPI-anchored proteins similar to that of mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Disrupted ZNT5 and ZNT7 genes in medaka fish show touch-insensitive phenotypes similar to those of zebrafish PIG mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and of protein quality control in the early secretory pathway.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Risa Sato

LAB HEAD: Yasuo Uchida

PROVIDER: PXD032172 | Pride | 2022-05-04

REPOSITORIES: Pride

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Project description:Somatic mutation in the X-linked phosphatidylinositol glycan class A (PIG-A) gene causes glycosylphosphatidylinositol (GPI) anchor deficiency in humans with Paroxysmal Nocturnal Hemoglobinuria (PNH). Clinically, patients with PNH have intravascular hemolysis, venous thrombosis and bone marrow failure. We produced a conditional Pig-a knock-out mouse model specifically inactivating the Pig-a gene in hematopoietic cells to study the role of PIG-A deficiency in PNH pathophysiology. We used Affymetrix Mouse Genome 430 2.0 chips to investigate the gene expression pattern in the mouse model of targeted Pig-a deletion. Experiment Overall Design: We performed microarray analysis on 3 pools of sorted GPI-deficient (GPI-) and GPI normal (GPI+) bone marrow cells derived from the same Pig-a knock-out animals, and identified 1275/669 genes of the 45,101 transcripts potentially available for screening using 2-fold change, <10% false discovery rate (FDR) and percentage of present calls as selection criteria. The major representative genes belong to the category of immune response. We tested whether the molecular differences between GPI- and GPI+ bone marrow cells could be preserved when GPI- cells were transplanted in lethally-irradiated wild type mice (C57BL/6). Microarray analysis was performed using sorted bone marrow cells from 4 animals transplanted with GPI-deficient bone marrow cells (T-GPI-) from Pig-a knock-out donors, 3 animals transplanted with GPI-normal bone marrow cells from C57BL/6 donors (T-GPI+), and GPI-normal bone marrow cells from 4 wild-type C57BL/6 mice (WT-GPI+). We found 296 probesets with 2-fold change cutoff, of which T-GPI- cells had 145 up-regulated genes in comparison to WT-GPI+ cells, and had 123 genes differentially expressed when compared to T-GPI+ cells. The gene expression of the GPI-deficient cells was very similar between the two sets of microarray experiments affirming the maintenance of the phenotype before and after bone marrow cell transplantation.

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Zinc supply mediated by ZNT5-ZNT6 and ZNT7 is critical to glycosylphosphatidylinositol-anchored protein expression

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