and early-onset seizure, suggesting the important role of PIGG in the neurological development5,6). Based on these evidences, we tried to elucidate the mechanism of neuronal abnormalities in the PIGG deficiencies. Glycosylphosphatidylinositols (GPIs) are glycolipids used as membrane anchors of many eukaryotic cell surface proteins. The structures of GPI anchors were first determined in rat Thy-1 and trypanosome variant surface glycoprotein in 1980s, and it was shown that the backbone structure is completely conserved among all eukaryotes. Its basic structure is EthN-P-6-Man-α1,2-Man-α1,6-Man-α1,4-GlcN-α1,6-myoInositol-phospholipid, where GlcN, Man, and EthN-P are glucosamine, mannose, and ethanolamine phosphate. Biosynthesis of GPI-anchored proteins (GPI-APs) occurs in the endoplasmic reticulum (ER). GPI anchor precursors and precursor proteins are synthesized separately, and GPI transamidase complex recognizes the C-terminal GPI attachment signal of the precursor proteins, processes and attaches them to EthN-P on the third Mannose (Man3) of GPI via an amide bond. Thus, this EthN-P is called “bridging EthN-P”. Both glycan and lipid moieties of GPI-APs are further modified in the ER and the Golgi before expression on the cell surface. In mammalian cells, at least 30 genes, termed PIG (Phosphatidylinositolglycan) and PGAP (Post GPI attachment to proteins) genes, are involved in the biosynthesis and maturation of GPI-APs1). More than 150 human proteins including enzymes, receptors, adhesion molecules and complement regulatory proteins are GPI-anchored to the cell surface. Many GPI-APs play important roles in neuronal tissues. Defects in PIG and PGAP genes cause inherited GPI deficiency (IGD), the main symptoms of which are intellectual disability, developmental delay, and seizures2). At present, IGD caused by defects in 24 genes, including recently identified ARV13), in GPI biosynthesis and the GPI-AP maturation pathway were reported4). As complete deficiency of most of these genes leads to embryonic death, most patients are partially deficient. Among them, we focused on the PIGG deficiency5). PIGG is an enzyme involved in the attachment of the EthN-P to the second Mannose (Man2) in the precursor GPI, believed to be removed soon after GPI is attached to the precursor protein by PGAP5. In mammalian PIGG knockout cells, GPI-APs such as CD59 and DAF are normally transported and expressed with the normal structure at normal level, so physiological significance of this transient modification has been unclear. However, the PIGG deficient individuals showed intellectual disability, hypotonia, published ：EMBO Rep. 2022 Jul 5;23(7):e54352. PIGG-dependent, low-level expression of GPI-anchored CD59 and DAF on PIGO KO cells and PIGB KO cellsAs mentioned above, PIGG-dependent, Man2-linked EthN-P is a transient structure that is removed soon after attachment of protein moiety to the Man3-linked EthN-P. In support of this notion, expression levels of CD59 and DAF, GPI-APs, on PIGG-KO HEK 293 cells were similar to those on wild type cells. PIGO is involved in the attachment of the bridging EthN-P to Man3, which is transferred by PIGB. The bridging EthN-P, which PIGO attaches, is covalently linked to the carboxyl terminal of the precursor proteins. We found that the HEK293 cells with complete knockout of PIGO or PIGB gene unexpectedly showed partial expression of GPI-APs, such as CD59 and DAF, which are disappeared completely after further knockout of PIGG, suggesting that PIGG plays a role in the residual expression (Figure 1A and B). This indicated that the EthN-P on Man2 can be used to bridge CD59 and DAF in a low efficiency.Mass spectrometry revealed attachment of CD59 to Man2-linked EthN-P in PIGB-KO cells and also in small fraction of wild type cellsTo clarify structural basis of PIGG-dependent CD59, we established the HEK293 cells highly expressing HFGF-CD59 (CD59 fused with epitope tags and GST for easy purification) and knocked out PIGB in these cells and isolated KO cell clones. HFGF-CD59 proteins were solubilized by PI-PLC-treatment from the wild type and the PIGB-KO cells, affinity-purified from the supernatants and subjected to SDS-PAGE and in-gel trypsin digestion. The C-terminal peptide linked to GPI glycan was determined by LC-ESI-mass spectrometry (MS). In a sample from the PIGB-KO cells, precursor ions corresponding to the 94A novel structure of GPI anchored proteins andits physiological roleYoshiko MurakamiResearch Institute for Microbial Diseases, Osaka University
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