Ethanolamine‐phosphate on the second mannose is a preferential bridge for some GPI‐anchored proteins

• Published in: EMBO reports Vol. 23; no. 7; pp. e54352 - n/a
• Main Authors: Ishida, Mizuki, Maki, Yuta, Ninomiya, Akinori, Takada, Yoko, Campeau, Philippe, Kinoshita, Taroh, Murakami, Yoshiko
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 05.07.2022; Springer Nature B.V; John Wiley and Sons Inc
• Subjects: Animals; Attachment; CD59 antigen; CD73; Cell surface; EMBO20; EMBO24; Ethanolamine; Ethanolamines - metabolism; Glycan; Glycolipids; Glycosylphosphatidylinositol; Glycosylphosphatidylinositols - genetics; Glycosylphosphatidylinositols - metabolism; GPI-Linked Proteins - genetics; inherited GPI deficiency; Localization; Mammals - metabolism; Mannose; Mannose - metabolism; mass spectrometry; Mutation; Nucleotidase; Phosphates; PIGB; PIGG; Proteins; PIGB; mass spectrometry; CD73; inherited GPI deficiency; PIGG
• ISSN: 1469-221X; 1469-3178; 1469-3178
• DOI: 10.15252/embr.202154352

Glycosylphosphatidylinositols (GPIs) are glycolipids that anchor many proteins (GPI‐APs) on the cell surface. The core glycan of GPI precursor has three mannoses, which in mammals, are all modified by ethanolamine‐phosphate (EthN‐P). It is postulated that EthN‐P on the third mannose (EthN‐P‐Man3) is the bridge between GPI and the protein and the second (EthN‐P‐Man2) is removed after GPI‐protein attachment. However, EthN‐P‐Man2 may not be always transient, as mutations of PIGG, the enzyme that transfers EthN‐P to Man2, result in inherited GPI deficiencies (IGDs), characterized by neuronal dysfunctions. Here, we show that EthN‐P on Man2 is the preferential bridge in some GPI‐APs, among them, the Ect‐5’‐nucleotidase and Netrin G2. We find that CD59, a GPI‐AP, is attached via EthN‐P‐Man2 both in PIGB ‐knockout cells, in which GPI lacks Man3, and with a small fraction in wild‐type cells. Our findings modify the current view of GPI anchoring and provide a mechanistic basis for IGDs caused by PIGG mutations. Synopsis This study challenges the dogma that GPI‐anchored proteins are always attached to the third mannose in GPI via an ethanolamine‐phosphate (EthN‐P) bridge by providing evidence for an alternative attachment to the second mannose via EthN‐P. Some proteins preferentially use the alternative mode of attachment to GPI, highlighting the importance of the EthN‐P transferred to the second mannose by PIGG. In cells that lack either the third mannose in GPI (PIGB‐KO) or the EthN‐P linked to the third mannose (PIGO‐KO), the GPI‐anchored proteins CD59 and DAF are attached to EthN‐P linked to the second mannose, allowing their membrane localization at low levels. Approximately 10% of CD59 on wild‐type HEK293 cells are similarly attached to the second mannose via EthN‐P. NT5E and NetrinG2, in contrast to CD59, are preferentially attached to the EthN‐P linked to the second mannose. Graphical Abstract This study challenges the dogma that GPI‐anchored proteins are always attached to the third mannose in GPI via an ethanolamine‐phosphate (EthN‐P) bridge by providing evidence for an alternative attachment to the second mannose via EthN‐P. Some proteins preferentially use the alternative mode of attachment to GPI, highlighting the importance of the EthN‐P transferred to the second mannose by PIGG.