# Preferential use of alkyl-acyl phosphatidylinositol for GPI biosynthesis and diagnostic potential of lipidomics for inherited GPI deficiencies

## TL;DR

This study shows that specific lipid structures are preferentially used in making GPI anchors and can help diagnose inherited GPI deficiencies.

## Contribution

The study reveals the preferential use of alkyl-acyl phosphatidylinositol in GPI biosynthesis and its diagnostic potential for inherited GPI deficiencies.

## Key findings

- 1-alkyl-2-acyl PIs are preferentially used in GPI biosynthesis despite being minor cellular components.
- Disruption of GNPAT or AGPS leads to GPI-APs with only diacylglycerol.
- Lipidomic profiling can diagnose inherited GPI deficiencies by identifying specific intermediates.

## Abstract

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are attached to the cell surface via a glycolipid anchor, GPI, whose conserved core is synthesized from phosphatidylinositol (PI) in the endoplasmic reticulum through a series of enzymatic reactions. Most PI species in mammalian cells contain diacylglycerol, whereas GPI-APs predominantly possess 1-alkyl-2-acylglycerol. The basis for this characteristic lipid structure has remained unclear. Lipidomic analysis revealed that 1-alkyl-2-acyl PIs, although minor components of cellular PI, are preferentially used by GPI-N-acetylglucosaminyltransferase, which catalyzes the first step of GPI biosynthesis. GPI intermediates containing 1-alkyl-2-acylglycerol were further enriched in subsequent biosynthetic steps, resulting in mature GPIs primarily harboring this lipid species. We demonstrate that a 1-alkyl-containing precursor lipid derived from peroxisomes, likely 1-alkyl-glyceronephosphate, contributes to the formation of 1-alkyl-2-acyl PIs. Disruption of glyceronephosphate O-acyltransferase (GNPAT) or alkylglycerone phosphate synthase (AGPS), the first two enzymes of the peroxisomal ether-lipid pathway, abolished 1-alkyl-2-acyl PI, yielding GPI-APs containing only diacylglycerol. Lipidomic profiling of GPI biosynthetic intermediates in GPI-defective cells revealed accumulation of defective-step-specific intermediates, enabling the use of this approach for diagnosing inherited GPI deficiency (IGD).