Recent advances in sequencing technologies have led to the discovery of the genetic causes of many new diseases. Here, we will focus on Disorders of Glycosphingolipid and GPI-Anchor Glycosylation. Defects in the glycosylphosphatidylinositol biosynthesis pathway can result in congenital disorders of glycosylation. CD BioGlyco provides comprehensive and deep insights into disorders of GPI anchor biosynthesis, such as Phosphosphingolipid Analysis, Ganglioside Analysis, and Sulfoglycosphingolipid Analysis.

Overview of GPI Anchor Biosynthesis

Glycosylphosphatidylinositols (GPIs) are membrane-anchored glycolipids for a variety of cell surface proteins. The GPI-anchor biosynthetic pathway allows the covalent attachment of glycolipids to the C-terminus of nascent proteins, which is a post-translational modification.

GPI-anchor biosynthesis can be divided into three stages. The first stage is the biosynthesis of GPI anchors. In the first part, a common core structure of GPI anchors was synthesized in the endoplasmic reticulum through 10 steps. The structure is composed of a phosphatidylinositol (PI) molecule and a glycan moiety containing glucosamine, three mannoses, and one ethanolamine phosphate. This leads to the stepwise construction of GPI anchor precursors. The second stage is the attachment of proteins and GPI anchors. In this stage, the preassembled GPI anchor is transferred to the carboxy terminus of the protein, which is a transamidase complex consisting of five components (PIGK, hGAA1, PIGS, PIGT, and PIGU) substance-mediated. The result is that GPIs attach to newly synthesized proteins in the lumen of the endoplasmic reticulum. The third is the remodeling of glycosylphosphatidylinositol-anchored proteins. Protein-bound GPI anchors are transported from the endoplasmic reticulum through the Golgi apparatus to the cell surface. During this process, some modification of the glycan, lipid moiety, inositol-linked acyl chain, and ethanolamine phosphate occurs on the second mannose.

Fig.1 A scheme for the overall GPI- anchored proteins biosynthetic pathway, structural remodeling and transport. (Wu, et al., 2020)

About 150 proteins are GPI-anchored, including enzymes, structural molecules, receptors, and regulatory proteins. Misregulation of GPI-anchored proteins occurs as a result of mutations in the GPI biosynthetic pathway, resulting in multiple phenotypes observed in inherited GPI deficiencies (IGDs).

Fig.2 GPI biosynthesis pathway. (Carmody, et al., 2020)

Description of Disorders of GPI Anchor Biosynthesis

Defects in the GPI-anchor biosynthetic pathway can lead to congenital disorders of glycosylation (CDG) in IGD, a relatively new subclass of (CDG). IGD is the result of a mutation in less than 30 genes encoding parts of the GPI biosynthetic pathway. To date, defects in 22 genes in the GPI biosynthetic pathway have been found in IGD.

Defects in GPI-anchor biosynthesis lead to rare genetic disorders including neurological symptoms, especially developmental delay, intellectual disability, epilepsy, deformities, hypotonia, hearing loss, elevated alkaline phosphatase, and many other congenital abnormalities. Substate mutations in multiple genes in this pathway lead to partial reductions in GPI-anchored proteins. Non-neurological phenotypes include renal abnormalities, cleft palate, cardiac defects, and hypertelorism. In a minority of individuals, symptoms manifest include iron deposition, hepatosplenomegaly, and portal vein thrombosis. Furthermore, complete deletion of the GPI pathway in mice resulted in embryonic lethality.

GPI-Anchored Protein Analysis to Improve the Diagnosis of IGD

Imaging techniques can be used to observe the lateral mobility of GPI-anchored proteins on the cell surface. Fluorescently labeled inactive toxin aerolysin (FLAER) is a useful fluorescent reagent that binds directly to GPI anchors in the cytoplasmic membrane. FLAER can be labeled with Alexa Fluor 488 dye and bind to the glycan moiety of the GPI anchor. Flow cytometry analysis is used to determine the presence or absence of GPI-anchored proteins on the cell surface. Combining FLAER-based flow cytometry with other multiparameter flow cytometers can further improve the diagnosis of IGD.

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References:

1. Wu, T.; et al. The Glycosylphosphatidylinositol biosynthesis pathway in human diseases. Orphanet Journal of Rare Diseases. 2020, 15(1): 1-11.
2. Carmody, L.C.; et al. Significantly different clinical phenotypes associated with mutations in synthesis and transamidase+ remodeling glycosylphosphatidylinositol (GPI)-anchor biosynthesis genes. Orphanet journal of rare diseases. 2020, 15(1): 1-13.