Modeling Congenital Disorders of N-Linked Glycoprotein Glycosylation in Drosophila melanogaster

Abstract

Protein glycosylation, the enzymatic addition of N-linked or O-linked glycans to proteins, serves crucial functions in animal cells and requires the action of glycosyltransferases, glycosidases and nucleotide-sugar transporters, localized in the endoplasmic reticulum and Golgi apparatus. Congenital Disorders of Glycosylation (CDGs) comprise a family of multisystemic diseases caused by mutations in genes encoding proteins involved in glycosylation pathways. CDGs are classified into two large groups. Type I CDGs affect the synthesis of the dolichol-linked Glc₃Man₉GlcNac₂ precursor of N-linked glycosylation or its transfer to acceptor proteins. Type II CDG (CDG-II) diseases impair either the trimming of the N-linked oligosaccharide, the addition of terminal glycans or the biosynthesis of O-linked oligosaccharides, which occur in the Golgi apparatus. So far, over 100 distinct forms of CDGs are known, with the majority of them characterized by neurological defects including mental retardation, seizures and hypotonia. Yet, it is unclear how defective glycosylation causes the pathology of CDGs. This issue can be only addressed by developing animal models of specific CDGs. Drosophila melanogaster is emerging as a highly suitable organism for analyzing glycan-dependent functions in the central nervous system (CNS) and the involvement of N-glycosylation in neuropathologies. In this review we illustrate recent work that highlights the genetic and neurobiologic advantages offered by D. melanogaster for dissecting glycosylation pathways and modeling CDG pathophysiology.

Keywords: Drosophila; Golgi; congenital disorders; glycosylation; model organism.

FIGURE 1

N-linked glycosylation pathway. Biosynthesis of the N-linked precursor glycan begins on the cytoplasmic face of the ER where a GlcNAc residue is added in a pyrophosphate linkage to dolichol, an isoprenoid lipid. The GlcNAc-P-P-Dol is extended to form Man₅GlcNAc₂-P-P-Dol which is then flipped so that the glycan moiety is within the lumen of the ER. Further extension produces a Glc₃Man₉GlcNAc₂-P-P-Dol that is a substrate for the oligosaccharyltransferase (OST) complex, which transfers the precursor glycan en bloc to a nascent polypeptide. This figure depicts the glycosylation of a glycoprotein (brown) with 3 N-linked glycosylation sites (labeled 1, 2, and 3). Once transfered to protein, the glycan precursor is trimmed of its Glc residues during folding as part of the calnexin/calreticulin quality control cycle. CDG Type I mutations affect the biosynthesis of the precursor glycan, its transfer to protein, and early trimming steps. Once successfully folded, glycoproteins bearing high-Man glycans are transported to the Golgi apparatus where Man trimming occurs. In the early cis Golgi, high-Man glycans can be trimmed to Man₅GlcNAc₂ by complete removal of Man residues on the α3 arm and partial removal of Man residues on the α6 arm. In the medial Golgi, the first committed step toward production of a complex glycan is taken; GlcNAcT1 adds a GlcNAc to the α3 Man residue to form a hybrid type glycan (site 1 retains this structure). The GlcNAc-extended Man₅GlcNAc₂ glycan can be core fucosylated by the addition of a Fuc residue to the internal GlcNAc (site 2). In Drosophila and other arthropods, a second Fuc residue can be added (site 3). Additional Man trimming by Golgi mannosidases provide substrates for branching in the medial and trans Golgi (site 2). Subsequent extention with Gal and capping with sialic acid (shown here, as N-acetylneuraminic acid, NeuAc) completes the maturation of complex N-linked glycans. Hybrid glycans can also be extended on the α3 arm (site 1). The abundance of hybrid and complex glycans is reduced in Drosophila compared to vertebrate species due to the presence of an hexosaminidase that removes the GlcNAc added by GlcNAcT1, thereby blocking additional branching/extension and producing a paucimanose glycan (site 3). CDG Type II mutations impact the availability of substrates and the activity of enzymes that process N-glycans in the Golgi apparatus. Graphical representation of monosaccharide residues and glycan structures is consistent with the Symbol Nomenclature For Glycans (SNFG), which has been broadly adopted by the glycobiology community (Varki et al., 2015).

FIGURE 2

Drosophila melanogaster as a model system to study glycoprotein N-glycosylation (A) Representation of Drosophila and vertebrate N-glycome characteristics. N-linked glycans are scaled proportionally to their relative abundance. The major reason for the high-mannose and pauci-mannose dominance in the Drosophila N-linked glycan profile is the existence of an arthropod-specific, N-acetylhexosaminidase known as Fused lobes (Fdl), which converts the precursor for complex glycans (GlcNAc₁Man₃₋₅GlcNAc₂-Protein) to a paucimannose structure (Man₃₋₅GlcNAc₂-Protein) that cannot be extended further. Glycoprotein glycans that escape Fdl are fully capable of being processed into complex structures. (B) Human glycosylation disorders and phenotypic characteristics of the Drosophila model.