Protein O-mannosylation: one sugar, several pathways, many functions

Abstract

Recent research has unveiled numerous important functions of protein glycosylation in development, homeostasis, and diseases. A type of glycosylation taking the center stage is protein O-mannosylation, a posttranslational modification conserved in a wide range of organisms, from yeast to humans. In animals, protein O-mannosylation plays a crucial role in the nervous system, whereas protein O-mannosylation defects cause severe neurological abnormalities and congenital muscular dystrophies. However, the molecular and cellular mechanisms underlying protein O-mannosylation functions and biosynthesis remain not well understood. This review outlines recent studies on protein O-mannosylation while focusing on the functions in the nervous system, summarizes the current knowledge about protein O-mannosylation biosynthesis, and discusses the pathologies associated with protein O-mannosylation defects. The evolutionary perspective revealed by studies in the Drosophila model system are also highlighted. Finally, the review touches upon important knowledge gaps in the field and discusses critical questions for future research on the molecular and cellular mechanisms associated with protein O-mannosylation functions.

Keywords: Drosophila model system; matriglycan; protein O-mannosylation; protein O-mannosyltransferases; receptor protein tyrosine phosphatase.

Fig. 1

Three families of enzymes mediating POM in animal cells. POMT1/2 O-mannosylate α-DG, RPTPs, KIAA1549, and some other proteins, whereas TMTC1–4 specialize in attaching O-mannose to the EC domains of cadherins. TMEM260, a recently discovered O-mannosyltransferase, is responsible for modifying the IPT domains of plexins and transmembrane receptor tyrosine kinases RON and MET. All these POM-mediating enzymes work in the ER and use Dol-P-Man as a donor substrate. They have a similar molecular architecture of integral membrane proteins with multiple membrane-spanning helixes, catalytically important aspartic acid residues in the first luminal loop (blue circles), and include different functional domains that are thought to be involved in substrate interactions (such as MIR and TPR). The substrate recognition of the enzymes remains not well understood. Modified from Larsen et al. (2019).

Fig. 2

Biosynthesis of POM. POM is initiated in the ER by three families of O-mannosyltransferases: POMT1/2, TMTC1–4 (transmembrane O-mannosyltransferases targeting cadherins), and TMEM260. Depending on a protein substrate, the O-mannose attached to a protein can remain non-elongated (M0 structure), or undergo further modification, such as elongation in the Golgi with β1,2-GlcNAc by POMGnT1 (protein O-mannose β1, 2-N-acetylglucosaminyltransferase 1), which creates core M1 structure, and additional modification with β1,6-GlcNAc by MGAT5B (α1,6-Mannosylglycoprotein 6-β-N-Acetylglucosaminyltransferase B), which creates core M2. M1 and M2 are further modified by enzymes that are not specific for POM, such as a galactosyltransferase, a sialyltransferase, etc., which results in structures with terminal sialic acid, HNK-1 (human natural killer 1 carbohydrate HSO₃-3GlcAβ1-3Galβ1-4GlcNAc-), or Lewis^X (Galβ1-4(Fucα1-3)GlcNAc-) epitopes. As an alternative to M1/M2 biosynthesis, the O-mannose can be modified in the ER with β1,2-GlcNAc by POMGnT2 (protein O-mannose β1,4-N-acetylglucosaminyltransferase 2), which creates core M3 and allows for further modification by the enzymes involved in the biosynthesis of matriglycan: B3GALNT2, POMK, FKTN (ribitol-5-phosphate transferase), FKRP (ribitol-5-phosphate transferase), RXYLT1, B4GAT1 (β1,4-glucuronyltransferase 1), and LARGE (β1,3-glucuronyltransferase and α1,3-xylosyltransferase, a bifunctional glycosyltransferase-polymerase that creates a long chain of -3GlcAβ1-3Xylα1- disaccharide repeats).

Fig. 3

Modification of α-DG with matriglycan is essential for interaction of the DGC complex with extracellular ligands. Left panel: α-DG ligands imbedded in the ECM, such as Laminin, Agrin, etc., bind to matriglycan-modified O-mannosyl glycans. Inside the cell, DGC interacts with actin filaments via dystrophin, which creates a DGC-mediated bridge between the basal lamina outside the cell and the cytoskeleton inside the cell. Note that matriglycan is also specifically recognized by IIH6 IgM antibody in vitro and by some viruses (e.g. Lassa virus) that use binding to matriglycan as a mechanism for cell infection. In POMT mutants (right panel), the O-mannosylation of α-DG is abolished, which disrupts α-DG interactions with the ECM ligands, leading to muscular dystrophy phenotypes (dystroglycanopathy).

Fig. 4

Phylogenetic trees of animal POMT1–2 and TMTC1–4 enzymes. A, Phylogenetic tree of Drosophila, mouse, and human POMTs. RT (Rotated Abdomen), Drosophila POMT1; TW (Twisted), Drosophila POMT2 (modified from Nakamura et al. 2010a). (B) Phylogenetic tree of Drosophila, mouse, and human TMTCs. The trees were built based on multiple sequence alignments performed using the EMBL-EBI Clustal Omega server, followed by distance-based construction of phylogenic trees using a neighbor joining algorithm ((https://www.ebi.ac.uk/Tools/msa/clustalo/). The trees were visualized using the FigTree software (http://tree.bio.ed.ac.uk/software/figtree/). Scale bars, phylogenetic distance expressed as substitutions per site.