Drosophila Dystroglycan is a target of O-mannosyltransferase activity of two protein O-mannosyltransferases, Rotated Abdomen and Twisted

Abstract

Recent studies highlighted an emerging possibility of using Drosophila as a model system for investigating the mechanisms of human congenital muscular dystrophies, called dystroglycanopathies, resulting from the abnormal glycosylation of alpha-dystroglycan. Several of these diseases are associated with defects in O-mannosylation, one of the most prominent types of alpha-dystroglycan glycosylation mediated by two protein O-mannosyltransferases. Drosophila appears to possess homologs of all essential components of the mammalian dystroglycan-mediated pathway; however, the glycosylation of Drosophila Dystroglycan (DG) has not yet been explored. In this study, we characterized the glycosylation of Drosophila DG using a combination of glycosidase treatments, lectin blots, trypsin digestion, and mass spectrometry analyses. Our results demonstrated that DG extracellular domain is O-mannosylated in vivo. We found that the concurrent in vivo activity of the two Drosophila protein O-mannosyltransferases, Rotated Abdomen and Twisted, is required for O-mannosylation of DG. While our experiments unambiguously determined some O-mannose sites far outside of the mucin-type domain of DG, they also provided evidence that DG bears a significant amount of O-mannosylation within its central region including the mucin-type domain, and that O-mannose can compete with O-GalNAc glycosylation of DG. We found that Rotated Abdomen and Twisted could potentiate in vivo the dominant-negative effect of DG extracellular domain expression on crossvein development, which suggests that O-mannosylation can modulate the ligand-binding activity of DG. Taken together these results demonstrated that O-mannosylation of Dystroglycan is an evolutionarily ancient mechanism conserved between Drosophila and humans, suggesting that Drosophila can be a suitable model system for studying molecular and genetic mechanisms underlying human dystroglycanopathies.

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

“…In vitro studies with peptides have further supported this (24), and here we provided evidence that elimination of POMT-driven O-mannosylation of ␣-DG in HEK293 cells results in GalNAc glycosylation at sites normally occupied by O-Man glycans, as shown previously by in vivo studies in Drosophila melanogaster (50). In normal human cells, the two glycosylation processes are topologically separated in ER and Golgi, but in cancer the GalNAc-Ts may relocate to the ER and thus potentially compete with POMTs (51,52).…”

“…In contrast, ␣-DG O-Man glycans are located in the mucin-like (50). However, these O-Man glycosites were found on ␣-DG regions poorly conserved in higher species, e.g.…”

N-glycosylation requirements in neuromuscular synaptogenesis

“…1D) (Desai et al, 1994;Beumer et al, 2002). Similarly, Dystroglycan (DG) is reportedly the primary substrate for VVA labeling at the NMJ (Haines et al, 2007;Nakamura et al, 2010). Both HRP and VVA labeling are absent at Mgat1 null NMJs (Fig.…”

“…This result suggests that the N-glycan LacdiNAc is enriched at the NMJ, and that the terminal GalNAc expected on Oglycans/glycosphingolipids may be present on N-glycans in this synaptic context. Importantly, VVA labels Dystroglycan and loss of Dystroglycan glycosylation blocks extracellular ligand binding and complex formation in Drosophila (Haines et al, 2007;Nakamura et al, 2010), and causes muscular dystrophies in humans (Ervasti et al, 1997;Muntoni et al, 2008;Tran et al, 2012). This study shows that VVA-recognized Dystroglycan glycosylation is not required for protein stabilization or synaptic localization, but did not test functionality or complex formation, which probably requires MGAT1-dependent modification.…”

Golgi Glycosylation

“…O-mannose glycans are rare and concentrated in brain and other tissues that express a-dystroglycan. They are required on a-dystroglycan for it to functionally bind to the extracellular matrix, and disruptions of O-mannose glycan synthesis lead to muscular dystrophies (Moore and Hewitt 2009;Nakamura et al 2010).…”