Structure‐function analysis of endogenous lectin mind‐the‐gap in synaptogenesis

Abstract

Mind-the-Gap (MTG) is required for neuronal induction of Drosophila neuromuscular junction (NMJ) postsynaptic domains, including glutamate receptor (GluR) localization. We have previously hypothesized that MTG is secreted from the presynaptic terminal to reside in the synaptic cleft, where it binds glycans to organize the heavily-glycosylated, extracellular synaptomatrix required for trans-synaptic signaling between neuron and muscle. In this study, we test this hypothesis with MTG structure-function analyses of predicted signal peptide (SP) and carbohydrate-binding domain (CBD), by introducing deletion and point-mutant transgenic constructs into mtg null mutants. We show that the SP is required for MTG secretion and localization to synapses in vivo. We further show that the CBD is required to restrict MTG diffusion in the extracellular synaptomatrix and for postembryonic viability. However, CBD mutation results in elevation of postsynaptic GluR localization during synaptogenesis, not the mtg null mutant phenotype of reduced GluRs as predicted by our hypothesis, suggesting that proper synaptic localization of MTG limits GluR recruitment. In further testing CBD requirements, we show that MTG binds N-acetylglucosamine (GlcNAc) in a Ca2+-dependent manner, and thereby binds HRP-epitope glycans, but that these carbohydrate interactions do not require the CBD. We conclude that the MTG lectin has both positive and negative binding interactions with glycans in the extracellular synaptic domain, which both facilitate and limit GluR localization during NMJ embryonic synaptogenesis.

N-glycosylation requirements in neuromuscular synaptogenesis

“…MTG regulates VVA synaptomatrix labeling (Rushton et al, 2009), suggesting a mechanistic link between the VVA and MTG changes in Mgat1 mutants. The MTG elevation observed in Mgat1 nulls provides a plausible causative mechanism for strengthened functional differentiation (Rohrbough and Broadie, 2010;Rushton et al, 2012). Consistent with our recent glycosylation gene screen findings , Mgat1 nulls exhibit increased synaptic growth and structural overelaboration.…”

“…Conversely, the secreted endogenous lectin MTG is highly elevated in Mgat1 null synaptomatrix, probably owing to attempted compensation for complex and hybrid N-glycan losses that serve as MTG binding sites. MTG binds GlcNAc in a calcium-dependent manner and pulls down a number of HRP-epitope proteins by immunoprecipitation (Rushton et al, 2012), although the specific proteins have not been identified. It will be of interest to perform immunoprecipitation on Mgat1 samples to identify changes in HRP bands.…”

“…For example, the widely employed anti-HRP antibody binds fucosylated N-glycans in the presynaptic membrane, which require Mgat1 for fucose modification (Sarkar et al, 2006). Likewise, VVA lectin is a synaptic glycan marker at the Drosophila NMJ, which reportedly recognizes primarily postsynaptic Dystroglycan (Haines et al, 2007;Rushton et al, 2012). The endogenous MTG lectin patterns the extracellular glycosylated synaptomatrix (Rohrbough et al, 2007), modulates trans-synaptic signaling and is essential for functional synaptogenesis (Rushton et al, 2009;Rohrbough and Broadie, 2010).…”

“…Drosophila genetic analyses show synaptomatrix glycan modification/binding has core roles in structural and functional development of the neuromuscular junction (NMJ) synapse (Broadie et al, 2011;. Glycan-dependent synaptogenic events include presynaptic active zone (AZ) differentiation, postsynaptic glutamate receptor (GluR) localization and extracellular matrix (ECM) organization within the synaptic cleft, driven by the secreted endogenous Mind the gap (MTG) lectin, for example Long et al, 2008;Rushton et al, 2012). Synaptogenic events are regulated by bidirectional trans-synaptic signals that traverse the synaptomatrix, and glycosylation of both ligands and receptors alters localization and binding (Henríquez and Salinas, 2012;Patton, 2003).…”

Transmission, Development, and Plasticity of Synapses

“…Furthermore, Dlg, Pak, Dock, and Pix are either reduced or severely mislocalized from mtg mutant synapses, suggesting that MTG plays a critical role upstream of these proteins to regulate GluR localization (Rohrbough et al 2007). Interestingly, when mtg mutants are rescued with MTG lacking its carbohydrate-binding domain (CBD), postsynaptic GluR accumulation increases (Rushton et al 2012). Furthermore, elimination of the CBD does not affect the ability of MTG to bind to glycans, supporting an unexpected function of this domain in limiting the PSD.…”