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Galactose-specific C-type lectin: Biological Overview | References

The synaptic cleft manifests enriched glycosylation, with structured glycans coordinating signaling between presynaptic and postsynaptic cells. Glycosylated signaling ligands orchestrating communication are tightly regulated by secreted glycan-binding lectins. Using the Drosophila neuromuscular junction (NMJ) as a model glutamatergic synapse, this study identified a new Ca2+-binding (C-type) lectin, Lectin-galC1 (LGC1), which modulates presynaptic function and neurotransmission strength. LGC1 is enriched in motoneuron presynaptic boutons and secreted into the NMJ extracellular synaptomatrix. LGC1 limits locomotor peristalsis and coordinated movement speed, with a specific requirement for synaptic function, but not NMJ architecture. LGC1 controls neurotransmission strength by limiting presynaptic active zone (AZ) and postsynaptic glutamate receptor (GluR) aligned synapse number, reducing both spontaneous and stimulation-evoked synaptic vesicle (SV) release, and capping SV cycling rate. During high-frequency stimulation (HFS), mutants have faster synaptic depression and impaired recovery while replenishing depleted SV pools. Although LGC1 removal increases the number of glutamatergic synapses, this study found that LGC1-null mutants exhibit decreased SV density within presynaptic boutons, particularly SV pools at presynaptic active zones. Thus, LGC1 regulates NMJ neurotransmission to modulate coordinated movement (Bhimreddy, 2021).

Synapse assembly and subsequent neurotransmission strength are dependent on secreted bidirectional signaling pathways, which communicate between presynaptic and postsynaptic partners to coordinate synaptic development, function and plasticity. Both synaptic cleft and the surrounding extracellular perisynaptic space (the 'synaptomatrix') are characterized by heavy glycosylation, which has been shown over the last half-century with extensive lectin imaging. Synaptic signals are both mediated by, and regulated via, secreted glycoproteins (GPs), proteoglycans (PGs) and endogenous glycan-binding lectins. Disruption of these synaptic glycan-binding and modifying mechanisms is linked to numerous heritable neurological diseases, which include autism spectrum disorder (ASD) and intellectual disability (ID) states such as fragile X syndrome (FXS), movement and degeneration disorders such as hereditary spastic paraplegias (HSPs), and most broadly, the rapidly expanding congenital disorders of glycosylation. Secreted lectins play important roles in synaptic processes, including the secreted Ca2+-binding (C-type) lectins first identified by Kurt Drickamer. For example, the Drosophila Mind-the-Gap (MTG) patterns synaptomatrix glycans, regulates glutamate receptor (GluR) clustering, and modulates trans-synaptic signaling to control neurotransmission strength (Bhimreddy, 2021).

This study has identified a second C-type lectin, Lectin-galC1 (LGC1), in a whole-genome transgenic Drosophila RNAi screen for genes regulating synapse structure and function at the neuromuscular junction (NMJ). LGC1 is one of 33 C-type lectins encoded by the Drosophila genome, which are classified by their binding to extracellular β-galactoside glycans via a single characteristic Ca2+-dependent carbohydrate-binding domain (CBD). Similar to the many other lectins encoded by the Drosophila genome, LGC1 exists within a gene cluster with two other duplicated C-type lectin gene homologs. However, these two neighbors, with homologies of 59% and 24% respectively, show both differential expression and function (Tanji, 2006). Previous LGC1 studies have revealed roles in development and cellular defense, showing enhanced expression in imaginal discs during pupation, binding to surface polysaccharide chains on gram-negative bacteria, and upregulation in response to injury due to an upstream nuclear factor κB-binding site, which is a common regulator of immune defense genes. Consistent with other glycosylation mechanisms, LGC1 has a pleiotropic role in neurotransmission (Scott and Panin, 2014). The current RNAi screen identification of LGC1 indicated critical roles in the neuromusculature, and specifically at NMJ synapses, with increased neurotransmission strength following LGC1 knockdown (Dani, 2012). The goal of the current study was to pursue this lead by generating selective tools to test LGC1 requirements in coordinated movement and NMJ neurotransmission mechanisms (Bhimreddy, 2021).

This study generated a LGC1 loss-of-function null allele using CRISPR/Cas9 genome editing by creating a frame-shift near the beginning of the coding sequence. To characterize neuromuscular roles, this null was combined with a characterized LGC1 deficiency. A LGC1::GFP fusion and specific LGC1 antibody were generated to reveal LGC1 protein at the NMJ, in presynaptic boutons and secreted into the extracellular synaptomatrix. Consistent with this NMJ localization, it was found that loss of LGC1 caused faster locomotory muscle peristalsis and much more rapid coordinated movement. Previously, LGC1 RNAi was used to show increased NMJ excitatory junction current (EJC) transmission, but no change in synaptic architecture (Dani, 2012). The current study confirmed these preliminary findings in null mutants employing confocal imaging and two-electrode voltage-clamp (TEVC) electrophysiology. Although the loss of LGC1 did not alter NMJ architecture, it did increase synapse number. Consistently, LGC1 mutants showed higher spontaneous synaptic vesicle (SV) fusion rates as well as elevated motor nerve stimulation-evoked EJC amplitudes. To test mechanisms, FM1-43 dye imaging was used to reveal faster SV cycling dynamics with smaller SV pools in the mutants. With high-frequency stimulation (HFS), faster depression and slower recovery were found. Both confocal and electron microscopy revealed smaller membrane-associated SV pools at mutant active zones, consistent with faster activity-dependent SV cycling. Taken together, these results indicate that LGC1 plays a role in presynaptic SV release dynamics to cap NMJ neurotransmission strength and thus limit motor function (Bhimreddy, 2021).

Anterograde and retrograde signals secreted into the NMJ synaptomatrix by both motor neuron and muscle cells orchestrate intercellular communication at the synapse. The signaling ligands, their receptors and other molecules in the synaptomatrix environment are all very highly glycosylated, and mutation of many genes affecting glycosylation has strong effects on both coordinated movement and NMJ synaptic function. Glycan-binding lectins interact with the NMJ synaptomatrix at many levels, to effectively modulate NMJ neurotransmission strength. In the extracellular space, lectins serve to regulate the distribution of glycans (as scaffolds, co-receptors and signaling platforms), thereby directing secreted and transmembrane protein. For example, the secreted C-type lectin Mind-the-Gap (MTG) was extensively characterized at the Drosophila NMJ. MTG regulates glycan distribution, secreted trans-synaptic signaling ligands, synaptic position-specific (PS) integrins and the postsynaptic glutamate receptor (GluR) field, as well as the downstream internal Drosophila Pix–Pak–Dock signaling cascade and the critical DLG synaptic scaffold. These secreted MTG C-type lectin roles controlling postsynaptic function suggest that similar C-type lectin roles may govern presynaptic function (Bhimreddy, 2021).

In a previous systematic transgenic RNAi screen for NMJ modulator genes, a new secreted C-type lectin was uncovered that strongly controls neurotransmission strength (Dani, 2012). Lectin-galC1 (LGC1) is an ideal candidate for modulation of synaptic function due to its characterized Ca2+-binding (C-type) and glycan-binding properties as a secreted protein (Tanji, 2006; Haq, 1996). To create a LGC1-specific null mutation, CRISPR/Cas9 homology-directed DNA repair was used to create a targeted frame-shift mutation. This LGC1 null mutant was paired with a deletion removing the C-type lectin gene cluster containing LGC1 to characterize a new specific LGC1 antibody and to show that LGC1 is locally secreted at the NMJ. Then the effects were studied of LGC1 loss on coordinated movement, which intimately depends on both NMJ synaptic architecture and function. Consistent with the initial discovery of increased NMJ neurotransmission following LGC1 RNAi knockdown (Dani, 2012), and earlier studies demonstrating LGC1 Ca2+-dependent binding functions (Tanji, 2006; Haq, 1996), this study reports that LGC1-null mutants exhibit accelerated movement. LGC1 loss increases both the rate of locomotion and the speed to complete complex coordinated behaviors when challenged. These findings suggest LGC1 acting locally at the NMJ is important for modulating locomotion and, particularly, complex coordinated motor function (Bhimreddy, 2021).

LGC1 is secreted at the NMJ, as demonstrated using non-permeabilized labeling, and concentrated within the extracellular synaptomatrix surrounding synaptic boutons; a space characterized by striking glycan accumulation. LGC1-null mutants have absolutely no alterations in NMJ structure. This is surprising, since NMJ overelaboration is a common feature of glycosylation mutations (Rushton, 2020). For example, mutations in the key phosphomannomutase type 2 (PMM2) glycosylation enzyme strongly alter synaptomatrix pauci-mannose glycans to cause significant NMJ architectural expansion, including more synaptic branching and supernumerary boutons. Similarly, loss of UDP-sugar precursors, which are required for synaptic glycosylation also results in obvious NMJ structural overelaboration, with both increased branching and synaptic bouton number (Jumbo-Lucioni, 2016). However, synaptic structure and function are independently regulated, with separable glycan roles. Consistently, LGC1-null mutants exhibit an elevated number of synapses within otherwise normal NMJ boutons, thereby exhibiting greater synaptic density. LGC1 nulls have more presynaptic BRP-positive active zones (AZs) and more apposed postsynaptic GluR fields, suggesting that a higher level of synaptic connectivity contributes to elevated neurotransmission strength (Bhimreddy, 2021).

LGC1 limits NMJ function by reducing SV fusion and evoked transmission. An elevated LGC1 EJC amplitude reflects an increase in number of released vesicles (quantal content) in a more mobile readily releasable pool (RRP). Since spontaneous mEJC amplitude does not change in LGC1 nulls, an increase in postsynaptic GluR number or function is ruled out as a contributing factor. In addition to increased synapse density, it was hypothesized that there would be higher SV fusion probability from altered SV cycling in LGC1 mutants. FM1-43 dye imaging shows that LGC1 loss results in a smaller cycling SV pool with higher cycling rate. A smaller SV pool could negate the effects of increased synapse number and SV fusion probability. However, most SVs do not participate detectably in transmission cycling, and smaller SV pools can show high transmission due to increased synaptic density and SV cycling rates. Moreover, altered SV endocytosis–exocytosis cycling efficacy can cause altered synaptic depression/recovery with high-frequency stimulation (HFS). Stimulation frequency can change SV cycling dynamics, with higher frequencies triggering differential SV release and recovery mechanisms. Consequently, NMJ function (depression and recovery) was assayed during varying levels of frequency stimulation, to stress neurotransmission machinery, and thus gain insight into the LGC1 mutant SV cycling mechanisms (Bhimreddy, 2021).

In LGC1 nulls, HFS causes more rapid fatigue and slower recovery. Consistent with this, smaller SV cycling pools with higher turnover rates can increase basal transmission strength while impairing transmission maintenance during HFS demand. Thus, the smaller cycling SV pool in LGC1 mutants can maintain elevated basal transmission while exhibiting impaired replenishment of SV pools and maintenance of transmission during HFS. Other glutamatergic synapse classes maintain a small cycling RRP for high probability SV release during basal stimulation conditions, yet are unable to maintain transmission under conditions of greater demand. Likewise, LGC1 nulls exhibit reduced membrane-associated SV pools at both the light microscope and electron microscope levels, including lower SV density at AZs. Other Drosophila mutants with increased NMJ neurotransmission can similarly show presynaptic SV depletion in TEM ultrastructural studies. At the AZ, SVs near the presynaptic membrane make up the RRP and linked cycling pools, but internal SV pools can be held in 'reserve' or serve other functions apart from neurotransmitter release. Overall, these findings are consistent with LGC1 mutants exhibiting a smaller and faster SV cycling pool, with altered SV cycle dynamics during and following HFS stress (Bhimreddy, 2021).

Similar to what is seen with LGC1 mutants, the loss of a number of C-type lectin domain (CTLD) proteins causes synaptic defects. For example, Mincle-deficient mice suppress TNF induction, which lowers glutamate release from central synapses, resulting in a direct modulation of neurotransmission strength. Although LGC1 appears to have the opposite effect, the Drosophila TNF homolog (Eiger) downregulates excitatory amino acid transporters 1 and 2 (EAAT1/2), and the EAAT1 mutation impairs locomotory peristaltic movement. This show'that LGC1 limits locomotory peristaltic movement, suggesting a possible relationship between LGC1, Eiger and EAAT1 may be a useful avenue to explore in future studies. Another CTLD protein is Caenorhabditis elegans CLEC-38, which has been shown to regulate presynaptic organization and mediate changes in the integral SV protein Synaptobrevin. As the vesicle SNARE (V-SNARE), Synaptobrevin drives SV fusion with the presynaptic membrane AZ to directly mediate neurotransmitter release. Since LGC1 mutants display neurotransmission defects comparable to CLEC-38 mutants, such as similar increased SV fusion and impaired SV cycling, the exploration of downstream LGC1 interactions and Synaptobrevin regulation in the presynaptic terminal could offer more insight into the mechanisms by which LGC1 produces neurotransmission changes (Bhimreddy, 2021).

In conclusion, this study provides insight into the roles of a novel C-type lectin at a glutamatergic neuromuscular synapse. Beyond the previously characterized roles in development and immunity (Tanji, 2006; Haq, 1996), this study found LGC1 secreted at the NMJ synapse modulates presynaptic function. Along with acting to limit synapse number and neurotransmission strength, LGC1 also regulates SV cycling to increase SV availability during basal levels of use, but decrease SV availability during high levels of use. In resting basal conditions, LGC1 caps NMJ neurotransmission strength to limit coordinated motor function. With intense stimulation, LGC1 loss causes more fatigue and decreases the recovery rate, indicating impaired activity-dependent SV cycling. This study revealed functional defects with electrophysiology, and visualized SVs with both FM1-43 dye and TEM electron microscopy imaging. In future studies, it is planned to dissect LGC1 mechanisms by focusing on SV pools and trafficking, as well as testing the possible involvement in signaling via Eiger, EAAT1 and downstream SV Synaptobrevin changes. This work indicates that LGC1-dependent signaling fine-tunes the multiple presynaptic pools that exchange SVs differentially based on activity levels and release demand to control SV release probability. Beyond this LGC1 mechanism, this new model will allow further exploration of SV cycling-dependent NMJ function in neuromuscular disease states (Bhimreddy, 2021).

Participation of a galactose-specific C-type lectin in Drosophila immunity

A galactose-specific C-type lectin has been purified from a pupal extract of Drosophila melanogaster. This lectin gene, named DL1 (Drosophila lectin 1; Lectin-galC1), is part of a gene cluster with the other two galactose-specific C-type lectin genes, named DL2 (Drosophila lectin 2) and DL3 (Drosophila lectin 3). These three genes are expressed differentially in fruit fly, but show similar haemagglutinating activities. The present study characterized the biochemical and biological properties of the DL1 protein. The recombinant DL1 protein bound to Escherichia coli and Erwinia chrysanthemi, but not to other Gram-negative or any other kinds of microbial strains that have been investigated. In addition, DL1 agglutinated E. coli and markedly intensified the association of a Drosophila haemocytes-derived cell line with E. coli. For in vivo genetic analysis of the lectin genes, this study also established a null-mutant Drosophila. The induction of inducible antibacterial peptide genes was not impaired in the DL1 mutant, suggesting that the galactose-specific C-type lectin does not participate in the induction of antibacterial peptides, but possibly participates in the immune response via the haemocyte-mediated mechanism (Tanji, 2006).

Purification, characterization, and cDNA cloning of a galactose-specific C-type lectin from Drosophila melanogaster

A lectin was purified from a pupal extract of Drosophila melanogaster. This lectin agglutinated trypsinized and glutaraldehyde-fixed bovine red blood cells in the presence of calcium or magnesium. The hapten sugar of this lectin was galactose. The molecular mass of the intact lectin was determined to be 41 kDa, and it comprised 14- and 17-kDa subunits. The 17-kDa subunit was shown to be a glycosylated form of the 14-kDa subunit. Analysis of the cDNA for this lectin revealed that the 14-kDa subunit consists of 163 amino acid residues and contains all residues conserved in various C-type lectins. It was suggested that the Drosophila lectin and Sarcophaga lectin share some properties and function similarly in defense and development, but probably they are not structural homologues (Haq, 1996).

Search PubMed for articles about Drosophila Lectin-galC1

Bhimreddy, M., Rushton, E., Kopke, D. L. and Broadie, K. (2021). Secreted C-type lectin regulation of neuromuscular junction synaptic vesicle dynamics modulates coordinated movement. J Cell Sci 134(9). PubMed ID: 33973638

Dani, N., Nahm, M., Lee, S. and Broadie, K. (2012). A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling. PLoS Genet 8(11): e1003031. PubMed ID: 23144627

Fischler, W., Kong, P., Marella, S. and Scott, K. (2007). The detection of carbonation by the Drosophila gustatory system. Nature 448(7157): 1054-1057. PubMed ID: 17728758

Haq, S., Kubo, T., Kurata, S., Kobayashi, A. and Natori, S. (1996). Purification, characterization, and cDNA cloning of a galactose-specific C-type lectin from Drosophila melanogaster. J Biol Chem 271(33): 20213-20218. PubMed ID: 8702748

Jumbo-Lucioni, P. P., Parkinson, W. M., Kopke, D. L. and Broadie, K. (2016). Coordinated movement, neuromuscular synaptogenesis and trans-synaptic signaling defects in Drosophila galactosemia models. Hum Mol Genet 25(17): 3699-3714. PubMed ID: 27466186

Rushton, E., Kopke, D. L. and Broadie, K. (2020). Extracellular heparan sulfate proteoglycans and glycan-binding lectins orchestrate trans-synaptic signaling. J Cell Sci 133(15). PubMed ID: 32788209

Scott, H. and Panin, V. M. (2014). The role of protein N-glycosylation in neural transmission. Glycobiology 24(5): 407-417. PubMed ID: 24643084

Tanji, T., Ohashi-Kobayashi, A. and Natori, S. (2006). Participation of a galactose-specific C-type lectin in Drosophila immunity. Biochem J 396(1): 127-138. PubMed ID: 16475980

date revised: 15 August 2022

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