Final Report Summary - FLYGLYGAN (Systematic Isolation of Glycosyltransferases in Drosophila melanogaster Using The Toxicity of Fungal Lectins)
The project objective was to exploit the toxicity associated with the glycan-binding function of fungal lectins to uncover novel glycosyltransferases in the Drosophila system and to use the lectins to associate Drosophila glycan structures with their biosynthetic pathways.
One fungal lectin, MOA, showed significant toxicity when fed to Drosophila larvae. Other lectins screened (CCL2, PSL1, CGL2, CC1G_10077, XCL, AAL) were non-toxic or resulted in a subtle grow delay. To determine if the absence of toxicity was due to a lack of appropriate glycan targets for the lectins five lectins (MOA, CCL2, PSL1, CGL2 and CC1G_10077) were fluorescently labeled and used to stain fixed Drosophila tissue. Under these conditions specific binding was readily detected by microscopy analysis for all five lectins. This result indicates that Drosophila do encode the machinery necessary to generate and express target glycans for these lectins. To explain the lack of toxicity we examined the spatial distribution of the lectin staining and found that 3 non-toxic lectins (CCL2, PSL1, CGL2) that showed binding to Drosophila tissues failed to bind to larval gut indicating that a lack of glycan targets accessible to an ingested lectin as a likely explanation for the lack of toxicity. Other mechanisms must also be involved in the toxicity reaction as CC1G_10077 and MOA both readily stained larval gut tissue but only MOA was toxic.
Four lectins (CCL2, PSL1, MOA, CC1G_10077) were associated with one or more of the glycostransferease required to generate the lectin target glycan. CCL2 shown previously to bind the GlcNAcβ1,4[Fucα1,3]GlcNAc epitope (Schubert et. al., 2012) stained the larval neuromuscular junction, a known site of expression of this glycan. CCL2 staining was strongly reduced in zygotic MGat1 mutants that are unable to synthesize the GlcNAcβ1,4[Fucα1,3]GlcNAc epitope (Sarkar et. al., 2006). PSL1 has been shown to bind to sialic acid α2-6 structures (Kadirvelraj et. al., 2011) and we found staining with this lectin was restricted to a small number of cells in the central nervous system, consistent with the reported expression of dSiaT, the sole Drosophila homolog of the vertebrate sialytransferases (Koles et. al., 2004). Expression of an RNAi construct against dSiaT in the nervous system (elav-Gal4 driver) resulted in reduced staining of these cells with the lectin indicating this lectin recognizes glycan structures generated by dSiaT in flies. In Drosophila sialic acid has been identified attached to terminal β4Gal on N-glycans (Aoki et. al., 2007) and β4GalNAcTA has been suggested as the enzyme that generates the dSiaT substrate (Nakamura et. al., 2012). Accordingly the staining pattern on PSL1 in the β4GalNAcTA / β4GalNAcTB double mutant was tested and found to be identical to wild-type. This result suggests the presents of a novel β4GalT in Drosophila that lacks sequence homology to the vertebrate β4GalTs. MOA and CC1G_10077 both stained the larval gastic system and staining was reduced (CC1G_10077) or absent (MOA) in larval null mutant for the egghead gene. The requirement for egghead in generating glycosphingolipids (Wandall et. al., 2005) links these lectins to binding glycosphingolipid glycans.
MOA was found to be highly toxic to Drosophila and we find that MOA toxicity and tissue staining is dependent on enzymes that function in the initial steps of glycosphingolipid (GSL) biosynthesis (Egghead, Brainiac, β4GalNAcTA/ β4GalNAcTB), but is independent of the α4GalNAcTs that function in the 5th step in this pathway. These data indicate a branched structure carrying an α1,3-galactose is present on Drosophila GSLs and is targeted by MOA. Since MOA targets a similar lipid species in C. elegans (Wohlschlager et. al., 2011) both the C. elegans and Drosophila genomes encode a novel α3Gal-transferase that is active against the glycolipid β4GalNAc. To identify novel candidate proteins for a α3GalT activity we carried out a sequence independent search to identify protein domains that are conserved in the Drosophila and C. elegans genomes and combined this with additional experimental and bioinformatics data. This search resulted in the identification of a candidate protein family that does not show sequence homology to any known glycosyltransferases, but protein structure prediction (http://www.sbg.bio.ic.ac.uk/~phyre/) indicates a nucleotide-diphospho-sugar transferase fold, compatible with a glycosyltransferase activity. RNAi knockdown of the sole Drosophila homolog of this family interacts phenotypically with preceding genes in the GSL biosynthesis pathway, indicating that this locus functions in GSL biosynthesis. Targeted mutagenesis of this locus is currently underway (Genetic Services Inc.) and resistances of resulting mutants to MOA toxicity will demonstrate if this gene is required to build the MOA epitope.
This work has demonstrated the utility of fungal lectins for identified of glycan presents/ absence in the Drosophila tissue samples and the ability of genetics analysis to link genes encoding glycosyltransferases to the glycans they generate. We have demonstrated the use of conserved domain searches to identify novel proteins with candidate glycosyltransferase activity.
REFERENCES
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Kadirvelraj, R. , O. C. Grant, I. J. Goldstein, H. C. Winter, H. Tateno, E. Fadda, and R. J. Woods. 2011. Structure and binding analysis of Polyporus squamosus lectin in complex with the Neu5Acα2-6Galβ1-4GlcNAc human-type influenza receptor. Glycobiology. 21: 973-84
Koles, K., K.D. Irvine, and V.M. Panin. 2004. Functional characterization of Drosophila sialyltransferase. J Biol Chem. 279:4346-57.
Nakamura, M., D. Pandey, and V. M. Panin. 2012. Genetic Interactions Between Drosophila sialyltransferase and b1,4-N-acetylgalactosaminyltransferase-A Genes Indicate Their Involvement in the Same Pathway. G3 2:653-56.
Sarkar, M., P.A. Leventis, C.I. Silvescu, V.N. Reinhold, H. Schachter, and G.L. Boulianne. 2006. Null mutations in Drosophila N-acetylglucosaminyltransferase I produce defects in locomotion and a reduced life span. J Biol Chem. 281:12776-85.
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Wandall, H.H. S. Pizette, J.W. Pedersen, H. Eichert, S.B. Levery, U. Mandel, S.M. Cohen, and H. Clausen. 2005. Egghead and brainiac are essential for glycosphingolipid biosynthesis in vivo. J Biol Chem. 280:4858-63.
Wohlschlager, T., A. Butschi, K. Zurfluh, S.C. Vonesch, U. Auf dem Keller, P. Gehrig, S. Bleuler-Martinez, M.O. Hengartner, M. Aebi, and M. Kunzler. 2011. Nematotoxicity of Marasmius Oreades Agglutinin (MOA) Depends on Glycolipid-Binding and Cysteine Protease Activity. J Biol Chem.