Horseradish peroxidase (HRP) is a plant enzyme which cross-reacts with similarly structured N-glycans in Drosophila and other invertebrates. These N-glycans, collectively known as HRP epitopes, are normally expressed in a restricted set of embryonic tissues throughout the Drosophila life cycle.1 HRP epitope structures are responsible for activating neural development and are important for proper cell signaling, migration, adhesion, and synaptic functions expressed in developing and mature neural tissues.2
Mutations in Tollo, an ectodermally expressed Toll-like receptor, prevent HRP-epitope expression from occurring. Tollo alters N-linked glycosylation involved in the developing embryonic nervous system. Previous studies have shown Tollo is expressed and interacts with non-neural ectodermal cells that surround differentiating neurons and drives a cell signaling pathway ultimately leading to neuron-specific glycosylation; however, many of the mechanisms and components involved with tissue-specific glycosylation are unknown at this time.3
Baas et al. performed a random mutagenesis screen to identify genes involved in HRP-epitope embryonic expression.4 Mutations that altered expression of HRP epitopes without affecting N-linked glycosylation were targeted. Drosophila embryos from the F2 generation derived from mutagenized male founders were stained with anti-HRP antibody and examined for loss of HRP epitope expression. Mutant embryos that exhibited altered HRP-epitope expression were stained with Concanavalin A (ConA) as a control to ensure glycosylation was unaltered.
One mutation recovered from the screen exhibited a loss of all neural HRP-epitope expression. This mutation, initially designated as B22, contained residual ConA in late stages within the axon scaffold of the ventral nerve cord. Staining was also restricted to the dorsal aspect of the nerve cord and gave the appearance of a frosting-like layer of epitope, which brought about the name change from B22 to sugar-free frosting (sff).
To identify glycan expression changes induced by the sff mutation, total N-linked glycan profiles of sff mutant and wild-type embryos were analyzed by mass spectrometry (MS). Adult head lysates were reduced, alkylated, and digested with sequencing, followed by proteomic analysis using LC-MS/MS. Two-dimensional RP/RP-HPLC LC-MS/MS analysis was employed using an LTQ Orbitrap mass spectrometer (Discovery, Thermo Scientific) and a Surveyor MS pump plus (Thermo Scientific). MS/MS data were searched against the Fly database (Drosophila melanogaster, 8-31-09) from Swiss Prot.
Since HRP epitopes represent less than 1% of all N-linked glycans in the Drosophila embryo, glycan profiles were obtained using mass spectrometry (MS), which was performed on samples processed with PNGaseA followed by a digestion of tryptic/chymotryptic peptides as previously described.5 Full MS and MS/MS spectral data were obtained using nanospray ionization interfaced to a linear ion trap instrument (NSI-MSn on an LTQ Ion Trap, Thermo Scientific) and validated by The Total Ion Mapping (TIM) functionality of the instrument control and data acquisition software (Xcalibur, v2.0), with ambiguities resolved by MS3-MS4. 89.5% of the MS profile of mutant sff embryos contained high mannose and paucimannose glycans, which determined the dominant glycan-processing pathways are largely unaffected by the sff mutation.
From the combined experiments, which included immunohistochemistry, immunofluorescence and confocal colocalization, in situ hybridization, geotaxis qRT-PCR experiments in addition to the proteomics experiments, sff was determined to be the Drosophila homolog of SAD kinase, which functions to regulate synaptic vesicle tethering and neuronal polarity in nematodes and vertebrates.6,7 In addition to this, sff regulates vesicle tethering at Golgi membranes in the developing Drosophila embryo, and its expression is dependent on transcellular signaling through a non-neural toll-like receptor.
1. Jan, L.Y. and Jan, Y.N. (1982) ‘Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos‘, Proceedings of the National Academy of Sciences of the United States of America, 79 (8), (pp. 2700-2704)
2. Matani, P., Sharrow M., and Tiemeyer M. (2007) ‘Ligand, modulatory, and co-receptor functions of neural glycans‘, Frontiers in Bioscience. May 1 (12), (pp. 3852-3879)
3. Seppo, A., et al. (2003) ‘Induction of neuron-specific glycosylation by Tollo/Toll-8, a Drosophila toll-like receptor expressed in non-neural cells‘, Development, 130 (7), (pp. 1439-1448)
4. Baas, S., et al. (2011) ‘Sugar-free frosting, a homolog of SAD kinase, drives neural-specific glycan expression in the drosophila embryo‘, Development, 138 (3), (pp. 553-563)
5. Aoki, K., et al. (2007) ‘Dynamic Developmental Elaboration of N-linked Glycan Complexity in the Drosophila Melanogaster Embryo‘, The Journal of Biological Chemistry, 282 (12), (pp. 9127-9142)
6. Crump, J., et al. (2001) ‘The SAD-1 kinase regulates presynaptic vesicle clustering and axon termination‘, Neuron, 29 (1), (pp. 115-129)
7. Kishi, M., (2005) ‘Mammalian SAD kinases are required for neuronal polarization‘, Science, 307 (5711), (pp. 929-932)