Glycosylation Signatures in Drosophila: Fishing with Lectins

Abstract

Glycosylation is a co- and/or post-translational protein modification that generates enormous structural diversity among glycoproteins. In this study, immobilized lectins were used to capture glycoproteins with different glycan profiles from Drosophila melanogaster extracts. On the basis of previous results from glycan array analyses, the snowdrop (Galanthus nivalis) agglutinin (GNA), the tobacco (Nicotiana tabacum) lectin (Nictaba) and the Rhizoctoni solani agglutinin (RSA) were used to select for a broad range of N- and O-glycan structures. After different lectin affinity chromatographies, the glycoproteome of Drosophila was analyzed using LC-MS/MS and glycoprotein abundances were calculated by different label-free methods. Bioinformatics tools were used to annotate the identified glycoproteins and the glycoproteins were classified according to their molecular function or their involvement in a biological process. Subsequent enrichment analysis (using the DAVID database) was employed to find biological processes or molecular functions in Drosophila in which a particular glycan signature is overrepresented. The results presented here clearly demonstrate that next to the presence of high-mannose and pauci-mannose N-glycans, Drosophila is capable of synthesizing glycoproteins carrying extended hybrid and complex N-linked glycans. Furthermore, it was demonstrated that a specific glycosylation signature can be associated with a functionally related group of glycoproteins in Drosophila, both in terms of biological process and molecular function.

Diversity in Protein Glycosylation among Insect Species

“…However, the percentage of proteins retained on the GNA column was found to be less than 5% of the total protein for the different insect species, suggesting that the number of identified glycoproteins is probably an underestimation of the actual number of glycoproteins. One important reason to explain the low percentage of glycoproteins may be that glycoproteins containing complex glycan structures are more abundant in insects than currently believed, as was recently also shown for Drosophila [37]. In addition, the identification of glycoproteins also depends on the quality of the insect databases.…”

“…After collection from hives of an experimental apiary in Ghent, honeybee workers ( A. mellifera ) were kept at 34°C and 70% relative humidity in laboratory cages and fed with sugar water [40]. A continuous colony of D. melanogaster was maintained on a corn meal-based diet, and the pea aphid A. pisum was reared on broad beans ( Vicia faba ) at 23–25°C and 65–70% relative humidity [37], [41].…”

Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its interaction with insect tissues and cells

“…Future studies will be necessary to identify and characterize the exact binding target receptor(s) for SSA on the cell membrane and unravel the signal transduction cascade that is induced by SSA to cause cell death and insect mortality. In this context, modern and advanced proteomic tools to isolate and identify glycoproteins (Vandenborre et al, 2010) and the recent availability of the pea aphid genome (The International Aphid Genomics Consortium, 2010) and the crystal structure of SSA (Sulzenbacher et al, 2010) will certainly help to unravel the mechanisms behind the strong insecticidal action of this fungal lectin. concentrations of GalNAc (50 and 100 mM) or asialomucin (5 and 10 mg/ml), and a non-binding sugar mannose (100 mM) was used as a negative control.…”

N-Glycosylation in Regulation of the Nervous System

“…Recent glycoproteomic approaches characterized in detail the totality of Drosophila N-glycosylated proteins, identifying more than 450 glycoproteins expressed in the head (Koles et al 2007, Vandenborre et al 2010, Baycin-Hizal et al 2011). These proteins comprise ion channels, transporters, cell surface receptors, cell adhesion molecules, molecules involved in proteolysis and carbohydrate metabolism, and some other protein families, including a large proportion of proteins with unknown functions (Fig.…”