Figure 2. Screening strategy and validation of the primary screen for genes whose knockdown impairs glycosylation. (A) Outline of screen design. (B) Coomassie Brilliant Blue (CBB) staining of Chp affinity-purified with anti-Chp antibody and separated by SDS- PAGE. Chp could be purified as a single band (,150 kDa) by a single step of affinity purification. (C) Binding abilities of glycan probes to purified Chp. The signal intensities normalized to the amount of Chp are represented. (D) Anti-HRP antibody reactivity normalized to Chp levels was decreased in fucTA knockdown flies (fucTA KD) compared to that in control gfp knockdown flies (gfp KD). Results are the mean 6 SD of 4 experiments. ***p,0.005. (E) Glycan structures attached to N1012 of Chp revealed by mass spectrometry analysis. Percentages of each structure in the control (left, gfp KD) and fucTA knockdown conditions (right, fucTA KD) are listed. The glycans bearing a1,3-fucose are highlighted (gray). Schematic structures are represented in Figure S3. M, mannose; F, fucose; Gn, N-acetylglucosamine. ND, not detected. doi:10.1371/journal.pgen.1001254.g002 could be purified as a single band detected by Coomassie Brilliant Blue (CBB) staining. Chp is a Drosophila glycoprotein essential for rhabdomere formation in adult photoreceptor cells, which is mediated through cell adhesion activity [15,17]. N-glycosylation of Chp was recently shown to play a pivotal role in its stability, transport to the plasma membrane, and cell adhesion activity [18]. Chp N-glycan Large-Scale Screen for Glycosylation-Related Genes structures are classified as high-mannose type, pauci-mannose type, or complex type [16]. Glycan structures added to Chp are summarized in Figure S1. Interestingly, a1,3-fucose is added only at N1012, suggesting that Chp has a single difucosylated glycan at N1012. To detect these glycan structures, the binding of glycan probes such as lectins and anti-HRP antibody was examined. Immunopurified Chp was subjected to lectin blot or immunoblot analysis using ABA, ConA, DBA, DSA, LCA, Lotus, PHA-L4, PNA, SBA, UEA, and WGA lectins and anti-HRP antibody. The specificity of these lectins and anti-HRP antibody is summarized in Table S1. The binding abilities of these probes were quantified relative to the amount of Chp protein (Figure 2C). These analyses suggested that the WGA, ConA, LCA, and DSA lectins and anti- HRP antibody would be useful as glycan probes in our assay system. As represented in Figure S1, WGA and ConA detect N- acetylglucosamine (GlcNAc) and mannose moieties of N-glycan core regions, respectively. LCA preferentially binds to N-glycans with a1,6-fucose and even weakly recognizes the core regions of N- glycans. Anti-HRP antibody binds to the a1,3-fucose moieties attached to the core [1,19]. DSA recognizes GlcNAc at the non- reducing end of N-glycans. Chp protein purified from knockdown flies was subjected to lectin and immuno-dot blot analyses using the same glycan probes described above and an anti-Chp antibody. An example of the dot blot analysis is shown in Figure S2. The glycan moieties detected by these probes were quantified relative to Chp protein, and the z- scores for each knockdown experiment were thus calculated. For verification of this procedure, a dsRNA that targets FucTA was expressed. The resulting knockdown of fucTA decreased the affinity of Chp to the anti-HRP antibody (66.2611.8% of the control, Figure 2D). Given the results of our previous mass spectrometry study showing that a1,3-fucose is added to the Chp N1012 site [16], Chp was purified from fucTA knockdown eyes, and mass spectrometry analysis was performed. The glycan structures attached to N1012 are summarized in Figure S3. The number of glycans with two fucoses present at N1012 (M3F2Gn2 and M3F2Gn3) selectively decreased in fucTA knockdown eyes (Figure 2E). This finding is consistent with those of previous studies reporting that most single- or double-fucosylated N-glycans bear a1,6-fucose alone, or both a1,6- and a1,3-fucose, respectively, in embryos [20] and adults [6]. Identification of glycosylation-related genes A large-scale screen was performed in the current study using an RNAi library that was previously constructed in National Institute of Genetics, Japan (NIG, http://www.shigen.nig.ac.jp/fly/nigfly/ about/aboutRnai.jsp). With this library, the expression of 6923 Drosophila genes can be suppressed. From the primary screen followed by tests for reproducibility, 171 genes were identified as candidate genes, knockdown of which compromised glycosylation (Table S2). Since these candidate genes likely included false- positives due to the off-target effects of RNAi [21], the results were validated by repeating the knockdown experiments using second- ary sets of dsRNAs. During the construction of these secondary sets of dsRNAs, we ensured that the target regions did not overlap. Of the 171 primary candidate genes, only 80 could be tested because, in most cases, the induction of the secondary sets of dsRNA resulted in lethal or severe eye malformation or cDNAs were too short to permit the design of secondary target regions. Of the 80 genes tested successfully, 57 were verified to be involved in glycosylation. Sources of the RNAi strains used for the verification of the 57 genes are listed in Table S2 (see ‘‘Line ID or transformant ID used for validation’’). When the validation results were compared with the calculated off-target probability scores PLoS Genetics | www.plosgenetics.org 3 December 2010 | Volume 6 | Issue 12 | e1001254