Abstract
Mucin type O-glycosylation is a highly conserved form of post-translational modification initiated by the family of enzymes known as the polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs in mammals and PGANTs in Drosophila). To address the cellular functions of the many PGANT family members, RNA interference (RNAi) to each pgant gene was performed in two independent Drosophila cell culture lines. We demonstrate that RNAi to individual pgant genes results in specific reduction in gene expression without affecting the expression of other family members. Cells with reduced expression of individual pgant genes were then examined for changes in viability, morphology, adhesion, and secretion to assess the contribution of each family member to these cellular functions. Here we find that RNAi to pgant3, pgant6, or pgant7 resulted in reduced secretion, further supporting a role for O-glycosylation in proper secretion. Additionally, RNAi to pgant3 or pgant6 resulted in altered Golgi organization, suggesting a role for each in establishing or maintaining proper secretory apparatus structure. Other subcellular effects observed included multinucleated cells seen after RNAi to either pgant2 or pgant35A, suggesting a role for these genes in the completion of cytokinesis. These studies demonstrate the efficient and specific knockdown of pgant gene expression in two Drosophila cell culture systems, resulting in specific morphological and functional effects. Our work provides new information regarding the biological roles of O-glycosylation and illustrates a new platform for interrogating the cellular and subcellular effects of this form of post-translational modification.
Highlights
Mucin type O-linked glycosylation is an evolutionarily conserved post-translational modification found on secreted and membrane-bound proteins in many diverse species (1– 4)
Before embarking on the RNA interference (RNAi) experiments, we performed quantitative PCR to determine which pgant family members are expressed in each cell line
Our results demonstrated that only the targeted pgant gene showed a significant decrease in transcript level, indicating that the RNAi is specific to the desired gene and that there is no compensatory up or down-regulation of the transcription of other family members in either cell line
Summary
DsRNA Preparation—For the generation of dsRNA, cDNAs from each pgant were amplified using primers containing T7 RNA polymerase binding sites and gene-specific sequences to produce ϳ500-bp fragments containing T7 promoters at the 5Ј ends. The cells were incubated for 30 min at room temperature before adding 750 l of medium containing 10% FBS. Each experiment was performed with two independent dsRNAs to each pgant to verify gene specific knockdown and observed phenotypes. Quantitative cell adhesion assays were performed on S2Rϩ cells after incubation with dsRNA as described previously (17). Protein Secretion Assays—2 ϫ 105 S2Rϩ or S2 cells in 250 l of Schneider’s medium with 10% FBS were added to each well of a 24-well plate.
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