Reduction of a single E3 ubiquitin ligase alters neural-specific glycosylation in Drosophila melanogaster
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The regulatory pathways that control tissue-specific glycan expression are not well understood. A mutagenesis screen was undertaken in Drosophila to identify mutants that are deficient in the expression of a family of structurally related, neural-specific N-linked glycans known as HRP-epitopes. These epitopes are detectable by anti-HRP antibody staining. To date, three mutants have been recovered, each in different genes. Each mutant lacks HRP-epitope expression during embryonic stages and, as a group, the mutants exhibit genetic interactions with each other in regards to the loss of HRP-epitope. Additionally, all three mutants exhibit a similar neuromotor defect as adults. While mutant adults are motile and can right themselves when flipped over, they all fail to exhibit spontaneous climbing activity, which is a robust behavior in wildtype adults. One of these mutants, originally designated ms16, affects the expression level of an E3 ubiquitin ligase known as roc2, which has previously been demonstrated to be essential for neural development. In addition to altered HRP-epitope expression, the total glycan profile of roc2ms16 embryos was shifted toward increased abundance of complex glycans in comparison to wildtype embryos. In addition, Golgi compartmentation was also shifted such that the overlap between markers for early and late Golgi was increased, suggesting that augmented glycan complexity may arise from altered access of processing enzymes and substrates. Differential proteomic analysis, by LC-MS/MS and validated subsequently by orthogonal approaches, detected significantly increased (2-fold) expression of ATPalpha (Na+/K+ pump-alpha subunit) in roc2ms16 mutants compared to wildtype. Conversely, quantification of HRP-epitope expression by ELISA detected increased expression of the neural-specific glycan class in a deficiency line that partially reduced ATPalpha expression. Therefore, increased ATPalpha expression (roc2ms16) reduced HRP-epitope abundance while decreased ATPalpha expression (ATPalpha deficiency line) increased HRP-epitope abundance, indicating a role for ATPalpha in the regulation of glycoprotein glycosylation. Other components of the membrane excitability machinery were also impacted in roc2ms16. Notably, expression of the excitatory glutamate receptor at the neuromuscular junction, dGluRIII, was increased in roc2ms16 larvae, perhaps in response to altered ionic fluxes induced by increased ATPalpha activity. Thus, altered roc2 activity impacts neural-specific glycosylation and key functions of excitable cells.