Researcher(s)
- Abigail Stevens, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Erica Selva, Biological Sciences, University of Delaware
Abstract
The Wnt signaling pathway is an evolutionarily conserved signal transduction pathway that governs organismal development and adult tissue homeostasis in Drosophila melanogaster and all metazoans by regulating cell proliferation, polarity, and differentiation. Dysfunction of this pathway is implicated in human diseases such as colorectal and breast cancers, as well as cardiac and vascular disorders. Wnt proteins are secreted through a conserved pathway, regulated by Porcupine (Por) and Wntless (Wls) proteins. Por is a member of a family of O-acyltransferase proteins that are mainly found in the Endoplasmic Reticulum (ER) and lipid-modify Wnts to modulate their trafficking from the ER through the Golgi apparatus for cell surface release. Wls is important for Wnt trafficking from the ER through the secretory pathway to the plasma membrane for release and may play a role in Wnt co/post-translational modification required for Wnt function. During this process, Wnts are asparagine glycosylated (N-glycosylated), a post-translational modification important for protein folding and stability. The prototypical Drosophila Wnt, Wingless (Wg), requires glycosylation on two sites to produce a mature secreted ligand that binds to its cognate receptors Frizzled (Fz) and LRP5/6, culminating in β-catenin accumulation and downstream transcriptional activation of Wnt target genes in receiving cells. However, four possible asparagine glycosylation sites with the recognition site Asparagine-X-Serine or Threonine (Asn-X-Ser/Thr) are found in Wg at amino acid residues 49, 103, 108, and 414. The goal of my research is to confirm that N103 and N414 are the primary sites of Wg N-glycosylation and determine how conservative mutation of glycosylation sites to glutamine (Q) affects Wg glycosylation, secretion, and signaling activity. To do this, I examined three Wg mutants controlled by the constitutive actin-5c promoter, pAW-Wg N103Q, pAW-Wg N414Q, and pAW-Wg N103/414Q. These constructs were transfected into Drosophila S2R+ cells. The effect of these mutations was assessed using the Wnt reporter dual-luciferase reporter assay and β-Catenin accumulation assay to evaluate activity, as well as the Wg protein glycosylation pattern in cell lysates and media to determine the functional impact of these mutations. My findings indicate that N-linked glycosylation is not essential for the activity, secretion, or signaling of Wg.