The Role of N‐linked Glycosylation in Drosophila Development

Abstract

Asparagine‐linked or N‐linked glycosylation is an important post‐translational modification pathway that adds 14‐sugar oligosaccharide chains to the Asparagine of specific Asn‐Xaa‐Ser/Thr sequences in target proteins on the luminal side of the endoplasmic reticulum. These glycan “tags” are necessary for multiple cell functions including cell‐cell recognition, extracellular signal regulation, and proper protein folding. The focus of this study is to examine the effect of loss of function mutations in the N‐linked pathway during development. A group of human diseases, congenital disorders of glycosylation (CDG), arise from mutations in the genes involved in various steps of this pathway. CDGs display severe and pleotropic phenotypes, which reflect the universal importance of this protein modification, but most cases display phenotypes that arise from impaired neuronal function. The two specific genes under study in this project are alg9 and alg10. Each gene encodes a glycosyltranferase that adds a sugar residue to the growing oligosaccharide chain before it is transferred en masse to a target protein. The Drosophila eye is used as a model organ to study the effects of loss‐of‐function mutations in these genes as it serves as a surrogate for neuronal development. In adult flies, these mutations yield a small rough eye phenotype, which is more severe in alg9, as it acts five steps before alg10 in the pathway. In order to determine the molecular basis of this phenotype, larval eye discs that are homozygous mutate for alg9 and alg10 were dissected, stained for different glycoprotein and neuronal markers, and then imaged using confocal microscopy. Using ELAV, a neuron specific marker, we observed normally differentiated neurons in the eye disc. However, these mutations interrupt proper glycoprotein trafficking, as Chaoptin, a photoreceptor specific cell surface adhesion molecule, accumulates in the cell bodies of alg9 and alg10 mutant photoreceptors. Presence of cleaved Caspase‐3 later in eye development suggests that the accumulation of glycoprotein in the cell body eventually leads to photoreceptor apoptosis. Photoreceptor death likely continues through pupal development resulting in the reduced number of photoreceptors seen in alg10 adult eyes and an almost complete absence in alg9 adult eyes. These results indicate that CDG patients may have normal neuronal specification and differentiation, but experience neuronal deficits due to intracellular accumulation of glycoproteins leading to cell death. These data also suggest induction of the unfolded protein response (UPR) resulting in apoptosis may play a role in this process. Markers of endoplasmic reticulum stress and the unfolded protein response such as XBP1 and BiP will be studied in the future.Support or Funding InformationHoward Hughes Medical Institute Summer Fellowship, 4 University of Delaware Supply and Expense Grants