182 Oral - Cell Stress and Cell Death
Saturday April 09, 11:00 AM - 11:15 AM

Authors:
Hans Dalton ¹; Raghuvir Viswanatha ²; Roderick Brathwaite, Jr. ²; JaeSophia Zuno ¹; Stephanie Mohr ²; Norbert Perrimon ^2,3; Clement Chow ¹

Affiliations:
1) Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT, USA; 2) Department of Genetics, Harvard Medical School, Boston, MA, USA; 3) Howard Hughes Medical Institute, Boston, MA, USA

Keywords:
g. unfolded protein response; g. unfolded protein response

Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). Glycosylation is a broad category of sugar modifications on proteins and lipids, with functions ranging from complex post-translational modifications (e.g. N-glycosylation) to single additions involved in cell signaling (e.g. O-GlcNAc-ylation). CDGs have a range of symptoms, but commonly include severe epilepsy, developmental delay, and disability. CDG Type Ij is caused by loss-of-function mutations in DPAGT1 – the first step in N-glycosylation. Our goal is to better understand the pathways connected to DPAGT1 loss to develop potential treatment options.

We performed a CRISPR knockout screen using the drug tunicamycin (Tun), a potent inhibitor of DPAGT1 function, on Drosophila S2R+ cells. Loss of DPAGT1 impairs N-glycosylation and causes massive protein misfolding, leading to reduction of cell surface glycoproteins and endoplasmic reticulum (ER) stress. We introduced a whole genome guide RNA library into S2R+ cells expressing constitutive Cas9. Cells were grown under either vehicle or Tun selection. Final populations were sequenced to determine candidate genes causing resistance or sensitivity to DPAGT1 inhibition. Gene Ontology of these genes related to the hexosamine pathway and glycolysis, two pathways important for N-glycosylation. We also performed a Concanavalin A (ConA) screen to assay cell surface glycoproteins in an identical pool of cells. This assay found loss of genes in GPI anchor biosynthesis (e.g. PIG-A and PIG-H) could rescue cell surface glycoproteins under DPAGT1 inhibition.

We created an in vivo Drosophila DPAGT1 model where Alg7/DPAGT1 RNAi is driven in the eye to cause a degraded eye phenotype. Using this and another ER stress model, we validated candidate genes via RNAi knockdown. RNAi in these models revealed that loss of the mannosyltransferase Dpm1, involved in mannose addition in all downstream glycosylation pathways, could strongly rescue DPAGT1 inhibition and ER stress phenotypes. Testing its downstream pathways (O- and C-mannosylation [rt, CG6659]), N-glycosylation [Alg3], GPI biosynthesis [PIG-M]), we found loss of O-mannosylation, N-glycosylation, and GPI biosynthesis partially recapitulates the Dpm1 RNAi rescue of DPAGT1 inhibition and ER stress. These findings suggest impairment of Dpm1, or its downstream pathways, as key targets for potential therapeutics for DPAGT1 loss.