Abstract 2189: The Impact of N-butyldeoxynojirimycin and 1-deoxymannojirimycin on SARS-CoV-2 S1 Subunit and Angiotensin-Converting Enzyme 2 Interaction in Intestinal Epithelial Cells

Abstract

Protein aggregation in the form of amyloid is associated with neurological diseases such as Alzheimer's disease and Parkinson's disease. Amyloids also serve as the structural basis for mammalian prions, which are infectious, self-propagating ordered aggregates of functional protein. Prions are found in the baker's yeast Saccharomyces cerevisiae where they propagate to progeny cells through the action of molecular chaperone proteins. Three chaperones are fundamentally required for the propagation of the prions [PSI+] and [RNQ+] in S. cerevisiae. These are the disaggregase Hsp104, the Hsp70 Ssa, and the Jdomain protein Sis1. In the model plant Arabidopsis thaliana, we previously identified six functional orthologs of Sis1 and demonstrated their differing abilities to propagate distinct yeast prions when complementing a deletion of SIS1. The overall goal of this investigation is to compare amino acid sequences and structural characteristics between multiple orthologs to better understand the prion-specific propagating functions of chaperone proteins. Here we show that the J-domain and glycinerich domains of these orthologs are fully sufficient for their prion-propagating properties, dramatically reducing the sequence complexity that must be untangled to understand the biochemical basis of their prion-specific propagation functions. Additionally, we are also examining the ability of the functional ortholog of Hsp104 found in A. thaliana, Hsp101, to potentially replace Hsp104 in prion propagation. Hsp101 has already been shown to successfully cooperate with yeast Hsp70s to replace Hsp104 function in thermotolerance assays. Prion propagation by Hsp101 has never been successfully demonstrated in the literature, however, this may be due to the sensitivity of prions to both over-and underabundance of Hsp104 function. Here we examine three different expression systems with the intention to vary Hsp101 expression levels to determine if any amount of Hsp101 is capable of stable prion propagation. Preliminary results so far continue to indicate that Hsp101 may not be capable of replacing Hsp104 in the yeast system to propagate either [PSI+] or [RNQ+], although experiments to test this further are ongoing.
This work was supported by the Camille and Henry Dreyfus Foundation (Grant: TH-18-017), and by the NIGMS of the NIH (Grant: R15GM110606). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, or decision to publish. 104127, https://doi.org/10.1016/j.jbc.2023.104127

Abstract 2189
The Impact of N-butyldeoxynojirimycin and 1deoxymannojirimycin on SARS-CoV-2 S1 Subunit and Angiotensin-Converting Enzyme 2 Interaction in Intestinal Epithelial Cells

Marianne El Khoury, University of Veterinary Medicine Hannover
Dalanda Wanes, Maura Lynch-Miller, Hassan Naim The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets mainly the respiratory tract. In addition to respiratory symptoms, many extrapulmonary manifestations were observed in the gastrointestinal tract and reported by SARS-CoV-2 patients, including abdominal pain, nausea, and diarrhea. SARS-CoV-2 binds initially to angiotensin-converting enzyme 2 (ACE2) on the host cell surface via its spike (S) protein before it undergoes endocytosis and fusion with the lysosomal membrane. The spike protein of SARS-CoV-2 is a heavily Nand O-glycosylated trimer. Glycosylation is an essential posttranslational modification in the life cycle of membrane and secretory proteins that affects their structural and functional characteristics as well as their trafficking and sorting patterns. This study aimed at elucidating the impact of glycosylation modulation on the trafficking of both S1 subunit and ACE2 as well as their interaction at the cell surface of intestinal epithelial cells. For this purpose, the S1 protein was expressed in COS-1 cells and its glycosylation modified using N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of ER-located α-glucosidases I and II, and or 1-deoxymannojirimycin (dMM), an inhibitor of the Golgi-located α-mannosidase I. The intracellular and secreted S1 proteins were analyzed by endoglycosidase H treatment. Similarly, ACE2 trafficking to the brush border membrane of intestinal Caco-2 cells was also assessed in the presence or absence of the inhibitors. Finally, the interaction between the S1 protein and ACE2 was investigated at the surface of Caco-2 cells by co-immunoprecipitation. Our data show that NB-DNJ significantly reduced the secretion of S1 proteins in COS-1 cells, while dMM affected S1 secretion to a lesser extent. Moreover, NB-DNJ and dMM differentially affected ACE2 trafficking and sorting to the brush border membrane of intestinal Caco-2 cells. Strikingly, the interaction between S1 and ACE2 was significantly reduced when both proteins were processed by the glycosylation inhibitors, rendering glycosylation and its inhibitors potential candidates for SARS-CoV-2 treatment.
This work has been supported by a grant from the German Research Foundation (DFG) grant NA331/15-1 to HYN. M.K. was supported by a scholarship from the Hannover Graduate School for Veterinary Pathobiology, Neuroinfectiology, and Translational Medicine (HGNI) and by the DFG grant NA331/ 15-1.