@article{Wu2024, 
author = {Min Wu and Guijie Bai and Ziyi Zhang and Haixia Xiao and Wenliang Sun and Chaoguang Tian},
title = {Targeted protein editing technique in living mammalian cells by peptide-fused PNGase},
year = {2024},
journal = {hLife},
volume = {2},
number = {11},
pages = {576-591},
keywords = {N-glycosylated proteins, peptide:N-glycanase (PNGase), protein-targeting peptides, deglycosylation, protein sequence editing, protein degradation},
url = {https://www.sciopen.com/article/10.1016/j.hlife.2024.07.003},
doi = {10.1016/j.hlife.2024.07.003},
abstract = {Various precise gene editing techniques at the DNA/RNA level, driven by clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology, have gained significant prominence. Yet, research on targeted protein editing techniques remains limited. Only a few attempts have been made, including the use of specific proteases and de-O-glycosylating enzymes as editing enzymes. Here, we propose direct editing of N-glycosylated proteins using de-N-glycosylating enzymes to modify N-glycosylation and simultaneously alter the relevant asparagine residue to aspartate in living cells. Selective protein deglycosylation editors were developed by fusing high-affinity protein-targeting peptides with active peptide:N-glycanases (PNGases). Three crucial cell membrane proteins, programmed cell death protein-1 (PD-1), programmed cell death-1 ligand 1 (PD-L1), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein, were chosen to be tested as a proof of concept. N-linked glycans were removed, and the relevant sites were converted from Asn to Asp in living mammalian cells, destabilizing target proteins and accelerating their degradation. Further investigation focused on SARS-CoV-2 spike protein deglycosylation editing. The collaboration of LCB1-PNGase F (PNGF) effectively reduced syncytia formation, inhibited pseudovirus packaging, and significantly hindered virus entry into host cells, which provides insights for coronavirus disease 2019 (COVID-19) treatment. This tool enables editing protein sequences post-de-N-glycosylation in living human cells, shedding light on protein N-glycosylation functions, and Asn to Asp editing in organisms. It also offers the potential for developing protein degradation technologies.}
}