Targeted protein editing technique in living mammalian cells by peptide-fused PNGase

Min Wu^{¹^,²^,^#}, Guijie Bai^{¹^,^#}, Ziyi Zhang^{¹^,³}, Haixia Xiao^{²^,⁴}, Wenliang Sun^{¹^,²}, Chaoguang Tian^{¹^,²}()

Key Laboratory of Engineering Biology for Low-carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China

National Technology Innovation Center of Synthetic Biology, Tianjin, China

School of Pharmaceutical Sciences, Jilin University, Jilin, China

Laboraroty of Protein Engineering and Vaccines, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China

^#These authors contributed equally to this work

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HIGHLIGHTS

● Peptide-fused peptide:N-glycanase (PNGase) targets and edits N-glycosylated proteins of interest in living cells.

● The targeted deglycosylation and deamidation editing significantly decreased protein stability and accelerated degradation.

● LCB1-PNGase F (PNGF) edited SARS-CoV-2 spike protein and substantially reduced syncytia formation and virus entry.

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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.

Keywords

N-glycosylated proteins peptide:N-glycanase (PNGase)protein-targeting peptides deglycosylation protein sequence editing protein degradation

References

[1]

Anzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol 2020;38:824-44.