Protein post-translational modifications play key roles in higher organisms. Glycosylation is one of the most common protein modifications. In the eukaryotic endoplasmic reticulum, the asparagine residues in the consensus sequence (Asn-Xaa-Ser/Thr, Xaa ≠ Pro) of nascent polypeptides are modified with high-mannose oligosaccharides. Cytoplasmic peptide:N-glycanase (PNGase) can cleave the linkage between sugar and asparagine side-chain on N-linked glycoproteins. One of the PNGase function is to recruit N-linked glycoproteins into proteasome when the proteins become unnecessary. However, a convenient method for detecting PNGase activity in live cells has not been developed.

On the other hand, split inteins that mediate protein trans-splicing (PTS) are used in chemical biology and protein engineering. We engineered naturally split Npu DnaE intein with altered split position and conjugated it to NanoLuc luciferase. Using this split intein-NanoLuc conjugates, active NanoLuc luciferase can be generated by the PTS reaction. In the present study, we attempted to develop a convenient method to detect PNGase activity using the split intein and NanoLuc luciferase. We designed a 16-residue peptide consisting of a NanoLuc-derived 5-residhe peptide and an 11-residue intein peptide. N,N’-diacetylchitobiose (GlcNAc₂) was incorporated into the intein region of the peptide. The peptide bearing GlcNAc₂ can react very slowly with the C-terminal intein fragment (C-int). In contrast, when the peptide was treated with PNGase capable of cleaving the amide bond between the innermost GlcNAc and Asn side-chain, active NanoLuc was rapidly generated via the PTS reaction of the PNGase-product peptide with C-int. Thus, the PNGase activity was successfully detected by the PTS-based luciferase assay. Moreover, PNGase activity in live cells was also detected by this method using a cell-penetrating peptide.