Amino acid editing of NFE2L1 by PNGase causes abnormal mobility on SDS-PAGE

• Published in: Biochimica et biophysica acta. General subjects Vol. 1867; no. 12; p. 130494
• Main Authors: Tachida, Yuriko, Hirayama, Hiroto, Suzuki, Tadashi
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.12.2023
• Subjects: Amino acid editing; amino acids; Deglycosylation; family; NFE2L1; NGLY1; peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase; peptides; polyacrylamide gel electrophoresis; proteolysis; transcription (genetics); transcription factors; Deglycosylation; Amino acid editing; NFE2L1; NGLY1
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2023.130494

NFE2L1 (also known as NRF1) is a member of the nuclear erythroid 2-like family of transcription factors and is critical for counteracting various types of cellular stress such as oxidative, proteotoxic or metabolic stress. This unique transcription factor is also known to undergo changes, including post-translational modifications, limited proteolysis or translocation into the nucleus, before it exerts full transcriptional activity. As a result, there are various molecular forms with distinct sizes for this protein, while the precise nature of each form remains elusive. In this study, the N-glycosylated status of NFE2L1 in cells was examined. The findings revealed that when NFE2L1 was deglycosylated by PNGase F, the size-shift on SDS-PAGE was minimal. This was in contrast to deglycosylation by Endo H, which resulted in a clear size-shift, even though N-linked GlcNAc residues remained on the protein. It was found that this unusual behavior of PNGase-deglycosylated NFE2L1 was dependent on the conversion of the glycosylated-Asn to Asp, resulting in the introduction of more negative charges into the core peptide of NFE2L1. We also demonstrate that NGLY1-mediated deglycosylation and DDI2-mediated proteolytic processing of NFE2L1 are not strictly ordered reactions. Our study will allow us to better understand the precise structures as well as biochemical properties of the various forms of NFE2L1. •Deglycosylation of NFE2L1 by NGLY1 results in an aberrant migration pattern on SDS-PAGE.•Various molecular forms of NFE2L1, generated by the protease DDI2 and the glycosidase NGLY1, were characterized.•Deglycosylation by NGLY1 and proteolysis by DDI2 on NFE2L1 occur independently without a strict order.