ELISA-based highly sensitive assay system for the detection of endogenous NGLY1 activity

• Published in: Biochemical and biophysical research communications 2024-05, Vol.710, p.149826, Article 149826
• Main Authors: Fujihira, Haruhiko, Sato, Keiko, Nishiuchi, Yuji, Murase, Takefumi, Matsuda, Yuka, Yoshida, Yukiko, Kamei, Takayuki, Suzuki, Tadashi
• Format: Article
• Language: English
• Subjects: ELISA; Glycopeptide-tag; NGLY1; Sequence editing
• ISSN: 0006-291X; 1090-2104; 1090-2104
• DOI: 10.1016/j.bbrc.2024.149826

Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 μg of crude extract, which is the equivalent of 5 × 103 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency. •・A facile NGLY1 assay system was established based on its sequence editing function.•・The ELISA-based assay could provide an early diagnosis of NGLY1 deficiency.•・This assay system can detect NGLY1 activity from as little as 5000 cells.