胞质肽:N-聚糖酶(NGLY1,PNGase)是从错误折叠的糖蛋白切割N-聚糖的酶。2012年,人类遗传疾病,NGLY1缺乏症,最初报道是由NGLY1基因突变引起的。从那以后,NGLY1生物学取得了快速进展,基因治疗已被认为是NGLY1缺乏症的有希望的治疗选择。虽然血浆/尿液生物标志物也被开发用于这种疾病,检测NGLY1活性可能是早期诊断NGLY1缺乏症的另一种可行选择。到目前为止,已经报道了几种体外和细胞NGLY1检测方法,但是这些测定系统具有若干问题,必须解决这些问题以便开发与常规临床检查相容的测定系统。这里,我们展示了一个容易的,利用其序列编辑功能可用于检测NGLY1活性的高灵敏度体外检测系统,即糖基化Asn转化为Asp,然后通过抗HA抗体检测新产生的表位(HA)-标签。使用这种基于ELISA的测定法,我们在低至2μg的粗提物中检测到内源性NGLY1活性,相当于5×103个细胞。我们的系统还从NGLY1活性受损的细胞中检测NGLY1活性,例如来自患者样本的iPS细胞。该测定系统可用于未来的临床检查,以实现NGLY1缺乏症的早期诊断。
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.