肽N-糖苷酶的动力学分析方法

Methods for the kinetic analysis of peptide N-glycosidase

肽N-糖苷酶(peptide N-glycanase,PNGase,在人体中称为NGLY1)几乎存在于所有的活细胞中,可以催化切断糖蛋白或糖肽上天冬酰胺与N-糖链之间的糖苷键,被作为工具酶,广泛应用于糖蛋白组学研究中。同时,NGLY1在内质网相关的降解过程中是必不可少的,NGLY1缺乏可能会导致先天性糖基化障碍。酶促反应动力学的研究有助于发现或定向进化更丰富的工具酶以用于不同的分析策略,研究NGLY1突变型与疾病的关系,探索相关疾病机制或治疗策略。本文介绍了PNGase的发现、生理病理功能及其在糖蛋白组学中的应用,重点阐述各种PNGase动力学分析方法,包括比色法、高效液相色谱法、毛细管凝胶电泳法和液相色谱-质谱联用法,并总结了这些分析方法的优势和不足,为今后开发更简便、灵敏度更高的PNGase的动力学分析方法提供启发。

The peptide N-glycanase(PNGase,also known as NGLY1 in human)is an ubiquitous enzyme found in nearly all living cells.PNGase,which is capable of catalyzing the cleavage of the glycosidic bond between asparagine and N-glycans in glycoproteins or glycopeptides,has been widely applied as valuable tool enzyme in glycoproteomics research.In addition,NGLY1 plays a crucial role in the process of endoplasmic reticulum-associated degradation,and deficiency of NGLY1 may lead to congenital disorder of glycosylation.The investigation of enzymatic reaction kinetics is of great value for the discovery or directed evolution of more abundant tool enzymes suitable for various analysis strategies,examining the correlation between NGLY1 mutants and diseases,as well as exploring the mechanisms of related disease and developing strategies for their treatment.In this paper,the discovery,physiological and pathological functions and the application of PNGase in glycoproteomics are described.Kinetic analysis methods of PNGase,including colorimetric method,high performance liquid chromatography methods,sodium dodecyl sulfate-capillary gel electrophoresis methods,and liquid chromatography-mass spectrometry methods,are emphasized,and the advantages and disadvantages of the above methods are summarized.It provides inspiration for future advancements in the development of simpler and more sensitive kinetic analysis method for PNGase.