Developing exo- versus endo-Labeling Probes for β-Glucosidase Towards the Application in Proteomics
Date Issued
2007
Date
2007
Author(s)
Chen, Yu-Fen
DOI
zh-TW
Abstract
In organisms, saccharide derivatives are deemed as information carriers, involving biological functions, such as immune response, virus infection, cell-cell communication and signal transduction, etc.. Their biological importance makes glycobiology become a new focus of life sciences. Among them, glycosidases play important roles in controlling saccharide metabolism, anabolism and cell surface molecule recognization. Any imbalance in their activities may result in many kinds of diseases. So the study of this kind of enzymes will benefit to the clinical diagnosis/treatment, drug discovery and understanding pathology etc. In this thesis, the commercially available β-glucosidase from almonds is used as the research model. Various ABPs (activity-based probes) were developed to label the enzymes hoping to extend the strategy to study proteome. We have succeeded in developing photoaffinitity ABP B-1 (exo-labeling probes) and mechanism-based ABP C-1 (endo-labeling probes), which exhibit potential in labeling β-glucosidases. Probe B-1 consists of a S-linked β-D-glucose derivative, a photoreactive group, and a biotin tag. This trifunctional probe is designed to form the covalent bond by photoreacting group near the active site once the probe and the targeted protein were bound together specifically and the biotin tag could be regarded as output for facile purification. In the protein labeling experiment employing the probe B-1, it showed that the probe can recognize the active site of the targeted enzyme specifically via a covalent bond. The probe C-1 is composed of a epoxyalkyl glucoside derivative and an azido group. A covalent bond between the enzyme and epoxide reactive group forms first, and then an azido group could react with the tag by click reaction afterwards. The protein labeling experiment using control C-1a, which has no epoxide, displayed good labeling effect, despite that the labeling mechanism is not clear. Moreover, probe B-1, which has S-linked ligand, cannot specifically label β-glucosidases, whereas probe C-1, which has native O-linked ligand, can specifically label β-glucosidases. To summarize, the study has laid the foundation for the development of ABPP (activity-based protein profiling) probes. Moreover, the modules nature of the probe designs offer molecular simplicity and concision. The specificity can be improved either by changing the ligand or reactive groups.
Subjects
β-葡萄糖水解酶
蛋白質體學
ABPP
光親合性
以酵素催化機制為基礎的
β-glucosidase
proteomics
photoaffinity
mechanism-based
Type
thesis
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