Novel Methods For Fluorescence Imaging Detection Of Cellular Sialylation-Related Enzymes

Glycosylation is an important posttranslational modification of proteins.It plays critical roles in a variety of fundamental biological processes,and appears to be particularly sensitive to malignant transformation.Most of glycan biosynthesis occurs in the endoplasmic reticulum and Golgi compartments of the cell.However,enzymatic modification of glycan structures also occurs after glycoprotein biosynthesis,a process known as glycan remodeling.Two kinds of specific enzymes participate in glycan remodeling,glycosyltransferases and glycosidases,which synthesize and degrade the glycosidic linkage,respectively.Considering the degree of glycosylation of cells are generally related to diseases,especially cancer,glycosyltransferases and glycosidases are therefore of significant interest as potential drug targets and tumor markers for diagnosis.As one class of glycosylation,sialylation that can introduce sialic acid(N-acetylneuraminic acid)to the terminal position of glycan chain attached on protein or lipid have attracted much considerable attention.The expression of sialic acid is generally deregulated in cancer cells.The amount of sialic acids incorporated into glycoconjugates within the cell is regulated by sialyltransferases and neuraminidases.On most occasions,however,it has been noticed that the mechanisms of the altered sialylation in cancer cells and the correlation between the enzymatic activity and the appearance of sialylated glycans remain unknown.To elucidate the biological and pathological functions of sialyltransferases and neuraminidases and use them as diagnostic and therapeutic tools,sensitive methods for the assessment of sialyltransferases and neuraminidases activity have become urgent needs.In this thesis,which is based on chemical biology,cytology and molecular biology and combined the technology of fluorescence microscopy and nanomaterial,a series of novel nanoprobes and analytical methods have been developed for in situ noninvasive assay of sialyltransferases and neuraminidases activity.The dissertation includes the following two sections:1.Noninvasive imaging of sialyltransferase activity in living cells by chemoselective recognitionTo elucidate the biological and pathological functions of sialyltransferases(STs),intracellular ST activity evaluation is necessary.Focusing on the lack of noninvasive methods for obtaining the dynamic activity information,this work designs a sensing platform for in situ FRET imaging of intracellular ST activity and tracing of sialylation process.The system uses tetramethylrhodamine isothiocyanate labeled asialofetuin(TRITC-AF)as a ST substrate and fluorescein isothiocyanate labeled 3-aminophenylboronic acid(FITC-APBA)as the chemoselective recognition probe of sialylation product,both of which are encapsulated in a liposome vesicle for cellular delivery.The recognition of FITC-APBA to sialylated TRITC-AF leads to the FRET signal that is analyzed by FRET efficiency images.This strategy has been used to evaluate the correlation of ST activity with malignancy,cell cycle and cell surface sialylation,and the sialylation inhibition activity of inhibitors.This work provides a powerful noninvasive tool for glycan biosynthesis mechanism research,cancer diagnostics and drug development.2.A light-up imaging protocol for neutral pH-enhanced fluorescent detection of lysosomal neuraminidase activity in living cellsLysosomal neuraminidases(Lyso-Neus)are a class of glycosidases located in lysosomes responsible for the removal of sialic acid residues from carbohydrate portions of glycoproteins and glycolipids.This work proposes a light-up imaging strategy for neutral pH-enhanced fluorescent detection of Lyso-Neu activity in living cells with a Neu-specific and lysosome-accessing nanoprobe.The nanoprobe is prepared by assembling branched poly(ethyleneimine)(BPEI)and 4-methylumbelliferyl-N-acetylneuraminic acid(4MUNA)on 3-aminophenylboronic acid functionalized graphene oxide.Upon incubation with cells,the nanoprobe can be internalized through endocytic pathway and transported to lysosomes,where the non-fluorescent 4MUNA of the nanoprobe can be specifically recognized and cleaved by Lyso-Neus at the glycoside linkage site to generate free 4MU moieties.The released 4MU can then escape from acidic lysosomes into neutral cytosol environment due to the proton-sponge effect of BPEI.Owing to the pH-responsive fluorescence emission of 4MU,greatly enhanced fluorescent signal can be obtained and served as an indicator of Lyso-Neu activity.The proposed protocol can be further used to sensitively monitor the dynamic change of Lyso-Neu activity in living cells during biological processes,providing a valuable tool for understanding the biological roles of Neus.