Study Of The Function And Properties Of O-GalNAc Glycosylation-Associated Key Glycosyltransferases

Compared with small molecule drugs,recombinant protein drugs with strong targeting specificity and low immunogenicity,and have gradually become a hot spot concerned by all pharmaceutical companies.At present,most of the recombinant protein drugs used in clinical therapies are glycosylated proteins,glycosylation modification will greatly affect the properties and efficacy of drugs.O-GalNAc glycosylation is greatly associated with diverse diseases including cancers and viral infections.Thus,probing the function and properties of O-GalNAc glycosylation-related glycosyltransferase is of great significance for the development of therapeutic drugs.N-acetylaminogalactosyltransferase(polypeptideUDP-N-acetyl-α-D-galactosaminotransferase,ppGalNAc-Ts,EC:2.4.1.41)is a glycosyltransferase that initiates the O-GalNAc glycosylation,which is responsible for transferring N-acetylaminogalactose(GalNAc)residue to the Thr/Ser/Tyr glycosylation site of proteins to form Tn antigen structures;subsequently the glycosyltransferase β1,3-galactosyltransferase(C1GalT1.EC:2.4.1.122)catalyzes the transfer of galactose(Gal)residue to the Tn structure to form a T antigen structure,which can be further elongated to form the most common Core 1 structure with virous terminal structures.Therefore,ppGalNAc-Ts and C1GALT1 are of great significance for the study of O-GalNAc glycosylation mechanism and the production of O-glycoprotein drugs.At present,the ppGalNAc-Ts used for in vitro preparation of O-GalNAc modified glycoproteins or glycopeptides was human derived ppGalNAc-Ts produced in mammalian cells or insect cells,which had the drawbacks of low yield and high cost and could not meet the needs of industrial production of O-GalNAc glycoprotein drugs.As the most mature protein expression system,Escherichia coli has the potential to be transformed into an O-glycoprotein production platform with the advantages such as short production cycle,high yield,low cost and no interference from its own glycosylation.In this paper,several soluble and active ppGalNAc-T isoforms were obtained through co-expression with human protein disulfideisomerase PDI(PDI,EC:5.3.4.1)in E.coli.and the efficient soluble expression of ppGalNAcTs in E.coli was further improved by fusion expression with fusion tag proteins.This work provides a platform for producing efficient and soluble glycosyltransferases derived from advanced beings in E.coli.Unlike the C1GALT1 isforms in mammals.which relies on its specific chaperone COSMC to perform enzymatic activities,DmC1GalT1 derived from fruit flies could be used in the synthesis of T structure modified glycoproteins/glycopeptides because it is chaperoneindependent and has high activity.In this paper,the constructed E.coli system in Chapter 2 was successfully used to prepare DmC1GalT1 with enzymatic activity.In order to better explore the functions and properties of DmC1GalT1 and expand its application range,we carried out site-specific mutation modification of DmC1GalT1 through structural informatics analysis,and set up the one-pot enzymatic method to detect the sugar donor selectivity for the variants.We aimed to screen for DmC1GalT1 variants with more relaxed selections of nucleotide-activated monosaccharides,which might broaden the application of DmC1GalT1 in O-glycoprotein drug synthesis.The abnormal expression of ppGalNAc-Ts could promote the occurrence and development of tumors by affecting the glycosylation level of their substrate proteins and thereby regulating the expression,stability and function of substrate proteins.However,the mechanism how ppGalNAc-Ts and their specific O-glycosylated substrates regulate the tumorigenesis and tumor progression has not been elucidated.The ppGalNAc-T5 tends to be highly expressed in breast cancer patients with low survival rates.This paper constructs a series of breast cancer cell lines with the knockout of GALNT5 gene or the knockin of GALNT5 active-deletion mutant genes using CRISPR-Cas9 techniques.Cell experiments have confirmed that knocking out of GALNT5 gene does not affect the proliferation of breast cancer cells,but greatly inhibited the migration and invasion of cells.Furthermore,we found that the gene of GALNT5 didn’t affect the properties of breast cancer cells through the conventional PI3K-Akt pathway and MAPK pathway.Finally,through quantitative differential glycoproteomics analysis several potential specific substrates of ppGalNAc-T5 were identified and some of these proteins have been reported to be associated with the migration of cancers.In conclusion,an efficient E.coli system for the production of glycosyltransferases derived from advanced beings was constructed by co-expression of human protein disulfide isomerase PDI and fusion proteins.At the same time,the site-specific mutation of DmC1GalT1 was performed to screen for the variant with more relaxed sugar donor selectivity in order to expand its application in the synthesis of O-glycoprotein drugs in vitro.Furthermore,we used gene knockout and knockin methods and differential glycoproteomics technology to study the specific protein substrates of ppGalNAc-T5 in breast cancer,which provided new ideas for the role of O-GalNAc glycosylation modification in breast cancer and related drug development.