Enzymatic Synthesis Of Branched O-GalNAc Glycans

Glycosylation is a common post-translational modification of proteins in mammals that affects the structure and biological function of proteins,participating in life activities such as cell adhesion,molecule transport,and receptor activation.Based on the glycosidic bonds between proteins and sugar chains,protein glycosylation can be divided into four types:Nlinked glycosylation,O-linked glycosylation,C-linked glycosylation,and glycosylphosphatidylinositol anchor glycosylation.N-linked and O-linked glycosylation are the most common forms of protein glycosylation in nature.The O-glycosylation of proteins is highly diverse,including O-GalNAc,O-GlcNAc,O-Fucose,and O-Mannose,with O-GalNAc being the most abundant and diverse.O-GalNAc glycan is formed by connecting Nacetylgalactosamine(GalNAc)to the hydroxyl group of Ser and Thr through an alphaglycosidic bond to form Tn antigen,which is then extended and branched to form O-GalNAc glycans with different core structures(Core 1-Core 8).Based on the structural characteristics of the sugar chain,O-GalNAc glycans can be divided into eight different core structures,with Core 1 to Core 4 being more common and Core 5 to Core 8 being less common.The sugar chain skeleton can be linearly or branchedly extended by different glycosyltransferases catalyzing complex reactions,while there are also different site-specific fucosylation and sialylation modifications of the sugar chain,forming different glycan antigen determinants and expanding the diversity and complexity of protein functions,playing an important role in cell adhesion,vascular development,immune cell transport,and the occurrence and development of major diseases.The efficient acquisition of glycosylated compounds with defined chemical structures is a prerequisite for the study of biological functions and is also one of the major scientific issues that restricts the development of glycoscience.The synthesis of biological polysaccharides is a non-template-driven process that is influenced by multiple factors,and the O-GalNAc sugar chain is low in abundance and structurally heterogeneous,making it a great challenge to obtain them efficiently with defined chemical structures.Currently,the preparation of O-GalNAc glycans mainly relies on chemo-enzymatic synthesis strategies,which first synthesize OGalNAc core structures with branching sugar chains using chemical methods,and then extend the sugar chain skeleton with glycosyltransferases to obtain structurally diverse O-GalNAc glycans.The branching structure of O-GalNAc glycan can form sugar chain branching structures by β1-6GlcNAc modification,which is currently mainly dependent on chemical synthesis of the core structure with branching sugar chains due to the scarcity of enzyme components.Repeated protection and deprotection steps and the use of large amounts of organic solvents limit the efficient preparation of branching structure O-GalNAc glycans.This work focuses on the study of the enzymatic synthesis of O-GalNAc glycans,especially the core sugar chain Core 2 with branching structure and uses the Pichia pastoris protein expression system to achieve the secretion expression of the key enzyme component GCNT1(β1-6-Nacetylglucosaminyltransferase)and conducts enzymatic property research.The multi-enzyme cascade reaction achieved efficient preparation of the Core 2 trisaccharide with branching structure,providing a basis for subsequent series of O-GalNAc glycan enzymatic synthesis based on product structure.Chapter 2 describes the recombinant expression of GCNT1,a key enzyme component responsible for catalyzing the β1-6GlcNAc branching structure in O-GalNAc glycan biosynthesis,using the Pichia pastoris protein expression system.Activity assays of the expressed GCNT1 protein showed that it is a glycosyltransferase with N-glycosylation modification,which is necessary for the catalytic activity of the enzyme component.However,changes in the glycan type of N-glycosylation had little effect on the catalytic activity of the enzyme component.The P.pastoris expression system also enabled the expression of humanized glycosyltransferases,providing a reference for the in vitro synthesis of complex oligosaccharides and glycoconjugates.Although the P.pastoris system could not obtain a high purity GCNT1 enzyme,the crude enzyme still showed good catalytic activity,providing a lowcost approach for the synthesis of complex oligosaccharides.Subsequently,a one-pot multienzyme catalytic reaction was used to synthesize the Core 1 disaccharide of O-GalNAc,and as a catalytic reaction system for GCNT1,1.3 7 g of Core 2 trisaccharide was obtained with a yield of 77%,providing an economical and efficient solution for the in vitro enzymatic synthesis of O-GalNAc branched glycans.Chapter 3 of the thesis focuses on the enzymatic synthesis of O-GalNAc glycans with double-branching structures.Three different starting glycan compounds with double-branching Core 2 trisaccharides were synthesized.Seven multi-enzyme catalytic reaction modules were designed by coupling monosaccharide activation with enzymatic transfer reactions based on the specificity of glycosyltransferase-catalyzed reactions.Using the structure-oriented strategy,the double-branched O-GalNAc glycans were symmetrically or asymmetrically extended with twosugar chains to synthesize four long-chain bi-branched O-GalNAc glycans with PolyLacNAc structures.Furthermore,selective modifications were introduced into the synthesized glycans using reaction modules for fucosylation and sialylation,which resulted in the formation of different glycans with distinct antigenic determinants.In total,24 bi-branching O-GalNAc glycans were synthesized,which provide a foundation for further studies on the structureactivity relationships of O-GalNAc and its biological functions.Chapter 4 explored the application of enzyme-catalyzed reactions in the synthesis of multibranching glycans.Based on our previsou studies on β1-6 GlcNAc transferase,a multibranching glycans synthesis strategy was developed using double-branching O-GalNAc glycans as a starting substrate.Through the study of the catalytic properties of two enzymes responsible for catalyzing branching structures,a sequential reaction system was constructed to generate defiend tri-branching,tetra-branching,and multi-branching O-GalNAc glycans.The enzyme-catalyzed synthesis of these branching glycans broadened the application scope of glycosyltransferases and provided guidance for the synthesis of more complex oligosaccharides and glycoconjugates.Furthermore,selective extensions or modifications were introduced into the synthesized multi-branching O-GalNAc glycans using multi-enzyme catalytic modules,which resulted in the synthesis of 11 structurally defined multi-branching O-GalNAc glycans.In summary,this work developed an enzymatic synthesis strategy for branching OGalNAc glycans and explored the use of Pichia pastoris expression system for the expression of humanized glycosyltransferases.The synthesis of β1-6 GlcNAc branching glycans was achieved,and double-branching Core 2 trisaccharides were synthesized at gram scale.Structure-oriented multi-enzyme catalytic reaction system was designed,and 36 doublebranching,tri-branching,and multi-branching O-GalNAc glycans were synthesized,which provide important materials for the study of the structure-activity relationships of O-GalNAc and its biological functions.The enzyme-catalyzed synthesis strategy developed in this study could also guide the synthesis of other complex oligosaccharides with branching structures,which has significant scientific significance and application prospects.