Glycosyltransferases and Glycohydrolases for Glycan Synthesis and Glycoprotein Modification

Protein glycosylations are the most complicated post-translational modifications a protein can undergo. They are non-template-driven processes and are regulated by factors that differ greatly among cell types and organisms. Therefore, glycosylation patterns are highly complex and heterogeneous. To understand the significance of the structure diversity of glycosylation, it is important to gain the ability of generating precise glycan structures efficiently.;The synthesis of a structurally defined, complex glycan has always been a great challenge to biologists as well as chemists. Carbohydrates have multiple hydroxyl groups, some of which have similar reactivity. Therefore, it is very hard to control selectivity by traditional chemical synthesis. Enzymatic synthesis on the other hand, has shown great advantages thanking to its high stereo- and regioselectivity, mild reaction conditions, and high efficiency. The limitation of enzymatic synthesis is that good enzymes with high catalytic efficiency, good stability and good protein yields are always hard to get. The Chen group has been cloning and expressing carbohydrate-active enzymes from all sources. Many of them have been used in our chemoenzymatic one-pot multiple-enzyme synthesis strategy. Natural or modified monosaccharides were activated into respective sugar-nucleotides and transferred by glycosyltransferases to form desired glycans in one pot. We have been successful in making sialic acid containing structures and now we are trying to address more challenging question in the glycobiology field.;I have been interested in synthesizing O-glycosylated peptides and glycan remodeling of N-glycans on recombinant glycoproteins produced from plants and other sources. Chapter 2 describes the study of Enterococcus faecalis endo-alpha-N-acetylgalactosaminidase (EfENG) and mutants. EfENG is one of a few known hydrolase capable of removing Galbeta1-3GalNAcalpha- (Core 1) disaccharide from O-glycans. It also has transglycosylation activity, which transfers Core 1 structure to free hydroxyl groups. EfENG was successfully cloned and expressed in E. coli. Two mutants (D737A and D636A) have also been made in attempt to increase transglycosylation activity. However, the transglycosylation activity on peptides has yet to be confirmed.;In Chapter 3, a human N-acetylgalactosaminyltransferase (hGnT-I) was successfully cloned and expressed in E. coli. The hGnT-I naturally belongs to the human N-glycosylation pathway. The activity of hGnT-I was confirmed using Mass spectrometry when either oligomannose or RNase B which contains one high-mannose type N-glycosylation site was used as the substrates. Combining with other glycosyltransferases, the capability of in vitro humanizing N-glycans on recombinant glycoprotein can be expected in near future.;Chapters 4--6 describe my effort on developing chemoenzymatic methods for synthesizing structurally defined, homogeneous oligosaccharide analogs of chondroitin sulfate. In Chapter 4, to synthesize the donor precursors of glucuronic acid-containing oligosaccharides, an Arabidopsis thaliana glucuronokinase (AtGlcAK) was cloned and expressed in E. coli. It catalyzes the C-1 phosphorylation of glucuronic acid (k cat/Km = 19.5 mM-1s-1) at an optimal pH of 7.5. AtGlcAK has been successfully used for the synthesis of glucuronic acid-1-phosphate (GlcA-1-P) and galacturonic acid-1-phosphate (GalA-1-P).;Chapter 5 describes the study of a Bifidobacterium longum UDP-glucose/galactose pyrophosphorylase (BLUGP). It was cloned and expressed in E. coli. Its broad substrate specificity was later discovered and the enzyme was renamed UDP-sugar pyrophosphorylase (BLUSP). Its good expression level (167 mg/L culture) and high efficiency make it an excellent candidate for synthesizing diverse UDP-sugars and glycans. It has been used together with different monosaccharide-1-kinases and a Pasteurella multocida pyrophosphatase (PmPpA) in a one-pot three-enzyme system for the synthesis of a library of diverse UDP-sugars, including UDP-Gal, UDP-Glc, UDP-2-deoxyglucose (UDP-2deoxyGlc), UDP-Glucosamine (UDP-GlcNH2), UDP-2-azido-2-deoxyglucose (UDP-GlcN3), UDP-Mannose (UDP-Man), UDP-2-floro-2-deoxy-mannose (UDP-ManF), UDP-2-azido-2-deoxy-mannose (UDP-ManN 3) and others, with good to excellent yields in a wide pH range (4.5-7.5). More recently, GlcA was found to be a tolerable substrate by this enzyme. AtGlcAK and BLUSP have been used together with PmPpA for one-pot three-enzyme preparative-scale synthesis of UDP-GlcA.;In Chapter 6, a chondroitin synthases was cloned from Pasteurella multocida (PmCS) and used for the formation of a chondroitin disaccharide in small-scale one-pot multiple-enzyme (OPME) reactions.