Carbohydrates are involved in a wide array of biological roles. Carbohydrates and carbohydrate-derived drugs are used extensively in therapeutics ranging from inflammatory diseases, cardiovascular, and hematological treatments.
Gangliosides are sialic acid-containing glycosphingolipids that are ubiquitously found in the plasma membrane of vertebrate cells and are the major sialic acid-containing glycoconjugates in animal nervous systems. A human sialyltransferase ST3GAL II (hST3GAL II) was successfully expressed in Escherichia coli as an active soluble fusion protein with an N-terminal maltose-binding protein (MBP) and a C-terminal hexa-histidine tag. It was used as an efficient catalyst in a one-pot multienzyme (OPME) sialylation system for high-yield production of the glycans of ganglioside GM1b and highly sialylated brain gangliosides GD1a and GT1b. Further sialylation of GM1b and GD1a glycans using a bacterial alpha-2–8-sialyltransferase in another OPME sialylation reaction led to the formation of the glycans of GD1c and brain ganglioside GT1a, respectively. The lower reverse glycosylation activity of the recombinant hST3GAL II compared to its bacterial sialyltransferase counterpart simplifies the handling of enzymatic synthetic reactions and has an advantage for future use in automated chemoenzymatic synthetic processes. This work is described in Chapter 2.
Human milk oligosaccharides (HMOs) are the third most abundant solid component of human milk, after lactose and fat. Approximately 28% to 61% of HMOs are sialylated with alpha-2–3 and/or alpha-2–6 linkages. Human ST6GALNAC V was successfully cloned and expressed in E. coli as an active soluble recombinant enzyme. This enzyme was found to be able to synthesize disialyllacto-N-tetraose (DSLNT), an important disialylated HMO. The cloning and characterizations of hST6GALNAC V is described in Chapter 3.
Enzyme replacement therapy is widely used for treating lysosomal storage diseases (LSDs). However, LSDs due to deficiency of transmembrane enzymes cannot use this treatment. I worked on the efforts of engineering enzymes to bypass the function of heparan acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT) to find potential treatments for Mucopolysaccharidosis IIIC. Related background information is described in Chapter 4. In Chapter 5, the approaches that I used for enzymatic engineering of alpha-N-acetylglucosaminidase (NAGLU) and its homolog enzyme are described.
In summary, I worked on the cloning and expression of sialyltransferases for chemoenzymatic synthesis of gangliosides and HMOs, and used them for chemoenzymatic synthesis. These enzymes have high potential for automated synthesis of ganglioside glycans and HMOs. I also worked on protein engineering of NAGLU and its homolog enzyme for finding potential candidates for the treatment of mucopolysaccharidosis IIIC. The latter requires further research efforts.
