JAV2025_SushmaaDANGUDUBIYYAM.mp4

Aberrant cellular glycosylation has long been established as a hallmark for the development and progression of cancer and therefore there exists the need to study the various glycosylation changes that occur in cancer. The most widely occurring changes in glycosylation associated with cancer include an increase in overall sialylation – which could be an increase in silalylated tumor associated carbohydrate antigens, such as sialyl lewis x, sialyl lewis a, an increase in terminal α2,6-sialylated structures such as sialyl Tn, an increase in the α2,8-linked polysialic acid structures, and certain sialic acid containing glycosphingolipids. Other broadly occurring changes in glycosylation include, an enhancement of β1,6GlcNAc-branched structures and overexpression of core fucosylation. In my project, I are mainly interested in glycosyltransferases that are responsible for some of these changes. We are interested in two sialyltransferases, namely ST6Gal1 and ST6GalNAc1 and three fucosyltransferases, namely FUT6, 7 and FUT8.

The human genome encodes for twenty sialyltransferase genes, grouped under the GT-29 family. These enzymes catalyse the transfer of sialic acid from nucleotide activated sugar donor, such as CMP-NeuAc to the non-reducing end of a growing carbohydrate chain linked to a protein or a lipid generating alpha linkages. These enzymes have been classified into four families based on the type of linkage formed and the monosaccharide acceptor as ST3Gal, ST6Gal, ST6GalNAc and ST8Sia. We are interested in a member of the ST6Gal family, the ST6Gal1 enzyme, that transfers sialic acid on to a galactose or a GalNAc on a glycoprotein. The other ST we are interested in is the ST6GalNAc1, which catalyses the transfer of sialic acid onto GalNAc on a o-linked glycoprotein generating the sialylTn antigen.

Fucosyltransferases, there are 13 FTs encoded by the human genome, classified under various GT families. In general, they catalyse the transfer of a fucose residue from GDP-fucose onto a carbohydrate acceptor on a glycoprotein or a glycolipid. The FTs of interest here are, FUT6, which can transfer fucose via a alpha 1,3 linkage onto a sialylated or nonsialylated LacNAc giving rise to a Lewisx/ sialyllewis x structure implicated in liver, renal, bladder cancers and leukemia. FUT7 on the other hand, can transfer fucose onto only a sialylated LacNAc tructure and gives rise also to sialyl lewis x antigen. FUT8 is a unique core fucosyltransferase that transfers fucose onto the core GlcNAc linked directly to N-glycoprotein. 

Moving onto the Microplate Sialyltransferase assay, used to study sialyltranferase activity and kinetics. CMP-Neu5Ac is the natural donor of STs. Based on this a functionalized donor (suitable for biorthogonal labelling) was chemoenzymatically synthesized, which is CMP-SiaNAl with an alkyne modification on the N-acyl group of Neu5Ac. To perform the MPSA, we first coat the asialylated glycoprotein onto the microplate overnight. This is followed by a sialylation step with the functionalized donor and ST enzyme to generate the product with the alkynyl NeuAc. We follow this with biorthogonal labelling with azido biotin in the presence of CuSO, sodium ascorbate and BTTAA to form this complex, which is next detected by adding an anti-biotin antibody and a TMB reaction, from which the product is read at 620nm. So the amount of  sialylation  corresponds to the absorbance reading.

The MPSA was further expanded to all 13 STs working on glycoproteins and optimized the conditions for sialylation of all the enzymes, like the quantity of enzyme needed, time of reaction and concentration of donor substrate needed and studied their kinetic parameters such as apparent Km, apparent Vmax, Kcat and Kcat/ Km. This study allows us to systematically compare the activities of all the enzymes at the same time. This work had been submitted for publication.

So why these microplate assays for studying the glycosyltransferases? They are rapid and versatile (covers a range of STs for example). They are simple and do not require any expensive equipment. They are sensitive, for example requiring only 4ng of some enzymes to be assayed. They can be used to study the inhibition kinetics of these enzymes, which is something we are doing right now. They allow the systematic comparison between the various enzymes within the family, which is also relevant to check inhibition between the various enzymes.