Using Eukaryotic Expression Systems to Generate Human α1,3 Fucosyltransferases that Effectively Create Selectin-binding Glycans on Stem Cells

Asma S. Al-Amoodi, Kosuke Sakashita, Amal J. Ali, Ruoyu Zhou, Jae Man Lee, Muhammad Tehseen, Mo Li, Juan Carlos Izpisua Belmonte, Takahiro Kusakabe, Jasmeen Merzaban

Research output: Contribution to journalArticlepeer-review

Abstract

Recruitment of circulating cells towards target sites is primarily dependent on selectin/ligand adhesive interactions.Glycosyltransferases are involved in the creation of selectin ligands on proteins and lipids. α1,3-fucosylation is imperative for the creation of selectin ligands and a number of fucosyltransferases (FTs) are able to modify terminal lactosamines on cells to create these ligands. One FT, fucosyltransferase VI (FTVI) adds a fucose in an α1,3 configuration to N-acetylglucosamine to generate sialyl Lewis X (sLex) epitopes on proteins of live cells and enhances their ability to bind E-selectin. Although a number of recombinant human FTVIs have been purified, apart from limited commercial enzymes, they were not characterized for their activity on live cells. Here we focused on establishing a robust method to produce FTVI that is active on living cells (hematopoietic cells and mesenchymal stromal cells). To this end, we used two expression systems, Bombyx mori (silkworm) and Pichia pastoris (yeast), to produce significant amounts of N-terminally tagged FTVI and demonstrated that these enzymes have superior activity when compared to currently available commercial enzymes that are produced from various expression systems. Overall, we outline a scheme to attain large amounts of highly active FTVI that can be used for the applications of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.
Original languageEnglish (US)
JournalBiochemistry
DOIs
StatePublished - Sep 9 2020

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