RRC ID 64950
Author Al-Amoodi AS, Sakashita K, Ali AJ, Zhou R, Lee JM, Tehseen M, Li M, Belmonte JCI, Kusakabe T, Merzaban JS.
Title Using Eukaryotic Expression Systems to Generate Human α1,3-Fucosyltransferases That Effectively Create Selectin-Binding Glycans on Stem Cells.
Journal Biochemistry
Abstract Recruitment of circulating cells toward 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) can 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 for producing 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 for obtaining large amounts of highly active FTVI that can be used for the application of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.
Volume 59(39)
Pages 3757-3771
Published 2020-10-6
DOI 10.1021/acs.biochem.0c00523
PMID 32901486
MeSH Animals Bombyx / genetics Cell Line Cell Line, Tumor E-Selectin / metabolism* Fucosyltransferases / genetics Fucosyltransferases / metabolism* Gene Expression Humans Pichia / genetics Polysaccharides / metabolism* Recombinant Proteins / genetics Recombinant Proteins / metabolism Stem Cells / metabolism*
IF 2.865
Silkworms d17 p53