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

Abstract

Highlights• Human fucosyltransferases expressed and purified from both Pichia pastoris yeast and Bombyx mori silkworm display specific activities > 1000 pmol/min/g that efficiently create sialyl Lewis X epitopes on primary human stem cells capable of binding E-selectin.• Fusing BmApoLpIII to fucosyltransferases allows for the production of highly active human enzymes in large scale from Bombyx mori silkworm.

CD34+ HSPCs-derived exosomes contain dynamic cargo and promote their migration through functional binding with the homing receptor E-selectin

“…To further investigate the impact of sLe x formation on the ability of exosomes to interact with E-selectin, exosomes were isolated from K562 ( Al-Amoodi et al, 2020 ) cell line ( Supplementary Figure S5A ). K562 cells are not able to bind E-selectin, because their ligands are missing the necessary glycosylation.…”

“…K562 cells are not able to bind E-selectin, because their ligands are missing the necessary glycosylation. Using recombinant fucosyltransferase VI (FTVI), an enzyme, that is, not expressed in K562 cells but can aid in the creation of sLe x , E-selectin binding can be achieved ( Al-Amoodi et al, 2020 ). Like the cells they originate from, the E-selectin ligands, CD44, CD43 and PSGL-1 were created on K562-derived exosomes ( Supplementary Figure S5B ).…”

“…Fucosyltransferase VI treatment: K562-derived exosomes were isolated and treated with the recombinant human fucosyltransferase VI (rhFTVI) enzyme that was produced in our laboratory ( Al-Amoodi et al, 2020 ). The treatment was performed in a 96-well plate in rhFTVI using 100 μL of 2X reaction buffer [25 mM HEPES (pH 7.5) (Gibco Invitrogen), 0.1% human serum albumin (Sigma-Aldrich), 0.5 mM GDP-fucose (Sigma), and 5 mM MnCl 2 ], 100 μl of PBS containing 50 × 10 6 exosomes, and approximately 1 μg of purified rhFTVI enzyme.…”

Refining the migration and engraftment of short-term and long-term HSCs by enhancing homing-specific adhesion mechanisms

“…Therefore, we sought to engineer the expression of sLe x glycan structures on the surface of Flk2 − CD34 − and Flk2 − CD34 + HSCs using the (1,3) linkage-specific fucosyltransferase, rhFTVI. 27 This enzyme specifically places fucose onto a terminal type 2 lactosamine unit; if that lactosamine is capped with a (2,3)-linked sialic acid, sLe x is created. Flow cytometric analysis was performed to quantify sLe x expression using the HECA-452 antibody, and as illustrated in Figure 4A , rhFTVI treatment increased HECA-452 reactivity in both populations, indicating sLe x structures were created.…”

“…The fucosylation treatment was performed as described previously. 27 Briefly, both Flk2 − CD34 + and Flk2 − CD34 − HSCs were harvested, washed twice with HBSS, and resuspended at a density of 20 to 40 × 10 3 cells per mL in FTVI reaction buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)[pH 7.5]; Gibco Invitrogen), 0.1% human serum albumin (Sigma-Aldrich), 0.5 mM guanosine diphosphate-fucose (GDP-fucose) (Sigma), and 5 mM MnCl2 and 1 μg of purified rhFTVI enzyme in HBSS. Cells were incubated at 37°C for 30 minutes.…”