Targeting of active rat α2,3-sialyltransferase to the yeast cell wall by the aid of the hsp 150Δ-carrier: toward synthesis of sLex-decorated L-selectin ligands

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Abstract
Interactions between selectins and their oligosaccharide-decorated ligands play a crucial role in the initiation of leukocyte extravasation. We have shown that synthetic multivalent sialyl Lewis x glycans inhibit strongly the adhesion of lymphocytes to endothelium at sites of inflammation. However, enzyme-assisted synthesis of these oligosaccharides is hampered by the lack of sufficient amounts of specific glycosyltransferases. We report here the construction of Saccharomyces cerevisiae strains expressing the soluble catalytic ectodomain of rat Galβ1–3/4GlcNAc α2,3-sialyltransferase (ST3Ne) fused to the C-terminus of the hsp150Δ-carrier polypeptide. The hsp150Δ-carrier, which is an N-terminal fragment of a natural secretory protein of yeast, is able to confer secretion-competence to several heterologous proteins, which otherwise remain in the yeast endoplasmic reticulum. The ST3Ne portion of the hspl50Δ-ST3Ne fusion protein adopted an enzymatically active conformation and was N-glycosylated and disulfide-bonded. HSP150Δ-ST3Ne was secreted with a half-time of about 7.5 min and remained intercalated in the cell wall, which covers the yeast plasma membrane. About 110 mU of sialyl transferase per litre was produced in 16 h. Whole live yeast cells were able to transfer sialic acid from CMP-NeuNAc to N-acetyllactosamine yielding α2,3-sialyl-N-acetyllactosa-mine, as evidenced by paper chromatography, cleavage by linkage-specific sialidase, and NMR analysis. Our data suggest that yeast cells externalizing mammalian glycosyl-transferases with the aid of the hsp150Δ-carrier could provide a source of enzymes for synthesis of valuable oligosac-charides.

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