Coexpression of 1,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae

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Abstract
We have studied in vivo neo-galactosylation in Saccharomyces cerevisiae and analyzed the critical factors involved in this system. Two heterologous genes, gma12 + encoding α1,2-galactosyltransferase (α1,2 GalT) from Schizosaccharomyces pombe and UGT2 encoding UDP-galactose (UDP-Gal) transporter from human, were functionally expressed to examine the intracellular conditions required for galactosylation. Detection by fluorescence labeled α-galactose specific lectin revealed that 50% of the cells incorporated galactose to cell surface mannoproteins only when the gma12 + and hUGT2 genes were coexpressed in galactose media. Integration of both genes in the Δmnn1 background cells increased galactosylation to 80% of the cells. Correlation between cell surface galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter rather than α1,2 GalT played a key role for efficient galactosylation in S.cerevisiae . In addition, this heterologous system enabled us to study the in vivo function of S.pombe α1,2 GalT to prove that it transfers galactose to both N - and O -linked oligosaccharides. Structural analysis indicated that this enzyme transfers galactose to O -mannosyl residue attached to polypeptides and produces Galα1,2-Man1-O-Ser/Thr structure. Thus, we have successfully generated a system for efficient galactose incorporation which is originally absent in S.cerevisiae , suggesting further possibilities for in vivo glycan remodeling toward therapeutically useful galactose containing heterologous proteins in S.cerevisiae .