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

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 ∆mnn1 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.

Overexpression of HUT1 gene stimulates in vivo galactosylation by enhancing UDP–galactose transport activity in Saccharomyces cerevisiae

“…In this strain, both in vitro UDP-Gal transport activity and in vivo galactosylation at the cell surface were confirmed (Roy et al, 1998). A similar strain construct, MKY12, in which the gma12 + gene was functionally expressed under the background of a common laboratory strain W303-1A, showed a detectable level of in vitro UDP-Gal transport activity (Kainuma et al, 1999). In addition, we found that overexpression of human derived UDP-Gal transporter resulted in a significant increase of UDP-Gal transport activity as well as in vivo galactosylation on both N-and O-linked oligosaccharides.…”

“…Strains MKY6, MKY12, MKY13 and MKY14 were constructed as described previously (Kainuma et al, 1999). Briefly, MKY6 harbours an integrated gma12+ gene encoding a1,2-galactosyltransferase from Sz.…”

“…The preparation of chitinase samples, their separation by 7.5% SDS-PAGE, transfer to PVDF membrane and lectin blot analysis using biotinylated GS I-B4 were carried out as described previously (Kainuma et al, 1999), with the exception of using CDP-star (TROPIX) for more sensitive detection by chemiluminescence.…”

“…The pellet was resuspended in the same buffer. The samples were fractionated on a 12% SDS-PAGE and Western immunoblotting was carried out using anti-HA-monoclonal antibody as described previously (Kainuma et al, 1999).…”

“…The following components are essential for glycosylation in the Golgi: glycosyltransferases; sugar-nucleotides as donor substrates; oligosaccharides attached to proteins or lipids as acceptors; and sugar-nucleotide transporters to transport the substrate from the cytoplasm to the lumen of the Golgi. It has been shown that protein glycosylation can be regulated by the availability of various sugar-nucleotides in the Golgi lumen (Kainuma et al, 1999;Pels Rijcken et al, 1995;Toma et al, 1996).…”