Substrate specificities of α1,2- and α1,3-galactosyltransferases and characterization of Gmh1p and Otg1p in Schizosaccharomyces pombe

Abstract

In the fission yeast Schizosaccharomyces pombe, α1,2- and α1,3-linked D-galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases. Although the galactomannans are important for cell–cell communication in S. pombe (e.g., in non-sexual aggregation), the mechanisms underlying galactosylation in cells remain unclear. S. pombe has 10 galactosyltransferase-related genes: seven belonging to glycosyltransferase (GT) family 34 and three belonging GT family 8. Disruption of all 10 α-galactosyltransferases (strain Δ10GalT) has been shown to result in a complete lack of α-Gal residues. Here, we have investigated the function and substrate specificities of galactosyltransferases in S pombe by using strains expressing single α-galactosyltransferases in the Δ10GalT background. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides showed that two GT family 34 α1,2-galactosyltransferases (Gma12p and Gmh6p) and two GT family 8 α1,3-galactosyltransferases (Otg2p and Otg3p) are involved in galactosylation of O-linked oligosaccharide. Moreover, 1H-NMR of N-glycans revealed that three GT family 34 α1,2-galactosyltransferases (Gmh1p, Gmh2p, and Gmh3p) are required for galactosylation of N-linked oligosaccharides. Furthermore, HPLC and lectin-blot analysis revealed that Otg1p showed α1,3-galactosyltransferase activity under conditions of co-expression with Gmh6p, indicating that α-1,2-linked galactose is required for the galactosylation activity of Otg1p in S. pombe. In conclusion, eight galactosyltransferases have been shown to have activity in S. pombe with different substrate specificities. These findings will be useful for genetically tailoring the galactosylation of both N- and O- glycans in fission yeast.

Humanization of Yeasts for Glycan-Type End-Products

“…Inactivation of the OCH1 gene is often an effective approach to eliminate hypermannosylation in H. polymorpha (Fukunaga et al, 2021b ; Gallo et al, 2022 ), K. lactis (Park et al, 2011 ), P. pastoris (Ma et al, 2019 ), S. cerevisiae (Piirainen et al, 2022 ), and Y. lipolytica (Karbalaei et al, 2020 ), which results in the expressed proteins with short-chain glycan from Man 3 GlcNAc 2 to Man 14 GlcNAc 2 ( Table 1 ). Especially in H. polymorpha , the deletion of OCH1 and alg3, and/or alg3 and alg11 can produce the protein with Man 3 GlcNAc 2 (Gündüz Ergün et al, 2019 ; Fukunaga et al, 2021b ). The expression of alg6 in S. pombe also produces the short-chain glycoprotein (Hamilton et al, 2013 ).…”

“…Gene modification has been applied to construct a humanized N-linked glycosylation pathway in K. lactis . Besides the above conventional yeast species, other established yeast expression systems include Hansenula polymorpha (Gündüz Ergün et al, 2019 ; Fukunaga et al, 2021a , b ; Giorgetti et al, 2021 ; Gallo et al, 2022 ), Yarrowia lipolytica (Hamilton et al, 2013 ; Karbalaei et al, 2020 ; Jin et al, 2021 ), Schizosaccharomyces pombe (Kim et al, 2006 ; Katla et al, 2019 ; Lawton et al, 2021 ), Kluyveromyces marxianus (Lee et al, 2020 ), Candida albicans (Li et al, 2019 ), and Zygosaccharomyces cidri (Liu et al, 2009 ). The strategies to produce humanized glycosylated end-products were explored by using these yeast species.…”