Emerging facets of prokaryotic glycosylation

Abstract

Glycosylation of proteins is one of the most prevalent post-translational modifications occurring in nature, with a wide repertoire of biological implications. Pathways for the main types of this modification, the N- and O-glycosylation, can be found in all three domains of life-the Eukarya, Bacteria and Archaea-thereby following common principles, which are valid also for lipopolysaccharides, lipooligosaccharides and glycopolymers. Thus, studies on any glycoconjugate can unravel novel facets of the still incompletely understood fundamentals of protein N- and O-glycosylation. While it is estimated that more than two-thirds of all eukaryotic proteins would be glycosylated, no such estimate is available for prokaryotic glycoproteins, whose understanding is lagging behind, mainly due to the enormous variability of their glycan structures and variations in the underlying glycosylation processes. Combining glycan structural information with bioinformatic, genetic, biochemical and enzymatic data has opened up an avenue for in-depth analyses of glycosylation processes as a basis for glycoengineering endeavours. Here, the common themes of glycosylation are conceptualised for the major classes of prokaryotic (i.e. bacterial and archaeal) glycoconjugates, with a special focus on glycosylated cell-surface proteins. We describe the current knowledge of biosynthesis and importance of these glycoconjugates in selected pathogenic and beneficial microbes.

Keywords: glycan biosynthesis; glycoengineering; glycoproteins; prokaryotes; secondary cell-wall polymers; surface (S-) layer.

Figure 1

Scheme of cell envelopes of prokaryotic organisms, showing representative glycoconjugates (different scenarios of cell envelope architecture are separated by blue perpendicular lines), including S-layer glycoproteins (Messner, Schäffer and Kosma 2013), SCWPs (Messner, Schäffer and Kosma 2013), bacterial flagella (Cullen and Trent 2010; Mukherjee and Kearns 2014) and bacterial pili (Korotkov, Sandkvist and Hol 2012; Reunanen et al. 2012) and archaella and archaeal pili (Pohlschroder et al. 2011; Korotkov, Sandkvist and Hol 2012). CM, cytoplasmic membrane; PG, peptidoglycan; , peptidoglycan strand; OM, outer membrane; , outer membrane lipid A; , bacterial and archaeal membrane phospholipid; , archaeal membrane tetraetherlipid; , , , different S-layer glycans; SCWP, secondary cell wall polymer. In different archaeal species, S-layer (glyco)protein anchoring to the cell envelope has been suggested either by a protein transmembrane anchor (Lechner and Wieland 1989) or in an archaeosortase-dependent process by a lipid anchor (Abdul Halim et al. 2016). (Extended and modified from Messner, Schäffer and Kosma 2013. With permission from Elsevier).

Figure 2

Schematic depiction of S-layer glycoprotein glycans from Gram-positive and Gram-negative bacteria and archaea and of SCWPs of Gram-positive bacteria. Ac, CH₃COO⁻; CONH₂, amidyl-; -COOMe, methyl ester; Me, CH_3-; NAc, N-acetyl-; P, phosphate; Pyr, pyruvyl; R₁, -COOH; R₂, -CONHAc; R₃, -CONAc₂, S, sulfate;. Sugar symbols:◯, hexose;, hexuronic acid;, glucose;, N-acetylglucosamine;, glucuronic acid;, 3-N-acetylquinovosamine;, 2,3-di-N-acetylglucuronic acid;, 6-sulfoquinovose;, 2-amino-6-sulfo-2,6-dideoxy-quinovose;, galactose;, galactofuranose;, N-acetylgalactosamine;, galacturonic acid;, 3-O-methylgalacturonic acid;, mannose;, N-acetylmannosamine;, mannosaminuronic acid;, 6-N-amido-Thr mannosaminuronic acid;, l-rhamnose;, d-rhamnose;, l-fucose;, d-fucose;, 3-N-acetylfucosamine;, iduronic acid;, ribose;, d-glycero-d-manno-heptose;, xylose;, digitoxose;, 5-acetamidino-7-glycerolyl pseudaminic acid;, N-acetylmuramic acid. Monosaccharide symbols follow the SNFG (Symbol Nomenclature for Glycans) (Appendix-1B 2015). (Mescher and Strominger 1976; Koval and Jarrell 1987; Lechner and Sumper 1987; Sumper et al. 1990; Altman et al. 1991, 1992, 1995, 1996; Baumeister and Lembcke 1992; Christian et al. 1993; Kärcher et al. 1993; Möschl et al. 1993; Bock et al. 1994; Messner et al. 1995; Kosma et al. 1995a,b; Meier-Stauffer et al. 1996; Schäffer et al. 1999a,b, 2000a,b, 2004; Eichler 2000, 2013; Steindl et al. 2002, 2005; Pfoestl et al. 2003; Kählig et al. 2005; Voisin et al. 2005; Steiner et al. 2008; Veith et al. 2009; Peyfoon et al. 2010; Posch et al. 2011; Messner, Schäffer and Kosma 2013; Palmieri et al. 2013; Kandiba and Eichler 2014; Parente et al. 2014; Anzengruber et al. 2014a; Appendix-1B 2015; Lu et al. 2015; Kandiba et al. 2016). (Extended and modified from Eichler 2013. With permission from Nature Publishing Group).

Figure 2

Schematic depiction of S-layer glycoprotein glycans from Gram-positive and Gram-negative bacteria and archaea and of SCWPs of Gram-positive bacteria. Ac, CH₃COO⁻; CONH₂, amidyl-; -COOMe, methyl ester; Me, CH_3-; NAc, N-acetyl-; P, phosphate; Pyr, pyruvyl; R₁, -COOH; R₂, -CONHAc; R₃, -CONAc₂, S, sulfate;. Sugar symbols:◯, hexose;, hexuronic acid;, glucose;, N-acetylglucosamine;, glucuronic acid;, 3-N-acetylquinovosamine;, 2,3-di-N-acetylglucuronic acid;, 6-sulfoquinovose;, 2-amino-6-sulfo-2,6-dideoxy-quinovose;, galactose;, galactofuranose;, N-acetylgalactosamine;, galacturonic acid;, 3-O-methylgalacturonic acid;, mannose;, N-acetylmannosamine;, mannosaminuronic acid;, 6-N-amido-Thr mannosaminuronic acid;, l-rhamnose;, d-rhamnose;, l-fucose;, d-fucose;, 3-N-acetylfucosamine;, iduronic acid;, ribose;, d-glycero-d-manno-heptose;, xylose;, digitoxose;, 5-acetamidino-7-glycerolyl pseudaminic acid;, N-acetylmuramic acid. Monosaccharide symbols follow the SNFG (Symbol Nomenclature for Glycans) (Appendix-1B 2015). (Mescher and Strominger 1976; Koval and Jarrell 1987; Lechner and Sumper 1987; Sumper et al. 1990; Altman et al. 1991, 1992, 1995, 1996; Baumeister and Lembcke 1992; Christian et al. 1993; Kärcher et al. 1993; Möschl et al. 1993; Bock et al. 1994; Messner et al. 1995; Kosma et al. 1995a,b; Meier-Stauffer et al. 1996; Schäffer et al. 1999a,b, 2000a,b, 2004; Eichler 2000, 2013; Steindl et al. 2002, 2005; Pfoestl et al. 2003; Kählig et al. 2005; Voisin et al. 2005; Steiner et al. 2008; Veith et al. 2009; Peyfoon et al. 2010; Posch et al. 2011; Messner, Schäffer and Kosma 2013; Palmieri et al. 2013; Kandiba and Eichler 2014; Parente et al. 2014; Anzengruber et al. 2014a; Appendix-1B 2015; Lu et al. 2015; Kandiba et al. 2016). (Extended and modified from Eichler 2013. With permission from Nature Publishing Group).

Figure 2

Schematic depiction of S-layer glycoprotein glycans from Gram-positive and Gram-negative bacteria and archaea and of SCWPs of Gram-positive bacteria. Ac, CH₃COO⁻; CONH₂, amidyl-; -COOMe, methyl ester; Me, CH_3-; NAc, N-acetyl-; P, phosphate; Pyr, pyruvyl; R₁, -COOH; R₂, -CONHAc; R₃, -CONAc₂, S, sulfate;. Sugar symbols:◯, hexose;, hexuronic acid;, glucose;, N-acetylglucosamine;, glucuronic acid;, 3-N-acetylquinovosamine;, 2,3-di-N-acetylglucuronic acid;, 6-sulfoquinovose;, 2-amino-6-sulfo-2,6-dideoxy-quinovose;, galactose;, galactofuranose;, N-acetylgalactosamine;, galacturonic acid;, 3-O-methylgalacturonic acid;, mannose;, N-acetylmannosamine;, mannosaminuronic acid;, 6-N-amido-Thr mannosaminuronic acid;, l-rhamnose;, d-rhamnose;, l-fucose;, d-fucose;, 3-N-acetylfucosamine;, iduronic acid;, ribose;, d-glycero-d-manno-heptose;, xylose;, digitoxose;, 5-acetamidino-7-glycerolyl pseudaminic acid;, N-acetylmuramic acid. Monosaccharide symbols follow the SNFG (Symbol Nomenclature for Glycans) (Appendix-1B 2015). (Mescher and Strominger 1976; Koval and Jarrell 1987; Lechner and Sumper 1987; Sumper et al. 1990; Altman et al. 1991, 1992, 1995, 1996; Baumeister and Lembcke 1992; Christian et al. 1993; Kärcher et al. 1993; Möschl et al. 1993; Bock et al. 1994; Messner et al. 1995; Kosma et al. 1995a,b; Meier-Stauffer et al. 1996; Schäffer et al. 1999a,b, 2000a,b, 2004; Eichler 2000, 2013; Steindl et al. 2002, 2005; Pfoestl et al. 2003; Kählig et al. 2005; Voisin et al. 2005; Steiner et al. 2008; Veith et al. 2009; Peyfoon et al. 2010; Posch et al. 2011; Messner, Schäffer and Kosma 2013; Palmieri et al. 2013; Kandiba and Eichler 2014; Parente et al. 2014; Anzengruber et al. 2014a; Appendix-1B 2015; Lu et al. 2015; Kandiba et al. 2016). (Extended and modified from Eichler 2013. With permission from Nature Publishing Group).

Figure 2

Schematic depiction of S-layer glycoprotein glycans from Gram-positive and Gram-negative bacteria and archaea and of SCWPs of Gram-positive bacteria. Ac, CH₃COO⁻; CONH₂, amidyl-; -COOMe, methyl ester; Me, CH_3-; NAc, N-acetyl-; P, phosphate; Pyr, pyruvyl; R₁, -COOH; R₂, -CONHAc; R₃, -CONAc₂, S, sulfate;. Sugar symbols:◯, hexose;, hexuronic acid;, glucose;, N-acetylglucosamine;, glucuronic acid;, 3-N-acetylquinovosamine;, 2,3-di-N-acetylglucuronic acid;, 6-sulfoquinovose;, 2-amino-6-sulfo-2,6-dideoxy-quinovose;, galactose;, galactofuranose;, N-acetylgalactosamine;, galacturonic acid;, 3-O-methylgalacturonic acid;, mannose;, N-acetylmannosamine;, mannosaminuronic acid;, 6-N-amido-Thr mannosaminuronic acid;, l-rhamnose;, d-rhamnose;, l-fucose;, d-fucose;, 3-N-acetylfucosamine;, iduronic acid;, ribose;, d-glycero-d-manno-heptose;, xylose;, digitoxose;, 5-acetamidino-7-glycerolyl pseudaminic acid;, N-acetylmuramic acid. Monosaccharide symbols follow the SNFG (Symbol Nomenclature for Glycans) (Appendix-1B 2015). (Mescher and Strominger 1976; Koval and Jarrell 1987; Lechner and Sumper 1987; Sumper et al. 1990; Altman et al. 1991, 1992, 1995, 1996; Baumeister and Lembcke 1992; Christian et al. 1993; Kärcher et al. 1993; Möschl et al. 1993; Bock et al. 1994; Messner et al. 1995; Kosma et al. 1995a,b; Meier-Stauffer et al. 1996; Schäffer et al. 1999a,b, 2000a,b, 2004; Eichler 2000, 2013; Steindl et al. 2002, 2005; Pfoestl et al. 2003; Kählig et al. 2005; Voisin et al. 2005; Steiner et al. 2008; Veith et al. 2009; Peyfoon et al. 2010; Posch et al. 2011; Messner, Schäffer and Kosma 2013; Palmieri et al. 2013; Kandiba and Eichler 2014; Parente et al. 2014; Anzengruber et al. 2014a; Appendix-1B 2015; Lu et al. 2015; Kandiba et al. 2016). (Extended and modified from Eichler 2013. With permission from Nature Publishing Group).

Figure 3

Working model of S-layer glycan biosynthesis in Pa. alvei CCM 2051^T. The initial transfer of a Gal residue from UDP-α-d-Gal to a lipid carrier is catalysed by WsfP (A). The adaptor saccharide is formed by the α1,3-linkage of an l-Rha residue from dTDP-β-l-Rha to the linkage sugar d-Gal possibly performed by WsfG, followed by the transfer of two additional α1,3-linked l-Rha residues possibly by the action of WsfF (B). The glycan chain would be elongated by the activity of the aminosugar transferase WsfE and the tripartite transferase WsfC. WsfE may form the β1,4-linkage of a ManNAc residue from UDP-ManNAc to the third rhamnose residue. WsfC putatively adds a single glycerol phosphate from CDP-glycerol to the ManNAc residue of the adaptor oligosaccharide and may form the β1,3-linkage of a ManNAc residue to the third rhamnose residue as well as the β1,4-linkage of a Gal to the ManNAc residues of the repeating units. The glycan chain would be recognised by the carboxy-terminal part of Wzt and exported by the ABC transporter system through the cytoplasmic membrane (C). The transfer of cytoplasmic Glc to the lipid carrier would be carried out by WsfH and, after reorientation, is used at the external face of the cytoplasmic membrane by WsfD for α1,6-linkage of the Glc residues to ManNAc residues of the repeating units (D). The final transfer of the completed S-layer glycan to certain tyrosine residues of the S-layer protein is predicted to occur cosecretionally upon catalysis of the O-OST WsfB (E). Eventually, the mature S-layer glycoprotein would be self-assembled at the cell surface (F). Please note that so far only the WsfP protein has been experimentally verified to perform its predicted role. (Adapted from Zarschler et al. 2010b. With permission from Oxford University Press).