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Crossroads between Bacterial and Mammalian Glycosyltransferases.

Brockhausen I - Front Immunol (2014)

Bottom Line: Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity.A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria.The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Queen's University , Kingston, ON , Canada ; Department of Biomedical and Molecular Sciences, Queen's University , Kingston, ON , Canada.

ABSTRACT
Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen-Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures.

No MeSH data available.


Biosynthesis of N-glycosylated glycoproteins in eukaryotes. N-glycosylation is initiated at the endoplasmic reticulum (ER) membrane using nucleotide sugar donor substrates and a membrane-bound acceptor phospholipid with multiple isoprenyl units (dolichol-phosphate, P-Dol). The first sugar (GlcNAc) is transferred as GlcNAc-phosphate from UDP-GlcNAc by GlcNAc-P-transferase, resulting in GlcNAc-diphosphate-dolichol (GlcNAc-PP-Dol). This step can be inhibited by the UDP-GlcNAc analog tunicamycin. On the outside face of the ER membrane, another GlcNAc is added to form chitobiose, followed by five Man residues to form a heptasaccharide (Man5GlcNAc2)-PP-Dol. This heptasaccharide is flipped to the inside of the ER where the chain grows by transfer of sugars from membrane-bound Man-P-Dol and Glc-P-Dol. The completed saccharide Glc3Man9GlcNAc2 is then transferred by an oligosaccharyltransferase complex (OST) to the Asn residue in an Asn-x-Ser/Thr sequon of nascent proteins. After trimming of sugar residues in the ER by removal of Glc and Man residues to the Man8GlcNAc2 structure, glycoproteins are exported to the Golgi where further trimming occurs by mannosidases. Many N-glycan chains are processed to the complex type by the addition of GlcNAc residues by GlcNAc-transferases I to V (MGAT1 to 5). Chains grow further by the addition of Gal-GlcNAc sequences and termination by sialyl-, Fuc-, Gal-, GlcNAc-, and GalNAc-transferases, which are all highly specific for both the donor and the acceptor substrates and with few exceptions form only one type of linkage between sugars. This creates a multitude of hundreds of different structures and epitopes with many possible functions, depending on the final destination of the glycoprotein, e.g., in the cell membrane or in secretions. Glycoprotein biosynthesis is regulated at many different levels, e.g., by the synthesis and delivery of nucleotide sugar substrates, the expression, activities and localization of glycosyltransferases and trimming hydrolases, the competition of enzymes for common substrates, levels of metal ion activating factors, localization of enzymes involved, and rate of transport of glycoproteins.
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Figure 1: Biosynthesis of N-glycosylated glycoproteins in eukaryotes. N-glycosylation is initiated at the endoplasmic reticulum (ER) membrane using nucleotide sugar donor substrates and a membrane-bound acceptor phospholipid with multiple isoprenyl units (dolichol-phosphate, P-Dol). The first sugar (GlcNAc) is transferred as GlcNAc-phosphate from UDP-GlcNAc by GlcNAc-P-transferase, resulting in GlcNAc-diphosphate-dolichol (GlcNAc-PP-Dol). This step can be inhibited by the UDP-GlcNAc analog tunicamycin. On the outside face of the ER membrane, another GlcNAc is added to form chitobiose, followed by five Man residues to form a heptasaccharide (Man5GlcNAc2)-PP-Dol. This heptasaccharide is flipped to the inside of the ER where the chain grows by transfer of sugars from membrane-bound Man-P-Dol and Glc-P-Dol. The completed saccharide Glc3Man9GlcNAc2 is then transferred by an oligosaccharyltransferase complex (OST) to the Asn residue in an Asn-x-Ser/Thr sequon of nascent proteins. After trimming of sugar residues in the ER by removal of Glc and Man residues to the Man8GlcNAc2 structure, glycoproteins are exported to the Golgi where further trimming occurs by mannosidases. Many N-glycan chains are processed to the complex type by the addition of GlcNAc residues by GlcNAc-transferases I to V (MGAT1 to 5). Chains grow further by the addition of Gal-GlcNAc sequences and termination by sialyl-, Fuc-, Gal-, GlcNAc-, and GalNAc-transferases, which are all highly specific for both the donor and the acceptor substrates and with few exceptions form only one type of linkage between sugars. This creates a multitude of hundreds of different structures and epitopes with many possible functions, depending on the final destination of the glycoprotein, e.g., in the cell membrane or in secretions. Glycoprotein biosynthesis is regulated at many different levels, e.g., by the synthesis and delivery of nucleotide sugar substrates, the expression, activities and localization of glycosyltransferases and trimming hydrolases, the competition of enzymes for common substrates, levels of metal ion activating factors, localization of enzymes involved, and rate of transport of glycoproteins.

Mentions: In eukaryotic cells, N-glycans are assembled first on a dolichol-phosphate (P-Dol) intermediate on the cytoplasmic side of the endoplasmic reticulum (ER) membrane (7, 12) (Figure 1). GlcNAc-phosphate is transferred by GlcNAc-phosphotransferase in a reversible reaction, inhibited by tunicamycin, to P-Dol, followed by the transfer of another GlcNAc residue to form chitobiose. This is followed by five Man residues, all transferred from nucleotide sugar donor substrates to form the common N-glycan core structure Man5-chitobiose linked to PP-Dol. This heptasaccharide is flipped across the membrane and further addition of sugars occurs on the inside of the ER lumen through transfer from Man-P-Dol and Glc-P-Dol donor substrates. After completion of the lipid-linked N-glycan, it is transferred en bloc to the Asn residue(s) of Asn-X-Ser/Thr sequons in a glycoprotein by the oligosaccharyltransferase complex (OST), and Glc and Man residues are selectively cleaved by glycosidases. After transfer to the Golgi, further removal of Man residues occurs, and GlcNAc-transferase I (GnT I, MGAT1) adds the first of the N-glycan antennae in β1–2 linkage to the Manα1–3 arm of the core. This can then be followed by several steps that depend on the presence of this first GlcNAc antenna and the expression of processing enzymes, which remove two Man residues from the Manα1–6 arm, add Fucα1–6 to the inner chitobiose GlcNAc, and add further antennae to form complex type N-glycans. N-glycans can be extended by repeating Gal-GlcNAc residues to form type 1 or type 2 chains (Table 1); they may be branched by GlcNAcβ1–6 linkages and may be decorated with specific functional epitopes and blood group determinants (7). This results in hundreds of different N-glycan structures, depending on the glycosylation potential of the cell.


Crossroads between Bacterial and Mammalian Glycosyltransferases.

Brockhausen I - Front Immunol (2014)

Biosynthesis of N-glycosylated glycoproteins in eukaryotes. N-glycosylation is initiated at the endoplasmic reticulum (ER) membrane using nucleotide sugar donor substrates and a membrane-bound acceptor phospholipid with multiple isoprenyl units (dolichol-phosphate, P-Dol). The first sugar (GlcNAc) is transferred as GlcNAc-phosphate from UDP-GlcNAc by GlcNAc-P-transferase, resulting in GlcNAc-diphosphate-dolichol (GlcNAc-PP-Dol). This step can be inhibited by the UDP-GlcNAc analog tunicamycin. On the outside face of the ER membrane, another GlcNAc is added to form chitobiose, followed by five Man residues to form a heptasaccharide (Man5GlcNAc2)-PP-Dol. This heptasaccharide is flipped to the inside of the ER where the chain grows by transfer of sugars from membrane-bound Man-P-Dol and Glc-P-Dol. The completed saccharide Glc3Man9GlcNAc2 is then transferred by an oligosaccharyltransferase complex (OST) to the Asn residue in an Asn-x-Ser/Thr sequon of nascent proteins. After trimming of sugar residues in the ER by removal of Glc and Man residues to the Man8GlcNAc2 structure, glycoproteins are exported to the Golgi where further trimming occurs by mannosidases. Many N-glycan chains are processed to the complex type by the addition of GlcNAc residues by GlcNAc-transferases I to V (MGAT1 to 5). Chains grow further by the addition of Gal-GlcNAc sequences and termination by sialyl-, Fuc-, Gal-, GlcNAc-, and GalNAc-transferases, which are all highly specific for both the donor and the acceptor substrates and with few exceptions form only one type of linkage between sugars. This creates a multitude of hundreds of different structures and epitopes with many possible functions, depending on the final destination of the glycoprotein, e.g., in the cell membrane or in secretions. Glycoprotein biosynthesis is regulated at many different levels, e.g., by the synthesis and delivery of nucleotide sugar substrates, the expression, activities and localization of glycosyltransferases and trimming hydrolases, the competition of enzymes for common substrates, levels of metal ion activating factors, localization of enzymes involved, and rate of transport of glycoproteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202792&req=5

Figure 1: Biosynthesis of N-glycosylated glycoproteins in eukaryotes. N-glycosylation is initiated at the endoplasmic reticulum (ER) membrane using nucleotide sugar donor substrates and a membrane-bound acceptor phospholipid with multiple isoprenyl units (dolichol-phosphate, P-Dol). The first sugar (GlcNAc) is transferred as GlcNAc-phosphate from UDP-GlcNAc by GlcNAc-P-transferase, resulting in GlcNAc-diphosphate-dolichol (GlcNAc-PP-Dol). This step can be inhibited by the UDP-GlcNAc analog tunicamycin. On the outside face of the ER membrane, another GlcNAc is added to form chitobiose, followed by five Man residues to form a heptasaccharide (Man5GlcNAc2)-PP-Dol. This heptasaccharide is flipped to the inside of the ER where the chain grows by transfer of sugars from membrane-bound Man-P-Dol and Glc-P-Dol. The completed saccharide Glc3Man9GlcNAc2 is then transferred by an oligosaccharyltransferase complex (OST) to the Asn residue in an Asn-x-Ser/Thr sequon of nascent proteins. After trimming of sugar residues in the ER by removal of Glc and Man residues to the Man8GlcNAc2 structure, glycoproteins are exported to the Golgi where further trimming occurs by mannosidases. Many N-glycan chains are processed to the complex type by the addition of GlcNAc residues by GlcNAc-transferases I to V (MGAT1 to 5). Chains grow further by the addition of Gal-GlcNAc sequences and termination by sialyl-, Fuc-, Gal-, GlcNAc-, and GalNAc-transferases, which are all highly specific for both the donor and the acceptor substrates and with few exceptions form only one type of linkage between sugars. This creates a multitude of hundreds of different structures and epitopes with many possible functions, depending on the final destination of the glycoprotein, e.g., in the cell membrane or in secretions. Glycoprotein biosynthesis is regulated at many different levels, e.g., by the synthesis and delivery of nucleotide sugar substrates, the expression, activities and localization of glycosyltransferases and trimming hydrolases, the competition of enzymes for common substrates, levels of metal ion activating factors, localization of enzymes involved, and rate of transport of glycoproteins.
Mentions: In eukaryotic cells, N-glycans are assembled first on a dolichol-phosphate (P-Dol) intermediate on the cytoplasmic side of the endoplasmic reticulum (ER) membrane (7, 12) (Figure 1). GlcNAc-phosphate is transferred by GlcNAc-phosphotransferase in a reversible reaction, inhibited by tunicamycin, to P-Dol, followed by the transfer of another GlcNAc residue to form chitobiose. This is followed by five Man residues, all transferred from nucleotide sugar donor substrates to form the common N-glycan core structure Man5-chitobiose linked to PP-Dol. This heptasaccharide is flipped across the membrane and further addition of sugars occurs on the inside of the ER lumen through transfer from Man-P-Dol and Glc-P-Dol donor substrates. After completion of the lipid-linked N-glycan, it is transferred en bloc to the Asn residue(s) of Asn-X-Ser/Thr sequons in a glycoprotein by the oligosaccharyltransferase complex (OST), and Glc and Man residues are selectively cleaved by glycosidases. After transfer to the Golgi, further removal of Man residues occurs, and GlcNAc-transferase I (GnT I, MGAT1) adds the first of the N-glycan antennae in β1–2 linkage to the Manα1–3 arm of the core. This can then be followed by several steps that depend on the presence of this first GlcNAc antenna and the expression of processing enzymes, which remove two Man residues from the Manα1–6 arm, add Fucα1–6 to the inner chitobiose GlcNAc, and add further antennae to form complex type N-glycans. N-glycans can be extended by repeating Gal-GlcNAc residues to form type 1 or type 2 chains (Table 1); they may be branched by GlcNAcβ1–6 linkages and may be decorated with specific functional epitopes and blood group determinants (7). This results in hundreds of different N-glycan structures, depending on the glycosylation potential of the cell.

Bottom Line: Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity.A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria.The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Queen's University , Kingston, ON , Canada ; Department of Biomedical and Molecular Sciences, Queen's University , Kingston, ON , Canada.

ABSTRACT
Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen-Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures.

No MeSH data available.