Crossroads between Bacterial and Mammalian Glycosyltransferases

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.

Keywords: glycan mimics; glycoprotein epitopes; glycosyltransferases; protein structure; specificities.

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.

Figure 2

Biosynthesis of lipopolysaccharides in Gram-negative bacteria by the polymerase-dependent pathway. Many steps of the complex sequences and controls in the biosynthesis of LPS in Gram-negative bacteria are similar to those in mammalian glycoprotein biosynthesis. The inner membrane serves as the site of glycan biosynthesis, and the membrane-bound acceptor is undecaprenol-phosphate (P-Und) having 11 isoprenyl units, which is less than those found in eukaryotic Dol. Nucleotide sugars are synthesized in the cytosol and used for most glycosylation reactions. As in the N-glycan biosynthesis, the first sugar is transferred as sugar-phosphate by membrane-bound WecA to synthesize GalNAc/GlcNAc-PP-Und. This step can also be blocked by tunicamycin. It is possible that a 4-epimerase is involved. Subsequently, sugars are added individually to form the repeating unit of the O antigen. The glycosyltransferases that transfer sugars from nucleotide sugars usually have a high specificity for their donor and acceptor substrates and are associated with the membrane. After Wzx transports the repeating units to the periplasm, they are polymerized by Wzy by addition of repeating units to the reducing end of the growing polysaccharide linked to PP-Dol. The O antigen can be further processed and modified to form completed O antigens and the biosynthesis is usually terminated with Wzz. The O polysaccharide is then transferred to a sugar of the core oligosaccharide linked to lipid A by a ligase, forming the LPS, which is exported to the outer membrane by the Lpt complex. The O antigenic polysaccharide is then exposed to the environment on the outer membrane. Although many bacterial enzymes involved in LPS synthesis have been cloned, the individual steps of LPS synthesis are not well understood, mainly because of the major challenge to find the appropriate enzyme substrates and conditions to assay enzymes. The example shows the biosynthesis of the E. coli O104 antigen. The repeating unit tetrasaccharide contains the cancer-associated T antigen (Galβ1-3GalNAc), as well as the sialyl-T antigen (sialylα2-3Galβ1-3GalNAc). The WbwA sialyltransferase and the WbwB Gal-transferase remain to be characterized.

Figure 3

Biosynthesis of polysialic acids in E. coli using the ABC transporter pathway. The biosynthesis of homopolymeric O antigens (or capsules) is initiated at the cytosolic side of the inner membrane. Polysialic acids (PSA) of E. coli are proposed to be assembled in a processive fashion as shown, based on undecaprenol-phosphate. Membrane-associated polysialyltransferase (PST) transfers many units of sialic acid from CMP-sialic acid to the growing polysialic acid. The enzyme can act on a number of acceptor substrates to form repeated sialylα2–8 linkages. A termination reaction stops the growth of the long PSA chain. The PSA is then transported to the periplasmic space by the Wzm exporter, which is associated with the ATP-binding Wzt. Further processing occurs in the periplasm. The completed PSA is ligated to the core-lipid A and then translocated by export proteins to the outer membrane to serve as a highly charged and hydrophilic protective coat. Other homopolymeric O antigens such as poly-d-Mannose or poly-d-Rhamnose are processed in a similar fashion by the ABC transporter pathway.