Biochemical Properties of Neisseria gonorrhoeae LgtE

Abstract

A fragment of chromosomal DNA encoding the lgtE gene of Neisseria gonorrhoeae strain F62 was amplified by PCR and cloned into the expression vector pET15b. Functional LgtE was purified and its biochemical properties were determined. The purified enzyme was maximally active in buffer containing manganese; minimal activity was obtained in buffer containing other divalent cations. LgtE was only able to mediate the addition of UDP-galactose into neisserial lipooligosaccharides (LOSs). We used a variety of genetically defined and chemically verified LOS structures to determine acceptor specificity. LgtE was able to mediate the addition of galactose into a variety of LOS structures, indicating the this enzyme possesses broad acceptor specificity. Furthermore, it was able to add multiple galactose residues onto LOS. We also determined that this enzyme was capable of adding galactose onto both the ␣ and ␤ chains of neisserial LOS.Lipooligosaccharide (LOS) is an important virulence determinant of the pathogenic neisseriae (12,16). It consists of an oligosaccharide component that is attached to lipid A via a Kdo (3-deoxy-D-manno-octulosonic acid) linkage. The genes involved in the synthesis of the oligosaccharide portion of this molecule from a variety of species have been identified and characterized (1,2,11,13,17,28). A common feature of LOS expression in all of these species is the expression of multiple phase-variable LOS structures. Most of this variability is attributed to changes in the carbohydrate composition of the molecule. The genetic basis for this variation has been well characterized. Key genes in the biosynthetic pathway contain homopolymeric runs of guanine (2, 11). Changes in the number of guanines result in reading frame shifts, with the end result being the truncation or elongation of a particular LOS molecule, depending on the nature of the starting reading frame (4,5,37).The data presented in Fig. 1 summarize the genetic potential and reported carbohydrate structures that have been identified in Neisseria gonorrhoeae (7,9,10,15,18,(34)(35)(36). The genes responsible for the addition of most of these sugars have been defined genetically; loss of gene function results in the truncation of an LOS structure. Biochemical characterization of several of these gene products has been performed by measuring the incorporation of sugars from various UDP-sugars into a variety of synthetic carbohydrates. LgtA possessed broad substrate specificity towards ␣ and ␤ galactosides. Depending on the acceptor, this enzyme could mediate the transfer of GlcNAc from UDP-GlcNAc and GalNAc from UDP-GalNAc (3). However, this broad specificity was not seen in vivo (27). The biochemical properties of LgtB and LgtC have been examined similarly (21,30,31). Both enzyme possessed the predicted galactosyltransferase activities.Genetic evidence supporting the function of lgtE as encoding a glycosyl transferase responsible for the addition of galactose ␤-1,4 to glucose has been reported in a variety of publications (6,11,14,26). However...

Biochemical Analysis of Lpt3, a Protein Responsible forPhosphoethanolamine Addition to Lipooligosaccharide ofPathogenic Neisseria

“…Genetic studies on LOS biosynthesis in strain F62 identified a gene cluster lgtA-E (10) that was responsible for the addition of most of the sugars found on the ␣ chain. Biochemical and genetic analysis have confirmed the functions of each of these genes (3,24,38,40) and their involvement in the synthesis of the ␣ chain. Additional genes needed to synthesize the ␤ and ␥ chain have also been identified (1,18), and most of the biochemical properties of these gene products have been defined (41).…”

The lgtABCDE Gene Cluster, Involved in Lipooligosaccharide Biosynthesis in Neisseria gonorrhoeae , Contains Multiple Promoter Sequences

“…Simultaneous production of multiple LOS epitopes is mediated by production of limiting amounts of the LgtA, LgtD, or LgtE. This occurs via transcriptional or translational strand slippage (11), via regulation of transcriptional expression (the present study), and via the low kinetic efficiencies of specific glycosyl transferases (36). In addition, recombination between glycosyl transferases is seen in some strains of Neisseria and these recombinant loci may consequently invoke one or more of the above mechanisms of phase variation (5,36 …”

Glycomimicry: display of fucosylation on the lipo-oligosaccharide of recombinant Escherichia coli K12

“…The resulting strain was termed LPS1. Five glycosyltransferase genes necessary for the display of the Lewis Y antigen from different bacterial sources were expressed heterologously: lgtE, encoding a β1,4-galactosyltransferase of Neisseria gonorrhoea reported to transfer to glucose in the LOS structure of N. gonorrhoea [31]; lgtA from Neisseria meningitidis encoding a β1,3-N-acetylglucosaminyltranferase with galactose as an acceptor, and lgtB, encoding a β1,3-galactosyltranferase with N-acetylglucosamine as an acceptor, also from N. meningitidis [32]. In addition, futA and futC, encoding α1,3-and α1,2- (Fig.…”