High-level expression of the Neisseria meningitidis lgtA gene in Escherichia coli and characterization of the encoded N-acetylglucosaminyltransferase as a useful catalyst in the synthesis of GlcNAc 1->3Gal and GalNAc 1->3Gal linkages

Abstract

We have expressed the Neisseria meningitidis lgtA gene at a high level in Escherichia coli. The encoded beta-N-acetylglucosaminyltransferase, referred to as LgtA, which in the bacterium is involved in the synthesis of the lacto-N-neo-tetraose structural element of the bacterial lipooligosaccharide, was obtained in an enzymatically highly active form. This glycosyltransferase appeared to be unusual in that it displays a broad acceptor specificity toward both alpha- and beta-galactosides, whether structurally related to N- or O-protein-, or lipid-linked oligosaccharides. Product analysis by one- and two-dimensional 400 MHz 1H- and 13C-NMR spectroscopy reveals that LgtA catalyzes the introduction of GlcNAc from UDP-GlcNAc in a beta 1-->3-linkage to accepting Gal residues. The enzyme can thus be characterized as a UDP-GlcNAc:Gal alpha/beta-R beta 3-N-acetylglucosaminyltransferase. Although lactose is a highly preferred acceptor substrate the recombinant enzyme also acts efficiently on monomeric and dimeric N-acetyllactosamine revealing its potential value in the synthesis of polylactosaminoglycan structures in enzyme assisted procedures. Furthermore, LgtA shows a high donor promiscuity toward UDP-GalNAc, but not toward other UDP-sugars, and can catalyze the introduction of GalNAc in beta 1-->3-linkage to alpha- or beta-Gal in the acceptor structures at moderate rates. LgtA therefore shows promise to be a useful catalyst in the preparative synthesis of both GlcNAc beta 1-->3Gal and GalNAc beta 1-->3Gal linkages.

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).…”

A Novel Glycosulfopeptide Binds to P-selectin and Inhibits Leukocyte Adhesion to P-selectin

“…Alternatively, GP-2 (0.04 mM) was sulfated for 18 h at 37°C using [ 35 S]PAPS (0.2 mM, 30300 cpm/nmol) (Sigma) and TPST-1 (0.7 nmol/h) in a total volume of 56 l. The conversion of GP-2 to 35 SO 3 -GSP-2 was Ͼ95%. GP-5 was sulfated in a similar fashion as GP-6 using [ Enzymatic Synthesis of GSP-6Ј-␤1,3-GlcNAcT-Extension of GP-2 was carried out using purified recombinant ␤1,3-GlcNAcT from Neisseria meningitidis IgtA (34). Acceptor GP-2 (0.1 mM) was incubated for 20 h at 25°C in the presence of 1 mM UDP-GlcNAc and 8 nmol/h of ␤1,3-GlcNAcT (activity assayed using lactose as an acceptor) in a total volume of 100 l in 100 mM sodium cacodylate, pH 7.5, containing 5 mM ATP, 15 mM MnCl 2 , and 0.5% bovine serum albumin.…”

“…It was synthesized by a series of steps as outlined under "Experimental Procedures." A key step in the synthesis of GSP-6Ј is the addition of GlcNAc in ␤1-3 linkage to the Gal residue in the core 1 O-glycan by a recombinant ␤1,3-GlcNAcT from N. meningitidis IgtA (34). This glycopeptide, designated GP-3Ј, was subsequently modified by the action of ␤1,4-GalT, ␣2,3-sialylT, and ␣1,3-FucT to generate a glycopeptide designated GP-6Ј, which has sLe x on the extended core 1 O-glycan.…”

Efficient production of human β-1,3-N-acetylglucosaminyltransferase-2 fused with green fluorescence protein in insect cell

“…These modifications contributed to a marked increase in extracellular ␤3GnT2 production from 0.017 [1] to 0.86 mU/ml. Moreover, ␤3GnT2 fused to its N-terminal with GFP uv exhibited ␤3GnT activity, with its specific activity being approximately 50-fold higher than that of a recombinant N-acetylglucosaminyltransferase (LgtA) from Neisseria meningitides [21]. Although its activity and specific activity cannot be directly compared because of the use of different substrates, GFP fusion techniques and the BES have been found to be suitable for the production of human ␤3GnT2.…”