Overexpression and biochemical characterization of beta-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus influenzae strain Rd

The lipopolysaccharide of capsule deficient Haemophilus influenzae strain Rd contains an N-acetylgalactosamine residue attached to the terminal globotriose moiety in the Hex5 glycoform. Genome analysis identified an open reading frame HI1578, referred to as lgtD, whose amino acid sequence shows significant level of similarity to a number of bacterial glycosyltransferases involved in lipopolysaccharide biosynthesis. To investigate its function, overexpression and biochemical characterization were performed. Most of the protein was obtained in a highly soluble and active form. By using standard glycosyltransferase assay and HPLC, we show that LgtD is an N-acetylgalactosaminyltransferase with high donor substrate specificity and globotriose is a highly preferred acceptor substrate for the enzyme. The K_m for UDP-GalNAc and globotriose are 58 μM and 8.6 mM, respectively. The amino acid sequence of the enzyme shows the conserved features of family II glycosyltransferases. This is the first N-acetylgalactosaminyltransferase identified from H. influenzae, which shows potential application in large-scale synthesis of globo-series oligosaccharides.     

Synthesis of a globotetraose trimer

The synthesis is described of a globotetraose trimer in 74% yield by the reaction of tris(2-aminoethyl)amine with the hydrophobic squaric decyl ester glycoside of globotetraose. The synthesis was readily monitored and purified using reversed phase HPLC. Unreacted squaric decyl ester globotetraoside was recovered rendering the method highly economical.