The final step in the enzymatic synthesis of the ABO(H) blood group A and B antigens is catalyzed by two closely related glycosyltransferases, an ␣-(133)-N-acetylgalactosaminyltransferase (GTA) and an ␣-(133)-galactosyltransferase (GTB). Of their 354 amino acid residues, GTA and GTB differ by only four "critical" residues. High resolution structures for GTB and the GTA/GTB chimeric enzymes GTB/G176R and GTB/G176R/ G235S bound to a Glycosyltransferases synthesize carbohydrate moieties of glycoconjugates by catalyzing the sequential addition of monosaccharides from specific donors to specific acceptors. The ubiquitous presence of glycolipids and glycoproteins in all living systems underlines the importance of the glycosyltransferases superfamily, and the DNA of all domains of life encode for a large number of these enzymes (1). To date, crystal structures of glycosyltransferases have displayed a high degree of structural similarity even when there is low sequence homology (2-4). As such, glycosyltransferases provide an excellent example of the preferential conservation of structural phenotype over the conservation of sequence identity (2), which indicates that the mechanism of glycosylation, although not yet fully understood, has been conserved.

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy.

The mammalian neuraminidase (NEU) enzymes are found in diverse cellular compartments. Members of the family, such as NEU2 and NEU1, are cytosolic or lysosomal, while NEU3 and NEU4 are membrane-associated. NEU enzymes that act on substrates in the plasma membrane could modulate cellular signaling, cell surface glycoforms and the composition of plasma membrane glycolipids. Therefore, their substrates and mechanism of action are of interest for discerning their physiological roles. We have studied the structure of the human NEU3 using molecular modeling to predict residues involved in the recognition and hydrolysis of glycolipid substrates. To test the model, we have used site-directed mutagenesis of the recombinant protein. Enzymatic studies of the relative activity of these mutants, as well as their pH profiles and inhibition by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, are reported. Using nuclear magnetic resonance spectroscopy, we confirmed that the enzyme is a retaining exo-sialidase, and we propose that the key catalytic residues of the enzyme consist of the general acid-base D50 and the nucleophilic Y370-E225 pair. Mutations of residues expected to interact directly with the sialic acid N5-acetyl (A160, M87, I105) and C7-C9 glycerol side-chain (E113, Y179, Y181) reduced enzymatic activity. We identified several active mutants of the enzyme which contain modifications at the periphery of the active site. Truncations at the N- or C-terminus of more than 10 residues abolished enzyme activity. We propose a catalytic mechanism consistent with the data and identify residues that contribute to glycolipid recognition.