X-NeuNAc

Km: 0.89 mM for neuraminidase

X-Neu5Ac is a new substrate for chromogenic assay of neuraminidase activity in bacterial expression systems.

Many studies have focused on the functional role of cell surface gangliosides, which are thought to facilitate antigenicity, cell-cell recognition and cell growth regulation. Nacetyl neuraminic acid (Neu5Ac) within gangliosides Plays a key function in these roles. Chromogenic substrates, such as Neu5Ac, are used routinely for cytologic, histologic and spectroscopic analyses of enzyme activity.

In vitro: X-Neu5Ac was hydrolyzed by neuraminidase to release a halogenated product undergoing rapid aerobic oxidation to form the dark blue pigment. Preliminary kinetic studies indicated that X-Neu5Ac was a good and stable substrate for neuraminidase. In addition, X-Neu5Ac would also be hydrolyzed mutant enzymes [1].

In vivo: To visualize extracellular sialidase activity on the membrane surface in the rat brain, acute brain slices were incubated with X-Neu5Ac at pH 7.3. After 1h, myelin-abundant regions showed intense fluorescence in the rat brain. Although the hippocampus showed weak fluorescence in the brain, mossy fiber terminals in the hippocampus showed relatively intense fluorescence. In addition, the fluorescence intensities caused by X-Neu5Ac was correlated with the sialidase activity. Therefore, staining with X-Neu5Ac was specific for sialidase and useful for quantitative analysis of sialidase activities [1].

Clinical trial: N/A

References:
[1] Fujii I,Iwabuchi Y,Teshima T,Shiba T,Kikuchi M.  X-Neu5Ac: a novel substrate for chromogenic assay of neuraminidase activity in bacterial expression systems. Bioorg Med Chem.1993 Aug;1(2):147-9.
[2] Minami A,Shimizu H,Meguro Y,Shibata N,Kanazawa H,Ikeda K,Suzuki T.  Imaging of sialidase activity in rat brain sections by a highly sensitive fluorescent histochemical method. Neuroimage.2011 Sep 1;58(1):34-40.

Glycolipid acceptor specificity of a human Gal beta(1-3/1-4) GlcNAc alpha 2,3-sialyltransferase.[Pubmed:8554608]

Biochem Biophys Res Commun. 1995 Dec 26;217(3):852-8.

A human Gal beta(1-3/1-4)GlcNAc alpha 2,3-sialyltransferase, called ST-4, is a sialyltransferase involved in the in vivo biosynthesis of sialyl Lewis X (NeuNAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc) determinant. The ST-4 enzyme could utilize nLc4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer) containing type 2 sugar chain, Lc4Cer (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer) containing type 1 sugar chain, Gg4Cer (Gal beta 1-3GalNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer), and LacCer as glycolipid acceptor substrates, but not other neutral glycolipids (GalCer, GlcCer, Gb3Cer, Gg3Cer, Gb4Cer) and gangliosides (GM1a, GM2, GM3, GD1a, GD1b, and GT1b) as substrates. The order of sialic acid incorporation into glycolipids for the enzyme was nLc4Cer > Gg4Cer > Lc4Cer > LacCer. The apparent Km values of ST-4 for nLc4Cer and Gg4Cer were 0.47 and 2.5 mM, respectively. Thus, the ST-4 could efficiently utilize both nLc4Cer and Gg4Cer as glycolipid acceptor substrates in vitro, suggesting that the substrate specificity of the enzyme may be similar to that of a glycolipid sialyltransferase (SAT-3), which is defined as the enzyme that uses both nLc4Cer and Gg4Cer as glycolipid acceptor substrates.

NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS.[Pubmed:12410816]

Plant J. 2002 Nov;32(3):391-400.

GUS continues to be the reporter of choice for many gene fusion applications, due to the unparalleled sensitivity of the encoded enzyme and the ease with which it can be quantified in cell-free extracts and visualized histochemically in cells and tissues. A compatible and functionally equivalent reporter gene would facilitate dual promoter studies and internal standardization of expression analyses in the same plant. A search for a candidate enzyme activity not found in plants, which might form the basis of a novel GUS-compatible reporter system, led us to investigate nanH, a Clostridium perfringens gene which encodes the so-called 'small' cytoplasmic sialidase. Expression of the native, AT-rich nanH gene in transgenic plants did not, however, result in detectable sialidase activity. For this reason, a codon-optimized derivative, NAN, was synthesized which possesses a GC content similar to that found in highly expressed plant genes. NAN enzyme activity was expressed at high levels in both stably and transiently transformed cells, possessed kinetic and stability properties similar to those of GUS, and showed optimal activity in GUS buffer. Moreover, NAN and GUS activity could be visualized simultaneously in polyacrylamide gels using the corresponding methylumbelliferone-based substrates, and in whole seedlings and tissue sections using the histochemical substrates 5-bromo-4-chloro-3-indolyl alpha-d-N-acetylneuraminic acid (X-NeuNAc) and 5-bromo-6-chloro-3-indolyl beta-d-glucuronide (X-GlucM), respectively.

Molecular cloning of a fourth member of a human alpha (1,3)fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x, and difucosyl sialyl Lewis x epitopes.[Pubmed:1339443]

J Biol Chem. 1992 Dec 5;267(34):24575-84.

We and others have previously described the isolation of three human alpha (1,3)fucosyltransferase genes which form the basis of a nascent glycosyltransferase gene family. We now report the molecular cloning and expression of a fourth homologous human alpha (1,3)fucosyltransferase gene. When transfected into mammalian cells, this fucosyltransferase gene is capable of directing expression of the Lewis x (Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc), sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4 [Fuc alpha 1-->3]GlcNAc), and difucosyl sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3 Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc) epitopes. The enzyme shares 85% amino acid sequence identity with Fuc-TIII and 89% identity with Fuc-TV but differs substantially in its acceptor substrate requirements. Polymerase chain reaction analyses demonstrate that the gene is syntenic to Fuc-TIII and Fuc-TV on chromosome 19. Southern blot analyses of human genomic DNA demonstrate that these four alpha (1,3)fucosyltransferase genes account for all DNA sequences that cross-hybridize at low stringency with the Fuc-TIII catalytic domain. Using similar methods, a catalytic domain probe from Fuc-TIV identifies a new class of DNA fragments which do not cross-hybridize with the chromosome 19 fucosyltransferase probes. These results extend the molecular definition of a family of human alpha (1,3)fucosyltransferase genes and provide tools for examining fucosyltransferase gene expression.

X-Neu5Ac is a substrate for chromogenic assay of neuraminidase activity in bacterial expression systems; with a K_m of 0.89 mM for neuraminidase.

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