Treatment of rabbits, rats, and mice with Dgalactosamine increased their sensitivity to the lethal effects of lipopolysaccharide several thousand fold. The susceptibility of the animals was highest when the lipopolysacharide was injected together with galactosamine and decreased successively when injection was carried out 1, 2, and 3 hr later. Sensitization was absent when the lipopolysaccharide was administered 1 hr before or 4 hr after galactosamine. The onset of lethality after treatment with galactosamine and lipopolysaccharide occurred faster than with lipopolysaccharide alone; usually all animals died 5-9 hr later. The galactosamine-induced sensitization to lipopolysaccharide could be reversed by uridine which is known to inhibit the early biochemical alterations induced by the amino sugar in the hepatocytes. Although galactosamine is known to exhibit hepatotoxic activity inducing ultimate necrosis of the hepatocytes, the data so far suggests that the sensitization to lipopolysaccharide is related only to the early metabolic effects ofthe hexosamine.
Modification of cell surface molecules with sialic acid is crucial for their function in many biological processes, including cell adhesion and signal transduction. Uridine diphosphate-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) is an enzyme that catalyzes an early, rate-limiting step in the sialic acid biosynthetic pathway. UDP-GlcNAc 2-epimerase was found to be a major determinant of cell surface sialylation in human hematopoietic cell lines and a critical regulator of the function of specific cell surface adhesion molecules.
Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. Sialylation is crucial for a variety of cellular functions, such as cell adhesion or signal recognition, and regulates the biological stability of glycoproteins. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase͞N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. We report that inactivation of the UDP-GlcNAc 2-epimerase by gene targeting causes early embryonic lethality in mice, thereby emphasizing the fundamental role of this bifunctional enzyme and sialylation during development. The need of UDP-GlcNAc 2-epimerase for a defined sialylation process is exemplified with the polysialylation of the neural cell adhesion molecule in embryonic stem cells.
Protein glycosylation is a complex form of posttranslational modification and has been shown to be crucial for the function of many proteins. Sialic acid is prominently positioned at the outer end of membrane glycoproteins. It plays a critical role for the regulation of a myriad of cellular functions and it forms a shield around the cell. Furthermore, it constantly interacts with the environment of cells and contributes to histocompatibility. [1] This makes studying sialylation an interesting field of research, but monitoring sialic acid in vivo is challenging. While proteins are routinely labeled by genetic methods, such as expression as GFP fusion proteins, comparable methods are not available for secondary gene products, such as glycans of glycoconjugates. Metabolic oligosaccharide engineering (MOE) is a successful new strategy to visualize the localization of glycans in vitro and in vivo. [2] In this approach, cells are cultivated in the presence of non-natural monosaccharide derivatives that carry a chemical reporter group and are nonetheless accepted by the biosynthetic machinery of a cell. For instance, peracetylated N-azidoacetylmannosamine (Ac 4 ManNAz) is taken up by the cell, deacetylated by cellular esterases, and owing to the promiscuity of the enzymes of sialic acid biosynthesis, is converted into N-azidoacetyl neuraminic acid and incorporated into sialoglycoconjugates. [3] Once presented on the cell surface, the azide-containing sialylated glycan can be visualized through a bioorthogonal ligation reaction. [4] Besides Ac 4 ManNAz, several monosaccharide derivatives of N-acetylgalactosamine, [5] N-acetylglucosamine, [6] and l-fucose [7] are suitable for MOE providing further insights into the role of cellular structures and functions of glycans in the cell.Currently, mainly Staudinger ligation [3] and azide-alkyne [3+2] cycloaddition (copper-catalyzed [8] or strain-promoted, [9] also known as the click reaction) are applied as ligation reactions in MOE. However, both of them rely on the reaction of azides and thus cannot be used for the concurrent detection of two different metabolically incorporated carbohydrates. A labeling strategy that can be carried out in the presence of azides and alkynes would significantly expand the scope of chemical labeling reactions in living cells and is thus highly desirable.Recently, it was shown that the Diels-Alder reaction with inverse electron demand (DARinv) of 1,2,4,5-tetrazines [10] with strained dienophiles, such as trans-cyclooctenes, [11] cyclobutenes, [12] norbornenes, [11d,f, 13] cyclooctynes, [11d,f] and substituted cyclopropenes, [14] fulfills the requirements of a bioorthogonal ligation reaction and furthermore is orthogonal to the azide-alkyne cycloaddition. However, these cyclic alkenes or kinetically stable tetrazines [15] are expected to be too large for being efficiently metabolized by the sialic acid biosynthetic pathway, starting from the corresponding Nacylmannosamine derivative. In search for smaller dienophiles suitable for MOE, we identifi...
N-Acetylneuraminic acid is the most prominent sialic acid in eukaryotes. The structural diversity of sialic acid is exploited by viruses, bacteria, and toxins and by the sialoglycoproteins and sialoglycolipids involved in cell-cell recognition in their highly specific recognition and binding to cellular receptors. The physiological precursor of all sialic acids is N-acetyl D-mannosamine (ManNAc). By recent findings it could be shown that synthetic N-acyl-modified D-mannosamines can be taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acids in vitro and in vivo. Successfully employed D-mannosamines with modified N-acyl side chains include N-propanoyl- (ManNProp), N-butanoyl- (ManNBut)-, N-pentanoyl- (ManNPent), N-hexanoyl- (ManNHex), N-crotonoyl- (ManNCrot), N-levulinoyl- (ManNLev), N-glycolyl- (ManNGc), and N-azidoacetyl D-mannosamine (ManNAc-azido). All of these compounds are metabolized by the promiscuous sialic acid biosynthetic pathway and are incorporated into cell surface sialoglycoconjugates replacing in a cell type-specific manner 10-85% of normal sialic acids. Application of these compounds to different biological systems has revealed important and unexpected functions of the N-acyl side chain of sialic acids, including its crucial role for the interaction of different viruses with their sialylated host cell receptors. Also, treatment with ManNProp, which contains only one additional methylene group compared to the physiological precursor ManNAc, induced proliferation of astrocytes, microglia, and peripheral T-lymphocytes. Unique, chemically reactive ketone and azido groups can be introduced biosynthetically into cell surface sialoglycans using N-acyl-modified sialic acid precursors, a process offering a variety of applications including the generation of artificial cellular receptors for viral gene delivery. This group of novel sialic acid precursors enabled studies on sialic acid modifications on the surface of living cells and has improved our understanding of carbohydrate receptors in their native environment. The biochemical engineering of the side chain of sialic acid offers new tools to study its biological relevance and to exploit it as a tag for therapeutic and diagnostic applications.
Abstract.A novel member of the cadherin family of cell adhesion molecules has been characterized by cloning from rat liver, sequencing of the corresponding cDNA, and functional analysis after heterologous expression in nonadhesive $2 cells, cDNA clones were isolated using a polyclonal antibody inhibiting Ca2+-dependent intercellular adhesion of hepatoma cells. As inferred from the deduced amino acid sequence, the novel molecule has homologies with E-, P-, and N-cadherins, but differs from these classical cadherins in four characteristics. Its extracellular domain is composed of five homologous repeated domains instead of four characteristic for the classical cadherins. Four of the five domains are characterized by the sequence motifs DXNDN and DXD or modifications thereof representing putative Ca2+-binding sites of classical cadherins. In its NH2-terminal region, this cadherin lacks both the precursor segment and the endogenous protease cleavage site RXKR found in classical cadherins. In the extracellular EC1 domain, the novel cadherin contains an AAL sequence in place of the HAV sequence motif representing the common cell adhesion recognition sequence of E-, P-, and N-cadherin. In contrast to the conserved cytoplasmic domain of classical cadherins with a length of 150-160 amino acid residues, that of the novel cadherin has only 18 amino acids. Examination of transfected $2 cells showed that despite these structural differences, this cadherin mediates intercellular adhesion in a Ca2+-dependent manner. The novel cadherin is solely expressed in liver and intestine and was, hence, assigned the name LI-cadherin. In these tissues, LI-cadherin is localized to the basolateral domain of hepatocytes and enterocytes. These results suggest that LI-cadherin represents a new cadherin subtype and may have a role in the morphological organization of liver and intestine.C ELL-cell interactions are of fundamental importance for the development and the maintenance of tissues and organs in multicellular organisms. The basic morphogenetic processes involved in organogenesis, including cellular aggregation, segregation, and migration, are mediated and controlled by an increasingly large and complex number of cell adhesion molecules that exhibit a well-regulated spatiotemporal pattern of expression during development and regeneration (for review see Simons and Fuller, 1985;Ekblom et al
Sialic acids are essential components of the cell surface receptors of many microorganisms including viruses. A synthetic, N-substituted D-mannosamine derivative has been shown to act as precursor for structurally altered sialic acid incorporated into glycoconjugates in vivo (Kayser, H., Zeitler, R., Kannicht, C., Grunow, D., Nuck, R., and Reutter, W. (1992) J. Biol. Chem. 267, 16934-16938). In this study we have analyzed the potential of three different sialic acid precursor analogues to modulate sialic acid-dependent virus receptor function on different cells. We show that treatment with these D-mannosamine derivatives can result in the structural modification of about 50% of total cellular sialic acid content. Treatment interfered drastically and specifically with sialic acid-dependent infection of two distinct primate polyoma viruses. Both inhibition (over 95%) and enhancement (up to 7-fold) of virus binding and infection were observed depending on the N-acyl substitution at the C-5 position of sialic acid. These effects were attributed to the synthesis of metabolically modified, sialylated virus receptors, carrying elongated N-acyl groups, with altered binding affinities for virus particles. Thus, the principle of biosynthetic modification of sialic acid by application of appropriate sialic acid precursors to tissue culture or in vivo offers new means to specifically influence sialic acid-dependent ligand-receptor interactions and could be a potent tool to further clarify the biological functions of sialic acid, in particular its N-acyl side chain.
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