Selective inhibition of β-1,4- and α-1,3-galactosyltransferases: donor sugar-nucleotide based approach

Takayama, Shuichi; Chung, Sang J.; Igarashi, Yasuhiro; Ichikawa, Yoshitaka; Sepp, Armin; Lechler, Robert I.; Wu, Jiangyue; Hayashi, Takashi; Siuzdak, Gary; Wong, Chi Huey

doi:10.1016/s0968-0896(98)00249-1

Cited by 55 publications

(43 citation statements)

References 52 publications

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“…Maximum extension (Å ) of the UDP-Glc molecule and theoretical coupling constants (Hz) averaged over the four molecular dynamics simulations and compared with NMR experimental data [18]. [36,37]. The present conformational study could help in the future in the understanding of the catalytic mechanism and therefore for further rational design of glycosyltransferase inhibitors.…”

Section: O N C L U S I O N Smentioning

confidence: 93%

Molecular dynamics simulations of solvated UDP–glucose in interaction with Mg²⁺ cations

Petrova

Koča

Imberty

2001

European Journal of Biochemistry

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Glycosyltransferases are key enzymes involved in biosynthesis of oligosaccharides. Nucleotide-sugars, the glycosyltransferase substrates, serve as activated donors of sugar residues during the enzymatic reaction Although very little is known about the catalytic mechanism of these enzymes, it appears that the catalytic activity in most glycosyltransferases is dependent upon the presence of a divalent cation, for example Mn 21 or Mg 21. It is not known whether the ion is bound to the enzyme before its interaction with the substrate, or if it binds the substrate before the enzymatic reaction to modify its conformation to fit better the active site of the enzyme. We have inspected the latter possibility by running four 2-ns molecular dynamics trajectories on fully solvated UDP-glucose in the presence of Mg 21 ions.Our results indicate that the divalent cation interacts strongly with the nucleotide-sugar in solution, and that it can alter its conformational behavior. It is also shown that a conformation of the pyrophosphate moiety that results in an eclipsed or almost eclipsed orientation of two of the oxygen atoms, and which is found in protein interacting with a nucleotide di-or tri-phosphate X-ray data, is energetically favored. The results are also discussed in light of existing NMR data, and are found to be in a good agreement with them.Keywords: molecular dynamics; Mg 21 ; UDP-glucose; solution conformation.The biosynthesis of oligosaccharides, polysaccharides and glycoconjugates has until recently remained one of the great unexplored domains of structural and mechanistic enzymology. It is only in the last two years that the first crystal structures of the enzymes involved in this process, i.e. the nucleotide-sugar dependent glycosyltransferase, have been solved. The 3D structures of the seven glycosyltransferases can be classified in two families based on their fold [1,2]. Nevertheless, in most cases, the catalytic mechanism of these enzymes is largely unknown and the first hypotheses have only appeared recently [3]. In most glycosyltransferases, the catalytic activity is dependent upon the presence of a divalent cation. For example, the fucosyltransferases FucT V has a preference for metal ions that can adopt an octahedral geometry and operates optimally with Mn 21[4]. X-ray crystallography studies established that, in glycosyltransferases adopting the most commonly observed fold, this divalent cation is sandwiched between the phosphate groups of the nucleotide-sugar and conserved negatively charged amino acids of the catalytic site [5 -9].A detailed description of the conformational behavior of the substrate is a prerequisite for the characterization and understanding of the mechanism of glycosyltransferases. The nucleotide-sugars are glycosyl esters of nucleoside di-or mono-phosphates and serve as an activated donor during the catalytic reaction. Very little is known about the 3D structure and conformational behavior of nucleotidesugars. Only one crystal structure, the sodium salt of UDPGlc is available [10...

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Section: O N C L U S I O N Smentioning

confidence: 93%

Molecular dynamics simulations of solvated UDP–glucose in interaction with Mg²⁺ cations

Petrova

Koča

Imberty

2001

European Journal of Biochemistry

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“…12 Later, it was demonstrated that per-acetylated forms of Gal␤1,4GlcNAc-␤-O-napthalenemethanol and GlcNAc␤1,3Gal-␤-O-Gal␤1,4GlcNAc-␤-Onapthalenemethanol at 50M can act as decoys/primers that block selectin-ligand formation. [13][14][15][16] Second, glycosyltransferase inhibitors are also in development based on the structure of the sugar-nucleotide transition-state analogs 17,18 and high throughput screens, 19 although testing of these reagents has largely been performed in cell-free enzymatic assays. Third, per-acetylated, modified monosaccharides have been applied to cells as these may compete with the natural monosaccharides.…”

Section: Introductionmentioning

confidence: 99%

Fluorinated per-acetylated GalNAc metabolically alters glycan structures on leukocyte PSGL-1 and reduces cell binding to selectins

Marathe

Buffone

Chandrasekaran³

et al. 2010

Blood

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Novel strategies to control the binding of adhesion molecules belonging to the selectin family are required for the treatment of inflammatory diseases. We tested the possibility that synthetic monosaccharide analogs can compete with naturally occurring sugars to alter the O-glycan content on human leukocyte cell surface selectin-ligand, P-selectin glycoprotein ligand-1 (PSGL-1). Resulting reduction in the sialyl Lewis-X-bearing epitopes on this ligand may reduce cell adhesion. Consistent with this hypothesis, 50Mper-acetylated 4F-GalNAc added to the growth media of promyelocytic HL-60 cells reduced the expression of the cutaneous lymphocyte associated-antigen (HECA-452 epitope) by 82% within 2 cell doubling cycles. Cell binding to all 3 selectins (L-, E-, and P-selectin) was reduced in vitro. 4F-GalNAc was metabolically incorporated into PSGL-1, and this was accompanied by an approximately 20% reduction in PSGL-1 glycan content. A 70% to 85% reduction in HECA-452 binding epitope and N-acetyl lactosamine content in PSGL-1 was also noted on 4F-GalNAc addition. Intravenous 4F-GalNAc infusion reduced leukocyte migration to the peritoneum in a murine model of thioglycolate-induced peritonitis. Thus, the compound has pharmacologic activity. Overall, the data suggest that 4F-GalNAc may be applied as a metabolic inhibitor to reduce O-linked glycosylation, sialyl Lewis-X formation, and leukocyte adhesion via the selectins. (Blood. 2010;115:1303-1312) IntroductionThe binding of adhesion molecules belonging to the selectin family to carbohydrate ligands facilitates the adhesion of blood leukocytes to activated endothelial cells, platelets, and other leukocytes in the human vasculature. 1,2 Such molecular interactions play an important role in regulating leukocyte recruitment at sites of inflammation, cancer metastasis, and various cardiovascular disorders. 3 Whereas numerous glycoproteins and glycolipids participate in selectin-mediated cell adhesion, interactions with carbohydrate epitopes expressed on the leukocyte glycoprotein P-selectin glycoprotein ligand-1 (PSGL-1, CD162) are particularly important because this ligand binds all 3 members of the selectin family (E-, P-, and L-selectin) with high affinity and under fluid flow conditions. Structural analysis of the glycans of PSGL-1 expressed on human promyelocytic leukemia HL-60 cells reveals that PSGL-1 is predominantly composed of core-2 based O-linked glycans. 4,5 The prototypic selectin-binding carbohydrate structure sialyl Lewis-X (NeuAc␣2,3Gal␤1,4(Fuc␣1,3)GlcNAc-, sLe X ; Figure 1A) is expressed on 2% to 14% of these O-glycans.There is active interest in developing antagonists that control/ block selectin-mediated cell adhesion using either competitive inhibitors or metabolic inhibitors. Competitive inhibitors attempt to block cell adhesion by regulating the ligand-binding epitope of either the selectin or its primary counter-receptor PSGL-1. Antagonists used for such inhibition include the tetrasaccharide sLe X and its glycomimetics, 6 humanized antibodies direc...

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“…1) but to-date there is no information about its structure or that of any other member of this GT-15 family. By analogy with retaining glycosidases, it has been postulated that the mechanism of action of retaining glycosyltransferases involves a double displacement with the transient formation of a covalent glycosyl enzyme intermediate (12)(13)(14). However, attempts to characterize such an intermediate for any retaining glycosyltransferase have so far been unsuccess-ful (15) and their exact catalytic mechanisms are still unknown even though the structures of several of these enzymes have recently been solved (16 -21).…”

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confidence: 99%

Structure of Kre2p/Mnt1p

Lobsanov

Romero

Sleno

et al. 2004

Journal of Biological Chemistry

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Kre2p/Mnt1p is a Golgi ␣1,2-mannosyltransferase involved in the biosynthesis of Saccharomyces cerevisiae cell wall glycoproteins. The protein belongs to glycosyltransferase family 15, a member of which has been implicated in virulence of Candida albicans. We present the 2.0 Å crystal structures of the catalytic domain of Kre2p/Mnt1p and its binary and ternary complexes with GDP/Mn 2؉ and GDP/Mn 2؉ /acceptor methyl-␣-mannoside. The protein has a mixed ␣/␤ fold similar to the glycosyltransferase-A (GT-A) fold. Although the GDPmannose donor was used in the crystallization experiments and the GDP moiety is bound tightly to the active site, the mannose is not visible in the electron density. The manganese is coordinated by a modified DXD motif (EPD), with only the first glutamate involved in a direct interaction. The position of the donor mannose was modeled using the binary and ternary complexes of other GT-A enzymes. The C1؆ of the modeled donor mannose is within hydrogen-bonding distance of both the hydroxyl of Tyr 220 and the O2 of the acceptor mannose. The O2 of the acceptor mannose is also within hydrogen bond distance of the hydroxyl of Tyr 220 . The structures, modeling, site-directed mutagenesis, and kinetic analysis suggest two possible catalytic mechanisms. Either a double-displacement mechanism with the hydroxyl of Tyr 220 as the potential nucleophile or alternatively, an S N i-like mechanism with Tyr 220 positioning the substrates for catalysis. The importance of Tyr 220 in both mechanisms is highlighted by a 3000-fold reduction in k cat in the Y220F mutant.

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Selective inhibition of β-1,4- and α-1,3-galactosyltransferases: donor sugar-nucleotide based approach

Cited by 55 publications

References 52 publications

Molecular dynamics simulations of solvated UDP–glucose in interaction with Mg²⁺ cations

Molecular dynamics simulations of solvated UDP–glucose in interaction with Mg²⁺ cations

Fluorinated per-acetylated GalNAc metabolically alters glycan structures on leukocyte PSGL-1 and reduces cell binding to selectins

Structure of Kre2p/Mnt1p

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Selective inhibition of β-1,4- and α-1,3-galactosyltransferases: donor sugar-nucleotide based approach

Cited by 55 publications

References 52 publications

Molecular dynamics simulations of solvated UDP–glucose in interaction with Mg2+ cations

Molecular dynamics simulations of solvated UDP–glucose in interaction with Mg2+ cations

Fluorinated per-acetylated GalNAc metabolically alters glycan structures on leukocyte PSGL-1 and reduces cell binding to selectins

Structure of Kre2p/Mnt1p

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Molecular dynamics simulations of solvated UDP–glucose in interaction with Mg²⁺ cations

Molecular dynamics simulations of solvated UDP–glucose in interaction with Mg²⁺ cations