Ginsenoside Rg3 suppresses FUT4 expression through inhibiting NF-κB/p65 signaling pathway to promote melanoma cell death.

Abnormal glycosylation is catalyzed by the specific glycosyltransferases and correlates with tumor cell apoptosis. Increased fucosyltransferase IV (FUT4) is seen in many types of cancer, and manipulating FUT4 expression through specific signaling pathway inhibits cell growth and induces apoptosis. NF-κB is known playing a vital role to control cell growth and apoptosis. Ginsenoside Rg3 is an herbal medicine with strong antitumor activity through inhibiting tumor growth and promoting tumor cell death. However, whether Rg3-induced inhibition on tumor development involves reduced NF-κB signaling and FUT4 expression remains unknown. In the present study, we found that Rg3 suppressed FUT4 expression by abrogating the binding of NF-κB to FUT4 promoter through inhibiting the expression of signaling molecules of NF-κB pathway, reducing NF-κB DNA binding activity and NF-κB transcription activity. NF-κB inhibitor (Bay 11-7082) or knocking down p65 expression by p65 siRNA also led to a significant decreased FUT4 expression. In addition, Rg3 induced apoptosis by activating both extrinsic and intrinsic apoptotic pathways. Moreover, in a xenograft mouse model, Rg3 downregulated FUT4 and NF-κB/p65 expression and suppressed melanoma cell growth and induced apoptosis without any noticeable toxicity. In conclusion, Rg3 induces tumor cell apoptosis correlated with its inhibitory effect on NF-κB signaling pathway-mediated FUT4 expression. Results suggest Rg3 might be a novel therapy agent for melanoma treatment.

Human alpha1,3/4-fucosyltransferases. II. A single amino acid at the COOH terminus of FucT III and V alters their kinetic properties.

An analysis of the acceptor substrate specificity of domain swap mutants of human alpha1,3/4-fucosyltransferases (FucTs) III and V has been carried out. The results demonstrate that changing Asp336 of FucT III to Ala (as in FucT V) produced a protein (III/V1) with a reduced activity with a variety of acceptors. An analysis of the kinetic properties of FucT III and the III/V1 mutant demonstrated that III/V1 had a 40-fold reduction in its affinity for the H-type 1 acceptor substrate (Fucalpha1,2Galbeta1,3GlcNAc) and 4-fold reduction in its affinity for GDP-fucose when compared with FucT III. Further, the overall catalytic efficiency of III/V1 was approximately 100-fold lower than that of FucT III with an H-type 1 acceptor substrate. The complementary domain swap resulting from the change of Ala349 of FucT V to Asp (V/III1) produced a FucT that had higher enzyme activity with a range of acceptor substrates and had a higher affinity for an H-type 2 acceptor substrate (Fucalpha1, 2Galbeta1,4GlcNAc) with an 8-fold higher overall catalytic efficiency than that of FucT V. No significant change occurred in the Km for GDP-fucose for this protein when compared with FucT V. Kinetic parameters of two other FucT domain swaps (III8/V and V8/III), resulting in proteins that differed from FucT III and V at the NH2 terminus of their catalytic domain, were not significantly different from those of the parental enzymes when H-type 1 and H-type 2 acceptor substrates were utilized. Thus, substitution of an acidic amino acid for a nonpolar amino acid (i.e. Asp versus Ala) at the COOH terminus of FucTs produces an enzyme with enhanced enzyme activities. These results, together with the results presented in the accompanying papers (Nguyen, A. T., Holmes, E. H., Whitaker, J. M., Ho, S., Shetterly, S., and Macher, B. A. (1998) J. Biol. Chem. 273, 25244-25249; Sherwood, A. L., Nguyen, A. T., Whitaker, J. M., Macher, B. A., and Holmes, E. H. (1998) J. Biol. Chem. 273, 25256-25260), provide new insights into the structure/function relationships of human alpha1,3/4-FucT enzymes.

Novel branched nod factor structure results from alpha-(1-->3) fucosyl transferase activity: the major lipo-chitin oligosaccharides from Mesorhizobium loti strain NZP2213 bear an alpha-(1-->3) fucosyl substituent on a nonterminal backbone residue.

Mesorhizobium loti has been described as a microsymbiont of plants of the genus Lotus. Lipo-chitin oligosaccharides (LCOs), or Nod factors, produced by several representative M. loti strains all have similar structures. Using fast-atom-bombardment tandem mass spectrometry and NMR spectroscopy, we have now examined the LCOs from the type strain NZP2213 and observed a much greater variety of structures than has been described for the strains of M.loti studied previously. Interestingly, we have identified as the major LCO a structure that bears a fucose residue alpha-1,3-linked to the GlcNAc residue proximal to the nonreducing terminal GlcNAc residue. This is the first time, to our knowledge, that substitution on an internal GlcNAc residue of the LCO backbone has been observed. This novel LCO structure suggests the presence of a novel fucosyltransferase activity in strain NZP2213. Since the presence of this extra structure does not have the effect of broadening the host range, we suggest that the modification of the LCOs with a fucose residue linked to a nonterminal GlcNAc residue might provide protection against degradation by a particular host plant enzyme (e.g., a chitinase) or alternatively represents adaptation to a particular host-specific receptor. The action of the alpha-(1-->3) fucosyltransferase seems to reduce significantly the activity of NodS, the methyltransferase involved in the addition of the N-methyl substituent to the nonreducing terminal GlcNAc residue. An additional novel LCO structure has been identified having only a GlcNAc2 backbone. This is to our knowledge the first description of such a minimal LCO structure.

Functional purification and characterization of a GDP-fucose: beta-N-acetylglucosamine (Fuc to Asn linked GlcNAc) alpha 1,3-fucosyltransferase from mung beans.

An alpha 1,3-fucosyltransferase was purified 3000-fold from mung bean seedlings by chromatography on DE 52 cellulose and Affigel Blue, by chromatofocusing, gelfiltration and affinity chromatography resulting in an apparently homogenous protein of about 65 kDa on SDS-PAGE. The enzyme transferred fucose from GDP-fucose to the Asn-linked N-acetylglucosaminyl residue of an N-glycan, forming an alpha 1,3-linkage. The enzyme acted upon N-glycopeptides and related oligosaccharides with the glycan structure GlcNAc2Man3 GlcNAc2. Fucose in alpha 1,6-linkage to the asparagine-linked GlcNAc had no effect on the activity. No transfer to N-glycans was observed when the terminal GlcNAc residues were either absent or substituted with galactose. N-acetyllactosamine, lacto-N-biose and N-acetylchito-oligosaccharides did not function as acceptors for the alpha 1,3-fucosyltransferase. The transferase exhibited maximal activity at pH 7.0 and a strict requirement for Mn2+ or Zn2+ ions. The enzyme's activity was moderately increased in the presence of Triton X-100. It was not affected by N-ethylmaleimide.