Structure-Function Analysis of Human alpha1[IMAGE]3Fucosyltransferases (original) (raw)
Related papers
Glycobiology, 2000
The α1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human α1,3/4fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to ∼40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the K m values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved Cterminal N-glycosylation sites for expression of full enzyme activity.
Delineation of fucosyltransferase activities with thiol reagents
The Biochemical journal, 1979
The thiol reagent dithiothreitol inhibits the activity of a core GDP-fucose-N-acetylglucosaminide alpha-6-fucosyltransferase in plasma and blood-cell homogenates, while promoting the activity of alpha-2- and alpha-3-fucosyltransferases. The latter enzymes catalyse transfer of fucose on to terminal galactose and subterminal N-acetylglucosamine residues respectively. A thiol-blocking reagent N-ethylmaleimide does not affect the activity of the alpha-6-fucosyltransferase, but inhibits the other two enzymes. These results indicate the presence of a critical disulphide linkage in the alpha-6-fucosyltransferase, and provide a means of delineation of different fucosyltransferases.
Acceptor specificity and tissue distribution of three human alpha-3-fucosyltransferases
European Journal of Biochemistry, 1990
Based on the capacity to transfer a-L-fucose onto type-1 and type-2 synthetic blood group H and sialylated acceptors, a comparison of the a-3-fucosyltransferase activities of different human tissues is shown. Three distinct acceptor specificity patterns are described: (I) myeloid a-3-fucosyltransferase pattern, in which leukocytes and brain enzymes transfer fucose actively onto H type-2 acceptor and poorly onto sialylated N-acetyllactosamine; (11) plasma a-3-fucosyltransferase (EC 2.4.1.152), in which plasma and hepatocyte enzymes transfer, in addition, onto the sialylated N-acetyllactosamine; (111) Lewis a-3/4-fucosyltransferase (EC 2.4.1.65), in which gall-bladder, kidney and milk enzymes transfer, in addition, onto type-1 acceptors. The small amount (< 10%) of a-3fucosyltransferase activity found in the plasma of an a-3-fucosyltransferase-deficient individual had a myeloidtype acceptor pattern, suggesting that this small proportion of the plasma enzyme is derived from leukocytes. In addition to the three acceptor specificity patterns, these enzyme activities can be differentiated by their optimum pH: 8.0-8.7 for the enzymes from myeloid cells and brain, 7.2-8.0 for liver enzymes and 6.0-7.2 for gallbladder enzymes. Milk samples had two a-3-fucosyltransferase activities, the Lewis or a-3/4-fucosyltransferase under control of the Lewis gene and an a-3-fucosyltransferase with plasma acceptor pattern which was independent of the control of the Lewis gene. The apparent affinity for GDP-fucose of the myeloid-like enzyme was weaker than those of the plasma and Lewis-like enzymes. The apparent affinities for H type 2 and sialylated Nacetyllactosamine were stronger for exocrine secretions as compared to the plasma and myeloid enzymes. The plasma type of a-3-fucosyltransferase activity was more sensitive to N-ethylmaleimide and heat inactivation than the samples with myeloid-like a-3-fucosyltransferase activity. The names X, Le", SSEA-1 and CD15 have been used to describe different properties of the same epitope, the terminal trisaccharide 3-fucosyl-N-acetyllactosamine or Galjl-+ 4(Fucal-f 3)GlcNAcP-, which is found in 0-and N-linked glycoproteins and in glycolipids [I]. Many monoclonal antibodies, raised against different cells, were shown to react specifically with this epitope [2, 31. The first monoclonal with this specificity [4] was called SSEA-1 (stage-specific embryonic antigen-1) because it reacted with 8-cell-stage mouse embryos, morulae and early blastocysts. However, monoclonal antibodies reacting with normal human granulocytes [5], myeloid cell lines [6], human neuroblastoma, fetal brain [7], human gastric, colon and lung cancers [8] also had the same trisaccharide specificity. The epitope at the cell surface of leukocytes has been termed CD15 (cluster of differentiation 15) because a group of monoclonal antibodies with this specificity was identified
Insect Biochemistry and Molecular Biology, 2012
Glycans of glycoproteins are often associated with IgE mediated allergic immune responses. Hymenoptera venoms, e.g., carry a1,3-fucosyl residues linked to the proximal GlcNAc of glycoproteins. This epitope, formed selectively by a1,3-fucosyltransferase (FucTA), is xenobiotic and as such highly immunogenic and it also shows cross-reactivity if present on different proteins. Production of posttranslationally modified proteins in insect cells is however commonly used and, thus, resulting glycoproteins can carry this highly immunogenic epitope with potentially significant side effects on mammals. To analyze mechanism, specificity and reaction kinetics of the key enzyme, we chose FucTA from Apis mellifera (honeybee) and characterized it by saturation transfer difference (STD) NMR and surface plasmon resonance (SPR) experiments. Specifically, we show here that the donor substrate, GDP-Fucose, binds mostly via its guanine and less so via pyrophosphate and fucosyl fragments and has a K D ¼ 37 mM. Affinity and kinetic studies with both the core a1,6-fucosylated and the unfucosylated octa-or heptasaccharides, respectively, as acceptor substrate revealed that honeybee FucTA prefers the latter structure with affinities of K D w 10 mM. Establishment of progress curve analysis using an explicit solution of the integrated MichaeliseMenten equation allowed for determination of key constants of the transfer reaction of the glycosyl residue. The dominant minimum acceptor substrate is an unfucosylated heptasaccharide with K m ¼ 420 mM and k cat ¼ 6 min À1 . Time-resolved NMR spectra as well as STD NMR allow molecular insights into specificity, activity and interaction of the enzyme with substrates and acceptors.
R-1,3-Fucosyltransferase V (FucT V) catalyzes the transfer of l-fucose from the donor sugar guanosine 5′-diphospho-l-fucose (GDP-Fuc) to an acceptor sugar. A secondary isotope effect on the fucosyltransfer reaction with guanosine 5′-diphospho-[1-2 H]-l-fucose (GDP-[1-2 H]-Fuc) as the substrate was observed and determined to be D V) 1.32 (0.13 and D V/K) 1.27 (0.07. Competitive inhibition of FucT V by guanosine 5′-diphospho-2-deoxy-2-fluoro-l-fucose (GDP-2F-Fuc) was observed with an inhibition constant of 4.2 µM which represents the most potent inhibitor of this enzyme to date. Incubation of GDP-2F-Fuc with FucT V and an acceptor molecule prior to the addition of GDP-Fuc had no effect on the potency of inhibition, indicating that GDP-2F-Fuc is neither an inactivator nor a slow substrate. Both the observed secondary isotope effect and the inhibition by GDP-2F-Fuc are consistent with a charged, sp 2-hybridized, transition-state structure. A convenient and efficient synthesis of GDP-[1-2 H]-Fuc and GDP-2F-Fuc and a nonradioactive, fluorescence assay for fucosyltransferase activity have been developed.
N-Glycosylations of human 1,3-fucosyltransferase IX are required for full enzyme activity
Glycobiology, 2013
Human α1,3-fucosyltransferase IX catalyzes the transfer of L-fucose from guanosine diphosphate-β-L-fucose to Nacetyllactosamine, generating a Lewis X epitope, and is thereby involved in the synthesis of fucosylated cell surface glycoconjugates. It contains three putative N-glycosylation sites (Asn62, Asn101 and Asn153). The current study considers the functional role of these potential Nglycosylations within the enzyme. We produced truncated variants of human fucosyltransferase IX containing the soluble extracellular catalytic domain. To analyze the relevance of each N-glycosylation site, several genomic mutant DNAs encoding a glutamine (Gln/Q) instead of the asparagine residue were created prosperously using site-directed mutagenesis and subsequently expressed in Spodoptera frugiperda cells applying a baculovirus expression system. After production and purification of these variants of human FucT IX, the wild-type (wt) enzyme and the variants were characterized regarding their activity and kinetic properties. The variants showed lower activity than the wt FucT, whereas the individual N-glycosylation sites had different effects on the enzyme activity and kinetic parameters. While the single variant N62Q still showed 60% of wt activity and N101Q retained 30% activity, replacement of Asn153 by glutamine led to an almost complete loss of enzymatic activity. The same could be observed for variants missing two or more putative N-glycosylation sites, which indicated the importance of N-glycosylation for enzyme stability and activity.
Mechanism and Specificity of Human R-1,3-Fucosyltransferase V
Human R-1,3-fucosyltransferase catalyzes the transfer of the L-fucose moiety from guanosine diphosphate-L-fucose (GDP-Fuc) to acceptor sugars to form biologically important fucoglycoconjugates, including sialyl Lewis x (SLe x). Evidence for a general base mechanism is supported by a pH-rate profile that revealed a catalytic residue with a pK a of 4.1. The characterized solvent kinetic isotope effect (D V) 2.9, D V/K) 2.1) in a proton inventory study indicates that only one-proton transfer is involved in the catalytic step leading to the formation of the transition state. Evidence for Mn 2+ as an electrophilic catalyst was supported by the observation that the nonenzymatic transfer of L-fucose from GDP-Fuc to the hydroxyl group of water in the presence of 10 mM MnCl 2 at 20 °C was accelerated from k obs) 3.5 × 10-6 to 3.8 × 10-5 min-1. Using the GDP-Fuc hydrolysis as the nonenzymatic rate, the enzymatic proficiency of FucT V, (k cat /K i,GDP-fuc ‚K m,LacNAc)/k non , was estimated to be 1.2 × 10 10 M-1 with a transition-state affinity of 8.6 × 10-11 M. The K m for Mn 2+ was determined to be 6.1 mM, and alternative divalent metal cofactors were identified as Ca 2+ , Co 2+ , and Mg 2+. Detailed kinetic characterization of the acceptor sugar specificity indicated that incorporation of hydrophobic functionality [e.g.-O-(CH 2) 5 CO 2 CH 3 ] to the reducing end of the acceptor sugar substantially decreased the K m,acceptor by over 100-fold. The role of the nucleotide was investigated by studying the inhibition of nucleotides, including the guanosine series. The inhibitory potency trend (GTP ≈ GDP > GMP >> guanosine) is consistent with bidentate chelation of Mn 2+ by GDP-Fuc. The role of charge and distance in the synergistic inhibitory effect by the combination of GDP, an aza sugar, and the acceptor sugar was probed. A mechanism for fucosyl transfer incorporating these findings is proposed and discussed.