Structure/function study of Lewis alpha3- and alpha3/4-fucosyltransferases: the alpha1,4 fucosylation requires an aromatic residue in the acceptor-binding domain

All vertebrate alpha3- and alpha3/4-FUTs possess the characteristic acceptor-binding motif VxxHH(W/R)(D/E). FUT6 and FUTb enzymes, harboring R in the acceptor-binding motif, transfer fucose in alpha1,3 linkage, whereas FUT3 and FUT5 enzymes with W at the candidate position can also transfer fucose in alpha1,4 linkage-FUT3 being more efficient than FUT5. To determine the involvement of the W/R residue in acceptor recognition, we produced 34 variants of human FUT3, FUT5, FUT6, and ox FUTb Lewis enzymes. Among the FUT3 variants where W(111) was replaced by the other amino acids, only enzymes with an aromatic residue at the candidate position kept about 50% of alpha1,4 activity and showed no changes in K(m) values for GDP-Fuc donor and H-type 1 acceptor substrates. All other substitutions produced enzymes with less than 20% of the alpha1,4 activity. Thus the ability of alpha3/4-FUTs to recognize type 1 substrates involves the aromatic character of W in the acceptor-binding domain. The alpha1,3 activity of FUT6 and FUTb significantly decreased when their R residue was substituted by basic or charged residues. Moreover, FUT3 and FUT5 variants with W-->R substitution had a better affinity for H-type 2 substrate and higher alpha1,3 activities. Therefore the optimal fucose addition in alpha1,3 linkage requires the R residue in the acceptor-binding motif of Lewis FUTs.     

Ovarian cancer alpha 1,3-L-fucosyltransferase. Differentiation of distinct catalytic species with the unique substrate, 3'-sulfo-N-acetyllactosamine in conjunction with other synthetic acceptors.

Several N-acetyllactosamine (LacNAc) derivatives were tested as acceptors for alpha 1,3-L-fucosyltransferase present in human ovarian cancer sera and ovarian tumor. The enzyme of the soluble fraction of tumor was purified to apparent homogeneity by chromatography on bovine IgG glycopeptide-Sepharose followed by Sephacryl S-200 (M(r) < 67,000). As compared with 2'-methyl LacNAc, 3'-sulfo LacNAc was about 5-fold more sensitive in measuring alpha 1,3-fucosyltransferase in sera (Km, 3'-sulfo LacNAc, 0.12 mM; 2'-methyl LacNAc, 6.67 mM). When ovarian cancer serum was the enzyme source, either the sulfate group or a sialyl moiety at C-3' of LacNAc enhanced the acceptor ability (341 and 242%, respectively), whereas the sulfate group at C-2' or C-6' reduced the activity (22-36%); sulfate at C-6 or fucose at C-2' increased the activity (172 and 253%). The beta-benzylation of the reducing end, in general, increased the activity 2-3-fold. The enzyme of the soluble fraction of tumor exhibited more activity toward 3'-sulfo LacNAc (447%), 2'-fucosyl-LacNAc (436%), and 6-sulfo LacNAc (272%). Very low activity was observed with 3'-sialyl LacNAc (12.4%), 2'-sulfo LacNAc (33%), and 6'-sulfo LacNAc (5%); Fuc alpha 1,2Gal beta 1,3GlcNAc beta-O-p-nitrophenyl (166%), 2-methyl Gal beta 1,3GlcNAc beta-O-benzyl (204%), and 3-sulfo Gal beta 1,3GlcNAc (415%) also acted as acceptors, indicating the coexistence of alpha 1,3- and alpha 1,4-fucosyltransferase. The tumor particulate enzyme behaved entirely different, exhibiting low activity with 3'-sulfo LacNAc (39%) and 2'-fucosyl-LacNAc (148%); 3'-sialyl, 6'-sulfo, 6-sulfo, or 2'-sulfo LacNAc were 3, 43, 53, and 10% active, respectively. Thus, the ovarian cancer serum alpha 1,3-fucosyltransferase acts equally well on H-type 2,3'-sialyl LacNAc and 3'-sulfo LacNAc, but not on H-type 1. The enzyme of soluble tumor fraction acts on H-type 2,3'-sulfo LacNAc as well as H-type 1 but poorly on 3'-sialyl LacNAc. The tumor particulate enzyme acts on H-type 2 but poorly on 3'-sulfo or 3'-sialyl LacNAc and is inactive with H-type 1. When normal serum was examined with synthetic acceptors, > 80% activity was found as alpha 1,2-fucosyltransferase and the rest as alpha 1,3-fucosyltransferase. A screening of 21 ovarian cancer and 3 normal sera (3'-sulfo LacNAc as acceptor) showed 17-572% increase (average increase, 188%) of alpha 1,3-fucosyltransferase activity in cancer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three bovine alpha2-fucosyltransferase genes encode enzymes that preferentially transfer fucose on Galbeta1-3GalNAc acceptor substrates

To investigate the synthesis of alpha2-fucosylated epitopes in the bovine species, we have characterized cDNAs from various tissues. We found three distinct alpha2-fucosyltransferase genes, named bovine fut1, fut2, and sec1 which are homologous to human FUT1, FUT2, and Sec1 genes, respectively. Their open reading frames (ORF) encode polypeptides of 360 (bovine H), 344 (bovine Se), and 368 (bovine Sec1) amino acids, respectively. These enzymes transfer fucose in alpha1,2 linkage to ganglioside GM(1)and galacto- N -biose, but not to the phenyl-beta-D-galactoside, type 1 or type 2 acceptors, suggesting that their substrate specificity is different and more restricted than the other cloned mammalian alpha2-fucosyltransferases. Southern blot analyses detected four related alpha2-fucosyltransferase sequences in the bovine genome while only three have been described in other species. The supernumerary entity seems to be related to the alpha2-fucosyltransferase activity which can also use type 1 and phenyl-beta-D-galactoside substrate acceptors. It was exclusively found in bovine intestinal tract. Our results show that, at least in one mammalian species, four alpha2-fucosyltransferases are present, three adding a fucose on alpha1,2 linkage on type 3/4 acceptor (Galbeta1-3GalNAc) and another able to transfer also fucose on phenyl-beta-D-galactoside and type 1 (Galbeta1-3GlcNAc) acceptors. The phylogenetic tree of the enzymes homologous to those encoded by the bovine fut1, fut2, and sec1 genes revealed two main families, one containing all the H-like proteins and the second containing all the Se-like and Sec1-like proteins. The Sec1-like family had a higher evolutionary rate than the Se-like family.     

Alpha1,4-fucosyltransferase activity: a significant function in the primate lineage has appeared twice independently

In the animal kingdom the enzymes that catalyze the formation of alpha1,4 fucosylated-glycoconjugates are known only in apes (chimpanzee) and humans. They are encoded by FUT3 and FUT5 genes, two members of the Lewis FUT5-FUT3-FUT6 gene cluster, which had originated by duplications of an alpha3 ancestor gene. In order to explore more precisely the emergence of the alpha1,4 fucosylation, new Lewis-like fucosyltransferase genes were studied in species belonging to the three main primate groups. Two Lewis-like genes were found in brown and ruffed lemurs (prosimians) as well as in squirrel monkey (New World monkey). In the latter, one gene encodes an enzyme which transfers fucose only in alpha1,3 linkage, whereas the other is a pseudogene. Three genes homologous to chimpanzee and human Lewis genes were identified in rhesus macaque (Old World monkey), and only one encodes an alpha3/4-fucosyltransferase. The ability of new primate enzymes to transfer fucose in alpha1,3 or alpha1,3/4 linkage confirms that the amino acid R or W in the acceptor-binding motif "HH(R/W)(D/E)" is required for the type 1/type 2 acceptor specificity. Expression of rhesus macaque genes proved that fucose transfer in alpha1,4 linkage is not restricted to the hominoid family and may be extended to other Old World monkeys. Moreover, the presence of only one enzyme supporting the alpha1,4 fucosylation in rhesus macaque versus two enzymes in hominoids suggests that this function occurred twice independently during primate evolution.     

C-terminal amino acids of Helicobacter pylori alpha1,3/4 fucosyltransferases determine type I and type II transfer

The alpha1,3/4 fucosyltransferase (FucT) enzyme from Helicobacter pylori catalyzes fucose transfer from donor GDP-beta-l-fucose to the GlcNAc group of two series of acceptor substrates in H. pylori lipopolysaccharide: betaGal1,3betaGlcNAc (Type I) or betaGal1,4betaGlcNAc (Type II). Fucose is added either in alpha1,3 linkage of Type II acceptor to produce Lewis X or in alpha1,4 linkage of Type I acceptor to produce Lewis A, respectively. H. pylori FucTs from different strains have distinct Type I or Type II substrate specificities. FucT in H. pylori strain NCTC11639 has an exclusive alpha1,3 activity because it recognizes only Type II substrates, whereas FucT in H. pylori strain UA948 can utilize both Type II and Type I acceptors; thus it has both alpha1,3 and alpha1,4 activity, respectively. To identify elements conferring substrate specificity, 12 chimeric FucTs were constructed by domain swapping between 11639FucT and UA948FucT and characterized for their ability to transfer fucose to Type I and Type II acceptors. Our results indicate that the C-terminal region of H. pylori FucTs controls Type I and Type II acceptor specificity. In particular, the highly divergent C-terminal portion, seven amino acids DNPFIFC at positions 347-353 in 11639FucT, and the corresponding 10 amino acids CNDAHYSALH at positions 345-354 in UA948FucT, controls the Type I and Type II acceptor recognition. This is the opposite of mammalian FucTs where acceptor preference is determined primarily by the N-terminal residues in the hypervariable stem domain.     

Acceptor specificity and tissue distribution of three human alpha-3-fucosyltransferases

Based on the capacity to transfer alpha-L-fucose onto type-1 and type-2 synthetic blood group H and sialylated acceptors, a comparison of the alpha-3-fucosyltransferase activities of different human tissues is shown. Three distinct acceptor specificity patterns are described: (I) myeloid alpha-3-fucosyltransferase pattern, in which leukocytes and brain enzymes transfer fucose actively onto H type-2 acceptor and poorly onto sialylated N-acetyllactosamine: (II) plasma alpha-3-fucosyltransferase (EC 2.4.1.152), in which plasma and hepatocyte enzymes transfer, in addition, onto the sialylated N-acetyllactosamine; (III) Lewis alpha-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 (less than 10%) of alpha-3-fucosyltransferase activity found in the plasma of an alpha-3-fucosyltransferase-deficient individual had a myeloid-type 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 alpha-3-fucosyltransferase activities, the Lewis or alpha-3/4-fucosyltransferase under control of the Lewis gene and an alpha-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 N-acetyllactosamine were stronger for exocrine secretions as compared to the plasma and myeloid enzymes. The plasma type of alpha-3-fucosyltransferase activity was more sensitive to N-ethylmaleimide and heat inactivation than the samples with myeloid-like alpha-3-fucosyltransferase activity.     

A single aromatic amino acid at the carboxyl terminus of Helicobacter pylori {alpha}1,3/4 fucosyltransferase determines substrate specificity

Fucosyltransferases (FucT) from different Helicobacter pylori strains display distinct Type I (Galbeta1,3GlcNAc) or Type II (Galbeta1,4GlcNAc) substrate specificity. FucT from strain UA948 can transfer fucose to the OH-3 of Type II acceptors as well as to the OH-4 of Type I acceptors on the GlcNAc moiety, so it has both alpha1,3 and alpha1,4 activities. In contrast, FucT from strain NCTC11639 has exclusive alpha1,3 activity. Our domain swapping study (Ma, B., Wang, G., Palcic, M. M., Hazes, B., and Taylor, D. E. (2003) J. Biol. Chem. 278, 21893-21900) demonstrated that exchange of the hypervariable loops, (347)DNPFIFC(353) in 11639FucT and (345)CNDAHYSALH(354) in UA948FucT, were sufficient to either confer or abolish alpha1,4 activity. Here we performed alanine scanning site-directed mutagenesis to identify which amino acids within (345)CNDAHYSALH(354) of UA948FucT confer Type I substrate specificity. The Tyr(350) --> Ala mutation dramatically reduced alpha1,4 activity without lowering alpha1,3 activity. None of the other alanine substitutions selectively eliminated alpha1,4 activity. To elucidate how Tyr(350) determines alpha1,4 specificity, mutants Tyr(350) --> Phe, Tyr(350) --> Trp, and Tyr(350) --> Gly were constructed in UA948FucT. These mutations did not decrease alpha1,3 activity but reduced the alpha1,4 activity to 66.9, 55.6, and 3.1% [corrected] of wild type level, respectively. Apparently the aromatic nature, but not the hydroxyl group of Tyr(350), is essential for alpha1,4 activity. Our data demonstrate that a single amino acid (Tyr(350)) in the C-terminal hypervariable region of UA948FucT determines Type I acceptor specificity. Notably, a single aromatic residue (Trp) has also been implicated in controlling Type I acceptor preference for human FucT III, but it is located in an N-terminal hypervariable stem domain.     

Characterization of a novel alpha1,2-fucosyltransferase of Escherichia coli O128:b12 and functional investigation of its common motif

The wbsJ gene from Escherichia coli O128:B12 encodes an alpha1,2-fucosyltransferase responsible for adding a fucose onto the galactose residue of the O-antigen repeating unit via an alpha1,2 linkage. The wbsJ gene was overexpressed in E. coli BL21 (DE3) as a fusion protein with glutathione S-transferase (GST) at its N-terminus. GST-WbsJ fusion protein was purified to homogeneity via GST affinity chromatography followed by size exclusion chromatography. The enzyme showed broad acceptor specificity with Galbeta1,3GalNAc (T antigen), Galbeta1,4Man and Galbeta1,4Glc (lactose) being better acceptors than Galbeta-O-Me and galactose. Galbeta1,4Fru (lactulose), a natural sugar, was furthermore found to be the best acceptor for GST-WbsJ with a reaction rate four times faster than that of lactose. Kinetic studies showed that GST-WbsJ has a higher affinity for lactose than lactulose with apparent Km values of 7.81 mM and 13.26 mM, respectively. However, the kcat/appKm value of lactose (6.36 M(-1) x min(-1)) is two times lower than that of lactulose (13.39 M(-1) x min(-1)). In addition, the alpha1,2-fucosyltransferase activity of GST-WbsJ was found to be independent of divalent metal ions such as Mn2+ or Mg2+. This activity was competitively inhibited by GDP with a Ki value of 1.41 mM. Site-directed mutagenesis and a GDP-bead binding assay were also performed to investigate the functions of the highly conserved motif H152xR154R155xD157. In contrast to alpha1,6-fucosyltransferases, none of the mutants of WbsJ within this motif exhibited a complete loss of enzyme activity. However, residues R154 and D157 were found to play critical roles in donor binding and enzyme activity. The results suggest that the common motif shared by both alpha1,2-fucosyltransferases and alpha1,6-fucosyltransferases have similar functions. Enzymatic synthesis of fucosylated sugars in milligram scale was successfully performed using Galbeta-O-Me and Galbeta1,4Glcbeta-N3 as acceptors.     

Molecular basis for acceptor substrate specificity of the human beta1,3-glucuronosyltransferases GlcAT-I and GlcAT-P involved in glycosaminoglycan and HNK-1 carbohydrate epitope biosynthesis, respectively

The human beta1,3-glucuronosyltransferases galactose-beta1,3-glucuronosyltransferase I (GlcAT-I) and galactose-beta1,3-glucuronosyltransferase P (GlcAT-P) are key enzymes involved in proteoglycan and HNK-1 carbohydrate epitope synthesis, respectively. Analysis of their acceptor specificity revealed that GlcAT-I was selective toward Galbeta1,3Gal (referred to as Gal2-Gal1), whereas GlcAT-P presented a broader profile. To understand the molecular basis of acceptor substrate recognition, we constructed mutants and chimeric enzymes based on multiple sequence alignment and structural information. The drastic effect of mutations of Glu227, Arg247, Asp252, and Glu281 on GlcAT-I activity indicated a key role for the hydrogen bond network formed by these four conserved residues in dictating Gal2 binding. Investigation of GlcAT-I determinants governing Gal1 recognition showed that Trp243 could not be replaced by its counterpart Phe in GlcAT-P. This result combined with molecular modeling provided evidence for the importance of stacking interactions with Trp at position 243 in the selectivity of GlcAT-I toward Galbeta1,3Gal. Mutation of Gln318 predicted to be hydrogen-bonded to 6-hydroxyl of Gal1 had little effect on GlcAT-I activity, reinforcing the role of Trp243 in Gal1 binding. Substitution of Phe245 in GlcAT-P by Ala selectively abolished Galbeta1,3Gal activity, also highlighting the importance of an aromatic residue at this position in defining the specificity of GlcAT-P. Finally, substituting Phe245, Val320, or Asn321 in GlcAT-P predicted to interact with N-acetylglucosamine (GlcNAc), by their counterpart in GlcAT-I, moderately affected the activity toward the reference substrate of GlcAT-P, N-acetyllactosamine, indicating that its active site tolerates amino acid substitutions, an observation that parallels its promiscuous substrate profile. Taken together, the data clearly define key residues governing the specificity of beta1,3-glucuronosyltransferases.     

Significance of each of three missense mutations, G484A, G667A, and G808A, present in an inactive allele of the human Lewis gene (FUT3) for α(1,3/1,4)fucosyltransferase inactivation

Recently, we found three novel missense mutations, G484A (Asp162Asn), G667A (Gly223Arg), and G808A (Val270Met), present in a Lewis-negative allele (le484,667,808) from an African (Xhosa) population. To define the relative contribution of each of the three mutations in the le484,667,808 allele for inactivation of the FUT3-encoded enzyme, we made chimeric FUT3 containing each of the three mutations. A transient expression study indicated that COS7 cells transfected with the FUT3 construct containing the G484A mutation expressed the Lewis antigen and had about 20% enzyme activity as compared with COS7 cells transfected with the wild type FUT3 allele, whereas COS7 cells transfected with the FUT3 construct containing either the G667A mutation or the G808A mutation did not express the Lewis antigen and showed no detectable (1,3/1,4)fucosyltransferase activity. These results suggest that the G667A and/or the G808A missense mutations of FUT3 alleles are responsible for the inactivation of the FUT3-encoded enzyme.     

Identification of a missense mutation (G329A;Arg(110)--> GLN) in the human FUT7 gene

The human FUT7 gene codes for the alpha1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLe(x)) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLe(x) and SLe(x)-related epitopes. One individual showed lower levels of SLe(x) expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg(110) --> Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. The FUT7 Arg(110) is conserved in all previously cloned vertebrate alpha 1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of < or =1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7 alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLe(x) and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sj?gren's syndrome but had no history of recurrent bacterial infections or leukocytosis.     

Human fucosyltransferase IX: specificity towards N-linked glycoproteins and relevance of the cytoplasmic domain in intra-Golgi localization

The alpha3-fucosyltransferase IX (FUT9) catalyses the transfer of fucose in an alpha3 linkage onto terminal type II (Galbeta4GlcNAc) acceptors, the final step in the biosynthesis of the Lewis(x) (Le(x)) epitope, in neurons. In this work, FUT9 cloned from NT2N neurons and overexpressed in HeLa cells (FUT9wt), was found to efficiently fucosylate asialoerythropoietin (asialoEPO), and bovine asialofetuin, but not sialylated EPO. Analysis by HPAEC-PAD and MALDI/TOF-MS revealed predominantly mono-fucosylation by FUT9wt of type II di-, tri- and tetraantennary N-glycans with proximal fucose, with and without N-acetylactosamine repeats from asialoEPO. Minor amounts of difucosylated structures were also found. The results suggested that FUT9 could fucosylate Le(x) carrier-glycoproteins in neurons. Furthermore, FUT9wt was found to be activated by Mn(2+) and it was capable of synthesizing Le(a), although to a lesser extent than Le(x) and Le(y). In vivo, HeLa cells transfected with FUT9wt expressed de novo Le(x), as detected by immunofluorescence microscopy. FUT9 was found to be a trans-Golgi and trans-Golgi network (TGN) glycosyltransferase from confocal immunofluorescence co-localization with the markers of the secretory pathway beta4-galactosyltransferase (trans-Golgi and TGN) and TGN-46 (TGN). Deletion of the cytoplasmic domain caused a shift to the cis-Golgi, thus suggesting that information for intra-Golgi localization is contained within the cytoplasmic domain.     

Identification of a new alpha1,2-fucosyltransferase involved in O-antigen biosynthesis of Escherichia coli O86:B7 and formation of H-type 3 blood group antigen

Escherichia coli O86 possesses high human blood group B activity because of its O-antigen structure, sharing the human blood group B epitope. In this study, the wbwK gene of E. coli O86:B7 was expressed and purified as the GST fusion protein. Thereafter, the wbwK gene was biochemically identified to encode an alpha1,2-fucosyltransferase through radioactivity assays, as well as mass spectrometry and NMR spectroscopy. WbwK shows strict substrate specificity and only recognizes Gal beta1,3GalNAc alpha-OR (T-antigen and derivatives) as the acceptor to generate the H-type 3 blood group antigen. In contrast to other alpha1,2-fucosyltransferases, WbwK does not display activity toward the simple substrate Gal beta-OMe. Comparison with another recently characterized alpha1,2-fucosyltransferase (WbsJ) of E. coli O128:B12 indicates a low level of amino acid identity between them; however, they share a common acceptor substrate, Gal beta1,3GalNAc alpha-OR. Domain swapping between WbwK and WbsJ revealed that the smaller variable domains located in the C-terminus determine substrate specificity, whereas the larger variable domain in the N-terminus might play a role in forming the correct conformation for substrate binding or for localization of the alpha1,2-fucosyltransferase involved in O-antigen biosynthesis. In addition, milligram scale biosynthesis of the H-type 3 blood group antigen was explored using purified recombinant WbwK. WbwK may have potential applications in masking T-antigen, the tumor antigen, in vivo.     

Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408-->Glu/Lys939-->Arg mutant retained significant activity, while an Asp407-->Glu/Lys939-->Arg mutant exhibited only marginal function. An Asp407-->Glu/Asp408-->Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407-->Glu/Asp408-->Glu/Lys939-->Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.     

Tumor necrosis factor alpha increases the expression of glycosyltransferases and sulfotransferases responsible for the biosynthesis of sialylated and/or sulfated Lewis x epitopes in the human bronchial mucosa.

There is increasing evidence that inflammation may affect glycosylation and sulfation of various glycoproteins. The present study reports the effect of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, on the glycosyl- and sulfotransferases of the human bronchial mucosa responsible for the biosynthesis of Lewis x epitope and of its sialylated and/or sulfated derivatives, which are expressed in human bronchial mucins. Fragments of macroscopically normal human bronchial mucosa were exposed to TNF-alpha at a concentration of 20 ng/ml. TNF-alpha was shown to increase alpha1,3-fucosyltransferase activity as well as expression of the two alpha1,3-fucosyltransferase genes expressed in the human airway, FUT3 and FUT4. It had no influence on alpha1,2-fucosyltransferase activity or FUT2 expression. It also increased alpha2,3-sialyltransferase activity and the expression of ST3Gal-III and, more importantly, ST3Gal-IV and both N-acetylglucosamine 6-O-sulfotransferase and galactose 3-O-sulfotransferase. These results are consistent with the observation of oversialylation and increased expression sialyl-Lewis x epitopes on human airway mucins secreted by patients with severe lung infection such as those with cystic fibrosis, whose airways are colonized by Pseudomonas aeruginosa. However, other cytokines may also be involved in this process.     

Biosynthesis of fucose containing lacto-series glycolipids in human colonic adenocarcinoma Colo 205 cells

Biosynthesis of fucose containing lacto-series glycolipids has been studied in human colonic adenocarcinoma Colo 205 cells. Transfer of fucose in both alpha 1----3 linkage to type 2 chain acceptors and alpha 1----4 linkage to type 1 chain acceptors was demonstrated with a Triton X-100 solubilized membrane fraction. The enzyme was found to be highly active over a broad pH range between 6.0 and 7.5. Kinetics of the transfer reactions were studied and indicated that the enzyme had an apparent Km for GDPfucose of 53 and 49 microM with acceptors nLc4 and Lc4, respectively. The apparent Km values for acceptors Lc4, nLc4, and IV3NeuAcnLc4 were determined to be 42, 18, and 26 microM, respectively. Transfer of fucose to the type 1 chain acceptor Lc4 alone and in the presence of increasing concentrations of the type 2 chain acceptor IV3NeuAcnLc4 or Gb3 suggested that both type 1 and 2 acceptors were alternate acceptors for a single enzyme. This was further established by the finding that IV3NeuAcnLc4 behaved as a competitive inhibitor of fucose transfer with respect to Lc4. Conditions were defined for preparative scale in vitro synthesis of fucosylated products of nLc6 catalyzed by the Colo 205 cell enzyme. Yields of the monofucosyl derivative of 2.5 mg (46%) and 1 mg (17%) of the difucosyl derivative were obtained from 5 mg of original nLc6. The structures of these biosynthetic products were carefully studied by 1H NMR, +FAB-MS, and methylation analysis. These studies revealed extremely high purity products composed of III3FucnLc6 and III3V3Fuc2nLc6. The significance of the nature of these products and enzymatic properties is discussed.     

Cloning and characterization of the alpha(1,3/4) fucosyltransferase of Helicobacter pylori

The gastric pathogen Helicobacter pylori can express the histo blood group antigens, which are on the surface of many human cells. Most H. pylori strains express the type II carbohydrates, Lewis X and Y, whereas a small population express the type I carbohydrates, Lewis A and B. The expression of Lewis A and Lewis X, as in the case of H. pylori strain UA948, requires the addition of fucose in alpha1,4 and alpha1,3 linkages to type I or type II carbohydrate backbones, respectively. This work describes the cloning and characterization of a single H. pylori fucosyltransferase (FucT) enzyme, which has the ability to transfer fucose to both of the aforementioned linkages in a manner similar to the human fucosyltransferase V (Fuc-TV). Two homologous copies of the fucT gene have been identified in each of the genomes sequenced. The characteristic adenosine and cytosine tracts in the amino terminus and repeated regions in the carboxyl terminus are present in the DNA encoding the two UA948fucT genes, but these genes also contain differences when compared with previously identified H. pylori fucTs. The UA948fucTa gene encodes an approximately 52-kDa protein containing 475 amino acids, whereas UA948fucTb does not encode a full-length FucT protein. In vitro, UA948FucTa appears to add fucose with a greater than 5-fold preference for type II chains but still retains significant activity using type I acceptors. The addition of the fucose to the type II carbohydrate acceptors, by UA948FucTa, does not appear to be affected by fucosylation at other sites on the carbohydrate acceptor, but the rate of fucose transfer is affected by terminal fucosylation of type I acceptors. Through mutational analysis we demonstrate that only FucTa is active in this H. pylori isolate and that inactivation of this enzyme eliminates expression of all Lewis antigens.     

Site-specific fucosylation of sialylated polylactosamines by alpha1,3/4-fucosyltransferases-V and -VI Is defined by amino acids near the N terminus of the catalytic domain

Fucose transfer from GDP-fucose to GlcNAc residues of the sialylated polylactosamine acceptor NeuAcalpha2-3Galbeta1-4Glc-NAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta1-ceramide leads to two isomeric monofucosyl antigens, VIM2 and sialyl-Le(x). Human alpha1,3/4-fucosyltransferase (FucT)-V catalyzes primarily the synthesis of VIM2, whereas human FucT-VI catalyzes primarily the synthesis of sialyl-Le(x). Thus, these two enzymes have distinct "site-specific fucosylation" properties. Amino acid sequence alignment of these enzymes showed that there are 24 amino acid differences in their catalytic domains. Studies were conducted to determine which of the amino acid differences are responsible for the site-specific fucosylation properties of each enzyme. Domain swapping (replacing a portion of the catalytic domain from one enzyme with an analogous portion from the other enzyme) demonstrated that site-specific fucosylation was defined within a 40-amino acid segment containing 8 amino acid differences between the two enzymes. Site-directed mutagenesis studies demonstrated that the site-specific fucosylation properties of these enzymes could be reversed by substituting 4 amino acids from one sequence with the other. These results were observed in both in vitro enzyme assays and flow cytometric analyses of Chinese hamster ovary cells transfected with plasmids containing the various enzyme constructs. Modeling studies of human FucT using a structure of a bacterial fucosyltransferase as a template demonstrated that the amino acids responsible for site-specific fucosylation map near the GDP-fucose-binding site. Additional enzyme studies demonstrated that FucT-VI has approximately 12-fold higher activity compared with FucT-V and that the Trp(124)/Arg(110) site in these enzymes is responsible primarily for this activity difference.