Gastrointestinal mucins of Fut2-null mice lack terminal fucosylation without affecting colonization by Candida albicans

Posttranslational modification of apomucins by the sequential action of glycosyltransferases is required to produce mature mucins. The Secretor gene (FUT2) encodes an alpha(1,2)fucosyltransferase (EC 2.4.1.69) that catalyzes addition of terminal alpha(1,2)fucose residues on mucins and other molecules in mucosal epithelium. Mutant mice containing targeted replacement of Fut2 with the bacterial reporter gene lacZ were studied to determine the affect of the loss of Fut2 on glycosylation of mucins in the gastrointestinal tract. By whole organ X-gal staining, lacZ activity is prominently expressed in the foveolar pit and chief cells of the glandular stomach, Brunner's glands of the duodenum, and goblet cells in the large intestine of Fut2-LacZ-null mice. Staining with Aleuria aurantia agglutinin demonstrates loss of L-fucosylated epithelial glycans throughout the gastrointestinal tract of Fut2-LacZ-null mice, however, histologic appearance of the tissues appears normal. Analysis of oligosaccharides released from insoluble colonic mucins, largely Muc2, by mass spectrometry shows complete lack of terminal fucosylation of O-linked oligosaccharides in Fut2-LacZ-null mice. Precursor glycans accumulate with no evidence of compensation by other fucosyltransferases or sialyltransferases on mucin glycosylation. Because Candida albicans has been reported to adhere to intestinal mucins creating a potential reservoir associated with vaginitis, Fut2-LacZ-null and wild-type mice were inoculated by gastric lavage with C. albicans. We observe no difference in colonization between genotypes suggesting mucin terminal fucosylation does not significantly influence C. albicans-host interaction in the intestine, highlighting that infections caused by the same organism at different mucosal surfaces are not equal.     

Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.     

Reaction mechanism and substrate specificity for nucleotide sugar of mammalian alpha1,6-fucosyltransferase--a large-scale preparation and characterization of recombinant human FUT8

FUT8, mammalian 1,6-fucosyltransferase, catalyzes the transferof a fucose residue from the donor substrate, guanosine 5'-diphosphate(GDP)-ß-L-fucose, to the reducing terminal GlcNAcof the core structure of asparagine-linked oligosaccharide viaan 1,6-linkage. FUT8 is a typical type II membrane protein,which is localized in the Golgi apparatus. We have previouslyshown that two neighboring arginine residues that are conservedamong 1,2-, 1,6-, and protein O-fucosyltransferases play animportant role in donor substrate binding. However, detailsof the catalytic and reaction mechanisms and the ternary structureof FUT8 are not understood except for the substrate specificityof the acceptor. To develop a better understanding of FUT8,we established a large-scale production system for recombinanthuman FUT8, in which the enzyme is produced in soluble formby baculovirus-infected insect cells. Kinetic analyses and inhibitionstudies using derivatives of GDP-ß-L-fucose revealedthat FUT8 catalyzes the reaction which depends on a rapid equilibriumrandom mechanism and strongly recognizes the base portion anddiphosphoryl group of GDP-ß-L-fucose. These resultsmay also be applicable to other fucosyltransferases and glycosyltransferases.     

A novel alpha1,2-fucosyltransferase (CE2FT-2) in Caenorhabditis elegans generates H-type 3 glycan structures

The 1,2-fucosyltransferase family (1,2FT) is the largest familyof glycosyltransferases in the genome of the free-living nematodeCaenorhabditis elegans, and early evidence suggests that eachmember may have a unique activity. Here we describe a C. elegansgene (designated CE2FT-2) encoding an 1,2FT that has the potentialto generate the sequence Fuc1-2Galβ1-3GalNAc-R, which isthe H-type 3 blood group structure. The CE2FT-2 cDNA encodesa putative transmembrane protein that shows 42% amino acid identityto a previously cloned C. elegans 1,2FT (termed CE2FT-1), buthas a very low identity (16–20%) to 1,2FT sequences inhumans, rabbits, and mice. A recombinant form of CE2FT-2 expressedin human 293T cells has a high 1,2FT activity toward Galβ1-3GalNAc-O-pNP,but unexpectedly, the enzyme is inactive toward the acceptorGalβ-O-phenyl. Thus, CE2FT-2 differs from all other 1,2FTspreviously described from animals that all utilize Galβ-O-phenyl.CE2FT-2 is expressed at all stages of worm development, butremarkably, promoter analysis of the CE2FT-2 gene using greenfluorescent protein reporter constructs indicates that the CE2FT-2is expressed exclusively in pharyngeal cells of the worm fromembryo to an adult stage. Because pharyngeal cells are knownto secrete their glycoconjugates to the nematode surface, theseresults may indicate that products of CE2FT-2 contribute tointeractions of the nematode with its environment or are usedas ligands for bacterial attachment. These findings, along withthose on other 1,2FTs in C. elegans, suggest that each 1,2FTin this organism may have a unique acceptor specificity, expressionpattern, and biological function.     

The study of the substrate specificity of rat-brain fucosyltransferase using synthetic acceptors

The substrate specificity of fucosyltransferase (FT) from rat forebrain and cerebellum was studied using synthetic acceptors. Of 16 acceptors tested, only those containing the Galβ1-4GlcNAcβ1-R fragment were subjected to enzymic fucosylation. The isomer with a 1–3 bond as well as lactose and oligosaccharides with an additional Neu5Ac residue attached to Gal or a Fuc residue attached to GlcNAc were not fucosylated, whereas Fucα1-2Galβ1-4GlcNAc displayed the same substrate properties as Galβ1-4GlcNAc. FT from the cerebellum and forebrain was shown to have a specificity similar to that of mammalian FT IV. The activity of the cerebellum FT with all types of substrates was higher than that of FT isolated from the forebrain, the specificity profiles being similar. This communication is dedicated to the 70th birthday of Prof. A.Ya. Khorlin.     

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.     

Glycosyltransferases involved in type 1 chain and Lewis antigen biosynthesis exhibit glycan and core chain specificity

Sialyl Lewis A (SLe(a)), Lewis A (Le(a)), and Lewis B (Le(b)) have been studied in many different biological contexts, for example in microbial adhesion and cancer. Their biosynthesis is complex and involves beta1,3-galactosyltransferases (beta3Gal-Ts) and a combined action of alpha2- and/or alpha4-fucosyltransferases (Fuc-Ts). Further, O-glycans with different core structures have been identified, and the ability of beta3Gal-Ts and Fuc-Ts to use these as substrates has not been resolved. Therefore, to examine the in vivo specificity of enzymes involved in SLe(a), Le(a), and Le(b) synthesis, we have transiently transfected CHO-K1 cells with relevant human glycosyltransferases and, on secreted reporter proteins, detected the resulting Lewis antigens on N- and O-linked glycans using western blotting and Le-specific antibodies. beta3Gal-T1, -T2, and -T5 could synthesize type 1 chains on N-linked glycans, but only beta3Gal-T5 worked on O-linked glycans. The latter enzyme could use both core 2 and core 3 precursor structures. Furthermore, the specificity of FUT5 and FUT3 in Le(a) and Le(b) synthesis was different, with FUT5 fucosylating H type 1 only on core 2, but FUT3 fucosylating H type 1 much more efficient on core 3 than on core 2. Finally, FUT1 and FUT2 were both found to direct alpha2-fucosylation on type 1 chains on both N- and O-linked structures. This knowledge enables us to engineer recombinant glycoproteins with glycan- and core chain-specific Lewis antigen substitution. Such tools will be important for investigations on the fine carbohydrate specificity of Le(b)-binding lectins, such as Helicobacter pylori adhesins and DC-SIGN, and may also prove useful as therapeutics.     

Fucosyltransferase expression in human platelets and leucocytes

The activities of -2-l-fucosyltransferase and -3-l-fucosyltransferase were measured in human platelets and leucocytes from normal donors, -2-l-Fucosyltransferase was found in platelets but not in leucocytes. In contrast -3-l-fucosyltransferase was not detected in platelets but was present in leucocytes where it was demonstrated in the neutrophil, monocyte and lymphocyte fractions.     

Factors affecting the substrate specificity of histone deacetylases

Histone deacetylases (HDACs) catalyze the deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones and thereby mediate changes in the chromatin structure and regulate gene expression in eukaryotic cells. So far, surprisingly little is known about the substrate specificities of different HDACs. Here, we prepared a library of fluorogenic tripeptidic substrates of the general format Ac-P(-2)-P(-1)-Lys(Ac)-MCA (P(-1), P(-2)=all amino acids except cysteine) and measured their HDAC-dependent conversion in a standard fluorogenic HDAC assay. Different HDAC subtypes can be ranked according to their substrate selectivity: HDAH > HDAC8 > HDAC1 > HDAC3 > HDAC6. HDAC1, HDAC3, and HDAC6 exhibit a similar specificity profile, whereas both HDAC8 and HDAH have rather distinct profiles. Furthermore, it was shown that second-site modification (e.g., phosphorylation) of substrate sequences as well as corepressor binding can modulate the selectivity of enzymatic substrate conversion.     

LacZ expression in Fut2-LacZ reporter mice reveals estrogen-regulated endocervical glandular expression during estrous cycle, hormone replacement, and pregnancy

The secretor gene (FUT2) encodes an alpha(1,2)fucosyltransferase (E.C. 2.4.1.69) that elaborates alpha(1,2)fucose residues on mucosal epithelium and secreted mucins. Though uterine alpha(1,2)fucosylated glycans have been proposed to be involved in embryo adhesion, mice with a homozygous null mutation of Fut2 displayed normal fertility. To help develop alternative hypotheses for function, the cell type and regulation of Fut2 expression during the estrous cycle, hormone replacement, and pregnancy was examined in Fut2-LacZ reporter mice containing targeted replacement of Fut2 with bacterial lacZ. LacZ expression in the reproductive tract of Fut2-LacZ mice is most prominent in the glandular epithelium of the endocervix during estrus and pregnancy. Nuclear LacZ expression identifies cell-specific expression of Fut2 in mucus-secreting cells of the endocervix, uterine glands, foveolar pit and chief cells of the stomach, and goblet cells of the colon. In ovariectomized Fut2-LacZ mice, estradiol treatment stimulates X-gal staining in endocervix and uterus but does not affect expression in stomach and colon. Northern blot analysis in wild-type mice shows 15-fold elevations of Fut2 steady-state mRNA with estradiol treatment, whereas Fut1 varies little. Fut2 levels in the glandular stomach and distal colon remain constant, and uterine Fut2 levels vary eightfold during the estrous cycle. These data represent the first demonstration of a glycosyltransferase gene under tissue-specific hormonal regulation in a LacZ reporter mouse model. Endocervical expression of Fut2 in estrus and pregnancy may modify cervical mucus barrier properties from microbial infection analogous to the potential role of mucosal glycans in humans.     

Molecular cloning and characterization of the Caenorhabditis elegans alpha1,3-fucosyltransferase family

The genome of Caenorhabditis elegans encodes five genes with homology to known alpha1,3 fucosyltransferases (alpha1,3FTs), but their expression and functions are poorly understood. Here we report the molecular cloning and characterization of these C. elegans alpha1,3FTs (CEFT-1 through -5). The open-reading frame for each enzyme predicts a type II transmembrane protein and multiple potential N-glycosylation sites. We prepared recombinant epitope-tagged forms of each CEFT and found that they had unusual acceptor specificity, cation requirements, and temperature sensitivity. CEFT-1 acted on the N-glycan pentasaccharide core acceptor to generate Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-Asn. In contrast, CEFT-2 did not act on the pentasaccharide acceptor, but instead utilized a LacdiNAc acceptor to generate GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc, which is a novel activity. CEFT-3 utilized a LacNAc acceptor to generate Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc without requiring cations. CEFT-4 was similar to CEFT-3, but its activity was enhanced by some divalent cations. Recombinant CEFT-5 was well expressed, but did not act on available acceptors. Each CEFT was optimally active at room temperature and rapidly lost activity at 37 degrees C. Promoter analysis showed that CEFT-1 is expressed in C. elegans eggs and adults, but its expression was restricted to a few neuronal cells at the head and tail. We prepared deletion mutants for each enzyme for phenotypic analysis. While loss of CEFT-1 correlated with loss of pentasaccharide core activity and core alpha1,3-fucosylated glycans in worms, loss of other enzymes did not correlate with any phenotypic changes. These results suggest that each of the alpha1,3FTs in C. elegans has unique specificity and expression patterns.     

Association between expression of the H histo-blood group antigen, alpha1,2fucosyltransferases polymorphism of wild rabbits, and sensitivity to rabbit hemorrhagic disease virus

RHDV (rabbit hemorrhagic disease virus) is a highly virulentcalicivirus that has become a major cause of mortality in wildrabbit populations (Oryctolagus cuniculus). It binds to thehisto-blood group antigen (HBGA) H type 2 which requires an1,2fucosyltransferase for its synthesis. In rabbit, three 1,2fucosyltransferasesgenes are known, Fut1, Fut2, and Sec1. Nonfunctional allelesat any of these loci could potentially confer resistance toRHDV, similar to human FUT2 alleles that determine the nonsecretorphenotype and resistance to infection by various NoV strains.In this study, we looked for the presence of H type 2 on buccalepithelial cells of wild rabbits from two geographic areas underRHDV pressure and from one RHDV-free area. Some animals withdiminished H type 2 expression were found in the three populations(nonsecretor-like phenotype). Their frequency markedly increasedaccording to the RHDV impact, suggesting that outbreaks selectedsurvivors with low expression of the virus ligand. Polymorphismsof the Fut1, Fut2, and Sec1 coding regions were determined amonganimals that either died or survived outbreaks. The Fut2 andSec1 genes presented a high polymorphism and the frequency ofone Sec1 allele was significantly elevated, over 6-fold, amongsurvivors. Sec1 enzyme variants showed either moderate, low,or undetectable catalytic activity, whereas all variant Fut2enzymes showed strong catalytic activity. This functional analysisof the enzymes encoded by each Fut2 and Sec1 allele suggeststhat the association between one Sec1 allele and survival mightbe explained by a deficit of 1,2fucosyltransferase expressionrather than by impaired catalytic activity.     

Determinants of substrate specificity in omega-aminotransferases

Ornithine aminotransferase and 4-aminobutyrate aminotransferase are related pyridoxal phosphate-dependent enzymes having different substrate specificities. The atomic structures of these enzymes have shown (i) that active site differences are limited to the steric positions occupied by two tyrosine residues in ornithine aminotransferase and (ii) that, uniquely among related, structurally characterized aminotransferases, the conserved arginine that binds the alpha-carboxylate of alpha-amino acids interacts tightly with a glutamate residue. To determine the contribution of these residues to the specificities of the enzymes, we analyzed site-directed mutants of ornithine aminotransferase by rapid reaction kinetics, x-ray crystallography, and 13C NMR spectroscopy. Mutation of one tyrosine (Tyr-85) to isoleucine, as found in aminobutyrate aminotransferase, decreased the rate of the reaction of the enzyme with ornithine 1000-fold and increased that with 4-aminobutyrate 16-fold, indicating that Tyr-85 is a major determinant of specificity toward ornithine. Unexpectedly, the limiting rate of the second half of the reaction, conversion of ketoglutarate to glutamate, was greatly increased, although the kinetics of the reverse reaction were unaffected. A mutant in which the glutamate (Glu-235) that interacts with the conserved arginine was replaced by alanine retained its regiospecificity for the delta-amino group of ornithine, but the glutamate reaction was enhanced 650-fold, whereas only a 5-fold enhancement of the ketoglutarate reaction rate resulted. A model is proposed in which conversion of the enzyme to its pyridoxamine phosphate form disrupts the internal glutamate-arginine interaction, thus enabling ketoglutarate but not glutamate to be a good substrate.     

Pharmacology,genomics, and the evolutionary biology of ageing

Aging is a multifold process affected by many genes and thus many biochemical pathways. This conclusion is underscored by the failure to find simple central controls for the aging process during the 20th Century. This situation poses a fundamental challenge to anti-aging medicine: how to develop effective therapies for a genomically complex pathology. We propose such a strategy. As a first step, we recommend the use of model systems in which significant genetic intervention is not proscribed or impractical. Second, we propose that work with such model systems begin with selected lines that have genetic enhancements that allow increased lifespan. Third, genomic methods should be used to identify a number of biochemical pathways for increasing lifespan. Fourth, biochemical pathways that have been identified in model systems would then be available for pharmaceutical development, first in rodents, eventually in a clinical human population. This may seem to be a cumbersome R&D strategy, but starting with human populations or inadequately pre-screened compounds would be unlikely to succeed because of the complexity of the aging problem.     

Pharmacology,Genomics, and the Evolutionary Biology of Ageing

Aging is a multifold process affected by many genes and thus many biochemical pathways. This conclusion is underscored by the failure to find simple central controls for the aging process during the 20th Century. This situation poses a fundamental challenge to anti-aging medicine: how to develop effective therapies for a genomically complex pathology. We propose such a strategy. As a first step, we recommend the use of model systems in which significant genetic intervention is not proscribed or impractical. Second, we propose that work with such model systems begin with selected lines that have genetic enhancements that allow increased lifespan. Third, genomic methods should be used to identify a number of biochemical pathways for increasing lifespan. Fourth, biochemical pathways that have been identified in model systems would then be available for pharmaceutical development, first in rodents, eventually in a clinical human population. This may seem to be a cumbersome R&D strategy, but starting with human populations or inadequately pre-screened compounds would be unlikely to succeed because of the complexity of the aging problem.     

Genetic mechanisms for the synthesis of fucosyl GM1 in small cell lung cancer cell lines

Fucosyl GM1 has been reported to be specifically expressed in small cell lung cancer (SCLC) cells. However, the genetic basis for the synthesis of fucosyl GM1 has not been investigated. We analyzed the glycosyltransferases responsible for the synthesis of fucosyl GM1 in SCLC cell lines. In four SCLC cell lines expressing fucosyl GM1, both FUT1 and FUT2 mRNAs were detected, indicating that either one or both of alpha1,2-fucosyltransferases may be involved in the expression of fucosyl GM1. However, three of these four lines contained function-loss mutations in the FUT2 coding region, suggesting that FUT1 is mainly involved in the alpha1,2-fucosylation of GM1. The expression levels of the GM1 synthase gene showed no correlation with those of fucosyl GM1, whereas the co-transfection of GM1 synthase cDNA with FUT1 or FUT2 into SK-LC-17 clearly enhanced the neo-expression of fucosyl GM1, indicating its essential role. In contrast, the co-transfection of GD3 synthase cDNA reduced the expression levels of fucosyl GM1 with FUT1 or FUT2. Consequently, FUT1 seems to mainly contribute to the expression of fucosyl GM1, although both FUT1 and FUT2 are capable of generating the antigen. These results should promote the functional analysis of fucosyl GM1 leading to the development of novel therapies for SCLC.     

CEACAM1, an adhesion molecule of human granulocytes, is fucosylated by fucosyltransferase IX and interacts with DC-SIGN of dendritic cells via Lewis x residues

The CEA-related cell adhesion molecule 1, CEACAM1, is a glycoprotein expressed on the surface of human granulocytes and lymphocytes, endothelia, and many epithelia. CEACAM1 is involved in the regulation of important biological processes, such as tumor growth, angiogenesis, and modulation of the immune response. CEACAM1, a member of the immunoglobulin superfamily carries several Lewis x (Lex) structures as we recently demonstrated by mass spectrometry of native CEACAM1 from human granulocytes. Since Lex residues of pathogens bind to the C-type lectin dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) expressed on human DCs, we hypothesized that Lex glycans of CEACAM1 are recognized by DC-SIGN. Here, we demonstrate that CEACAM1, the major carrier of Lex residues in human granulocytes, is specifically recognized by DC-SIGN via Lex residues mediating the internalization of CEACAM1 into immature DCs. Expression studies with CEACAM1 in combination with different fucosyltransferases (FUTs) revealed that FUTIX plays a key role in the synthesis of Lex groups of CEACAM1. As Lex groups on CEACAM1 are selectively attached and specifically interact with DC-SIGN, our findings suggest that CEACAM1 participates in immune regulation in physiological conditions and in pathological conditions, such as inflammation, autoimmune disease, and cancer.     

Prospects for manipulating the substrate specificity of ribulose bisphosphate carboxylase/oxygenase

Pierce, J. 1988. Prospects for manipulating the substrate specificity of ribulose bisphosphate carboxylase/oxygenase. - Physiol. Plant. 72: 690–698.
The idea of enhancing plant productivity by minimizing the apparently wasteful process of photorespiration has been an enduring one. Since the relative fluxes of carbon through the competing pathways of photosynthesis and photorespiration are determined by the kinetic properties of a single enzyme, ribulose bisphosphate carboxylase/oxygenase, it has been conjectured that genetic modification of this protein could provide more productive plants. Recent advances in techniques for studying ribulose bisphosphate carboxylase/oxygenase hold promise for determining whether such modifications will prove possible.