RESUMEN
Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara3), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara3. None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields.
Asunto(s)
Pruebas de Enzimas , Fucosiltransferasas , Glicosiltransferasas , Proteínas de Plantas , Apium/enzimología , Apium/genética , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/metabolismo , Pared Celular/química , Pared Celular/enzimología , Pared Celular/metabolismo , Pruebas de Enzimas/instrumentación , Pruebas de Enzimas/métodos , Fucosiltransferasas/análisis , Fucosiltransferasas/clasificación , Fucosiltransferasas/metabolismo , Glicosiltransferasas/análisis , Glicosiltransferasas/metabolismo , Espectrometría de Masas , Oryza/enzimología , Proteínas de Plantas/análisis , Proteínas de Plantas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismoRESUMEN
BACKGROUND: 2'-fucosyllactose (2'-FL) is one of the most abundant oligosaccharides in human milk. It constitutes an authorized functional additive to improve infant nutrition and health in manufactured infant formulations. As a result, a cost-effective method for mass production of 2'-FL is highly desirable. RESULTS: A microbial cell factory for 2'-FL production was constructed in Saccharomyces cerevisiae by expressing a putative α-1, 2-fucosyltransferase from Bacillus cereus (FutBc) and enhancing the de novo GDP-L-fucose biosynthesis. When enabled lactose uptake, this system produced 2.54 g/L of 2'-FL with a batch flask cultivation using galactose as inducer and carbon source, representing a 1.8-fold increase compared with the commonly used α-1, 2-fucosyltransferase from Helicobacter pylori (FutC). The production of 2'-FL was further increased to 3.45 g/L by fortifying GDP-mannose synthesis. Further deleting gal80 enabled the engineered strain to produce 26.63 g/L of 2'-FL with a yield of 0.85 mol/mol from lactose with sucrose as a carbon source in a fed-batch fermentation. CONCLUSION: FutBc combined with the other reported engineering strategies holds great potential for developing commercial scale processes for economic 2'-FL production using a food-grade microbial cell factory.
Asunto(s)
Bacillus cereus/enzimología , Fucosiltransferasas/genética , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trisacáridos/biosíntesis , Bacillus cereus/genética , Técnicas de Cultivo Celular por Lotes , Fermentación , Fucosiltransferasas/clasificación , Trisacáridos/genéticaRESUMEN
During pollen-pistil interactions in angiosperms, the male gametophyte (pollen) germinates to produce a pollen tube. To fertilize ovules located within the female pistil, the pollen tube must physically penetrate specialized tissues. Whereas the process of pollen tube penetration through the pistil has been anatomically well described, the genetic regulation remains poorly understood. In this study, we identify a novel Arabidopsis (Arabidopsis thaliana) gene, O-FUCOSYLTRANSFERASE1 (AtOFT1), which plays a key role in pollen tube penetration through the stigma-style interface. Semi-in vivo growth assays demonstrate that oft1 mutant pollen tubes have a reduced ability to penetrate the stigma-style interface, leading to a nearly 2,000-fold decrease in oft1 pollen transmission efficiency and a 5- to 10-fold decreased seed set. We also demonstrate that AtOFT1 is localized to the Golgi apparatus, indicating its potential role in cellular glycosylation events. Finally, we demonstrate that AtOFT1 and other similar Arabidopsis genes represent a novel clade of sequences related to metazoan protein O-fucosyltransferases and that mutation of residues that are important for O-fucosyltransferase activity compromises AtOFT1 function in vivo. The results of this study elucidate a physiological function for AtOFT1 in pollen tube penetration through the stigma-style interface and highlight the potential importance of protein O-glycosylation events in pollen-pistil interactions.
Asunto(s)
Proteínas de Arabidopsis/genética , Flores/genética , Fucosiltransferasas/genética , Tubo Polínico/genética , Polinización/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fertilización/genética , Flores/metabolismo , Fucosiltransferasas/clasificación , Fucosiltransferasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Mutación , Filogenia , Plantas Modificadas Genéticamente , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The core alpha1,6-fucosyltransferase (FUT8) catalyzes the transfer of a fucosyl moiety from GDP-fucose to the innermost asparagine-linked N-acetylglucosamine residue of glycoproteins. In mammals, this glycosylation has an important function in many fundamental biological processes and although no essential role has been demonstrated yet in all animals, FUT8 amino acid (aa) sequence and FUT8 activity are very well conserved throughout the animal kingdom. We have cloned the cDNA and the complete gene encoding the FUT8 in the Sf9 (Spodoptera frugiperda) lepidopteran cell line. As in most animal genomes, fut8 is a single-copy gene organized in different exons. The open reading frame contains 12 exons, a characteristic that seems to be shared by all lepidopteran fut8 genes. We chose to study the gene structure as a way to characterize the evolutionary relationships of the fut8 genes in metazoans. Analysis of the intron-exon organization in 56 fut8 orthologs allowed us to propose a model for fut8 evolution in metazoans. The presence of a highly variable number of exons in metazoan fut8 genes suggests a complex evolutionary history with many intron gain and loss events, particularly in arthropods, but not in chordata. Moreover, despite the high conservation of lepidoptera FUT8 sequences also in vertebrates and hymenoptera, the exon-intron organization of hymenoptera fut8 genes is order-specific with no shared exons. This feature suggests that the observed intron losses and gains may be linked to evolutionary innovations, such as the appearance of new orders.
Asunto(s)
Evolución Biológica , Fucosiltransferasas/genética , Proteínas de Insectos/genética , Animales , Secuencia de Bases , Clonación Molecular , Exones , Fucosiltransferasas/clasificación , Fucosiltransferasas/metabolismo , Genoma , Proteínas de Insectos/clasificación , Proteínas de Insectos/metabolismo , Insectos/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera/genéticaRESUMEN
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.
Asunto(s)
Caenorhabditis elegans/genética , Clonación Molecular , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Genes de Helminto , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Secuencia de Carbohidratos , Secuencia Conservada , Epítopos , Fucosiltransferasas/clasificación , Glicosilación , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
We previously described a bacterial fermentation process for the in vivo conversion of lactose into fucosylated derivatives of lacto-N-neotetraose Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LNnT). The major product obtained was lacto-N-neofucopentaose-V Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, carrying fucose on the glucosyl residue of LNnT. Only a small amount of oligosaccharides fucosylated on N-acetylglucosaminyl residues and thus carrying the LewisX group (Le(X)) was also produced. We report here a fermentation process for the large-scale production of Le(X) oligosaccharides. The two fucosyltransferase genes futA and futB of Helicobacter pylori (strain 26695) were compared in order to optimize fucosylation in vivo. futA was found to provide the best activity on the LNnT acceptor, whereas futB expressed a better Le(X) activity in vitro. Both genes were expressed to produce oligosaccharides in engineered Escherichia coli (E. coli) cells. The fucosylation pattern of the recombinant oligosaccharides was closely correlated with the specificity observed in vitro, FutB favoring the formation of Le(X) carrying oligosaccharides. Lacto-N-neodifucohexaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc represented 70% of the total oligosaccharide amount of futA-on-driven fermentation and was produced at a concentration of 1.7 g/L. Fermentation driven by futB led to equal amounts of both lacto-N-neofucopentaose-V and lacto-N-neofucopentaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, produced at 280 and 260 mg/L, respectively. Unexpectedly, a noticeable proportion (0.5 g/L) of the human milk oligosaccharide 3-fucosyllactose Gal(beta1-4)[Fuc(alpha1-3)]Glc was produced in futA-on-driven fermentation, underlining the activity of fucosyltransferase FutA in E. coli and leading to a reassessment of its activity on lactose. All oligosaccharides produced by the products of both fut genes were natural compounds of human milk.
Asunto(s)
Escherichia coli/metabolismo , Fucosiltransferasas/metabolismo , Helicobacter pylori/enzimología , Antígeno Lewis X/biosíntesis , Leche Humana/química , Oligosacáridos/biosíntesis , Ingeniería de Proteínas/métodos , Reactores Biológicos/microbiología , Técnicas de Cultivo de Célula/métodos , Activación Enzimática , Escherichia coli/genética , Fucosiltransferasas/química , Fucosiltransferasas/clasificación , Fucosiltransferasas/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica , Mejoramiento Genético/métodos , Glicoproteínas/química , Glicoproteínas/metabolismo , Helicobacter pylori/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Leche Humana/metabolismo , Oligosacáridos/química , Proteínas Recombinantes/metabolismoRESUMEN
The presence of three conserved peptide motifs shared by alpha2-fucosyltransferases, alpha6-fucosyltransferases, the protein-O-fucosyltransferase family 1 (POFUT1) and a newly identified protein-O-fucosyltransferase family 2 (POFUT2), together with evidence that the present genes encoding for these enzymes have originated from a common ancestor by duplication and divergent evolution, suggests that they constitute a new superfamily of fucosyltransferases.
Asunto(s)
Fucosiltransferasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Fucosiltransferasas/clasificación , Fucosiltransferasas/genética , Humanos , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de AminoácidoRESUMEN
Fucosyltransferases appeared early in evolution, since they are present from bacteria to primates and the genes are well conserved. The aim of this work was to study these genes in the bird group, which is particularly attractive for the comprehension of the evolution of the vertebrate genome. Twelve fucosyltransferase genes have been identified in man. The orthologues of theses genes were looked for in the chicken genome and cytogenetically localized by FISH. Three families of fucosyltransferases: alpha6-fucosyltransferases, alpha3/4-fucosyltransferases, and protein-O-fucosyltransferases, were identified in the chicken with their associated genes. The alpha2-fucosyltransferase family, although present in some invertebrates and amphibians was not found in birds. This absence, also observed in Drosophila, may correspond to a loss of these genes by negative selection. Of the eight chicken genes assigned, six fell on chromosome segments where conservation of synteny between human and chicken was already described. For the two remaining loci, FUT9 and FUT3/5/6, the location may correspond to a new small syntenic area or to an insertion. FUT4 and FUT3/5/6 were found on the same chicken chromosome. These results suggest a duplication of an ancestral gene, initially present on the same chromosome before separation during evolution. By extension, the results are in favour of a common ancestor for the alpha3-fucosyltransferase and the alpha4-fucosyltransferase activities. These observations suggest a general mechanism for the evolution of fucosyltransferase genes in vertebrates by duplication followed by divergent evolution.
Asunto(s)
Pollos/genética , Evolución Molecular , Fucosiltransferasas/genética , Sintenía , Animales , Mapeo Cromosómico , Fucosiltransferasas/clasificación , Duplicación de Gen , Humanos , Hibridación Fluorescente in Situ , Ratones , FilogeniaRESUMEN
We cloned three members of a GDP-fucose:beta-galactoside alpha1,2-fucosyltransferase (alpha1,2-fucosyltransferase) family, MFUT-I, -II, and -III, from a cDNA of murine small intestine, and determined their enzymatic properties after transfection of the genes into COS-7 cells, and their expression in murine tissues by Northern blotting. MFUT-I, -II, and -III exhibited sequence homology with the human H (78.4%), Se (79.0%), and Sec1 (74.9%) gene products, respectively. COS-7 cells transfected with MFUT-I and -II exhibited alpha1,2-fucosyltransferase activity and the best acceptor substrate for both gene products was GA1 to yield a fucosyl GA1 structure, but no activity was detected in COS-7 cells with MFUT-III. MFUT-II yielded a 3.5-kb mRNA transcript in several tissues, whereas MFUT-I and -III were predominantly expressed in epididymis and testis, respectively. The administration of microbes into germ-free mice resulted in a rapid increase of the MFUT-II gene (Se gene) for the synthesis of fucosyl GA1 in the intestine.
Asunto(s)
Fucosiltransferasas/genética , Intestinos/enzimología , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/química , Antígenos Helmínticos , Secuencia de Bases , Células COS , Clonación Molecular , ADN/análisis , Fucosiltransferasas/clasificación , Fucosiltransferasas/aislamiento & purificación , Proteínas del Helminto/química , Humanos , Intestinos/microbiología , Ratones , Datos de Secuencia Molecular , Proteínas Munc18 , Proteínas del Tejido Nervioso/química , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Transfección , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
On the basis of function and sequence similarities, the vertebrate fucosyltransferases can be classified into three groups: alpha-2-, alpha-3-, and alpha-6-fucosyltransferases. Thirty new putative fucosyltransferase genes from invertebrates and bacteria and six conserved peptide motifs have been identified in DNA and protein databanks. Two of these motifs are specific of alpha-3-fucosyltransferases, one is specific of alpha-2-fucosyltransferases, another is specific of alpha-6-fucosyltransferases, and two are shared by both alpha-2- and alpha-6-fucosyltranserases. Based on these data, literature data, and the phylogenetic analysis of the conserved peptide motifs, a model for the evolution offucosyltransferase genes by successive duplications, followed by divergent evolution is proposed, with either two different ancestors, one for the alpha-2/6-fucosyltransferases and one for the alpha-3-fucosyltransferases or a single common ancestor for the two families. The expected properties of such an hypothetical ancestor suggest that the plant or insect alpha-3-fucosyltransferases using chitobiose as acceptor might be the present forms of this ancestor, since fucosyltransferases using chitobiose as acceptor are expected to be of earlier appearance in evolution than enzymes using N -acetyllactosamine. However, an example of convergent evolution of fucosyltransferase genes is suggested for the appearance of the Leaepitopes found in plants and primates.
Asunto(s)
Evolución Molecular , Fucosiltransferasas/genética , Secuencia de Aminoácidos , Animales , Bacterias/enzimología , Bacterias/genética , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Secuencia Conservada , Fucosiltransferasas/clasificación , Humanos , Leishmania/enzimología , Leishmania/genética , Modelos Genéticos , Datos de Secuencia Molecular , Filogenia , Schistosoma/enzimología , Schistosoma/genética , Homología de Secuencia de AminoácidoRESUMEN
Fucosyltransferases are the enzymes transferring fucose from GDP-Fuc to Gal in an alpha1,2-linkage and to GlcNAc in alpha1,3-, alpha1,4-, or alpha1,6-linkages. Since all fucosyltransferases utilize the same nucleotide sugar, their specificity will probably reside in the recognition of the acceptor and in the type of linkage formed. A search of nucleotide and protein databases yielded more than 30 sequences of fucosyltransferases originating from mammals, chicken, nematode, and bacteria. On the basis of protein sequence similarities, these enzymes can be classified into four distinct families: (1) the alpha-2-fucosyltransferases, (2) the alpha-3-fucosyltransferases, (3) the mammalian alpha-6-fucosyltransferases, and (4) the bacterial alpha-6-fucosyltransferases. Nevertheless, using the sensitive hydrophobic cluster analysis (HCA) method, conserved structural features as well as a consensus peptide motif have been clearly identified in the catalytic domains of all alpha-2 and alpha-6-fucosyltranferases, from prokaryotic and eukaryotic origin, that allowed the grouping of these enzymes into one superfamily. In addition, a few amino acids were found strictly conserved in this family, and two of these residues have been reported to be essential for enzyme activity for a human alpha-2-fucosyltransferase. The alpha-3-fucosyltransferases constitute a distinct family as they lack the consensus peptide, but some regions display similarities with the alpha-2 and alpha-6-fucosyltranferases. All these observations strongly suggest that the fucosyltransferases share some common structural and catalytic features.
Asunto(s)
Fucosiltransferasas/química , Fucosiltransferasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Consenso , Secuencia Conservada , Evolución Molecular , Fucosiltransferasas/clasificación , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoAsunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Fucosiltransferasas/fisiología , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Animales , Antígenos de Carbohidratos Asociados a Tumores/biosíntesis , Antígeno CA-19-9 , Clonación Molecular , Fucosiltransferasas/clasificación , Fucosiltransferasas/genética , Gangliósidos/metabolismo , Humanos , Neoplasias/metabolismo , Oligosacáridos/metabolismo , Antígeno Sialil Lewis X , Especificidad por Sustrato , Distribución TisularRESUMEN
Seven human fucosyltransferase genes have been cloned and registered in the Genome Data Base (GDB) as FUT1 to FUT7. According to their acceptor specificity, two main groups of enzymes can be distinguished. The alpha-2-fucosyltransferases: FUT1 (H) of red cells and vascular endothelium and FUT2 (Se) of exocrine secretions. The alpha-3-fucosyltransferases: FUT3 (Lewis) of exocrine secretions; FUT4 (myeloid) of white cells and brain; FUT5 whose tissue distribution has not been defined as yet; FUT6 (plasma) present in plasma, renal proximal tubules and hepatocytes; FUT7 (leukocyte) found in neutrophils. A high DNA sequence homology has been detected among the genes within each of these two groups, while no homology has been detected between the genes of the two groups. Point mutations responsible of inactivating genetic polymorphisms have been found for FUT1, FUT2, FUT3 and FUT6, while FUT4 and FUT7 seem to be genetically monomorphic. FUT4 has been detected in all tissues of 5 to 10 weeks old human embryos suggesting that it may play a role in development. FUT7 is a candidate for the control of the synthesis of the receptors of selectin mediated cell adhesion.