RESUMO
Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of ß1,4-galactosyltransferase 7 (ß4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human ß4GalT7, we conducted a phylogenetic analysis of the ß1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila ß4GalT7-UDP as template. Two evolutionary conserved motifs, (163)DVD(165) and (221)FWGWGREDDE(230), are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type ß4GalT7 and selected mutants identified Trp(224) as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp(228) acting as general base in the reaction catalyzed by human ß4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human ß4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human ß4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics.
Assuntos
Galactosiltransferases/química , Galactosiltransferases/metabolismo , Glicosaminoglicanos/biossíntese , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Domínio Catalítico , Galactosiltransferases/genética , Humanos , Invertebrados/química , Invertebrados/classificação , Invertebrados/enzimologia , Invertebrados/genética , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Especificidade por Substrato , Vertebrados/classificação , Vertebrados/genética , Vertebrados/metabolismoRESUMO
Sialyltransferases are key enzymes in the biosynthesis of sialoglycoconjugates that catalyze the transfer of sialic residue from its activated form to an oligosaccharidic acceptor. ß-Galactoside α2,6-sialyltransferases ST6Gal I and ST6Gal II are the two unique members of the ST6Gal family described in higher vertebrates. The availability of genome sequences enabled the identification of more distantly related invertebrates' st6gal gene sequences and allowed us to propose a scenario of their evolution. Using a phylogenomic approach, we present further evidence of an accelerated evolution of the st6gal1 genes both in their genomic regulatory sequences and in their coding sequence in reptiles, birds, and mammals known as amniotes, whereas st6gal2 genes conserve an ancestral profile of expression throughout vertebrate evolution.
Assuntos
Antígenos CD/genética , Evolução Molecular , Genoma Humano , Filogenia , Sialiltransferases/genética , Animais , Humanos , Análise de Sequência de ProteínaRESUMO
BACKGROUND: The animal sialyltransferases, which catalyze the transfer of sialic acid to the glycan moiety of glycoconjugates, are subdivided into four families: ST3Gal, ST6Gal, ST6GalNAc and ST8Sia, based on acceptor sugar specificity and glycosidic linkage formed. Despite low overall sequence identity between each sialyltransferase family, all sialyltransferases share four conserved peptide motifs (L, S, III and VS) that serve as hallmarks for the identification of the sialyltransferases. Currently, twenty subfamilies have been described in mammals and birds. Examples of the four sialyltransferase families have also been found in invertebrates. Focusing on the ST8Sia family, we investigated the origin of the three groups of alpha2,8-sialyltransferases demonstrated in vertebrates to carry out poly-, oligo- and mono-alpha2,8-sialylation. RESULTS: We identified in the genome of invertebrate deuterostomes, orthologs to the common ancestor for each of the three vertebrate ST8Sia groups and a set of novel genes named ST8Sia EX, not found in vertebrates. All these ST8Sia sequences share a new conserved family-motif, named "C-term" that is involved in protein folding, via an intramolecular disulfide bridge. Interestingly, sequences from Branchiostoma floridae orthologous to the common ancestor of polysialyltransferases possess a polysialyltransferase domain (PSTD) and those orthologous to the common ancestor of oligosialyltransferases possess a new ST8Sia III-specific motif similar to the PSTD. In osteichthyans, we have identified two new subfamilies. In addition, we describe the expression profile of ST8Sia genes in Danio rerio. CONCLUSION: Polysialylation appeared early in the deuterostome lineage. The recent release of several deuterostome genome databases and paralogons combined with synteny analysis allowed us to obtain insight into events at the gene level that led to the diversification of the ST8Sia genes, with their corresponding enzymatic activities, in both invertebrates and vertebrates. The initial expansion and subsequent divergence of the ST8Sia genes resulted as a consequence of a series of ancient duplications and translocations in the invertebrate genome long before the emergence of vertebrates. A second subset of ST8sia genes in the vertebrate genome arose from whole genome duplication (WGD) R1 and R2. Subsequent selective ST8Sia gene loss is responsible for the characteristic ST8Sia gene expression pattern observed today in individual species.
Assuntos
Evolução Molecular , Duplicação Gênica , Família Multigênica , Sialiltransferases/genética , Vertebrados/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Bases de Dados de Ácidos Nucleicos , Humanos , Funções Verossimilhança , Modelos Lineares , Camundongos , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Peixe-Zebra/genéticaRESUMO
The beta1,3-glucuronosyltransferases are responsible for the completion of the protein-glycosaminoglycan linkage region of proteoglycans and of the HNK1 epitope of glycoproteins and glycolipids by transferring glucuronic acid from UDP-alpha-D-glucuronic acid (UDP-GlcA) onto a terminal galactose residue. Here, we develop phylogenetic and mutational approaches to identify critical residues involved in UDP-GlcA binding and enzyme activity of the human beta1,3-glucuronosyltransferase I (GlcAT-I), which plays a key role in glycosaminoglycan biosynthesis. Phylogeny analysis identified 119 related beta1,3-glucuronosyltransferase sequences in vertebrates, invertebrates, and plants that contain eight conserved peptide motifs with 15 highly conserved amino acids. Sequence homology and structural information suggest that Y84, D113, R156, R161, and R310 residues belong to the UDP-GlcA binding site. The importance of these residues is assessed by site-directed mutagenesis, UDP affinity and kinetic analyses. Our data show that uridine binding is primarily governed by stacking interactions with the phenyl group of Y84 and also involves interactions with aspartate 113. Furthermore, we found that R156 is critical for enzyme activity but not for UDP binding, whereas R310 appears less important with regard to both activity and UDP interactions. These results clearly discriminate the function of these two active site residues that were predicted to interact with the pyrophosphate group of UDP-GlcA. Finally, mutation of R161 severely compromises GlcAT-I activity, emphasizing the major contribution of this invariant residue. Altogether, this phylogenetic approach sustained by biochemical analyses affords new insight into the organization of the beta1,3-glucuronosyltransferase family and distinguishes the respective importance of conserved residues in UDP-GlcA binding and activity of GlcAT-I.
Assuntos
Aminoácidos/metabolismo , Glucuronosiltransferase/metabolismo , Mutação , Filogenia , Uridina Difosfato Ácido Glucurônico/metabolismo , Animais , Sítios de Ligação/genética , Sequência Conservada , Glucuronosiltransferase/genética , Humanos , Mutagênese Sítio-DirigidaRESUMO
Nucleotide sugar transporters (NST) establish the functional link of membrane transport between the nucleotide sugars synthesized in the cytoplasm and nucleus, and the glycosylation processes that take place in the endoplasmic reticulum (ER) and Golgi apparatus. The aim of the present work was to perform a phylogenetic analysis of 87 bank annotated protein sequences comprising all the NST so far characterized and their homologues retrieved by BLAST searches, as well as the closely related triose-phosphate translocator (TPT) plant family. NST were classified in three comprehensive families by linking them to the available experimental data. This enabled us to point out both the possible ER subcellular targeting of these transporters mediated by the dy-lysine motif and the substrate recognition mechanisms specific to each family as well as an important acceptor site motif, establishing the role of evolution in the functional properties of each NST family.
Assuntos
Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Nucleotídeos/genética , Sequência de Aminoácidos , Animais , Metabolismo dos Carboidratos , Carboidratos/química , Bases de Dados de Proteínas , Evolução Molecular , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/classificação , Proteínas de Transporte de Nucleotídeos/química , Proteínas de Transporte de Nucleotídeos/classificação , Filogenia , Homologia de Sequência de AminoácidosRESUMO
We report the cloning of three splice variants of the FUT10 gene, encoding for active alpha-l-fucosyltransferase-isoforms of 391, 419, and 479 amino acids, and two splice variants of the FUT11 gene, encoding for two related alpha-l-fucosyltransferases of 476 and 492 amino acids. The FUT10 and FUT11 appeared 830 million years ago, whereas the other alpha1,3-fucosyltransferases emerged 450 million years ago. FUT10-391 and FUT10-419 were expressed in human embryos, whereas FUT10-479 was cloned from adult brain and was not found in embryos. Recombinant FUT10-419 and FUT10-479 have a type II trans-membrane topology and are retained in the endoplasmic reticulum (ER) by a membrane retention signal at their NH(2) termini. The FUT10-479 has, in addition, a COOH-ER membrane retention signal. The FUT10-391 is a soluble protein without a trans-membrane domain or ER retention signal that transiently localizes to the Golgi and then is routed to the lysosome. After transfection in COS7 cells, the three FUT10s and at least one FUT11, link alpha-l-fucose onto conalbumin glycopeptides and biantennary N-glycan acceptors but not onto short lactosaminyl acceptor substrates as do classical monoexonic alpha1,3-fucosyltransferases. Modifications of the innermost core GlcNAc of the N-glycan, by substitution with ManNAc or with an opened GlcNAc ring or by the addition of an alpha1,6-fucose, suggest that the FUT10 transfer is performed on the innermost GlcNAc of the core chitobiose. We can exclude alpha1,3-fucosylation of the two peripheral GlcNAcs linked to the trimannosyl core of the acceptor, because the FUT10 fucosylated biantennary N-glycan product loses both terminal GlcNAc residues after digestion with human placenta alpha-N-acetylglucosaminidase.
Assuntos
Processamento Alternativo/fisiologia , Evolução Molecular , Fucosiltransferases/metabolismo , Glicoproteínas/metabolismo , Filogenia , Adulto , Motivos de Aminoácidos/fisiologia , Animais , Encéfalo/enzimologia , Células COS , Chlorocebus aethiops , Embrião de Mamíferos/enzimologia , Retículo Endoplasmático/enzimologia , Fucosiltransferases/genética , Glicoproteínas/genética , Complexo de Golgi/enzimologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Lisossomos/enzimologia , Sinais Direcionadores de Proteínas/fisiologia , Especificidade por Substrato/fisiologiaRESUMO
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.
Assuntos
Antígenos CD57/biossíntese , Epitopos/biossíntese , Glucuronosiltransferase/química , Glucuronosiltransferase/metabolismo , Glicosaminoglicanos/biossíntese , Sequência de Aminoácidos , Sítios de Ligação , Antígenos CD57/química , Antígenos CD57/imunologia , Glicosaminoglicanos/química , Glicosaminoglicanos/imunologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
The animal sialyltransferases are Golgi type II transmembrane glycosyltransferases. Twenty distinct sialyltransferases have been identified in both human and murine genomes. These enzymes catalyze transfer of sialic acid from CMP-Neu5Ac to the glycan moiety of glycoconjugates. Despite low overall identities, they share four conserved peptide motifs [L (large), S (small), motif III, and motif VS (very small)] that are hallmarks for sialyltransferase identification. We have identified 155 new putative genes in 25 animal species, and we have exploited two lines of evidence: (1) sequence comparisons and (2) exon-intron organization of the genes. An ortholog to the ancestor present before the split of ST6Gal I and II subfamilies was detected in arthropods. An ortholog to the ancestor present before the split of ST6GalNAc III, IV, V, and VI subfamilies was detected in sea urchin. An ortholog to the ancestor present before the split of ST3Gal I and II subfamilies was detected in ciona, and an ortholog to the ancestor of all the ST8Sia was detected in amphioxus. Therefore, single examples of the four families (ST3Gal, ST6Gal, ST6GalNAc, and ST8Sia) have appeared in invertebrates, earlier than previously thought, whereas the four families were all detected in bony fishes, amphibians, birds, and mammals. As previously hypothesized, sequence similarities among sialyltransferases suggest a common genetic origin, by successive duplications of an ancestral gene, followed by divergent evolution. Finally, we propose predictions on these invertebrates sialyltransferase-related activities that have not previously been demonstrated and that will ultimately need to be substantiated by protein expression and enzymatic activity assays.
Assuntos
Família Multigênica , Filogenia , Sialiltransferases/genética , Sequência de Aminoácidos , Animais , Evolução Molecular , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Sialiltransferases/química , beta-D-Galactosídeo alfa 2-6-Sialiltransferase , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
We have identified a homozygous G>A substitution in the donor splice site of intron 6 (IVS6 + 1G>A) of the cytidine monophosphate (CMP)-sialic acid transporter gene of Lec2 cells as the mutation responsible for their asialo phenotype. These cells were used in complementation studies to test the activity of the 2 CMP-sialic acid transporter cDNA alleles of a patient devoid of sialyl-Le(x) expression on polymorphonuclear cells. No complementation was obtained with either of the 2 patient alleles, whereas full restoration of the sialylated phenotype was obtained in the Lec2 cells transfected with the corresponding human wild-type transcript. The inactivation of one patient allele by a double microdeletion inducing a premature stop codon at position 327 and a splice mutation of the other allele inducing a 130-base pair (bp) deletion and a premature stop codon at position 684 are proposed to be the causal defects of this disease. A 4-base insertion in intron 6 was found in the mother and is proposed to be responsible for the splice mutation. We conclude that this defect is a new type of congenital disorder of glycosylation (CDG) of type IIf affecting the transport of CMP-sialic acid into the Golgi apparatus.
Assuntos
Monofosfato de Citidina/metabolismo , Complexo de Golgi/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Proteínas de Transporte de Nucleotídeos/genética , Proteínas de Transporte de Nucleotídeos/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Células CHO , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cricetinae , DNA Complementar , Deleção de Genes , Glicosilação , Íntrons/genética , Dados de Sequência Molecular , Pais , RNA Mensageiro/análiseRESUMO
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.
Assuntos
Fucosiltransferases/genética , Primatas/genética , Sequência de Aminoácidos , Animais , Southern Blotting , Evolução Molecular , Fucosiltransferases/metabolismo , Humanos , Dados de Sequência Molecular , Filogenia , Primatas/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
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.
Assuntos
Fucosiltransferases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência Conservada , Fucosiltransferases/classificação , Fucosiltransferases/genética , Humanos , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de AminoácidosRESUMO
On the basis of the analysis of 64 glycosyltransferases from 14 species we propose that several successive duplications of a common ancestral gene, followed by divergent evolution, have generated the mannosyltransferases and the glucosyltransferases involved in asparagine-linked glycosylation (ALG) and phosphatidyl-inositol glycan anchor (PIG or GPI), which use lipid-related donor and acceptor substrates. Long and short conserved peptide motifs were found in all enzymes. Conserved and identical amino acid positions were found for the alpha 2/6- and the alpha 3/4-mannosyltransferases and for the alpha 2/3-glucosyltransferases, suggesting unique ancestors for these three superfamilies. The three members of the alpha 2-mannosyltransferase family (ALG9, PIG-B, and SMP3) and the two members of the alpha 3-glucosyltransferase family (ALG6 and ALG8) shared 11 and 30 identical amino acid positions, respectively, suggesting that these enzymes have also originated by duplication and divergent evolution. This model predicts a common genetic origin for ALG and PIG enzymes using dolichyl-phospho-monosaccharide (Dol-P-monosaccharide) donors, which might be related to similar spatial orientation of the hydroxyl acceptors. On the basis of the multiple sequence analysis and the prediction of transmembrane topology we propose that the endoplasmic reticulum glycosyltransferases using Dol-P-monosaccharides as donor substrate have a multispan transmembrane topology with a first large luminal conserved loop containing the long motif and a small cytosolic conserved loop containing the short motif, different from the classical type II glycosyltransferases, which are anchored in the Golgi by a single transmembrane domain.
Assuntos
Dolicol Monofosfato Manose/metabolismo , Evolução Molecular , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Monossacarídeos de Poli-Isoprenil Fosfato/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência Conservada , Dolicol Monofosfato Manose/química , Glicosiltransferases/química , Humanos , Dados de Sequência Molecular , Filogenia , Monossacarídeos de Poli-Isoprenil Fosfato/química , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The product of the FUT8 gene transfers an alpha1-6 fucose on the innermost N-acetylglucosamine of the chitobiose core of N-glycans. Northern blot analysis shows four main transcripts of 3.0, 3.3, 3.9, and 4.2 kb in the embryo. The larger forms around 4-kb decrease in fetus and adult. Fourteen embryo transcripts of FUT8 were cloned. Twelve exons comprising two new 5'untranslated-exons (A and B) and two new 3'UT-ends (L1 and L2) and the complete genomic organization of the FUT8 gene (330 kb) are described. Transcripts starting with the 5'UT-exon A are always associated with exons C and D. Exon B initiates another series of transcripts associated to exon C and D or directly to exon D. A third series of transcripts starts at exon C. The data suggest an expression of FUT8 regulated by three different promoters, starting transcription in exons A, B, or C. The A or C series are better expressed than the B series. After transfection with these cDNA constructs the transcripts with 5'UT-exons A or C have higher expression of FUT8 transcripts and higher alpha6-fucosyltransferase activity, whereas the activity of the B series is about two-thirds lower for both parameters, suggesting that exon B reduces the expression of the transcripts.
Assuntos
Processamento Alternativo/genética , Desenvolvimento Embrionário e Fetal/fisiologia , Fucosiltransferases/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Transcrição Gênica/genética , Adulto , Sequência de Bases , Clonagem Molecular , Primers do DNA , Éxons , Fucosiltransferases/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Variação Genética , Proteínas de Fluorescência Verde , Humanos , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Especificidade de Órgãos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
During murine schistosomiasis, egg-derived glycoconjugates play a key role in skewing the immune response towards a Th2 phenotype. Among the candidates responsible for this effect, complex-type N-glycans containing the core alpha 3-fucose and core beta 2-xylose determinants, two glycan epitopes found in some invertebrate- and plant-derived allergens, may be important. Here, we show that core alpha 3-fucose and core beta 2-xylose determinants are expressed in the different developmental stages of Schistosoma mansoni, particularly in the excretory-secretory systems of schistosomula and adult worms and in eggs deposited in the liver. Glycosyltransferase assays confirmed the presence of core alpha 3-fucosyltransferase and core beta 2-xylosyltransferase activities in egg extracts. Using a model of immunization with pulsed dendritic cells, we show that egg-derived glycoproteins containing the core alpha 3-fucose and core beta 2-xylose determinants generate a strong Th2-biased cellular response in mice and that the glycan moieties of this extract are important in this effect. During murine infection, these complex-type N-glycans induce a glycan-specific Th2 cellular response and elicit T-dependent anti-core alpha 3-fucose and anti-core beta 2-xylose IgG1 (a Th2-associated isotype), but not IgG2b (a Th1-associated isotype) Ab. Taken together, our results point out the importance of core fucosylated/xylosylated N-glycans in the Th2 immune response during murine schistosomiasis.
Assuntos
Fucose/imunologia , Polissacarídeos/imunologia , Esquistossomose mansoni/imunologia , Células Th2/imunologia , Xilose/imunologia , Animais , Células Dendríticas/imunologia , Epitopos , Feminino , Glicoconjugados/imunologia , Glicoproteínas/imunologia , Peroxidase do Rábano Silvestre/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Óvulo/química , Schistosoma mansoni/químicaRESUMO
The underlying causes of type I congenital disorders of glycosylation (CDG I) have been shown to be mutations in genes encoding proteins involved in the biosynthesis of the dolichyl-linked oligosaccharide (Glc(3)Man(9)GlcNAc(2)-PP-dolichyl) that is required for protein glycosylation. Here we describe a CDG I patient displaying gastrointestinal problems but no central nervous system deficits. Fibroblasts from this patient accumulate mainly Man(9)GlcNAc(2)-PP-dolichyl, but in the presence of castanospermine, an endoplasmic reticulum glucosidase inhibitor Glc(1)Man(9)GlcNAc(2)-PP-dolichyl predominates, suggesting inefficient addition of the second glucose residue onto lipid-linked oligosaccharide. Northern blot analysis revealed the cells from the patient to possess only 10-20% normal amounts of mRNA encoding the enzyme, dolichyl-P-glucose:Glc(1)Man(9)GlcNAc(2)-PP-dolichyl alpha3-glucosyltransferase (hALG8p), which catalyzes this reaction. Sequencing of hALG8 genomic DNA revealed exon 4 to contain a base deletion in one allele and a base insertion in the other. Both mutations give rise to premature stop codons predicted to generate severely truncated proteins, but because the translation inhibitor emetine was shown to stabilize the hALG8 mRNA from the patient to normal levels, it is likely that both transcripts undergo nonsense-mediated mRNA decay. As the cells from the patient were successfully complemented with wild type hALG8 cDNA, we conclude that these mutations are the underlying cause of this new CDG I subtype that we propose be called CDG Ih.
Assuntos
Erros Inatos do Metabolismo dos Carboidratos/enzimologia , Glucosiltransferases/química , Alelos , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Western Blotting , Erros Inatos do Metabolismo dos Carboidratos/diagnóstico , Erros Inatos do Metabolismo dos Carboidratos/genética , Células Cultivadas , Clorofórmio/farmacologia , Cromatografia em Camada Fina , Códon de Terminação , Análise Mutacional de DNA , DNA Complementar/metabolismo , Fibroblastos/metabolismo , Glucosiltransferases/metabolismo , Glicosilação , Humanos , Lipídeos/química , Linfócitos/metabolismo , Dados de Sequência Molecular , Mutação , Oligossacarídeos/química , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Type I congenital disorders of glycosylation (CDG I) are diseases presenting multisystemic lesions including central and peripheral nervous system deficits. The disease is characterized by under-glycosylated serum glycoproteins and is caused by mutations in genes encoding proteins involved in the stepwise assembly of dolichol-oligosaccharide used for protein N-glycosylation. We report that fibroblasts from a type I CDG patient, born of consanguineous parents, are deficient in their capacity to add the eighth mannose residue onto the lipid-linked oligosaccharide precursor. We have characterized cDNA corresponding to the human ortholog of the yeast gene ALG12 that encodes the dolichyl-P-Man:Man(7)GlcNAc(2)-PP-dolichyl alpha6-mannosyltransferase that is thought to accomplish this reaction, and we show that the patient is homozygous for a point mutation (T571G) that causes an amino acid substitution (F142V) in a conserved region of the protein. As the pathological phenotype of the fibroblasts of the patient was largely normalized upon transduction with the wild type gene, we demonstrate that the F142V substitution is the underlying cause of this new CDG, which we suggest be called CDG Ig. Finally, we show that the fibroblasts of the patient are capable of the direct transfer of Man(7)GlcNAc(2) from dolichol onto protein and that this N-linked structure can be glucosylated by UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum.
Assuntos
Erros Inatos do Metabolismo dos Carboidratos/enzimologia , Manosiltransferases/genética , Sequência de Aminoácidos , Sequência de Bases , Erros Inatos do Metabolismo dos Carboidratos/diagnóstico , Erros Inatos do Metabolismo dos Carboidratos/genética , Células Cultivadas , Primers do DNA , Etiquetas de Sequências Expressas , Feminino , Glicosilação , Humanos , Recém-Nascido , Manosiltransferases/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
La glicosilación específica de los grupos sanguíneos ABH y Lewis, constituye el primer sistema de histocompatibilidad humana, intervienen fucosilaciones y sialilaciones terminales de glicoproteínas y glicolípidos. Modula la señalización intercelular en la respuesta inmune, la migración y la adhesión celular. Las α2 y α3/4-fucosiltransferasas actúan sobre la cadena de base y forman los antígenos (Ag) ABH y Lewis en numerosos tejidos, varían durante el desarrollo embriofetal humano y algunos Ag son re-expresados en diversos tipos de cáncer, adhieren al endotelio vascular y migran para desarrollar metástasis. Las células de cáncer de colon, HT29, son capaces de diferenciarse in vitro y reproducir el "switch" de fucosiltransferasas observado en la embriogénesis humana. El objetivo del presente trabajo fue abordar la expresión de Ag Lewis durante la proliferación y diferenciación celular in vitro. Se evaluó la expresión progresiva de Ag Lewis frente a diferentes anticuerpos determinando el porcentaje de células fluorescentes en función del tiempo. La expresión Lea aumenta mientras disminuyen Lea, sial-Lea y sial-Lex. El switch se produce en el período de confluencia. Durante la proliferación, están expresados mayoritaria y transitoriamente las estructuras de tipo 2 fucosiladas en alfa (1, 2) o (1, 3) como las estructuras H tipo 2 o Lex. Las estructuras sial-Lea están expresadas transitoriamente durante este período. Luego de la confluencia celular, la mayoría de las estructuras son de tipo 1 fucosiladas (Lea). Las estructuras de tipo 2 están débilmente expresadas.
Specific sialylated and fucosylated ABH and Lewis blood groups are the first human histocompatibility system. Normal glycosylation modula intercellular signals, migration and cellular adhesion. The α 2 and α3/4-fucosyltransferases modify chains of membrane glycolipids and glycoproteins to form ABH and Lewis antigens in different tissues: they vary during human embryonic development. Some antigens change their expression in cancer. Aberrant cell surface glycosylation is thought to have great importance in tumor malignancy. HT29 colon cancer cells are able to differentiate in vitro and reproduce fucosyltransferases switch observed in human embryonic development. The aim of this work was to evaluate Lewis antigens expression during cells culture. Antigen expression was evaluated by the reaction with different antibodies. Percentage of fluorescent cells was established progressively. Lea expression rises while Lex, sial-Lea and sial-Lex decrease. Switch take place in the confluence time. During proliferation type 2 fucosylated either alfa (l ,2) or (1 ,3) structures are expressed (H type 2 or Lex). Sial-Le structures are also expressed. After confluence period, main of structures are type 1 fucosiladas (Lea). Structures type 2 are weakly expressed.