RESUMO
Two major functions of the Golgi apparatus (GA) are formation of complex glycans and sorting of proteins destined for various subcellular compartments or secretion. To fulfill these tasks proper localization of the accessory proteins within the different sub-compartments of the GA is crucial. Here we investigate structural determinants mediating transition of the two glycosyltransferases beta-1,4- galactosyltransferase 1 (gal-T1) and the alpha-1,3-fucosyltransferase 6 (fuc-T6) from the trans-Golgi cisterna to the trans-Golgi network (TGN). Upon treatment with the ionophore monensin both glycosyltransferases are found in TGN-derived swollen vesicles, as determined by confocal fluorescence microscopy and density gradient fractionation. Both enzymes carry a signal consisting of the amino acids E(5)P(6) in gal-T1 and D(2)P(3) in fuc-T6 necessary for the transition of these glycosyltransferases from the trans-Golgi cisterna to the TGN, but not for their steady state localization in the trans-Golgi cisterna.
Assuntos
Fucosiltransferases/metabolismo , Galactosiltransferases/metabolismo , Complexo de Golgi/fisiologia , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Fucosiltransferases/genética , Galactosiltransferases/genética , Humanos , Immunoblotting , Microscopia de Fluorescência , Monensin , Transporte Proteico/fisiologiaRESUMO
The following review on galactosyltransferase (gal-T1) intends to cover genetic, biochemical, structural, biotechnological, cell biological and medical aspects of this enzyme in a comprehensive manner from discovery to the present day which have brought to light a genetic defect of this enzyme. Early work has only been included if it appeared relevant to ongoing issues. Following the evolution of a research topic over 40 years is in itself a fascinating endeavor as it permits to observe the ins and outs of hypotheses, fashions and errors. Gal-T1 is a beautiful example as it has been involved in almost every aspect of life science. Importantly, there is a future to this enzyme as a research topic, since many questions still remain unanswered: to which extent is it a representative Golgi protein? What is the role of the gene family of gal-Ts? Does gal-T1 exert any functions other than a catalytic one? Why is it phosphorylated? Does it form homodimers in vivo? Surely, there is room for further work, which is likely to reveal further insights into cellular trafficking and signaling and, in the context of the gene family, shall contribute to understanding development and morphogenesis.
Assuntos
Galactosiltransferases , Sequência de Aminoácidos , Animais , Biotecnologia , Galactosiltransferases/química , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Humanos , Imunoquímica , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Estrutura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de AminoácidosRESUMO
Deficiencies in the pathway of N-glycan biosynthesis lead to severe multisystem diseases, known as congenital disorders of glycosylation (CDG). The clinical appearance of CDG is variable, and different types can be distinguished according to the gene that is altered. In this report, we describe the molecular basis of a novel type of the disease in three unrelated patients diagnosed with CDG-I. Serum transferrin was hypoglycosylated and patients' fibroblasts accumulated incomplete lipid-linked oligosaccharide precursors for N-linked protein glycosylation. Transfer of incomplete oligosaccharides to protein was detected. Sequence analysis of the Lec35/MPDU1 gene, known to be involved in the use of dolichylphosphomannose and dolichylphosphoglucose, revealed mutations in all three patients. Retroviral-based expression of the normal Lec35 cDNA in primary fibroblasts of patients restored normal lipid-linked oligosaccharide biosynthesis. We concluded that mutations in the Lec35/MPDU1 gene cause CDG. This novel type was termed CDG-If.
Assuntos
Defeitos Congênitos da Glicosilação/genética , Mutação , Proteínas Repressoras/genética , Sequência de Aminoácidos , Células Cultivadas , Mapeamento Cromossômico , Feminino , Fibroblastos/metabolismo , Glicosilação , Humanos , Masculino , Dados de Sequência Molecular , Oligossacarídeos/biossíntese , Proteínas Repressoras/químicaRESUMO
The potential of surface glycoengineering for biomaterials and biosensors originates from the importance of carbohydrate-protein interactions in biological systems. The strategy employed here utilises carbene generated by illumination of diazirine to achieve covalent bonding of carbohydrates. Here, we describe the synthesis of an aryl diazirine containing a disaccharide (lactose). Surface analysis techniques [X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS)] demonstrate its successful surface immobilisation on polystyrene (PS). Results are compared to those previously obtained with an aryl diazirine containing a monosaccharide (galactose). The biological activity of galactose- or lactose-modified PS samples is studied using rat hepatocytes, Allo A lectin and solid-phase semi-synthesis with alpha-2,6-sialyltransferase. Allo A shows some binding to galactose-modified PS but none to lactose-modified surfaces. Similar results are obtained with rat hepatocytes. In contrast, sialylation of lactose-modified PS is achieved but not with galactose-modified surfaces. The different responses indicate that the biological activity depends not only on the carbohydrate per se but also on the structure and length of the spacer.
Assuntos
Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Dissacarídeos/farmacologia , Monossacarídeos/farmacologia , Poliestirenos/química , Animais , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/metabolismo , Diazometano/química , Dissacarídeos/química , Dissacarídeos/metabolismo , Microanálise por Sonda Eletrônica , Galactose/metabolismo , Galactose/farmacologia , Hepatócitos/química , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Lactose/metabolismo , Lactose/farmacologia , Lectinas/metabolismo , Monossacarídeos/química , Monossacarídeos/metabolismo , Ratos , Sialiltransferases/metabolismo , Espectrometria de Massa de Íon Secundário , Propriedades de SuperfícieRESUMO
The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.
Assuntos
Bioquímica/métodos , Enzimas/química , Epitopos/química , Trissacarídeos/síntese química , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Galactosiltransferases/química , Glucosiltransferases/química , Dados de Sequência Molecular , N-Acetil-Lactosamina Sintase/química , Polietilenoglicóis/química , UDPglucose 4-Epimerase/químicaRESUMO
beta-1,4-galactosyltransferase 1 (beta4gal-T1, EC 2.4.1.38) transfers galactose from UDP-galactose to free N-acetyl-D-glucosamine or bound N-acetyl-D-glucosamine-R. Soluble beta4gal-T1, purified from human milk has been refractory to structural studies by X-ray or NMR. In a previous study (Malissard et al. 1996, Eur. J. Biochem. 239, 340-348) we produced in the yeast Saccaromyces cerevisiae an N-deglycosylated form of soluble beta4gal-T1 that was much more homogeneous than the human enzyme, as it displayed only two isoforms when analysed by IEF as compared to 13 isoforms for the native beta4gal-T1. The propensity of recombinant beta4gal-T1 to aggregate at concentrations > 1 mg.mL(-1) prevented structural and biophysical studies. In an attempt to produce a beta4gal-T1 form suitable for structural studies, we combined site-directed mutagenesis and heterologous expression in Escherichia coli. We produced a mutated form of the catalytic domain of beta4gal-T1 (sfbeta4gal-T1mut) in which seven mutations were introduced at nonconserved sites (A155E, N160K, M163T, A168T, T242N, N255D and A259T). Sfbeta4gal-T1mut was shown to be much more soluble than beta4gal-T1 expressed in S. cerevisiae (8.5 mg.mL(-1) vs. 1 mg.mL(-1)). Catalytic activity and kinetic parameters of sfbeta4gal-T1mut produced in E. coli were shown not to differ to any significant extent from those of the native enzyme.
Assuntos
Aminoácidos/química , Galactosiltransferases/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Bovinos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Glicosilação , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Isoformas de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
Previously, we demonstrated that beta 1,4galactosyltransferase (gal-T1) reversibly segregates from alpha 2,6sialyltransferase (ST6Gal) to swollen vesicles after monensin treatment of the cells. To further explore this phenomenon, we investigated the response to monensin of various Golgi proteins. Within 30 min of monensin treatment, gal-T1 moved from the Golgi apparatus, as defined by localization of giantin, to swollen vesicles whereas ST6Gal, alpha 2,3(N)sialyltransferase, mannosidase II, and N-acetylgalactosaminyltransferase 2 remained associated with the Golgi apparatus. Stably transfected CHO cells exhibited a similar phenomenon of monensin-induced displacement of recombinant gal-T1 to swollen vesicles while recombinant ST6Gal remained colocalized with endogenously expressed giantin. Gal-T1 and the cation-insensitive mannose 6-phosphate receptor colocalized in swollen vesicles as observed at both light and electron microscopic levels. When monensin was replaced by chloroquine, gal-T1 remained arrested in swollen vesicles. Brefeldin A treatment known to cause relocation of Golgi-associated gal-T1 to the endoplasmic reticulum had no effect on gal-T1 trapped in swollen vesicles. This evidence suggests that monensin blocks gal-T1 trafficking in post-Golgi structures and argues against swelling of gal-T1-containing trans Golgi cisternae as previously assumed.
Assuntos
N-Acetil-Lactosamina Sintase/análise , Receptor IGF Tipo 2/análise , Rede trans-Golgi/química , Rede trans-Golgi/enzimologia , Animais , Células CHO , Células Cultivadas , Cricetinae , Fibroblastos/citologia , Regulação Enzimológica da Expressão Gênica , Humanos , Ionóforos/farmacologia , Microscopia Imunoeletrônica , Monensin/farmacologia , N-Acetil-Lactosamina Sintase/genética , Transporte Proteico/efeitos dos fármacos , Transfecção , Rede trans-Golgi/ultraestruturaRESUMO
Human beta1,4-galactoside alpha2,6-sialyltransferase I (ST6GalI) recognition of glycoprotein acceptors has been investigated using various soluble forms of the enzyme deleted to a variable extent in the N-terminal half of the polypeptide. Full-length and truncated forms of the enzyme have been investigated with respect to their specificity for a variety of desialylated glycoproteins of known complex glycans as well as related proteins with different carbohydrate chains. Differences in transfer efficiency have been observed between membrane and soluble enzymatic forms, indicating that deletion of the transmembrane fragment induces loss of acceptor preference. No difference in substrate recognition could be observed when soluble enzymes of similar peptide sequence were produced in yeast or mammalian cells, confirming that removal of the membrane anchor and heterologous expression do not alter enzyme folding and activity. When tested on free oligosaccharides, soluble ST6GalI displayed full ability to sialylate free N-glycans as well as various N-acetyllactosaminyl substrates. Progressive truncation of the N terminus demonstrated that the catalytic domain can proceed with sialic acid transfer with increased efficiency until 80 amino acids are deleted. Fusion of the ST6GalI catalytic domain to the N-terminal half of an unrelated transferase (core 2 beta1,6-N-acetylglucosaminyltransferase) further showed that a chimeric form of broad acceptor specificity and high activity could also be engineered in vivo. These findings therefore delineate a peptide region of approximately 50 amino acids within the ST6GalI stem region that governs both the preference for glycoprotein acceptors and catalytic activity, thereby suggesting that it may exert a steric control on the catalytic domain.
Assuntos
Sialiltransferases/química , Sialiltransferases/metabolismo , Animais , Sítios de Ligação , Células CHO , Domínio Catalítico , Clonagem Molecular , Cricetinae , Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Variação Genética , Humanos , Cinética , Orosomucoide/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Sialiltransferases/genética , Especificidade por Substrato , Tireotropina/metabolismo , Transfecção , Transferrina/metabolismo , alfa-Fetoproteínas/metabolismo , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.
Assuntos
Fibroblastos/metabolismo , Complexo de Golgi/metabolismo , Glicoproteínas de Membrana , Transporte Proteico , Fenômenos Fisiológicos da Pele , Animais , Anticorpos , Linhagem Celular , Fibroblastos/ultraestrutura , Congelamento , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde , Humanos , Processamento de Imagem Assistida por Computador , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica , Microscopia Imunoeletrônica , Coelhos , Proteínas Recombinantes/metabolismo , Pele/metabolismo , Pele/ultraestrutura , Proteínas do Envelope Viral/metabolismoRESUMO
We previously showed that HL 60 leukemia cells exhibit various changes in their cellular glycans after phorbol 12-myristate 13-acetate (PMA) treatment. These changes could originate largely from changes in one or several glycosyltransferases. In this report, we show using enzymatic measures, fluorescence microscopy, immunoblotting and Northern blot that beta-(1 --> 4)-galactosyltransferase I (GalT I) activity was higher (> x 2) in PMA-treated compared with untreated HL 60 cells. Immunoblotting showed an increased intensity of the GalT I band at 49 kDa and Northern blot a weak increase of the GalT I transcript band, after PMA treatment. Moreover, Northern blot performed after actinomycin-D treatment of the cells, which inhibits transcription, suggests that the observed increase of GalT I expression could originate, in part, from increase of the stability of GalT I transcripts.
Assuntos
Galactosiltransferases/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Northern Blotting , Galactosiltransferases/genética , Galactosiltransferases/imunologia , Células HL-60 , Humanos , Immunoblotting , Microscopia de Fluorescência , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Surface glycosylation of endothelial cells is relevant to various processes including coagulation, inflammation, metastasis, and lymphocyte homing. One of the essential sugars involved in these processes is fucose linked alpha1-->3 to N-acetylglucosamine. A family of alpha1,3-fucosyltransferases (FucTs) called FucT-III, IV, V, VI, VII, and IX is able to catalyze such fucosylations. Reverse transcription-PCR analysis revealed that human umbilical vein endothelial cells express all of the FucTs except FucT-IX. The predominant activity, as inferred by acceptor specificity of enzyme activity in cell lysates, is compatible with the presence of FucT-VI. By using an antibody to recombinant soluble FucT-VI, the enzyme colocalized with beta4-galactosyltransferase-1 to the Golgi apparatus. By using a polyclonal antiserum raised against a 17-aa peptide of the variable (stem) region of the FucT-VI, immunocytochemical staining of FucT-VI was restricted to Weibel-Palade bodies, as determined by colocalization with P-selectin and von Willebrand factor. SDS/PAGE immunoblotting and amino acid sequencing of internal peptides confirmed the identity of the antigen isolated by the peptide-specific antibody as FucT-VI. Storage of a fucosyltransferase in Weibel-Palade bodies suggests a function independent of Golgi-associated glycosylation.
Assuntos
Fucosiltransferases/análise , Corpos de Weibel-Palade/enzimologia , Animais , Células CHO , Cricetinae , Endotélio Vascular/citologia , Técnica Indireta de Fluorescência para Anticorpo , Fucosiltransferases/genética , Expressão Gênica , Humanos , Coelhos , Análise de SequênciaRESUMO
Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndrome, represent a family of genetic diseases with variable clinical presentations. Common to all types of CDG characterized to date is a defective Asn-linked glycosylation caused by enzymatic defects of N-glycan synthesis. Previously, we have identified a mutation in the ALG6 alpha1,3 glucosyltransferase gene as the cause of CDG-Ic in four related patients. Here, we present the identification of seven additional cases of CDG-Ic among a group of 35 untyped CDG patients. Analysis of lipid-linked oligosaccharides in fibroblasts confirmed the accumulation of dolichyl pyrophosphate-Man9GlcNAc2 in the CDG-Ic patients. The genomic organization of the human ALG6 gene was determined, revealing 14 exons spread over 55 kb. By polymerase chain reaction amplification and sequencing of ALG6 exons, three mutations, in addition to the previously described A333 V substitution, were detected in CDG-Ic patients. The detrimental effect of these mutations on ALG6 activity was confirmed by complementation of alg6 yeast mutants. Haplotype analysis of CDG-Ic patients revealed a founder effect for the ALG6 allele bearing the A333 V mutation. Although more than 80% of CDG are type Ia, CDG-Ic may be the second most common form of the disease.
Assuntos
Defeitos Congênitos da Glicosilação/genética , Proteínas de Membrana , Alelos , Sequência de Bases , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/enzimologia , Primers do DNA/genética , Éxons , Teste de Complementação Genética , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicosilação , Haplótipos , Humanos , Dados de Sequência Molecular , Mutação , Oligossacarídeos/genética , Saccharomyces cerevisiae/genéticaRESUMO
The radical C-glycosidation of (-)-(1S,4R,5R, 6R)-6-endo-chloro-3-methylidene-5-exo-(phenylseleno)-7-ox abi cyclo[2. 2.1]heptan-2-one ((-)-4) with 2,3,4, 6-tetra-O-acetyl-alpha-D-mannopyranosyl bromide gave (+)-(1S,3R,4R, 5R,6R)-6-endo-chloro-5-exo-(phenylseleno)-3-endo-(1',3',4', 5'-tetra-O-acetyl-2', 6'-anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)-7-oxabi cyc lo[ 2.2.1]hept-2-one ((+)-5) that was converted into (+)-(1R,2S,5R, 6R)-5-acetamido-3-chloro-2-hydroxy-6-(1',3',4',5'-tetra-O-acetyl)-2', 6'-anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)cyclohex -3-en- 1-yl acetate ((+)-10) and into (+)-(1R,2S,5R, 6S)-5-bromo-3-chloro-2-hydroxy-6-(1',3',4',5'-tetra-O-acetyl-2', 6'-anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)cyclohex -3-en- 1-yl acetate ((+)-19). Ozonolysis of (+)-10 and further transformations provided 2-acetamido-2,3-dideoxy-3-C-(2', 6'-anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)-D-galac tos e (alpha-C(1-->3)-D-mannopyranoside of N-acetylgalactosamine (alpha-D-Manp-(1-->3)CH(2)-D-GalNAc): 1). Displacement of the bromide (+)-19 with NaN(3) in DMF provided the corresponding azide ((-)-20) following a S(N)2 mechanism. Ozonolysis of (-)-20 and further transformations led to 2-acetamido-2,3-dideoxy-3-C-(2', 6'-anhydro-7'-deoxy-D-glycero-D-manno-heptitol-7'-C-yl)-D-talose (alpha-C(1-->3)-D-mannopyranoside of N-acetyl D-talosamine (alpha-D-Manp-(1-->3)CH(2)-D-TalNAc): 2). The neutral C-disaccharide 1 inhibits several glycosidases (e.g., beta-galactosidase from jack bean with K(i) = 7.5 microM, alpha-L-fucosidase from human placenta with K(i) = 28 microM, beta-glucosidase from Caldocellum saccharolyticum with K(i) = 18 microM) and human alpha-1, 3-fucosyltransferase VI (Fuc-TVI) with K(i) = 120 microM whereas it 2-epimer 2 does not. Double reciprocal analysis showed that the inhibition of Fuc-TVI by 1 displays a mixed pattern with respect to both the donor sugar GDP-fucose and the acceptor LacNAc with K(i) of 123 and 128 microM, respectively.
Assuntos
Dissacarídeos/síntese química , Inibidores Enzimáticos/síntese química , Fucosiltransferases/antagonistas & inibidores , Glicosídeo Hidrolases/antagonistas & inibidores , Manose/síntese química , Animais , Configuração de Carboidratos , Dissacarídeos/química , Dissacarídeos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Indicadores e Reagentes , Cinética , Manose/química , Manose/farmacologia , Modelos Moleculares , EstereoisomerismoRESUMO
We have previously reported the molecular cloning of beta1, 3-galactosyltransferase-V (beta3GalT-V), which catalyzes the transfer of Gal to GlcNAc-based acceptors with a preference for the core3 O-linked glycan GlcNAc(beta1,3)GalNAc structure. Further characterization indicated that the recombinant beta3GalT-V enzyme expressed in Sf9 insect cells also utilized the glycolipid Lc3Cer as an efficient acceptor. Surprisingly, we also found that beta3GalT-V catalyzes the transfer of Gal to the terminal GalNAc unit of the globoside Gb4, thereby synthesizing the glycolipid Gb5, also known as the stage-specific embryonic antigen-3 (SSEA-3). The SSEA-3 synthase activity of beta3GalT-V was confirmed in vivo by stable expression of the human beta3GalT-V gene in F9 mouse teratocarcinoma cells, as detected with the monoclonal antibody MC-631 by flow cytometry analysis and immunostaining of extracted glycolipids. The biological relation between SSEA-3 formation and beta3GalT-V was further documented by showing that F9 cells treated with the differentiation-inducing agent retinoic acid induced the expression of both the SSEA-3 epitope and the endogenous mouse beta3GalT-V gene. This study represents the first example of a glycosyltransferase, which utilizes two kinds of sugar acceptor substrates without requiring any additional modifier molecule.
Assuntos
Galactosiltransferases/metabolismo , Glicoesfingolipídeos/metabolismo , Animais , Antígenos Glicosídicos Associados a Tumores , Sequência de Bases , Primers do DNA , Galactosiltransferases/genética , Humanos , Camundongos , Dados de Sequência Molecular , Antígenos Embrionários Estágio-Específicos , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndromes, lead to diseases with variable clinical pictures. We report the delineation of a novel type of CDG identified in 2 children presenting with severe developmental delay, seizures, and dysmorphic features. We detected hypoglycosylation on serum transferrin and cerebrospinal fluid beta-trace protein. Lipid-linked oligosaccharides in the endoplasmic reticulum of patient fibroblasts showed an accumulation of the dolichyl pyrophosphate Man(5)GlcNAc(2) structure, compatible with the reduced dolichol-phosphate-mannose synthase (DolP-Man synthase) activity detected in these patients. Accordingly, 2 mutant alleles of the DolP-Man synthase DPM1 gene, 1 with a 274C>G transversion, the other with a 628delC deletion, were detected in both siblings. Complementation analysis using DPM1-null murine Thy1-deficient cells confirmed the detrimental effect of both mutations on the enzymatic activity. Furthermore, mannose supplementation failed to improve the glycosylation status of DPM1-deficient fibroblast cells, thus precluding a possible therapeutic application of mannose in the patients. Because DPM1 deficiency, like other subtypes of CDG-I, impairs the assembly of N-glycans, this novel glycosylation defect was named CDG-Ie.
Assuntos
Defeitos Congênitos da Glicosilação/enzimologia , Defeitos Congênitos da Glicosilação/genética , Manosiltransferases/deficiência , Manosiltransferases/genética , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígenos CD59/metabolismo , Sequência de Carboidratos , Proteínas de Transporte/genética , Células Cultivadas , Pré-Escolar , Defeitos Congênitos da Glicosilação/complicações , Defeitos Congênitos da Glicosilação/patologia , Retículo Endoplasmático/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Proteínas Fúngicas/genética , Glicosilação , Humanos , Lactente , Oxirredutases Intramoleculares/líquido cefalorraquidiano , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/metabolismo , Lipocalinas , Masculino , Manose/metabolismo , Manose/farmacologia , Manosiltransferases/metabolismo , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Mutação , Oligossacarídeos/metabolismo , Antígenos Thy-1/biossíntese , Transferrina/metabolismoRESUMO
The palette of transfer vectors available for generation of recombinant baculoviruses based on transposition-mediated recombination has been enlarged by constructing the pFmel-protA vector. The pFmel-protA plasmid includes the honeybee melittin secretion signal and a Staphylococcus aureus protein A fusion protein tag, which allows the secretion and purification of recombinant proteins. Using this system, the human beta1-4 galactosyltransferase-I protein was expressed in Sf9 insect cells at a level ranging from 22 to 28 U (4.8 to 6.0 mg)/L. The protein A tag enabled a simple monitoring of recombinant protein expression by enzyme-linked immunosorbent assay and Western blotting. Single step purification was achieved by immunoglobulin G affinity chromatography achieving a recovery yield of 28% and a specific activity of 1.9 U per mg of recombinant protein.
Assuntos
Baculoviridae/genética , Elementos de DNA Transponíveis/genética , Insetos/enzimologia , Meliteno/genética , N-Acetil-Lactosamina Sintase/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Abelhas , Western Blotting , Linhagem Celular , Cromatografia de Afinidade , Clonagem Molecular , Meios de Cultivo Condicionados/química , Ensaio de Imunoadsorção Enzimática , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos/genética , Insetos/citologia , Dados de Sequência Molecular , N-Acetil-Lactosamina Sintase/genética , N-Acetil-Lactosamina Sintase/metabolismo , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteína Estafilocócica A/genéticaRESUMO
The cDNAs encoding soluble forms of human beta-1, 4-galactosyltransferase I (EC 2.4.1.22), alpha-2,6-sialyltransferase (EC 2.4.99.1), and alpha-1,3-fucosyltransferase VI (EC 2.4.1.65), respectively, have been expressed in the methylotrophic yeast Pichia pastoris. The vector pPIC9 was used, which contains the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway. The recombinant enzymes had similar kinetic properties as their native counterparts. Their identity was confirmed by Western blotting. Recombinant enzymes may be used for in vitro synthesis of oligosaccharides.
Assuntos
Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Lactose Sintase/genética , Lactose Sintase/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Clonagem Molecular/métodos , Humanos , Cinética , Fator de Acasalamento , Peptídeos/genética , Peptídeos/fisiologia , Pichia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Solubilidade , Especificidade por Substrato , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
We have previously reported that the elevated activities of serum alpha 1,3fucosyltransferase reverted to normal levels after curative removal of the tumors. To determine the origin of elevated serum alpha 1,3fucosyltransferase, blood samples were obtained from both the drainage vein and the artery in patients with different stages of colorectal cancer at surgery. The enzyme levels in all samples from the drainage vein were found to be higher than the levels in the artery that fed the tumor. Hence, the origin of elevated alpha1,3fucosyltransferase in serum was thought to be the tumor rather than the liver that is the normal source of serum alpha1,3fucosyltransferase. When serum samples not only from colorectal cancer patients but also from patients with gastric, liver, lung, pancreas, bladder and esophagus cancer were treated with anti-FUTVI antibody, the measured activities of alpha1,3fucosyltransferase were markedly reduced. Further, secretion of alpha1,3fucosyltransferase from human colorectal carcinoma cells was also detected in the culture medium by Western immuno-blot analysis with anti-FUTVI antibody.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Colorretais/sangue , Fucosiltransferases/sangue , Idoso , Anticorpos/imunologia , Biomarcadores Tumorais/imunologia , Western Blotting , Divisão Celular , Neoplasias Colorretais/patologia , Feminino , Fucosiltransferases/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Células Tumorais CultivadasRESUMO
Glycosyltransferases are increasingly being used for in vitro synthesis of oligosaccharides. Since these enzymes are difficult to purify from natural sources, expression systems for soluble forms of the recombinant enzymes have been developed. This review focuses on the current state of development of yeast expression systems. Two yeast species have mainly been used, i.e. Saccharomyces cerevisiae and Pichia pastoris. Safety and ease of fermentation are well recognized for S. cerevisiae as a biotechnological expression system; however, even soluble forms of recombinant glycosyltransferases are not secreted. In some cases, hyperglycosylation may occur. P. pastoris, by contrast, secrete soluble orthoglycosylated forms to the supernatant where they can be recovered in a highly purified form. The review also covers some basic features of yeast fermentation and describes in some detail those glycosyltransferases that have successfully been expressed in yeasts. These include beta1,4galactosyltransferase, alpha2,6sialyltransferase, alpha2,3sialyltransferase, alpha1,3fucosyltransferase III and VI and alpha1,2mannosyltransferase. Current efforts in introducing glycosylation systems of higher eukaryotes into yeasts are briefly addressed.
Assuntos
Glicosiltransferases/biossíntese , Proteínas Recombinantes/biossíntese , Leveduras/enzimologia , Biotecnologia , Fermentação , Expressão Gênica , Vetores Genéticos , Glicosiltransferases/genética , Engenharia de Proteínas , Proteínas Recombinantes/genéticaRESUMO
The idiopathic Tn-syndrome, formerly called 'permanent mixed-field polyagglutinability', is a rare hematological disorder characterized by the expression of the Tn-antigen on all blood cell lineages. The immunodominant epitope of the Tn-antigen is terminal alpha-N-acetylgalactosamine, O-glycosidically linked to protein. Normally this residue is 3'-substituted by 5-galactose thereby forming the core 1 structure known as the Thomsen-Friedenreich (TF) antigen (Galbeta1 ==> 3GalNAcalpha1 ==> Thr/Ser). The cause of the exposure of the Tn-antigen appears to be due to the silencing of the gene expression of beta1,3galactosyltransferase, since treatment of deficient Tn(+) lymphocyte T clones with 5'azacytidine or Na butyrate leads to reexpression of enzyme activity and the sialylated TF-antigen. The Tn-syndrome is acquired and permanent and affects both sexes at any age. Its origin is unknown. Pluripotent stem cells are affected since all lineages are involved but each one to a variable extent. Therefore, normal cells co-exist with Tn-transformed cells. Clinically, patients suffering from the Tn-syndrome appear healthy. Laboratory findings usually reveal moderate thrombocyto- and leukopenia and some signs of hemolytic anemia not warranting any treatment.