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1.
Glycobiology ; 11(4): 283-95, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11358877

RESUMO

Skp1 is a subunit of SCF-E3 ubiquitin ligases and other protein complexes in the nucleus and cytoplasm of yeast and mammalian cells. In Dictyostelium, Skp1 is partially modified by an unusual pentasaccharide O-linked to hydroxyproline143. This modification was found to be susceptible to known prolyl hydroxylase inhibitors based on M(r)-shift analysis using SDS-polyacrylamide gel electrophoresis/Western blotting. In addition, Dictyostelium Skp1 consists of 2 genetic isoforms, Skp1A and Skp1B, which differ by a single amino acid and appear to be expressed throughout the life cycle based on reverse-transcription polymerase chain reactions. The significance of these structural variations was examined by expressing myc-tagged Skp1s and mutants that lacked the glycosylation site. Gel-based M(r)-shift studies showed that Skp1A and Skp1B are both nearly completely glycosylated during growth and early development, and mass spectrometry of glycopeptides showed that they were glycosylated similarly. Skp1 expressed later in prespore cells was not glycosylated, unlike bulk Skp1 persisting from earlier in development, but became glycosylated after return to growth medium. Skp1A and Skp1B were each concentrated in the nucleus and regions of the cytoplasm, based on immunofluorescence localization. However, when Skp1 glycosylation was blocked by mutation, prolyl hydroxylase inhibitors, or expression in prespore cells, nuclear concentration of Skp1 was not detected. Furthermore, nuclear concentration occurred in a mutant that attached only the core disaccharide to Skp1. Overall, there was no evidence for differential Skp1 isoform expression, glycosylation variants in the bulk Skp1 pool, or regulation of nuclear localization. However, these studies uncovered evidence that the glycosylation pathway is developmentally regulated and can function posttranslationally, and that core glycosylation is required for Skp1's nuclear concentration.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Dictyostelium/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Proteínas de Ciclo Celular/genética , Núcleo Celular/química , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Dictyostelium/citologia , Dictyostelium/genética , Dictyostelium/crescimento & desenvolvimento , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Expressão Gênica , Glicosilação/efeitos dos fármacos , Estágios do Ciclo de Vida/genética , Ligases/química , Ligases/metabolismo , Mutação/genética , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Subunidades Proteicas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA de Protozoário/análise , RNA de Protozoário/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases Associadas a Fase S , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esporos/citologia , Esporos/crescimento & desenvolvimento , Esporos/metabolismo
2.
EMBO J ; 19(17): 4439-48, 2000 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10970838

RESUMO

Myotonic dystrophy (DM1) is an autosomal dominant neuromuscular disorder associated with a (CTG)(n) expansion in the 3'-untranslated region of the DM1 protein kinase (DMPK) gene. To explain disease pathogenesis, the RNA dominance model proposes that the DM1 mutation produces a gain-of-function at the RNA level in which CUG repeats form RNA hairpins that sequester nuclear factors required for proper muscle development and maintenance. Here, we identify the triplet repeat expansion (EXP) RNA-binding proteins as candidate sequestered factors. As predicted by the RNA dominance model, binding of the EXP proteins is specific for dsCUG RNAs and proportional to the size of the triplet repeat expansion. Remarkably, the EXP proteins are homologous to the Drosophila muscleblind proteins required for terminal differentiation of muscle and photoreceptor cells. EXP expression is also activated during mammalian myoblast differentiation, but the EXP proteins accumulate in nuclear foci in DM1 cells. We propose that DM1 disease is caused by aberrant recruitment of the EXP proteins to the DMPK transcript (CUG)(n) expansion.


Assuntos
Proteínas de Drosophila , Distrofia Miotônica/genética , Proteínas Nucleares/genética , Repetições de Trinucleotídeos , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/metabolismo , DNA/metabolismo , Primers do DNA , Drosophila , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Homologia de Sequência de Aminoácidos
3.
J Biol Chem ; 274(51): 36392-402, 1999 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-10593934

RESUMO

Skp1 is a cytoplasmic and nuclear protein required for the ubiquitination of cell cycle regulatory proteins and transcriptional factors. In Dictyostelium, Skp1 is modified by a linear pentasaccharide, Galalpha1-6Galalpha1-Fucalpha1-2Galbeta1-3Glc NAc, attached to a hydroxyproline (HyPro) residue at position 143. To study the formation of the GlcNAc-HyPro linkage, an assay was developed for the transfer of [(3)H]GlcNAc from UDP-[(3)H]GlcNAc to Skp1-HyPro-143 or a synthetic Skp1 4-HyPro peptide. The cytosolic but not the particulate fraction of the cell mediated transfer in a time-, concentration-, and HyPro-dependent fashion. Incorporated radioactivity was alkali-resistant and was recovered as GlcNH(2) after acid hydrolysis, consistent with linkage of GlcNAc to HyPro. The GlcNAc-transferase activity was purified 130,000-fold as a single component with a recovery of 5%. Key to the purification was the synthesis of a novel affinity resin linking UDP-GlcNAc at its 5-uridyl position. The purified activity had an apparent M(r) of approximately 45,000 by gel filtration, required dithiothreitol and a divalent cation, and consisted predominantly of a M(r) 51,000 band after SDS-polyacrylamide gel electrophoresis that was photoaffinity labeled with 5-(125)I-[3-(p-azidosalicylamido)-1-propenyl-UDP-GlcNAc in a UDP-GlcNAc-sensitive fashion. Its apparent K(m) values for UDP-GlcNAc and Skp1 were submicromolar. The presence of the enzyme in the cytosolic fraction, its dependence on a reducing environment, and its high affinity for UDP-GlcNAc strongly suggest that Skp1 is glycosylated by a HyPro GlcNAc-transferase that resides in the cytoplasm.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Dictyostelium/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Animais , Citoplasma/enzimologia , N-Acetilglucosaminiltransferases/isolamento & purificação , Proteínas Quinases Associadas a Fase S
4.
J Biol Chem ; 273(29): 18242-9, 1998 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-9660787

RESUMO

SKP1 is involved in the ubiquitination of certain cell cycle and nutritional regulatory proteins for rapid turnover. SKP1 from Dictyostelium has been known to be modified by an oligosaccharide containing Fuc and Gal, which is unusual for a cytoplasmic or nuclear protein. To establish how it is glycosylated, SKP1 labeled with [3H]Fuc was purified to homogeneity and digested with endo-Lys-C. A single radioactive peptide was found after two-dimensional high performance liquid chromatography. Analysis in a quadrupole time-of-flight mass spectrometer revealed a predominant ion with a novel mass. Tandem mass spectrometry analysis yielded a set of daughter ions which identified the peptide and showed that it was modified at Pro-143. A second series of daughter ions showed that Pro-143 was hydroxylated and derivatized with a potentially linear pentasaccharide, Hex-->Hex-->Fuc-->Hex-->HexNAc-->(HyPro). The attachment site was confirmed by Edman degradation. Gas chromatography-mass spectrometry analysis of trimethylsilyl-derivatives of overexpressed SKP1 after methanolysis showed the HexNAc to be GlcNAc. Exoglycosidase digestions of the glycopeptide from normal SKP1 and from a fucosylation mutant, followed by matrix-assisted laser desorption time-of-flight mass spectrometry analysis, showed that the sugar chain consisted of D-Galpalpha1-->6-D-Galpalpha1-->L-Fucpalpha1-->2-D- Galpbeta1--> 3GlcNAc. Matrix-assisted laser-desorption time-of-flight mass spectrometry analysis of all SKP1 peptides resolved by reversed phase-high performance liquid chromatography showed that SKP1 was only partially hydroxylated at Pro-143 and that all hydroxylated SKP1 was completely glycosylated. Thus SKP1 is variably modified by an unusual linear pentasaccharide, suggesting the localization of a novel glycosylation pathway in the cytoplasm.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Dictyostelium/metabolismo , Hidroxiprolina/metabolismo , Oligossacarídeos/metabolismo , Animais , Configuração de Carboidratos , Proteínas de Ciclo Celular/química , Citoplasma/metabolismo , Glicosilação , Oligossacarídeos/química , Proteínas Quinases Associadas a Fase S , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
Gene ; 200(1-2): 1-10, 1997 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-9373134

RESUMO

FP21 is a glycoprotein within the cytosolic compartment of Dictyostelium which carries an unusual carbohydrate modification(s) including the sugars fucose, galactose and N-acetylglucosamine. The soluble pool of FP21 from crude extracts resolves chromatographically into two fractions that differ in their glycosylation. Previous gene-mapping studies indicating the existence of two loci suggested that the FP21 fractions might be encoded by different genes. To address this issue, the two genes were cloned and sequenced, leading to the prediction that the protein products would differ by only a single amino acid, Ser or Ala, at codon 39. Protein sequence data on CNBr fragments of purified FP21 showed that both gene products are found in both fractions of the soluble pool. After further purification, the two fractions were no longer chromatographically resolvable, and there was no evidence for charge heterogeneity as determined by 2-D gel electrophoresis of whole cells. Thus, the initial separation of the different soluble subpopulations of this protein appears to be due to distinct molecular complexes, possibly related to differential glycosylation, and is not the result of the genetically-encoded amino acid polymorphism.


Assuntos
Dictyostelium/genética , Proteínas Fúngicas/genética , Glicoproteínas/genética , Polimorfismo Genético , Proteínas de Protozoários , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Genes Fúngicos , Biblioteca Genômica , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicosilação , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico
6.
J Biol Chem ; 271(20): 12024-35, 1996 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-8662604

RESUMO

A novel fucosyltransferase (cFTase) activity has been enriched over 10(6)-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galbeta1,3 GlcNAc beta-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent Mr of 95 X 10(3), was Mg2+-dependent with a neutral pH optimum, and exhibited a Km for GDP-fucose of 0.34 microM, a Km for pNP-LNB of 0.6 mM, and a Vmax for pN-P-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent Mr of 85 X 10(3), which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-125I-salicylate. Based on substrate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucalpha1, 2Galbeta1,3GIcNAcbeta-pNP. The cFTase preferred substrates with a Galbeta1,3linkage, and thus its acceptor substrate specificity resembles the human Secretor-type alpha1,2- FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K(m) of 0.21 microM and an apparent V(max) of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucalpha1,2Galbeta1,3GlcNAcbeta-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.


Assuntos
Dictyostelium/enzimologia , Fucosiltransferases/isolamento & purificação , Glicoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Citosol/enzimologia , Fucosiltransferases/metabolismo , Guanosina Difosfato/metabolismo , Cinética , Dados de Sequência Molecular , Galactosídeo 2-alfa-L-Fucosiltransferase
7.
J Am Soc Nephrol ; 7(2): 260-74, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8785396

RESUMO

Structurally and functionally distinct populations of intercalated cells have been described in the collecting duct of both rat and rabbit. However, little is known about these cells in the mouse kidney. The study presented here examines ultrastructural and immunological characteristics of different types of intercalated cells in the mouse. Kidneys of two strains of normal female mice, C57BL/6 and IBR, were preserved by in vivo perfusion with 1% glutaraldehyde or paraformaldehyde-picric acid fixatives and processed for morphological evaluation or light and electron microscopic immunohistochemistry, respectively. The avidin-biotin-horseradish peroxidase procedure was performed on was sections using antibodies against carbonic anhydrase II, H+ -ATPase and Band 3 protein. Immunogold cytochemistry was performed on Lowicryl sections using antibodies to H+ -ATPase and Band 3 protein. Colocalization of H+ -ATPase and Band 3 protein was performed by double labeling using an immunogold technique with silver enhancement. Intercalated cells identified by positive staining for H+ -ATPase and carbonic anhydrase II constituted 35% to 40% of all cells in the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD). Type A intercalated cells identified by positive Band 3 staining constituted 16%, 24%, and 33% of the total cell population in the CNT, CCD, and OMCD, respectively. Electron microscopy and immunogold cytochemistry demonstrated three distinct populations of intercalated cells. Type A intercalated cells with apical H+ -ATPase and basolateral Band 3 immunoreactivity were present in all segments examined, and had prominent apical microprojections and characteristic tubulovesicular structures beneath the apical surface, both coated with studs on the cytoplasmic face. Type B intercalated cells with basolateral and cytoplasmic H+-ATPase and no Band 3 immunoreactivity were most frequently observed in the initial collecting tubule, but were present also in the CNT and early CCD. Type B intercalated cells had a fairly smooth apical surface, a gray zone free of organelles beneath the apical plasma membrane, and small cytoplasmic vesicles without studs throughout the cell. A third type of intercalated cell with apical and cytoplasmic H+-ATPase, but no basolateral Band 3 protein, was observed exclusively in the CNT and the initial collecting tubule. This type of cell was large, with numerous mitochondria, and vesicles coated with studs were present throughout the cell. It resembled a third type of intercalated cell described previously in the rat. It is concluded that three morphologically and immunologically distinct types of intercalated cells are present in the mouse kidney.


Assuntos
Túbulos Renais Coletores/citologia , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Feminino , Imuno-Histoquímica , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Microscopia Eletrônica , ATPases Translocadoras de Prótons/metabolismo
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