RESUMO
The E6 Zn(2+)-binding protein of high-risk human papillomaviruses (HPVs) is one of the major transforming proteins encoded by these tumor viruses. A bacterial system was used to express wild type and truncated forms of HPV-16 E6 linked to GST. The recombinant proteins were released from GST through cleavage of a factor Xa site. Functional analysis of these proteins demonstrated that amino acids 2--142 comprise the minimal domain of E6 required to promote the degradation of p53 in vitro in a rabbit reticulocyte lysate. This purified protein, E6(Delta 143--151), required a high salt concentration for maximum solubility, eluted as a monomer on gel filtration, and was shown to bind two Zn(2+) ions by atomic absorption analysis. An N-terminal subdomain of E6 (amino acids 2--77, E6-N) was similarly purified. Unlike E6(Delta 143--151), E6--N was very soluble in low-salt buffers and hence was highly amenable to biophysical characterization. E6-N was shown to bind one Zn(2+) ion by electrospray mass spectrometry and by atomic absorption analysis. UV--visible spectroscopic analysis of Co(2+)-substituted E6--N revealed that four cysteine residues coordinate the metal ion. Mutational studies of all the cysteine residues in E6--N substantiated a critical role for Cys 30, 33, 63, and 66 in Zn(2+) binding and in proper folding of the subdomain. Equilibrium sedimentation of E6-N demonstrated that it is a monomer, like E6(Delta 143--151), at low concentrations, but dimerization occurs at high concentrations (K(d) = 0.1 mM). Finally, circular dichroism studies revealed significant secondary structure for both E6(Delta 143--151) and E6--N. The results support a model of monomeric E6 possessing two functionally critical Zn(2+)-binding motifs.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/isolamento & purificação , Papillomaviridae/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Repressoras , Zinco/química , Sequência de Aminoácidos , Proteínas de Transporte/genética , Cisteína/química , Escherichia coli/genética , Humanos , Ligantes , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Fragmentos de Peptídeos/genética , Ligação Proteica/genética , Dobramento de Proteína , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Deleção de Sequência , SolubilidadeRESUMO
Mannose trimming is not only essential for N-glycan maturation in mammalian cells but also triggers degradation of misfolded glycoproteins. The crystal structure of the class I alpha1, 2-mannosidase that trims Man(9)GlcNAc(2) to Man(8)GlcNAc(2 )isomer B in the endoplasmic reticulum of Saccharomyces cerevisiae reveals a novel (alphaalpha)(7)-barrel in which an N-glycan from one molecule extends into the barrel of an adjacent molecule, interacting with the essential acidic residues and calcium ion. The observed protein-carbohydrate interactions provide the first insight into the catalytic mechanism and specificity of this eukaryotic enzyme family and may be used to design inhibitors that prevent degradation of misfolded glycoproteins in genetic diseases.
Assuntos
Manosidases/química , Manosidases/metabolismo , Sequência de Carboidratos , Cristalografia por Raios X , Retículo Endoplasmático/metabolismo , Manosidases/genética , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Conformação Proteica , Controle de Qualidade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
Class I alpha-1,2-mannosidases are a family of Ca2+-dependent enzymes that have been conserved through eukaryotic evolution. These enzymes contain a conserved putative EF hand Ca2+-binding motif and nine invariant acidic residues. The catalytic domain of the alpha-1, 2-mannosidase from Saccharomyces cerevisiae was expressed in Pichia pastoris and was shown by atomic absorption and equilibrium dialysis to bind one Ca2+ ion with high affinity (KD = 4 x 10(-)7 M). Ca2+ protected the enzyme from thermal denaturation. Mutation of the 1st and 12th residues of the putative EF hand Ca2+ binding loop (D121N, D121A, E132Q, E132V, and D121A/E132V) had no effect on Ca2+ binding, demonstrating that the EF hand motif is not the site of Ca2+ binding. In contrast, three invariant acidic residue mutants (D275N, E279Q, and E438Q) lost the ability to bind 45Ca2+ following nondenaturing polyacrylamide gel electrophoresis whereas D86N, E132Q, E503Q, and E526Q mutants exhibited binding of 45Ca2+ similar to the wild-type enzyme. The wild-type enzyme had a Km and kcat of 0.5 mM and 12 s-1, respectively. The Km of E526Q was greatly increased to 4 mM with a small reduction in kcat to 5 s-1 whereas the kcat values of D86N and E132Q(V) were greatly reduced (0.005-0.007 s-1) with a decrease in Km (0.07-0.3 mM). The E503Q mutant is completely inactive. Asp275, Glu279, and Glu438 are therefore required for Ca2+ binding whereas Asp86, Glu132, and Glu503 are required for catalysis.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Manosidases/metabolismo , Fragmentos de Peptídeos/metabolismo , Saccharomyces cerevisiae/enzimologia , Asparagina/genética , Asparagina/metabolismo , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação/genética , Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Catálise , Estabilidade Enzimática , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Glutamina/genética , Glutamina/metabolismo , Temperatura Alta , Cinética , Manosidases/química , Manosidases/genética , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
The alpha 1,2-mannosidase from Saccharomyces cerevisiae catalyzes the conversion of Man9GlcNAc2 to Man8GlcNAc2 during the formation of N-linked oligosaccharides and is a member of the Class 1 alpha 1,2-mannosidases conserved from yeast to mammals. The enzyme is a type II membrane protein and a recombinant form of the alpha 1,2-mannosidase from S. cerevisiae, lacking the transmembrane domain, has been expressed in Pichia pastoris and crystallized using the hanging drop vapor diffusion technique. The crystals grow as flat plates, with unit cell dimensions a = 57.5 A, b = 84.1 A, c = 107.1 A, alpha = beta = gamma = 90 degrees. The crystals exhibit the symmetry of space group P2(1)2(1)2(1) and diffract to a minimum d-spacing of 3.5 A resolution. On the basis of density calculations one monomer is estimated to be present in the asymmetric unit (Vm = 2.08 A3 Da-1). This is the first report of the crystallization of any glycosidase involved in N-glycan biosynthesis.
Assuntos
Manosidases/química , Saccharomyces cerevisiae/enzimologia , Sequência de Carboidratos , Cristalografia por Raios X/métodos , Manosidases/isolamento & purificação , Manosidases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Polissacarídeos/biossíntese , Polissacarídeos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por SubstratoRESUMO
The Saccharomyces cerevisiae alpha1,2-mannosidase, which removes one specific mannose residue from Man9GlcNAc2 to form Man8GlcNAc2, is a member of a family of alpha1,2-mannosidases with similar amino acid sequences. The yeast alpha1,2-mannosidase contains five cysteine residues, three of which are conserved. Recombinant yeast alpha1, 2-mannosidase, produced as the soluble catalytic domain, was shown to contain two disulfide bonds and one free thiol group using 2-nitro-5-thiosulfobenzoate and 5,5'-dithiobis(2-nitrobenzoate), respectively. Cys485 contains the free thiol group, as demonstrated by sequencing of labeled peptides following modification with [3H]ICH2COOH and by high performance liquid chromatography/mass spectrometry tryptic peptide mapping. A Cys340-Cys385 disulfide was demonstrated by sequencing a purified peptide containing this disulfide and by tryptic peptide mapping. Cys468 and Cys471 were not labeled with [3H]ICH2COOH and a peptide containing these two residues was identified in the tryptic peptide map, showing that Cys468 and Cys471 form the second disulfide bond. The alpha1, 2-mannosidase loses its activity in the presence of dithiothreitol with first order kinetics, suggesting that at least one disulfide bond is essential for activity. Mutagenesis of each cysteine residue to serine showed that Cys340 and Cys385 are essential for production of recombinant enzyme, whereas Cys468, Cys471, and Cys485 are not required for production and enzyme activity. These results indicate that the sensitivity to dithiothreitol is due to reduction of the Cys340-Cys385 disulfide. Since Cys340 and Cys385 are conserved residues, it is likely that this disulfide bond is important to maintain the correct structure in the other members of the alpha1, 2-mannosidase family.
Assuntos
Cisteína , Manosidases/química , Manosidases/metabolismo , Polissacarídeos/biossíntese , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Sequência Conservada , Dissulfetos , Ácido Ditionitrobenzoico , Ditiotreitol/farmacologia , Drosophila , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Mapeamento de Peptídeos , Reação em Cadeia da Polimerase , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The yeast-specific alpha 1,2-mannosidase, Mns1p, converts Man,GlcNAc2 to a single isomer of Man8GlcNAc2 during N-linked oligosaccharide processing in Saccharomyces cerevisiae. Mns1p is a 68 kDa type II integral membrane glycoprotein with a very short amino terminal cytoplasmic tail of only two amino acids and a large carboxy-terminal catalytic region that is homologous to class 1 alpha 1,2-mannosidases from mammalian and other species. We have used immunofluorescence and immunoelectron microscopy to demonstrate that Mns1p is localized in the endoplasmic reticulum in Saccharomyces cerevisiae. As Mns1p contains none of the known endoplasmic reticulum retrieval motifs (HDEL, KK or RR), these results suggest that Mns1p is localized in the endoplasmic reticulum by a different retentin mechanism.
Assuntos
Retículo Endoplasmático/química , Manosidases/análise , Saccharomyces cerevisiae/química , Anticorpos Monoclonais , Sequência de Bases , Retículo Endoplasmático/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Manosidases/ultraestrutura , Microscopia Imunoeletrônica , Testes de Precipitina , Saccharomyces cerevisiae/ultraestrutura , Transformação GenéticaRESUMO
A simple and versatile spectrophotometric assay for alpha-mannosidase activity, which can be used with unlabelled natural substrate or synthetic substrates, was developed. The reducing mannose released from the substrate by the enzyme is quantitated using glucose oxidase, peroxidase and o-dianisidine. Using recombinant alpha 1,2-mannosidase obtained from Saccharomyces cerevisiae and Man9, GlcNAc, the spectrophotometric assay yielded values of 0.3 mM for Km and 15 mU/microgram for V(max), which are comparable to those obtained using the traditional radiochemical assay. The assay was also used to evaluate some alternative oligosaccharides as substrates for the enzyme. Man5-O(CH2)8-COOCH3 shows potential as an alternative synthetic substrate as the enzyme retained its specificity for a single alpha 1,2-mannose residue. Kinetic results suggest that the lower 1,3 linked arm of Man9GlcNAc is more critically involved in substrate recognition than the upper 1,6 linked arm.
Assuntos
Manosidases/análise , Espectrofotometria/métodos , Sequência de Carboidratos , Estudos de Avaliação como Assunto , Cinética , Espectroscopia de Ressonância Magnética , Mananas/química , Manosidases/metabolismo , Manosídeos/química , Dados de Sequência Molecular , Oligossacarídeos/química , Radioquímica , Proteínas Recombinantes/análise , Saccharomyces cerevisiae/enzimologia , Especificidade por SubstratoRESUMO
The alpha 1,2-mannosidase from Saccharomyces cerevisiae, which removes one specific alpha 1,2-linked mannose residue from Man9GlcNAc2, is a member of the Class 1 alpha 1,2-mannosidase family conserved from yeast to mammals. Although Class 1 alpha 1,2-mannosidases are essential for the maturation of N-linked oligosaccharides in mammalian cells, nothing is known about their mechanism of action. The availability of sufficient quantities of recombinant yeast alpha 1,2-mannosidase and its homology with the mammalian enzymes make it a good model to study the catalytic mechanism of this family of alpha 1,2-mannosidases. The stereochemical course of hydrolysis of Man9GlcNAc by the yeast enzyme was followed by proton nuclear magnetic resonance spectroscopy. It was observed that beta-D-mannose is related from the oligosaccharide substrate, thereby demonstrating that the enzyme is of the inverting type.
Assuntos
Glicosídeo Hidrolases/metabolismo , Manosidases/metabolismo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologia , Sequência de Carboidratos , Hidrólise , Espectroscopia de Ressonância Magnética , Mananas/química , Mananas/metabolismo , Dados de Sequência Molecular , EstereoisomerismoRESUMO
The Saccharomyces cerevisiae processing alpha 1,2-mannosidase, which trims Man9GlcNAc to Man8GlcNAc, has a lumenally oriented catalytic domain and an N-terminal transmembrane domain. To obtain sufficient protein to study the structure and mechanism of action of this enzyme, the sequence encoding the catalytic domain was inserted downstream of the alpha-factor promoter and signal peptide in a high-copy vector for expression in S. cerevisiae as a secreted protein. Using oligosaccharide substrate (Glc1Man9GlcNAc or Man9GlcNAc), the medium of cells transformed with this plasmid showed an increase in alpha-mannosidase activity that was directly related to the increase in cell density, whereas no alpha-mannosidase activity was detected in cells transformed with vector alone. SDS-PAGE of the medium showed the presence of a doublet of 63 and 60 kDa that was revealed by Coomassie Blue staining and by Western blotting with antibodies to the endogenous solubilized alpha-mannosidase. The recombinant alpha-mannosidase was present in the medium at a level of approximately 1 mg/l and was purified in a single step by chromatography on S-Sepharose. High-resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc in the presence of the recombinant enzyme proved that it retained its specificity and removed only one specific alpha 1,2-mannose residue of the alpha 1,3 branch. Endoglycosidase H treatment decreased the molecular mass of both components of the doublet by approximately 5 kDa, showing that the heterogeneity is not due to differential N-glycosylation. EDTA inhibited the activity of the recombinant enzyme, but the inhibition was reversed by the addition of divalent cations.(ABSTRACT TRUNCATED AT 250 WORDS)