RESUMEN
We present an analysis of the conformational and aggregative properties of an Aß concatemer (Con-Alz) of interest for vaccine development against Alzheimer's disease. Con-Alz consists of 3 copies of the 43 residues of the Aß peptide separated by the P2 and P30 T-cell epitopes from the tetanus toxin. Even in the presence of high concentrations of denaturants or fluorinated alcohols, Con-Alz has a very high propensity to form aggregates which slowly coalesce over time with changes in secondary, tertiary and quaternary structure. Only micellar concentrations of SDS were able to inhibit aggregation. The increase in the ability to bind the fibril-binding dye ThT increases without lag time, which is characteristic of relatively amorphous aggregates. Confirming this, electron microscopy reveals that Con-Alz adopts a morphology resembling truncated protofibrils after prolonged incubation, but it is unable to assemble into classical amyloid fibrils. Despite its high propensity to aggregate, Con-Alz does not show any significant ability to permeabilize vesicles, which for fibrillating proteins is taken to be a key factor in aggregate cytotoxicity and is attributed to oligomers formed at an early stage in the fibrillation process. Physically linking multiple copies of the Aß-peptide may thus sterically restrict Con-Alz against forming cytotoxic oligomers, forcing it instead to adopt a less well-organized assembly of intermeshed polypeptide chains.
Asunto(s)
Péptidos beta-Amiloides/química , Amiloide/química , Fragmentos de Péptidos/química , Enfermedad de Alzheimer , Secuencia de Aminoácidos , Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Dicroismo Circular , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Multimerización de ProteínaRESUMEN
Maltose O-acetyltransferase (Mac) is a member of the hexapeptide-repeat family of enzymes, which contains proteins with left-handed parallel beta-helix architecture forming homotrimers. Diffraction data for four well diffracting crystal forms were collected. Crystal form I diffracted beyond 1.53 A resolution but was perfectly merohedrally twinned with an apparent space group P622. Crystal forms II and III (space groups R3 and C2, respectively) could be obtained under very similar conditions by adjusting the buffer pH differently. Crystal forms II and III had several monomers in the asymmetric unit and were difficult to derivatize. However, during soaking with trimethyl lead acetate, the form III crystals dissolved and crystals with a different habit and space group grew in their place (form IV). In three of the crystal forms, a ladder of peaks was visible in the native Patterson maps along the c axis. These peaks were interpreted as corresponding to the vectors between the beta-strands in the turns of the beta-helix. Crystal form IV is suitable for structure determination of Mac exploiting the anomalous scattering of lead.
Asunto(s)
Acetiltransferasas/química , Escherichia coli/enzimología , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Polímeros/química , Conformación ProteicaRESUMEN
The Brassica napus gene, Cel16, encodes a membrane-anchored endo-1,4-beta-glucanase with a deduced molecular mass of 69 kD. As for other membrane-anchored endo-1,4-beta-glucanases, Cel16 consists of a predicted intracellular, charged N terminus (methionine(1)-lysine(70)), a hydrophobic transmembrane domain (isoleucine(71)-valine(93)), and a periplasmic catalytic core (lysine(94)-proline(621)). Here, we report the functional analysis of Delta(1-90)Cel16, the N terminally truncated Cel16, missing residues 1 through 90 and comprising the catalytic domain of Cel16 expressed recombinantly in the methylotrophic yeast Pichia pastoris as a soluble protein. A two-step purification protocol yielded Delta(1-90)Cel16 in a pure form. The molecular mass of Delta(1-90)Cel16, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was about 130 kD and about 60 kD after enzymatic removal of N-glycans, fitting the expected molecular mass of 59 kD. Delta(1-90)Cel16 was highly N glycosylated as compared with the native B. napus Cel16 protein. Delta(1-90)Cel16 had a pH optimum of 6.0. The activity of Delta(1-90)Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium. Delta(1-90)Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose. It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1-->3),(1-->4)-beta-D-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose. Size exclusion analysis of Delta(1-90)Cel16-hydrolyzed carboxymethylcellulose showed that Delta(1-90)Cel16 is a true endo-acting glucanase.
Asunto(s)
Brassica napus/enzimología , Celulasa/metabolismo , Celulosa/metabolismo , Proteínas de la Membrana/metabolismo , Pichia/genética , Secuencia de Aminoácidos , Anticuerpos Heterófilos , Brassica napus/genética , Brassica napus/crecimiento & desarrollo , Calcio/farmacología , Carboximetilcelulosa de Sodio , Catálisis , Membrana Celular/enzimología , Celulasa/genética , Celulasa/aislamiento & purificación , Clonación Molecular , Ácido Edético/farmacología , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Solubilidad , Especificidad por Sustrato , Zinc/farmacologíaRESUMEN
The amino acid sequences of serine carboxypeptidase I (CPD-I) and II (CPD-II), respectively, from Aspergillus niger have been determined by conventional Edman degradation of the reduced and vinylpyridinated enzymes and peptides hereof generated by cleavage with cyanogen bromide, iodobenzoic acid, glutamic acid cleaving enzyme, AspN-endoproteinase and EndoLysC proteinase. CPD-I consists of a single peptide chain of 471 amino acid residues, three disulfide bridges and nine N-glycosylated asparaginyl residues, while CPD-II consists of a single peptide chain of 481 amino acid residues, has three disulfide bridges, one free cysteinyl residue and nine glycosylated asparaginyl residues. The enzymes are closely related to carboxypeptidase S3 from Penicillium janthinellum. Both Ca2+ and Mg2+ stabilize CPD-I as well as CPD-II, at basic pH values, Ca2+ being most effective, while the divalent ions have no effect on the activity of the two enzymes.
Asunto(s)
Aspergillus niger/enzimología , Carboxipeptidasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Calcio/farmacología , Cationes Bivalentes/farmacología , Estabilidad de Enzimas , Glicosilación , Concentración de Iones de Hidrógeno , Isoenzimas/química , Magnesio/farmacología , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Análisis de Secuencia , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
cDNA clones encoding three additional serine carboxypeptidases (Ser-CPs) have been isolated from a gibberellic acid-induced barley aleurone cDNA library. The three deduced Ser-CPs belong to the two-chain subfamily of Ser-CPs; they are synthesized as precursors with a putative signal peptide, propeptide, and linker peptide between the A and B chains. Their identification provides the proof for the existence of more than three Ser-CPs in cereal grains, and, based on their sequences, they may exhibit new substrate specificities. The expression of these and of the three previously isolated Ser-CPs from barley grains (CP-MI, CP-MII, and CP-MIII) has been investigated by Northern and Western analysis and RNA PCR. CP-MII is the only Ser-CP to be expressed and accumulate in the developing grain and is stored in its active form in the mature grain. All six Ser-CPs are expressed de novo in the germinating grain, in the scutellum, and/or in the aleurone. Furthermore, at least CP-MI, CP-MII, and CP-MIII are secreted into the endosperm. In addition, all Ser-CPs (except CP-MI) are also expressed in the roots and shoots of the growing seedling. This enzyme family thus appears to be ubiquitous in the barley plant, which suggests that Ser-CPs play additional roles besides their participation in the mobilization of storage proteins.
Asunto(s)
Carboxipeptidasas/biosíntesis , Regulación Enzimológica de la Expresión Génica , Hordeum/fisiología , Isoenzimas/biosíntesis , Semillas/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Carboxipeptidasas/aislamiento & purificación , Carboxipeptidasas/metabolismo , Clonación Molecular , Sondas de ADN , Biblioteca de Genes , Genes de Plantas , Hordeum/enzimología , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN/biosíntesis , ARN/aislamiento & purificación , Semillas/enzimología , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
A procedure was developed to prepare in large amounts two carboxypeptidases, CPD-I and CPD-II, from Aspergillus niger. They were each shown to be serine proteases and single-chain monomers with molecular masses of ca. 81 kDa and containing 22% carbohydrates. Amino acid analysis, carbohydrate determination, and N-terminal sequencing (20 to 25 residues) were performed on each enzyme. CPD-I showed sequence homologies with malt carboxypeptidase II, while the N terminus of CPD-II was different from that of any known serine carboxypeptidase. Like carboxypeptidase Y from Saccharomyces cerevisiae and carboxypeptidase III from malt, CPD-II contained a free sulfhydryl group that could play a role in catalysis. Both A. niger enzymes had pH optima of about 4 and were unstable above pH 7. Their specificities for substrate positions P1 and P'1 were characterized by use of, as substrates, a series of N-blocked amino acid esters and dipeptides. Both enzymes were specific for Arg, Lys, and Phe in P1. CPD-I preferred hydrophobic residues in P'1, while CPD-II was highly specific for Arg and Lys in this position. Each displayed an original specificity when P1 and P'1 were considered together. The specificities were also studied by analyzing the time course of the release of amino acids from eight different peptides of various lengths. CPD-I and CPD-II appeared to be quite suitable for C-terminal sequence studies as well as for the synthesis of peptide bonds. The latter was studied with two peptide esters as aminolysis substrates and a series of amino acid amides as nucleophiles.(ABSTRACT TRUNCATED AT 250 WORDS)