RESUMEN
Substantial evidence suggests that the fibrillation of alpha-synuclein is a critical step in the development of Parkinson's disease. In vitro, alpha-synuclein forms fibrils with morphologies and a staining characteristic similar to those extracted from disease-affected brain. Monomeric alpha-synuclein is an intrinsically disordered protein, with three Tyr residues in the C-terminal region, one in the N-terminus, and lacking Trp. It is thought that interactions between the C-terminus and the central portion of the molecule may prevent or minimize aggregation/fibrillation. To test this hypothesis we examined the importance of the Tyr residues on the propensity for alpha-synuclein to fibrillate in vitro. Fibril formation of alpha-synuclein was completely inhibited, in the timescale over which measurements were made, by replacing the three C-terminal Tyr residues with Ala. In addition, substitution of Tyr133 by Ala also resulted in the absence of fibrillation, whereas the individual Y125A and Y136A mutants showed limited inhibition. Replacement of Tyr39 by Ala also resulted in substantial inhibition of fibrillation. Structural analysis showed that the Y133A mutant had a substantially different conformation, rich in alpha-helical secondary structure, as compared with the wild-type and other mutants, although the formation of any tertiary structure has not been observed as can be judged from near-UV-CD spectra. These observations suggest that the long-range intramolecular interactions between the N- and C-termini of alpha-synuclein are likely to be crucial to the fibrillation process.
Asunto(s)
Sustitución de Aminoácidos , Amiloide/genética , Mutación Missense , alfa-Sinucleína/genética , Alanina/genética , Amiloide/química , Amiloide/metabolismo , Dicroismo Circular , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Enfermedad de Parkinson/genética , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de Fourier , Tirosina/genética , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
The human ABO(H) blood group A and B antigens are generated by the homologous glycosyltransferases A (GTA) and B (GTB), which add the monosaccharides GalNAc and Gal, respectively, to the cell-surface H antigens. In the first comprehensive structural study of the recognition by a glycosyltransferase of a panel of substrates corresponding to acceptor fragments, 14 high resolution crystal structures of GTA and GTB have been determined in the presence of oligosaccharides corresponding to different segments of the type I (alpha-l-Fucp-(1-->2)-beta-D-Galp-(1-->3)-beta-D-GlcNAcp-OR, where R is a glycoprotein or glycolipid in natural acceptors) and type II (alpha-l-Fucp-(1-->2)-beta-D-Galp-(1-->4)-beta-d-GlcNAcp-OR) H antigen trisaccharides. GTA and GTB differ in only four "critical" amino acid residues (Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268). As these enzymes both utilize the H antigen acceptors, the four critical residues had been thought to be involved strictly in donor recognition; however, we now report that acceptor binding and subsequent transfer are significantly influenced by two of these residues: Gly/Ser-235 and Leu/Met-266. Furthermore, these structures show that acceptor recognition is dominated by the central Gal residue despite the fact that the L-Fuc residue is required for efficient catalysis and give direct insight into the design of model inhibitors for GTA and GTB.
Asunto(s)
Regulación de la Expresión Génica , Sistema del Grupo Sanguíneo ABO , Antígenos/química , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Electrones , Galactosiltransferasas/química , Glicina/química , Glucolípidos/química , Glicosiltransferasas/química , Humanos , Cinética , Leucina/química , Modelos Químicos , Modelos Moleculares , Monosacáridos/química , Oligosacáridos/química , Unión Proteica , Proteínas/química , Trisacáridos/químicaRESUMEN
The human ABO(H) blood group antigens are carbohydrate structures generated by glycosyltransferase enzymes. Glycosyltransferase A (GTA) uses UDP-GalNAc as a donor to transfer a monosaccharide residue to Fuc alpha1-2Gal beta-R (H)-terminating acceptors. Similarly, glycosyltransferase B (GTB) catalyzes the transfer of a monosaccharide residue from UDP-Gal to the same acceptors. These are highly homologous enzymes differing in only four of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. Blood group O usually stems from the expression of truncated inactive forms of GTA or GTB. Recently, an O(2) enzyme was discovered that was a full-length form of GTA with three mutations, P74S, R176G, and G268R. We showed previously that the R176G mutation increased catalytic activity with minor effects on substrate binding. Enzyme kinetics and high resolution structural studies of mutant enzymes based on the O(2) blood group transferase reveal that whereas the P74S mutation in the stem region of the protein does not appear to play a role in enzyme inactivation, the G268R mutation completely blocks the donor GalNAc-binding site leaving the acceptor binding site unaffected.