RESUMEN
Acetylcholinesterase (AChE) has been found to be associated with the core of senile plaques. We have shown that AChE interacts with the amyloid beta-peptide (Abeta) and promotes amyloid fibril formation by a hydrophobic environment close to the peripheral anionic binding site (PAS) of the enzyme. Here we present evidence for the structural motif of AChE involved in this interaction. First, we modeled the docking of Abeta onto the structure of Torpedo californica AChE, and identified four potential sites for AChE-Abeta complex formation. One of these, Site I, spans a major hydrophobic sequence exposed on the surface of AChE, which had been previously shown to interact with liposomes [Shin et al. (1996) Protein Sci. 5, 42-51]. Second, we examined several AChE-derived peptides and found that a synthetic 35-residue peptide corresponding to the above hydrophobic sequence was able to promote amyloid formation. We also studied the ability to promote amyloid formation of two synthetic 24-residue peptides derived from the sequence of a Omega-loop, which has been suggested as an AChE-Abeta interacting motif. Kinetic analyses indicate that only the 35-residue hydrophobic peptide mimics the effect of intact AChE on amyloid formation. Moreover, RP-HPLC analysis revealed that the 35-residue peptide was incorporated into the growing Abeta-fibrils. Finally, fluorescence binding studies showed that this peptide binds Abeta with a K(d) = 184 microM, independent of salt concentration, indicating that the interaction is primarily hydrophobic. Our results indicate that the homologous human AChE motif is capable of accelerating Abeta fibrillogenesis.
Asunto(s)
Acetilcolinesterasa/química , Péptidos beta-Amiloides/química , Placa Amiloide/química , Acetilcolinesterasa/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Química Encefálica , Bovinos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Placa Amiloide/ultraestructura , Conformación Proteica , Solubilidad , TorpedoRESUMEN
The structure of the glycan moiety of the glycosylphosphatidylinositol (GPI) membrane anchor from Torpedo californica electric organ acetylcholinesterase was solved using nuclear magnetic resonance (NMR), methylation analysis, and chemical and enzymic microsequencing. Two structures were found to be present: Glc alpha 1-2 Man alpha 1-2 Man alpha 1-6 Man alpha 1-4 GlcN alpha 1-6myo-inositol, and Glc alpha 1-2 Man alpha 1-2 Man alpha 1-6 (GalNAc beta 1-4) Man alpha 1-4 GlcN alpha 1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule.
Asunto(s)
Acetilcolinesterasa/biosíntesis , Órgano Eléctrico/química , Glicosilfosfatidilinositoles/química , Torpedo , Animales , Secuencia de Carbohidratos , Cromatografía de Gases y Espectrometría de Masas , Espectroscopía de Resonancia Magnética , Datos de Secuencia MolecularRESUMEN
The structure of the glycan moiety of the glycosylphosphatidylinositol (GPI) membrane anchor from Torpedo californica electric organ acetylcholinesterase was solved using nuclear magnetic resonance (NMR), methylation analysis, and chemical and enzymic microsequencing. Two structures were found to be present: Glcalfa1-2 Manalfa1-2 Manalfa1-6 Manalfa1-4 GlcNalfa1-6myo-inositol, and Glcalfa1-2 Manalfa1-2 Manalfa1-6 (GalNAcß1-4) Manalfa1-4 GlcNalfa1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule