title: Interaction structure of the complex between neuroprotective factor humanin and Alzheimer's β-amyloid peptide revealed by affinity mass spectrometry and molecular modeling.
abstract: Humanin (HN) is a linear 24-aa peptide recently detected in human Alzheimer's disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß-amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1-40) and characterization of the interaction structure through a molecular modeling study. Wild-type HN and HN-sequence mutations were synthesized by SPPS and the HPLC-purified peptides characterized by MALDI-MS. The interaction epitopes between HN and Aß(1-40) were identified by affinity-MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity-bound peptides. The affinity-MS analyses revealed HN(5-15) as the epitope sequence of HN, whereas Aß(17-28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1-40) and by ELISA with Aß(1-40) and Aß-partial sequences as ligands to immobilized HN. The specificity and affinity of the HN-Aß interaction were characterized by direct ESI-MS of the HN-Aß(1-40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a K(D) of the complex of 610 nm. A molecular dynamics simulation of the HN-Aß(1-40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1-40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN.
authors.affiliation: Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz, 78457, Konstanz, Germany.
authors.forename: Madalina
authors.initials: M
authors.lastname: Maftei
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Tian
authors.forename: Marilena
authors.initials: M
authors.lastname: Manea
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Exner
authors.forename: Daniel
authors.initials: D
authors.lastname: Schwanzar
authors.forename: Christine A F
authors.initials: CA
authors.lastname: von Arnim
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D047908:Intracellular Signaling Peptides and Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D008958:Models, Molecular; D018696:Neuroprotective Agents / Q000737:chemistry*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D016229:Amyloid beta-Peptides; D047908:Intracellular Signaling Peptides and Proteins; D018696:Neuroprotective Agents; C427211:humanin