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.

An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

“…The last mL of the washing fraction was collected as a control, and the bound epitope peptides eluted by 4 × 15 min incubation with 0.1% trifluoroacetic acid. Fractions were lyophilized and purified using the ZipTip procedure [17]. For affinity testing of the synthetic epitope peptides, the samples were loaded onto the affinity columns in PBS and incubated for 2 h at 37°C.…”

“…Epitope peptides were immobilized on gold chips as previously described [17,18] and different concentrations of the HD6 antibody injected. K D determinations were performed by using OriginPro 7.5 software with the FitMaster plug-in, providing a pseudo first order kinetic constant (k obs ).…”

“…For epitope identification, an epitope-excision MS experiment was performed [17][18][19][20]. HLA-B27 2 was immobilized on the antibody column and digested with trypsin, and the affinity-eluted fractions subjected to MALDI-MS (Figure 2).…”

Exploring the ‘aggregation-prone’ core of human Cystatin C: A structural study

“…Specific interactions between Cystatin C and Ab (Sastre et al, 2004) and Cystatin C and SAA (Bokarewa et al, 2007) had been found experimentally and, thus, had introduced two novel Cystatin C partners. Consecutive structural experiments were performed using selective proteolytic excision and high resolution mass spectrometry (Juszczyk et al, 2009;Maftei et al, 2012;Tian et al, 2007) in order to identify the crucial binding 'hotspots' both in Ab and SAA. Surprisingly, the identified interaction site of Fb is located in the 101 IYAVPWQGTMTLSKSTC 117 hCC fragment (Juszczyk et al, 2009), whereas the identified epitope on SAA is located in 96 FCSFQIY 102 fragment (Spodzieja et al, 2013;Spodzieja et al, 2012).…”

Affinity-Mass Spectrometry Approaches for Elucidating Structures and Interactions of Protein–Ligand Complexes

“…The identifi cation and quantifi cation of Aß peptides bound to specifi c anti-Aß antibodies were provided by the newly introduced online bioaffi nity-electrospray mass spectrometry [ 5 ]. In several further studies, proteolytic excision/extraction-mass spectrometry were successfully used for the identification of interacting Aß epitope sequences with aggregation-inhibiting polypeptides [17- Reprinted with author permission from [ 14 ] such as cystatin C [ 16 ], humanin [ 24 ], and Aß-nanobodies (single chain llama anti-ß-amyloid antibodies) providing also a potential for developing new diagnostic tools for AD as well as for designing new potential immunotherapeutic agents [ 34 ].…”