Development of human antibody fragments directed towards synaptic acetylcholinesterase using a semi-synthetic phage disp
Current Alzheimer’s disease therapies suppress acetylcholine hydrolysis by inhibiting acetylcholinesterase (AChE) at cholinergic synapses. However, anticholinesterases promote alternative splicing changing the composition of brain AChE variants. To study
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c. Flores-Flores!, A. Nissim--', S. Shochat', and H. Soreq' D epartment of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel 2Bone and Joint Research Unit, St. Bartholomew's and Royal London School of Medicine and Dentistry, Queen Mary, London , United Kingdom 3Felsenstein Medical Re search Center, Sackler Faculty of Medicine, Tel A viv University, Rabin Medical Center, Belinson Campus, Petach Tikva, Israel 1
Summary. Current Alzheimer's disease therapies suppress acetylcholine hydrolysis by inhibiting acetylcholinesterase (AChE) at cholinergic synapses. However, anticholinesterases promote alternative splicing changing the composition of brain AChE variants. To study this phenomenon we developed monoclonal antibodies to acetylcholinesterase synaptic peptide (ASP), a synthetic peptide with the C-terminal sequence unique to the human synaptic variant AChE-S. Screening of a phage display human antibody library allowed the isolation of single-chain Fv (scFv) antibodies that were highly specific for ASP, and displayed closely related third complementarity determining regions of the variable heavy chain domain (V H-CDR3). BIAcore analysis demonstrated dissociation constants at the micromolar range: 1.6 X 10- 6 and 2.0 x 10- 6 M for ASP and the complete AChE-S protein, respectively . The anti-ASP antibodies provide a novel tool for studying th e synaptic AChE-S variant, the expression of which is altered in ageing and dementia.
Abbreviations
AChE acetylcholinesterase; AChE-E erythrocytic variant; AChE-R readthrough variant; A ChE-S synaptic variant; ARP acetylcholinesterase readthrough peptide; ASP acetylcholinesterase synaptic peptide; ASP C-ter acetylcholinesterase synaptic peptide C-terminal 23 amino acid residues; antiASP anti-acetylcholinesterase synaptic peptide (antibody); BSA bovine serum albumin; ELISA enzyme linked immunoadsorbent assay; PBS phosphate buffered saline; PCR polymerase chain reaction; RD, resonance unit; scFv single-chain fragment variable; SPR surface plasmon resonance; t.u. transforming unit; V H -CD R3 variable heavy chain third complementarity determining region.
K. A. Jellinger et al. (eds.), Ageing and Dementia Current and Future Concepts © Springer-Verlag/Wein 2002
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Introduction
Acetylcholinesterase (AChE) hydrolyzes the neurotransmitter acetylcholine at cholinergic synapses, but also exercises morphogenic activities (Soreq and Seidman, 2001). Three C-terminal variants are generated by alternative splicing of the single ACHE gene transcript. "Synaptic" AChE-S constitutes the principal variant protein in brain and muscle, and plays an important role in growth-regulating processes affecting neurons, independently of its catalytic activity (Sternfeld et al., 1998). "Readthrough" AChE-R is a secretory, nonsynaptic form of the enzyme that is expressed in embryonic and tumor cells (Karpel et al., 1994), and is induced under psychological and chemical stressors, including therapeutic anticholinesterases (Friedman et al., 1996; Kaufer et
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