Modulation of Alzheimer's beta-amyloid neurotoxicity by site-directed single-chain antibody.
A single-chain antibody was constructed from variable regions of heavy and light genes of the parental anti-beta-amyloid peptide IgM 508 antibody. This antibody exhibits anti-aggregating properties, leading to disaggregation of Alzheimer beta-amyloid (betaA) fibrils and prevents its toxic effect on cultured PC-12 cells. Sequencing of the small antibody, namely 508 (Fv), revealed that the V(L) domain contained a cysteine residue in the complementary determining region (CDR)3 (residue 96) which affects its solubility and stability. The cysteine codon was replaced using SOE PCR, and one of the mutants obtained, namely 508F(Fv) (containing phenylalanine instead of cysteine), showed an increased storage stability and higher affinity compared to the wild type. Antibody 508F(Fv) prevents the neurotoxicity of betaA (90% cell viability) and disrupts the fibril structure of beta-amyloid (62% decrease in ThT fluorescence). The ability of antibody 508F(Fv) to dissolve already-formed betaA fibrils makes it a good candidate for intracellular expression and modulation of APP processing as the first step towards the production of therapeutic protection molecules for Alzheimer's disease treatment.
Frenkel
D
D
Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Tel-Aviv, Israel.
Solomon
B
B
Benhar
I
I
D000544:Alzheimer Disease / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000494:pharmacology*; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D003001:Cloning, Molecular; D006801:Humans; D006825:Hybridomas; D007128:Immunoglobulin Fragments / Q000235:genetics; D007075:Immunoglobulin M / Q000235:genetics / Q000494:pharmacology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D016716:PC12 Cells; D010446:Peptide Fragments / Q000494:pharmacology; D051381:Rats
D016229:Amyloid beta-Peptides; D000906:Antibodies; D007128:Immunoglobulin Fragments; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D009498:Neurotoxins; D010446:Peptide Fragments; C055966:immunoglobulin Fv