title: An ultra-specific avian antibody to phosphorylated tau protein reveals a unique mechanism for phosphoepitope recognition.
abstract: Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.
authors.affiliation: Global Biotherapeutics Technologies, Pfizer Global Research & Development, Cambridge, Massachusetts 02140, USA.
authors.forename: Heather H
authors.initials: HH
authors.lastname: Shih
authors.forename: Chao
authors.initials: C
authors.lastname: Tu
authors.forename: Wei
authors.initials: W
authors.lastname: Cao
authors.forename: Anne
authors.initials: A
authors.lastname: Klein
authors.forename: Renee
authors.initials: R
authors.lastname: Ramsey
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Fennell
authors.forename: Matthew
authors.initials: M
authors.lastname: Lambert
authors.forename: Deirdre
authors.initials: D
authors.lastname: Ní Shúilleabháin
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Autin
authors.forename: Eugenia
authors.initials: E
authors.lastname: Kouranova
authors.forename: Sri
authors.initials: S
authors.lastname: Laxmanan
authors.forename: Steven
authors.initials: S
authors.lastname: Braithwaite
authors.forename: Leeying
authors.initials: L
authors.lastname: Wu
authors.forename: Mostafa
authors.initials: M
authors.lastname: Ait-Zahra
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Milici
authors.forename: Jo Ann
authors.initials: JA
authors.lastname: Dumin
authors.forename: Edward R
authors.initials: ER
authors.lastname: LaVallie
authors.forename: Maya
authors.initials: M
authors.lastname: Arai
authors.forename: Christopher
authors.initials: C
authors.lastname: Corcoran
authors.forename: Janet E
authors.initials: JE
authors.lastname: Paulsen
authors.forename: Davinder
authors.initials: D
authors.lastname: Gill
authors.forename: Orla
authors.initials: O
authors.lastname: Cunningham
authors.forename: Joel
authors.initials: J
authors.lastname: Bard
authors.forename: Lydia
authors.initials: L
authors.lastname: Mosyak
authors.forename: William J J
authors.initials: WJ
authors.lastname: Finlay
mesh_terms: D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001921:Brain / Q000378:metabolism; D002645:Chickens; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016875:tau Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000906:Antibodies; D000939:Epitopes; D007074:Immunoglobulin G; C054369:MAPT protein, human; D016875:tau Proteins