- Castillo-Carranza D.L.
- Sengupta U.
- Guerrero-Muñoz M.J.
- Lasagna-Reeves C.A.
- Gerson J.E.
- Singh G.
- Estes D.M.
- Barrett A.D.
- Dineley K.T.
- Jackson G.R.
- Kayed R.
Results
Oligomer formation

- Kayed R.
- Head E.
- Sarsoza F.
- Saing T.
- Cotman C.W.
- Necula M.
- Margol L.
- Wu J.
- Breydo L.
- Thompson J.L.
- Rasool S.
- Gurlo T.
- Butler P.
- Glabe C.G.
- Kayed R.
- Head E.
- Sarsoza F.
- Saing T.
- Cotman C.W.
- Necula M.
- Margol L.
- Wu J.
- Breydo L.
- Thompson J.L.
- Rasool S.
- Gurlo T.
- Butler P.
- Glabe C.G.
M204 antibody binding to tau oligomers


- Kayed R.
- Head E.
- Sarsoza F.
- Saing T.
- Cotman C.W.
- Necula M.
- Margol L.
- Wu J.
- Breydo L.
- Thompson J.L.
- Rasool S.
- Gurlo T.
- Butler P.
- Glabe C.G.
Design of the single-chain M204 antibody


M204-scFv inhibits aggregation recombinant tau


X-ray crystal structure of the M204-scFv monomer, dimer, and trimer
Monomer_M204 | Dimer_M204 | Trimer_M204 | |
---|---|---|---|
Wavelength (Å) | 0.7749 | 0.9792 | 0.9791 |
Resolution range | 39.9-2.20 (2.26-2.20) | 90.41-2.91 (2.99-2.91) | 65.8-3.6 (3.69-3.60) |
Space group | P 41 2 2 | P 21 21 21 | P 43 2 2 |
Unit cell | 60.4 60.4 159.4 | 60.6 105.3 176.5 | 60.2 60.2 526.4 |
90 90 90 | 90 90 90 | 90 90 90 | |
Total reflections | 55,416 (4091) | 93,397 (5737) | 24,601 (1765) |
Unique reflections | 15,422 (1120) | 25,041 (1651) | 10,789 (795) |
Multiplicity | 3.6 (3.7) | 3.7 (3.5) | 2.3 (2.2) |
Completeness (%) | 97.9 (99.0) | 98.0 (92.11) | 87.4 (91.1) |
Mean I/σ (I) | 11.1 (1.9) | 6.7 (1.2) | 3.7 (0.9) |
Wilson B-factor | 41.2 | 56.9 | 72.0 |
Rmerge | 0.072 (0.644) | 0.171 (0.975) | 0.240 (0.923) |
Rmeas | 0.084 (0.752) | 0.198 (1.114) | 0.276 (1.021) |
Rpim | 0.050 (0.435) | 0.123 (0.722) | 0.191 (0.712) |
CC1/2 | 99.9 (73.6) | 98.8 (48.1) | 95.2 (44.8) |
Reflections used in refinement | 15,417 (1,503) | 25,026 (2,310) | 9,710 (715) |
Reflections used for Rfree | 1,542 (151) | 2,504 (231) | 1,079 (79) |
Rwork | 0.194 (0.267) | 0.186 (0.331) | 0.252 (0.381) |
Rfree | 0.235 (0.359) | 0.238 (0.338) | 0.283 (0.365) |
Number of nonhydrogen atoms | |||
Macromolecules | 1,707 | 6,790 | 5,103 |
Ligands | 3 | 20 | 0 |
Solvent | 104 | 32 | 0 |
Protein residues | 226 | 899 | 675 |
RMS (bonds) (Å) | 0.010 | 0.007 | 0.008 |
RMS (angles) (°) | 1.1 | 1.2 | 1.50 |
Ramachandran favored (%) | 94.6 | 95.5 | 95.2 |
Ramachandran allowed (%) | 5.0 | 4.0 | 4.7 |
Ramachandran outliers (%) | 0.4 | 0.6 | 0.2 |
Rotamer outliers (%) | 2.7 | 6.6 | 0.15 |
Clashscore | 1.78 | 2.84 | 3.38 |
Average B-factor (Å2) | |||
Macromolecules | 42.9 | 56.8 | 94.3 |
Ligands | 51.0 | 101. | NA |
Solvent | 44.5 | 39.6 | NA |

Discussion
- Kayed R.
- Head E.
- Sarsoza F.
- Saing T.
- Cotman C.W.
- Necula M.
- Margol L.
- Wu J.
- Breydo L.
- Thompson J.L.
- Rasool S.
- Gurlo T.
- Butler P.
- Glabe C.G.
Experimental procedures
Monoclonal M204 expression and purification
M204 antibody engineering, expression, and purification
Tau protein expression
Preparation and purification of tau oligomers
ThT fluorescence assay
ELISA
Immunoblotting
Crystallization
X-ray data collection and structure solution
Tau biosensor cell maintenance and seeding
Preparation of crude and Sarkosyl-insoluble brain-derived tau seeds
Dot blot
- Krotee P.
- Griner S.L.
- Sawaya M.R.
- Cascio D.
- Rodriguez J.A.
- Shi D.
- Philipp S.
- Murray K.
- Saelices L.
- Lee J.
- Seidler P.
- Glabe C.G.
- Jiang L.
- Gonen T.
- Eisenberg D.S.
Epitope mapping
Statistical analysis
Data availability
Acknowledgments
Supplementary Material
References
- Worldwide trends in the prevalence of dementia.J. Neurol. Sci. 2017; 379 (28716255): 259-260
- Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-associated protein tau.Proc. Natl. Acad. Sci. U.S.A. 1988; 85 (3131773): 4051-4055
- Tau and Aβ imaging, CSF measures, and cognition in Alzheimer's disease.Sci. Transl. Med. 2016; 8: 338-366
- Short fibrils constitute the major species of seed-competent Tau in the brains of mice transgenic for human P301S Tau.J. Neurosci. 2016; 36: 762-772
- Invited review: Frontotemporal dementia caused by microtubule-associated protein tau gene (MAPT) mutations: a chameleon for neuropathology and neuroimaging.Neuropathol. Appl. Neurobiol. 2015; 41 (25556536): 24-46
- Tau in neurodegenerative disease.Ann. Transl. Med. 2018; 6 (29951497): 175
- Prion-like transmission of protein aggregates in neurodegenerative diseases.Nat. Rev. Mol. Cell Biol. 2010; 11 (20308987): 301-307
- The role of tau oligomers in the onset of Alzheimer's disease neuropathology.ACS Chem. Neurosci. 2014; 5 (25268947): 1178-1191
- The widespread alteration of neurites in Alzheimer's disease may be unrelated to amyloid deposition.Ann. Neurol. 1989; 26 (2557796): 771-778
- The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.Science. 2002; 297 (12130773): 353-356
- Soluble protein oligomers as emerging toxins in Alzheimer's and other amyloid diseases.IUBMB Life. 2007; 59 (17505973): 332-345
- Tau oligomers impair memory and induce synaptic and mitochondrial dysfunction in wild-type mice.Mol. Neurodegener. 2011; 6 (21645391): 39
- Alzheimer brain-derived tau oligomers propagate pathology from endogenous tau.Sci. Rep. 2012; 2 (23050084): 700
- Characterization of prefibrillar Tau oligomers in vitro and in Alzheimer disease.J. Biol. Chem. 2011; 286 (21550980): 23063-23076
- Identification of oligomers at early stages of tau aggregation in Alzheimer's disease.FASEB J. 2012; 26 (22253473): 1946-1959
- Extracellular Tau oligomers produce an immediate impairment of LTP and memory.Sci. Rep. 2016; 6 (26786552): 19393
- Internalized Tau oligomers cause neurodegeneration by inducing accumulation of pathogenic Tau in human neurons derived from induced pluripotent stem cells.J. Neurosci. 2015; 35 (26490863): 14234-14250
- Production of recombinant tau oligomers in vitro.Methods Cell Biol. 2017; 141 (28882311): 45-64
- Structure-based inhibitors of tau aggregation.Nat. Chem. 2018; 10 (29359764): 170-176
- Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation.Nature. 2011; 475 (21677644): 96-100
- Macrocyclic β-sheet peptides that inhibit the aggregation of a tau-protein-derived hexapeptide.J. Am. Chem. Soc. 2011; 133 (21319744): 3144-3157
- Out-of-register β-sheets suggest a pathway to toxic amyloid aggregates.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (23213214): 20913-20918
- Atomic structures of amyloid cross-beta spines reveal varied steric zippers.Nature. 2007; 447 (17468747): 453-457
- Passive immunization with Tau oligomer monoclonal antibody reverses tauopathy phenotypes without affecting hyperphosphorylated neurofibrillary tangles.J. Neurosci. 2014; 34 (24647946): 4260-4272
- Conformation dependent monoclonal antibodies distinguish different replicating strains or conformers of prefibrillar Aβ oligomers.Mol. Neurodegener. 2010; 5 (21144050): 57
- Suppression and dissolution of amyloid aggregates using ionic liquids.Biophys. Rev. 2018; 10 (29696571): 853-860
- Ionic liquid-induced formation of the α-helical structure of β-lactoglobulin.J. Phys. Chem. B. 2013; 117 (23926920): 10142-10148
- Structure and function of proteins in hydrated choline dihydrogen phosphate ionic liquid.Phys. Chem. Chem. Phys. 2012; 14 (22089924): 790-801
- Toward a unified scheme for the aggregation of tau into Alzheimer paired helical filaments.Biochemistry. 2002; 41 (12475237): 14885-14896
- Preparation of stable tau oligomers for cellular and biochemical studies.Anal. Biochem. 2019; 566 (30315761): 67-74
- Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers.Mol. Neurodegener. 2007; 2 (17897471): 18
- Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis.Science. 2003; 300 (12702875): 486-489
- Passive immunization in JNPL3 transgenic mice using an array of phospho-Tau specific antibodies.PLoS ONE. 2015; 10 (26270821): e0135774
- Tau prion strains dictate patterns of cell pathology, progression rate, and regional vulnerability in vivo.Neuron. 2016; 92 (27974162): 796-812
- Development trends for therapeutic antibody fragments.Nat. Biotechnol. 2009; 27 (19352366): 331-337
- Single-chain antigen-binding proteins.Science. 1988; 242 (3140379): 423-426
- Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains.J. Mol. Biol. 2008; 377 (18262543): 478-488
- Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp.Front. Cell Infect. Microbiol. 2013; 3: 72
- Factors influencing the dimer to monomer transition of an antibody single-chain Fv fragment.Biochemistry. 1998; 37 (9737871): 12918-12926
- Human anti-self antibodies with high specificity from phage display libraries.EMBO J. 1993; 12 (7679990): 725-734
- Neuropathological alterations in Alzheimer disease.Cold Spring Harb. Perspect. Med. 2011; 1 (22229116): a006189
- Diversity in the CDR3 region of V(H) is sufficient for most antibody specificities.Immunity. 2000; 13 (10933393): 37-45
- Crystal structure of a conformation-dependent rabbit IgG Fab specific for amyloid prefibrillar oligomers.Biochim. Biophys. Acta. 2012; 1820 (22940003): 1908-1914
- Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies.Acta Crystallogr. D Biol. Crystallogr. 2013; 69 (24311579): 2381-2394
- Specific chemical and structural damage to proteins produced by synchrotron radiation.Proc. Natl. Acad. Sci. U.S.A. 2000; 97 (10639129): 623-628
- Influence of molecular size on tissue distribution of antibody fragments.MAbs. 2016; 8 (26496429): 113-119
- Binding efficiency of protein-protein complexes.Biochemistry. 2012; 51 (23088250): 9124-9136
- Propagation of Tau aggregates and neurodegeneration.Annu. Rev. Neurosci. 2017; 40 (28772101): 189-210
- Structures of filaments from Pick's disease reveal a novel tau protein fold.Nature. 2018; 561 (30158706): 137-140
- Novel tau filament fold in chronic traumatic encephalopathy encloses hydrophobic molecules.Nature. 2019; 568 (30894745): 420-423
- Monoclonal antibodies against Aβ42 fibrils distinguish multiple aggregation state polymorphisms in vitro and in Alzheimer disease brain.J. Biol. Chem. 2014; 289 (25281743): 32131-32143
- Conformational Abs recognizing a generic amyloid fibril epitope.Proc. Natl. Acad. Sci. U.S.A. 2002; 99 (11818542): 1485-1490
- Dementia from Alzheimer disease and mixed pathologies in the oldest old.JAMA. 2012; 307 (22550192): 1798-1800
- Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat. Methods. 2009; 6 (19363495): 343-345
- Probing the N-terminal β-sheet conversion in the crystal structure of the human prion protein bound to a nanobody.J. Am. Chem. Soc. 2014; 136 (24400836): 937-944
- The Alzheimer-like phosphorylation of tau protein reduces microtubule binding and involves Ser-Pro and Thr-Pro motifs.FEBS Lett. 1992; 307 (1644173): 199-205
- Xds.Acta Crystallogr. D Biol. Crystallogr. 2010; 66 (20124692): 125-132
- Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40 (19461840): 658-674
- PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66 (20124702): 213-221
- Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66 (20383002): 486-501
- NIH Image to ImageJ: 25 years of image analysis.Nat. Methods. 2012; 9 (22930834): 671-675
- Tau seeding activity begins in the transentorhinal/entorhinal regions and anticipates phospho-tau pathology in Alzheimer's disease and PART.Acta Neuropathol. 2018; 136 (29752551): 57-67
- Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity.Elife. 2017; 6: e19273
- Common fibrillar spines of amyloid-β and human islet amyloid polypeptide revealed by microelectron diffraction and structure-based inhibitors.J. Biol. Chem. 2018; 293 (29282295): 2888-2902
Article info
Publication history
Footnotes
This article contains supporting information.
Author contributions—R. A., M. R. S., and D. S. E. conceptualization; R. A., P. M. S., M. R. S., D. C., T. P. Y., P. L. F., R. N., and C. G. G. data curation; R. A., P. M. S., M. R. S., and D. S. E. formal analysis; R. A., M. R. S., and D. S. E. supervision; R. A., P. M. S., M. R. S., D. C., S. P., C. K. W., K. L. N., B. G., M. A. D., D. W. D., H. V. V., P. L. F., R. N., C. G. G., and D. S. E. validation; R. A., P. M. S., M. R. S., and D. S. E. investigation; R. A., P. M. S., R. N., and D. S. E. methodology; R. A. writing-original draft; R. A., P. M. S., and D. S. E. writing-review and editing; S. P., C. K. W., K. L. N., B. G., M. A. D., D. W. D., H. V. V., P. L. F., and C. G. G. resources; D. S. E. funding acquisition; D. S. E. visualization.
Funding and additional information—This work was supported by Grants 1R01 AG029430 (to D. S. E.), RF1 AG054022 (to D. S. E.), RF1AG056507 (to C. G. G.), and PHS P30-AG010133 (to B. G.) from the NIA National Institutes of Health, Grant 1F32 NS095661 from the NINDS, National Institutes of Health (to P. M. S.), Grant A2016588F from the BrightFocus Foundation (to P. M. S.), and the Howard Hughes Medical Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Conflict of interest—D. S. E. is SAB chair and an equity holder of ADRx, Inc.
Abbreviations—The abbreviations used are: AD
Identification
Copyright
User license
Creative Commons Attribution (CC BY 4.0) |
Permitted
- Read, print & download
- Redistribute or republish the final article
- Text & data mine
- Translate the article
- Reuse portions or extracts from the article in other works
- Sell or re-use for commercial purposes
Elsevier's open access license policy