Extensive deamidation at asparagine residue 279 accounts for weak immunoreactivity of tau with RD4 antibody in Alzheimer’s disease brain

Abstract

BackgroundIntracytoplasmic inclusions composed of filamentous tau proteins are defining characteristics of neurodegenerative tauopathies, but it remains unclear why different tau isoforms accumulate in different diseases and how they induce abnormal filamentous structures and pathologies. Two tau isoform-specific antibodies, RD3 and RD4, are widely used for immunohistochemical and biochemical studies of tau species in diseased brains.ResultsHere, we show that extensive irreversible post-translational deamidation takes place at asparagine residue 279 (N279) in the RD4 epitope of tau in Alzheimer’s disease (AD), but not corticobasal degeneration (CBD) or progressive supranuclear palsy (PSP), and this modification abrogates the immunoreactivity to RD4. An antiserum raised against deamidated RD4 peptide specifically recognized 4R tau isoforms, regardless of deamidation, and strongly stained tau in AD brain. We also found that mutant tau with N279D substitution showed reduced ability to bind to microtubules and to promote microtubule assembly.ConclusionThe biochemical and structural differences of tau in AD from that in 4R tauopathies found in this study may therefore have implications for prion-like propagation of tau.

Deep clinical and neuropathological phenotyping of Pick disease

“…This technique allows for increased visualization of neuropathology and enhances study of neuroanatomic structures. 13, 15 Standard 6 μm sections were stained with Thioflavin-S (ThS) to detect tau inclusions with amyloid-dye binding properties, and IHC using primary MAbs specific for phospho-tau (12E8; Elan Pharmaceuticals, San Francisco, CA) 16 , ubiquitin (UBQ) (1510; Millipore Billerica, MA) 17 , 4R tau (RD4; Millipore 18 ; Anti-4R Tau; (Cosmo Bio, Carlsbad, CA) 19 , and C-terminally truncated tau (TauC3; Dr. LI Binder) 20 using methods as described 9, 21, 22 . Finally, double-label immunofluorescence experiments were performed using AT-8, RD4 or 3R tau (RD3; Millipore) 18 with MAbs specific for glial fibrillary associated protein (GFAP; Dako Carpinteria, CA) 23 or microtubule-associated protein 2 (MAP2) (17028; CNDR) 24 and Alexa Fluor 488 and 594 species-specific secondary antibodies (Molecular Probes) as described 21, 22 .…”

Cryo-EM structures of tau filaments from Alzheimer’s disease

“…In addition, to confirm that R2 is not part of the pronase-resistant core, we used Anti-4R, an antibody raised against V 275 -C 291 of R2 (with D 279 ) 26 . In agreement with a pronase-resistant R3:R4 core, Anti-4R detected both PHFs and SFs before, but not after, pronase treatment.…”

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

“…These studies have reported loss of stability, activity of proteins, and also increased aggregation properties as shown in crystallins, 7,8,11,12,23,24,[51][52][53] superoxide dismutase, 49 and immunoglobins. 14,34,54 Reported properties of mutC suggest that this protein has retained the secondary and tertiary structure with a partial loss in activity but with a significant improvement in the ability to withstand multiple heat cycles.…”

“…5,6 Deamidation results in an addition of negative charge to the residue resulting in alterations in protein interactions, which could affect the activity or refolding. 7,8 Interest in deamidation has gained importance since the extent of deamidation was associated with aging, 9,10 cataract formation, 9,11,12 immune recognition, 13 shelf-life of protein pharmaceuticals such as antibodies, 9,13,14 and so on. Deamidation of asparagines is initiated by the cyclization reaction, which occurs between the carboxyl group of the asparagine and amide nitrogen of carboxyl side amino acid (n11) followed by deamination resulting in the formation of succinimide intermediate.…”