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

Ayaho Dan,Takao Arai,Shigeo Murayama,Haruhiko Akiyama,Hiroyuki Hatsuta,Takashi Nonaka,Hiromi Kondo,Masami Masuda-Suzukake,David Ma Mann,Muneaki Takahashi,Masato Hasegawa,Fuyuki Kametani,Yuko Saito

doi:10.1186/2051-5960-1-54

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.

Highlights

Intracytoplasmic 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
We show that the irreversible post-translational deamidation takes place at N279 (N279D) 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
Low immunoreactivities of tau in AD and tau deamidated at N279 to RD4 When Sarkosyl-insoluble fractions of tau from AD, PSP and CBD brains were analyzed by immunoblotting with T46 and RD4, we noticed a lower immunoreactivity of

Summary

Introduction

Intracytoplasmic 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. 6 tau isoforms are expressed as a result of mRNA splicing. Isoform-specific tau antibodies are useful tools for immunohistochemical and biochemical studies of tau species in diseased brains. RD3 and RD4 [4], which are specific antibodies to 3R and 4R tau isoforms, respectively, have been widely used to investigate tau pathologies [5,6,7]. One of the present authors (M.H.) had found that the asparagine residue at position 279 (N279), located in the RD4 epitope, was detected mostly as aspartic acid owing to deamidation of asparagine when PHF-tau in AD brains was subjected to protein sequencing and LC/MS/

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Results

Conclusion