Dimeric Transthyretin Variant Assembles into Spherical Neurotoxins

Matsubara, Kimiaki; Mizuguchi, Mineyuki; Igarashi, Kouhei; Shinohara, Yoshinori; Takeuchi, Makoto; Matsuura, Atsushi; Saitoh, Takayuki R.; Mori, Yoshihiro; Shinoda, Hiroyuki; Kawano, Keiichi

doi:10.1021/bi048838c

Cited by 25 publications

(23 citation statements)

References 37 publications

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“…2a). The difference in mass between the labeled and unlabeled protein was ϳ3 kDa, enabling us to resolve these species in spectra, with peaks corresponding to the 14ϩ to 16ϩ charge states of 12 C-14 N and 13 C-15 N intact homotetramers. In addition, peaks at high m/z values were assigned to a range of higher oligomers.…”

Section: Mass Spectrometry Of An Isotopically Labeled Transthyretinmentioning

confidence: 99%

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L55P Transthyretin Accelerates Subunit Exchange and Leads to Rapid Formation of Hybrid Tetramers

Keetch¹,

Bromley²,

McCammon

et al. 2005

Journal of Biological Chemistry

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Transthyretin is a tetrameric protein associated with the commonest form of systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry, we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, L55P. Wild-type subunit exchange occurs via both monomers and dimers, whereas exchange via dimers is the dominant mechanism for the L55P variant. Because patients with the L55P mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and L55P tetramers. We found that hybrid tetramers containing two or three L55P subunits dominate in the early stages of the reaction. Surprisingly, we also found that, in the presence of L55P transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidosis.

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Section: Mass Spectrometry Of An Isotopically Labeled Transthyretinmentioning

confidence: 99%

“…Support for this hypothesis comes from investigations of a dimeric variant that is prone to amyloid formation (11) and another that forms cytotoxic spherical aggregates (12). Spin labeling experiments have demonstrated that many of the interactions found in the native transthyretin dimer are maintained in fibrils (13).…”

mentioning

confidence: 99%

L55P Transthyretin Accelerates Subunit Exchange and Leads to Rapid Formation of Hybrid Tetramers

Keetch¹,

Bromley²,

McCammon

et al. 2005

Journal of Biological Chemistry

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“…Furthermore, to more accurately model the properties of human TTR, it is desirable to remove the polyhistidine tag prior to experimentation since it can alter protein structure and influence aggregation properties, particularly when divalent cations are present. Biophysical studies using 2-mercaptoethanol-bound and polyhistidine-tagged rTTR [11,18,19] may yield results that deviate significantly from identical studies conducted on native sequence rTTR containing a free thiol group. In addition, our results suggest that mass spectrometric analysis of any expression product is essential when using reductants to optimize the yield of recombinant systems.…”

Section: Discussionmentioning

confidence: 82%

Expression, purification, and in vitro cysteine-10 modification of native sequence recombinant human transthyretin

Kingsbury

Klimtchuk

Théberge

et al. 2007

Protein Expression and Purification

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Transthyretin (TTR) is a serum protein that is also a prominent component of deposits in two different types of systemic amyloid disease, senile systemic and familial TTR amyloidoses. Studies of recombinant TTR (rTTR) have provided many insights into the relationship between protein structure and amyloidogenicity. Yet, there is no existing recombinant system that results in high yield production of a protein that is identical in primary structure to human TTR. To date, most published studies have generated rTTR using the human gene sequence, which is poorly expressed in E. coli. In addition, the gene sequence has been flanked by a 3′ AUG start codon to initiate translation, resulting in the expression of a protein containing an N-terminal methionine residue not present in the human protein. We present an improved technique which can be used to generate large quantities of human native sequence TTR. Our recombinant system utilizes a gene containing codons altered for efficient expression in E. coli and an N-terminal polyhistidine tag for simplified purification. Optimization of this system was accomplished by generating a modified polyhistidine tag that was efficiently removed by dipeptidyl aminopeptidase I (DAPase). This is the first report detailing an effective and useful method for producing rTTR containing an amino acid sequence identical to human TTR. Furthermore, we describe the thiol modification of the recombinant protein to achieve exact replication of the several prominent post-translationally modified forms of TTR that have been identified in human serum.

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“…Tissue-deposited ATTR was extracted as previously described (11 ). Recombinant TTRs were expressed and purified as described previously (12 ). We analyzed samples using 3 different ProteinChip surfaces: a 7 Nonstandard abbreviations: FAP, familial amyloidotic polyneuropathy; ATTR, amyloidogenic transthyretin; TTR, transthyretin; MS, mass spectrometry; WT, wild-type; CSF, cerebrospinal fluid.…”

Section: Resultsmentioning

confidence: 99%

SELDI-TOF Mass Spectrometry Evaluation of Variant Transthyretins for Diagnosis and Pathogenesis of Familial Amyloidotic Polyneuropathy

Ueda¹,

Misumi

Mizuguchi

et al. 2009

Clinical Chemistry

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Dimeric Transthyretin Variant Assembles into Spherical Neurotoxins

Cited by 25 publications

References 37 publications

L55P Transthyretin Accelerates Subunit Exchange and Leads to Rapid Formation of Hybrid Tetramers

L55P Transthyretin Accelerates Subunit Exchange and Leads to Rapid Formation of Hybrid Tetramers

Expression, purification, and in vitro cysteine-10 modification of native sequence recombinant human transthyretin

SELDI-TOF Mass Spectrometry Evaluation of Variant Transthyretins for Diagnosis and Pathogenesis of Familial Amyloidotic Polyneuropathy

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