Post-translational Modification of Transthyretin in Plasma

doi:10.1006/bbrc.1998.9097

Biochemical and Biophysical Research Communications

Volume 249, Issue 1, 10 August 1998, Pages 26-30

https://doi.org/10.1006/bbrc.1998.9097 Get rights and content

Abstract

To determine the behavior of transthyretin (TTR) in blood circulation, TTR purified from normal subjects’ plasma was injected to rats, and blood and urine were collected time dependently. Although TTR in plasma was proven to be a predominantly cysteine (Cys) conjugated form by electrospray ionization mass spectrometry (ESI-MS) analysis, it was gradually converted into free, 32 Da (dihydroxylation), 80 Da (phosphorylation), and 306 Da (glutathionylation), increased forms in molecular weight of TTR. The plasma levels of TTR were decreased in a time-dependent manner with the half life of 72.4 min. No secretion of TTR into the urine was observed by ESI-MS. In conclusion, this method can be simply performed without loading a radioactive molecule to the targeted protein. It offers a possibility to determine natural protein behaviors in the blood stream.

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Cited by (35)

A chemometric approach for characterization of serum transthyretin in familial amyloidotic polyneuropathy type I (FAP-I) by electrospray ionization-ion mobility mass spectrometry
2018, Talanta
Citation Excerpt :
TTR is a homotetrameric protein composed of four non-covalently associated subunits. Each monomer consists of 127 amino acid residues (approximately 14 kDa) with different post-translational modifications (PTMs) [20–24]. TTR, which is highly abundant in serum (200–400 µg TTR mL−1) and cerebrospinal fluid (CSF) (10–40 µg TTR mL−1), is predominantly expressed in the liver and in the choroid plexus of the brain, and transports thyroxine and retinol [25].
In this study, we describe a chemometric data analysis approach to assist in the interpretation of the complex datasets from the analysis of high-molecular mass oligomeric proteins by ion mobility mass spectrometry (IM-MS). The homotetrameric protein transthyretin (TTR) is involved in familial amyloidotic polyneuropathy type I (FAP-I). FAP-I is associated with a specific TTR mutant variant (TTR(Met30)) that can be easily detected analyzing the monomeric forms of the mutant protein. However, the mechanism of protein misfolding and aggregation onset, which could be triggered by structural changes in the native tetrameric protein, remains under investigation. Serum TTR from healthy controls and FAP-I patients was purified under non-denaturing conditions by conventional immunoprecipitation in solution and analyzed by IM-MS. IM-MS allowed separation and characterization of several tetrameric, trimeric and dimeric TTR gas ions due to their differential drift time. After an appropriate data pre-processing, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the complex datasets. A group of seven independent components being characterized by their ion mobility profiles and mass spectra were resolved to explain the observed data variance in control and patient samples. Then, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were considered for exploration and classification. Only four out of the seven resolved components were enough for an accurate differentiation. Furthermore, the specific TTR ions identified in the mass spectra of these components and the resolved ion mobility profiles provided a straightforward insight into the most relevant oligomeric TTR proteoforms for the disease.
On-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using Fab´antibody fragments for the analysis of serum transthyretin
2017, Talanta
Citation Excerpt :
In this paper, an IA-SPE-CE-MS method using a lab-made IA sorbent with Fab′ antibody fragments is described for the analysis of serum TTR. TTR is a homotetrameric protein composed of four identical monomers (MO) (Mr~14,000) with different proteoforms (isoforms and post-translational modifications (PTMs)) [27–29]. TTR is known to misfold and aggregate as stable insoluble amyloid fibrils, which are characteristic of different neurodegenerative diseases known as amyloidosis [30,31].
This paper describes an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using an immunoaffinity sorbent with Fab′ antibody fragments (Fab′-IA) for the analysis of serum transthyretin (TTR), a homotetrameric protein (M_r~56,000) involved in different types of amyloidosis. The IA sorbent was prepared by covalent attachment of Fab′ fragments obtained from a polyclonal IgG antibody against TTR to succinimidyl silica particles. The Fab′-IA-SPE-CE-MS methodology was first established analyzing TTR standard solutions. Under optimized conditions, repeatability and reproducibility were acceptable, the method was linear between 1 and 25 µg mL⁻¹, limits of detection (LODs) were around 0.5 µg mL⁻¹ (50-fold lower than by CE-MS, ~25 µg mL⁻¹) and different TTR conformations were observed (folded and unfolded). The applicability of the developed method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was evaluated analyzing serum samples from healthy controls and FAP-I patients. For the analysis of sera, the most abundant proteins were precipitated with 5% (v/v) of phenol before Fab′-IA-SPE-CE-MS. The current method enhanced our previous results for the analysis of TTR using intact antibodies immobilized on magnetic beads. It allowed a slight improvement on LODs (2-fold), the detection of proteoforms found at lower concentrations and the preparation of microcartridges with extended durability.
Comparison of capillary electrophoresis and capillary liquid chromatography coupled to mass spectrometry for the analysis of transthyretin in human serum
2016, Journal of Chromatography A
Citation Excerpt :
In the present study, CE-MS and CapLC–MS were compared for the analysis of serum transthyrethin (TTR). TTR is a homotetramer composed of four identical monomers (MO) (relative molecular mass (Mr) ∼14,000) with different proteoforms (isoforms and PTMs) [25–27]. TTR is known to misfold and aggregate as stable insoluble fibrils due to mutations and conformational changes, causing different neurodegenerative diseases known as amyloidosis [28,29].
Capillary electrophoresis and capillary liquid chromatography coupled to mass spectrometry (CE-MS and CapLC–MS, respectively) are nowadays very suitable techniques for the separation and characterization of intact proteins in biological fluids. In this paper, we compare the performance of both techniques for the analysis of transthyretin (TTR), which is a homotetrameric protein (relative molecular mass (M_r) ∼56,000) involved in different types of amyloidosis. Furthermore, it is also presented a novel sample pretreatment based on immunoprecipitation (IP) using Protein A Ultrarapid Agarose™ (UAPA) magnetic beads (MBs) to purify TTR from serum samples. This novel IP based on MBs allowed the detection of TTR monomeric proteoforms that were not possible to analyze by conventional IP in solution. In addition, UAPA MBs provided many other desirable advantages including higher selectivity and minimal unspecific binding of other proteins. CE-MS and CapLC–MS were applied to analyze serum samples from healthy controls and familial amyloidotic polyneuropathy type I (FAP-I) patients, who suffered from the most common hereditary systemic amyloidosis. Both techniques allowed detecting the same TTR proteoforms, including the mutant TTR (Met 30) variant (variation in relative molecular mass (ΔM_r) was +32.07, from wild-type TTR). Migration/retention times and relative quantitation of the different proteoforms were similar and reproducible in both cases, but the limits of detection (LODs) achieved by CE-MS were slightly lower (2–2.5-fold). Some other differences were also found on separation selectivity (migration orders and separation of antibody), peak efficiency, total analysis time, calibration ranges and experimental M_r accuracy.
Characterisation of serum transthyretin by electrospray ionisation-ion mobility mass spectrometry: Application to familial amyloidotic polyneuropathy type I (FAP-I)
2015, Talanta
Citation Excerpt :
Its structure is a homotetramer composed of four non-covalently associated subunits [7–10]. Each monomer consists of 127 amino acid residues (approximately 14 kDa) with different post-translational modifications (PTMs) [10–13]. While SSA is a non-hereditary cardiomyopathy affecting 25% of the male population over the age of 80, in which deposits are formed from wild-type TTR, FAP is associated with around 73 of the about 100 point mutations known in the TTR-gene [5,6].
Transthyretin (TTR) is a homotetrameric protein which is known to misfold and aggregate causing different types of amyloidosis, such as familial amyloidotic polyneuropathy type I (FAP-I). FAP-I is associated with a specific TTR mutant variant (TTR (Met30)) that can be easily detected analysing the monomeric forms of the mutant protein. Meanwhile, the mechanism of protein aggregation onset, which could be triggered by structural changes on the native tetrameric protein complex, remains uncertain. We developed and described herein a new sample pretreatment based on immunoprecipitation (IP) to purify TTR from serum under non-denaturing conditions. Later, a nano-electrospray ionization-ion mobility mass spectrometry (nano-ESI-IM-MS or IM-MS) method was optimised to analyse the protein complexes in serum samples from healthy controls and FAP-I patients. IM-MS allowed separation and characterisation of tetrameric, trimeric and dimeric TTR gas ions due to their differential drift time, which is related to ion size and charge. The tetramer-to-dimer abundance ratio was differential between healthy controls and FAP-I patients (asymptomatic, symptomatic and an iatrogenic patient originally without the mutation who received a liver transplant from an FAP-I patient), and was also indicative of the effectiveness of liver transplantation as a treatment for FAP-I.
Quantitative analysis of post-translational modifications in human serum transthyretin associated with familial amyloidotic polyneuropathy by targeted LC-MS and intact protein MS
2015, Journal of Proteomics
Citation Excerpt :
However, lower TTR levels have been described for TTR-amyloidotic patients [16,17]. It occurs as a very heterogeneous protein where variability is not only due to point mutations in the encoding gene but also to post-translational modifications (PTMs) at Cys-10, the single Cys residue in the protein sequence [18,19]. Only around 10–15% of the circulating TTR in plasma remains unmodified at this residue and the most common PTMs at Cys-10 are the S-sulfonation (S-Sulfo), S-glycinylcysteinylation (S-CysGly), S-cysteinylation (S-Cys) and S-glutathionylation (S-GSH) [20].
Transthyretin (TTR) is an amyloidogenic tetrameric protein, present in human plasma, associated with several familial amyloidoses. Variability of TTR is not only due to point mutations in the encoding gene but also to post-translational modifications (PTMs) at Cys10, being the most common PTMs the S-sulfonation, S-glycinylcysteinylation, S-cysteinylation and S-glutathionylation. It is thought that PTMs at Cys10 may play an important biological role in the onset and pathological process of the amyloidosis. We report here the development of a methodology for quantification of PTMs in serum samples, as well as for the determination of serum TTR levels, from healthy (wt) and TTR-amyloidotic (V30M mutation) individuals. It involves an enrichment step by immunoprecipitation followed by mass spectrometry analysis of (i) the intact TTR protein and (ii) targeted LC–MS analysis of peptides carrying the PTMs of interest. Analysis of serum samples by the combination of the two methods affords complementary information on the relative and absolute amounts of the selected TTR PTM forms. It is shown that methods based on intact protein are biased for specific PTMs since they assume constant response factors, whereas the novel targeted LC–MS method provides absolute quantification of PTMs and total TTR variants.
The study of TTR has a high clinical relevance since it is responsible for diverse familial polyneuropathies. In particular, more than 80 point mutations have been described through genetic studies. However, genetic heterogeneity alone fails to explain the diverse onset and pathological process of the TTR related amyloidosis. The use of proteomic characterization is required to gather information about the PTMs variants present in serum, which have been suggested to be relevant for the amyloidotic pathology. This article is part of a Special Issue entitled: HUPO 2014.
Protein glutathionylation in health and disease
2013, Biochimica et Biophysica Acta - General Subjects
It is now recognized that protein cysteines exist not only as free thiols or intramolecular disulfides, that help maintain the 3D structure of proteins, but can also undergo different types of oxidation, one of which is glutathionylation, or the formation of mixed disulfides with glutathione (GSH).
We will discuss how proteins can undergo glutathionylation and how this can affect the protein characteristics/function. Glutathionylation is reversible and de-glutathionylation can be catalysed by protein thiol–disulfide oxidoreductases. Genetic modification of the expression of these enzymes, particularly glutaredoxin, using overexpression, knockout mice or siRNA, is becoming an important tool to study the role of protein glutathionylation. While in the past this post-translational modification was mainly known in the context of oxidative stress, measurement of glutathionylated proteins in patients is pointing out a potential importance if this modification in pathogenesis and could identify new biomarkers. We also wanted to point out the main findings in the role of glutathionylation in diseases and drug action.
We identify two major open problems in the field, namely the complexity of the mechanisms responsible for glutathionylation and de-glutathionylation, as well as what makes a protein susceptible to glutathionylation.
This review underlines the peculiarities of this post-translational modification and their biological role. This article is part of a Special Issue entitled Cellular functions of glutathione.

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^☆: Abbreviations used: FAP, familial amyloidotic polyneuropathy; TTR, transthyretin, Cys, cysteine; MALDI/TOFS, matrix-assisted laser desorption ionization/time-of-flight mass spectrometry; ESI-MS, electrospray ionization mass spectrometry; HPLC, high performance liquid chromatography; Fr. IV, fraction IV

^☆☆: Rijswijk, M. H.

¹: To whom correspondence should be addressed at First Department of Internal Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-0811, Japan. Fax: +81 96 371 0582. E-mail:[email protected].

²: Dr. Yukio Ando worked temporarily as a visiting professor at Department of Medicine, Umeå University Hospital.

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Regular ArticlePost-translational Modification of Transthyretin in Plasma☆,☆☆

Abstract

Biochem. Biophys. Res. Commun.

FEBS Lett.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Am. Soc. Mass. Spectrom.

Biochem. Biophys. Res. Commun.

Neurosci. Lett.

J. Biol. Chem.

J. Biol. Chem.

Mech. Ageing Dev.

Neuromusc. Disord.

Regular Article
Post-translational Modification of Transthyretin in Plasma☆,☆☆