Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis

Abstract

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe → Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.

Transthyretin (TTR, formerly called prealbumin) is one of at least 20 human proteins known to be associated with amyloidosis. Transthyretin circulates in plasma as a homotetramer. Each subunit is composed of 127 amino acid residues. It is predominantly synthesized in the liver. However, the choroid plexus and the eye are also sites of production. In plasma, TTR binds to and transports the hormone thyroxine, as well as the retinol-binding protein-vitamin A complex.

Transthyretin is associated with familial transthyretin amyloidosis (ATTR) and senile systemic amyloidosis (SSA; Falk et al. 1997^; Benson 2001). The ATTR is an autosomal dominant disorder associated with the deposition of variant TTR, wild-type protein, and TTR fragments as amyloid fibrils in tissues and organs (Costa et al. 1978; Skinner and Cohen 1981; Pras et al. 1983). Certain amino acid substitutions appear to diminish the overall stability of the tetramer, leading to the formation of amyloidogenic intermediates that self-associate into amyloid fibrils (Lai et al. 1996; Lashuel et al. 1998; Quintas et al. 1999). More than 80 TTR variants have been identified (Connors et al. 2000; Saraiva 2001). The majority of these variants are pathologic. In contrast, SSA is a nonhereditary disorder that affects ~25% of individuals more than 80 years old (Cornwell et al. 1983). In SSA, amyloid fibrils are usually composed of wild-type TTR and its fragments and are found mainly in the heart (Cornwell et al. 1983; Westermark et al. 1990).

The clinical characteristics of ATTR are related to specific mutations distinctive to certain ethnic groups. Usually, the main clinical features of ATTR are peripheral and autonomic neuropathy, cardiomyopathy, and vitreous opacities. The disease is usually fatal within 7–15 years after the appearance of symptoms (Bergethon et al. 1996). The most effective treatment of ATTR is liver transplantation (Holmgren et al. 1991; Bergethon et al. 1996). ATTR is diagnosed in symptomatic individuals who have biopsy proven amyloid disease with the detection of amyloid deposits in tissue and identification of a pathological variant TTR in plasma.

Current techniques used to identify circulating TTR variants and gene mutations include isoelectric focusing (IEF) and genetic mutation analyses such as restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), and direct DNA sequence analysis. However, each technique has some limitations. The IEF analysis is a good initial protein screening procedure but cannot detect electrophoretically silent or low abundance variants such as Gly6Ser, Asp18Glu, Tyr69His, and Tyr114Cys. The SSCP analysis is helpful as a DNA screening test but can produce false negatives (Nelis et al. 1996). The DNA sequence analysis is the standard procedure for defining a DNA mutation, but results are occasionally ambiguous. The RFLP analysis detects a mutation in the DNA when the mutation is present in the restriction site and is a good technique for screening family members for a known mutation. Furthermore, genetic analyses provide information at the DNA level, but they do not yield any information on posttranslational modifications.

We have developed a mass spectrometry-based approach in combination with direct DNA sequence analysis to fully characterize variant forms of TTR (Lim et al. 2002). Our methodology consists of using immunoprecipitation to isolate TTR from serum, as well as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), in combination with proteolytic digestions, to identify variant TTR (Fig. 1 ▶). The ESI MS of the intact TTR provides masses of TTR-related components in the sample and identifies whether there is a modification in the TTR. An aliquot of the immunoprecipitated TTR sample is digested with endoproteinase Asp-N, endoproteinase Glu-C, lysyl endopeptidase Lys-C, or trypsin. The MALDI MS of the digest narrows the location of the modification to the span of an individual peptide derived from the variant protein. Unambiguous identification of the amino acid substitutions in the variant protein is then performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis of the appropriate TTR gene coding region. In addition, MS and MS/MS analyses provide information on posttranslational modifications. Using this approach, we have recently identified a novel pathologic Phe33Cys variant TTR. We have also determined that this variant is extensively modified at the novel site. The wild-type TTR has one Cys residue at position 10. The Phe33Cys variant has two Cys residues. Like the Cys10 present in the wild type and in this variant, the Cys33 residue was found to be both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.

Figure 1.

A schematic diagram showing the general strategy used in the analysis of TTR variants.

Results

Preliminary amyloid testing

Biopsies of kidney, abdominal fat, and bone marrow tissues stained with Congo red (Puchtler et al. 1962) confirmed amyloid deposits. Tests for a plasma cell dyscrasia yielded negative results, suggesting the amyloid type was not of the primary (AL), immunoglobulin light chain type. The IEF screening of the patient’s serum was performed to test for the presence of a variant TTR. The IEF results are shown in Figure 2 ▶. Normally, only a single band corresponding to wild-type TTR is displayed (Fig. 2 ▶, lane B). The appearance of a second band in the IEF gel indicates the presence of a variant TTR. Serum from an individual known to be heterozygous for the ATTR Val30Met mutation is shown in Figure 2 ▶, lane C. The IEF analysis of the serum from the patient (ATTR-00084) showed the presence of wild-type and variant TTR bands (Fig. 2 ▶, lane A), suggesting that the patient has a variant TTR. To fully characterize this variant TTR, we used a method consisting of mass spectrometric peptide mapping and direct DNA sequence analysis (see Fig. 1 ▶).

Figure 2.

The IEF analysis of serum samples obtained from patient ATTR-00084 (lane A), a normal control individual (lane B), and a patient diagnosed with Val30Met ATTR (lane C).

Determination of the mass of intact TTR using ESI MS

The reversed-phase high-performance liquid chromatography (HPLC) chromatogram of TTR immunoprecipitated from the patient’s serum is displayed in the inset of Figure 3A ▶. The TTR-related components eluted within the usual time window at 16.0–17.4 min (52.0%–54.1% acetonitrile, peaks a and b). A peak corresponding to S-cysteinylated TTR eluted at 16.4 min (52.6% acetonitrile, peak a), and another corresponding to S-sulfonated TTR eluted at 17.1 min (53.6% acetonitrile, peak b). However, in this sample, additional components eluted at 15.3–15.7 min (50.8%–51.6% acetonitrile), centering at 15.5 min (51.2% acetonitrile, doublet peak c).

Figure 3.

Deconvoluted ESI mass spectra of intact TTR, immunoprecipitated and purified from the serum of patient ATTR-00084. All masses shown are average values. (A) ESI mass spectrum of the components in the HPLC fraction containing peaks a and b, eluting between 16.0 and 17.4 min (52.0%–54.1% acetonitrile). The TTR-related components usually elute during this time. (B) ESI mass spectrum of the components in the HPLC fraction containing doublet peak c, eluting between 15.3 and 15.7 min (50.8%–51.6% acetonitrile). (Inset) HPLC chromatogram of the reversed-phase HPLC purification of TTR, immunoprecipitated from the serum of patient ATTR-00084.

Assignments of the components in the HPLC fraction that was obtained by combining peaks a and b were verified using ESI MS. The deconvoluted ESI mass spectrum showed peaks corresponding to wild-type TTR only (Fig. 3A ▶). The peak at 13,761 Da contained the unmodified wild-type TTR. This observed mass agreed with the mass calculated (M_cal) from the amino acid sequence of unmodified wt TTR (Kanda et al. 1974). The peaks at 13,841 Da, 13,880 Da, 13,937 Da, and 14,066 Da contained wild-type TTR conjugated to sulfite (M_cal = 13,840 Da), cysteine (M_cal = 13,881 Da), cysteinylglycine (M_cal = 13,938 Da), and glutathione (M_cal = 14,067 Da), respectively (Kishikawa et al. 1996, 1999a; Théberge et al. 1999). No variant TTR was detected in this HPLC fraction.

Similarly, an aliquot of the HPLC fraction containing doublet peak c was analyzed using ESI MS. The deconvoluted ESI mass spectrum is shown in Figure 3B ▶. The overall pattern of the peaks is similar to that observed for the wild-type TTR (Fig. 3A ▶), but the peaks are shifted 75 Da higher in mass, suggesting the presence of a variant.

Identification of the region containing the modification using MALDI MS peptide mapping

An aliquot of the HPLC fraction containing peaks a and b was treated with Lys-C. The resulting peptides were analyzed using MALDI MS. All the peaks contained peptides generated from the Lys-C cleavage of wild-type TTR only, supporting the ESI MS data mentioned, that this HPLC fraction (peaks a and b) did not contain any variant TTR. For example, Figure 4A ▶ displays the MALDI mass spectrum of this Lys-C digest, showing the m/z 2070–2500 region. The peaks detected at [M+H]⁺ m/z 2149.6 and [M+H]⁺ m/z 2456.8 corresponded to wild-type peptides containing residues 16–35 (calculated [M+H]⁺ m/z 2149.6) and 49–70 (calculated [M+H]⁺ m/z 2456.6), respectively.

Figure 4.

The MALDI mass spectra, showing m/z 2070–2500, of the Lys-C digests of aliquots of the TTR-related components from the HPLC fraction (A) containing peaks a and b and (B) doublet peak c (see Fig. 3A ▶ inset). (C) To confirm the presence of disulfide linkages, an aliquot of the Lys-C digest of the TTR-related components from the HPLC fraction containing doublet peak c (see Fig. 3A ▶ inset) was treated with DTT. The S-sulfonation and S-cysteinylation of Cys33 are lost after reduction with DTT. All m/z values shown are average.

Similarly, an aliquot of the HPLC fraction containing doublet peak c was treated with Lys-C and analyzed using MALDI MS. In the MALDI mass spectrum (Fig. 4B ▶), the peak at [M+H]⁺ m/z 2456.8 corresponded to the wild-type peptide containing residues 49–70 (calculated [M+H]⁺ m/z 2456.6). The peak at [M+H]⁺ m/z 2105.6 does not match to any known [M+H]⁺ m/z values of TTR Lys-C peptides. The peak at [M+H]⁺ m/z 2224.9 is 119.3 u higher than m/z 2105.6. This +119 u mass shift could indicate S-cysteinylation of a Cys residue (Lim et al. 2001). Furthermore, the peak at [M+H]⁺ m/z 2185.7 is 80.1 u higher than m/z 2105.6, suggesting S-sulfonation of a Cys residue (Kishikawa et al. 1999a; Théberge et al. 1999). In addition, the presence of these peaks ([M+H]⁺ m/z 2105.6, 2185.7, and 2224.9; Fig. 4B ▶) in association with the peak at [M+H]⁺ m/z 2149.6 (Fig. 4A ▶) suggests that there is a modification in the region containing residues 16–35.

To verify whether the peaks at [M+H]⁺ m/z 2185.7 and 2224.9 represented disulfide-linked peptides, an aliquot of the Lys-C digest was treated with dithiothreitol (DTT) and reanalyzed using MALDI MS. The disappearance of these peaks and the increase in abundance of the peak at [M+H]⁺ m/z 2105.8 (Fig. 4C ▶) confirmed that the peaks at [M+H]⁺ m/z 2185.7 and 2224.9 (Fig. 4B ▶) contained disulfide-linked peptides and that the modifications were most likely S-sulfonation and S-cysteinylation, respectively. These results also suggest that there is a Cys residue in the primary sequence of the peptide containing residues 16–35. Because wild-type TTR has only one Cys residue (at position 10), the presence of the Cys residue in the region containing residues 16–35 most likely results from a mutation in the TTR gene.

S-Cysteinylated peptides often experience the facile loss of the S-cysteinyl group due to prompt fragmentation in the MALDI ion source. In the spectrum shown in Figure 4B ▶, the facile loss of the S-cysteinyl group from the peak at [M+H]⁺ m/z 2224.9 would produce the peak at [M+H]⁺ m/z 2105.6. The ESI mass spectrum in Figure 3B ▶ showed a +75-Da mass shift. This mass shift of +75 Da could correspond to S-cysteinylation (+119 Da) of a Phe → Cys amino acid substitution (−44 Da). The only possible Cys amino acid substitution that would give a mass shift of −44 Da is a Phe → Cys substitution. In the region containing residues 16–35, there is one Phe residue, located at position 33. Thus, the peak at [M+H]⁺ m/z 2105.6 most likely represents a peptide with a Phe → Cys substitution at position 33 (calculated [M+H]⁺ m/z 2105.5).

To further support these results, an aliquot of each of the HPLC fractions (peaks a–b and c) was mixed in equal volume and treated with Lys-C. The MALDI mass spectrum of this digest is shown in Figure 5 ▶. All of the peaks discussed were seen in this MALDI mass spectrum. The peak at [M+H]⁺ m/z 2105.7 is 44.4 u lower than m/z 2150.1 and likely corresponds to the peptide with residues 16–35 resulting from the Lys-C cleavage of the variant TTR. This peptide would contain the Phe → Cys substitution at position 33. S-Sulfonation and S-cysteinylation of this peptide were detected at [M+H]⁺ m/z 2185.5 and 2224.9 (calculated [M+H]⁺ m/z 2184.6 and 2224.7), respectively (Fig. 5 ▶).

Figure 5.

The MALDI mass spectrum of the Lys-C digest of an aliquot of the TTR-related components from the combined HPLC fractions containing peaks a-c (see Fig. 3A ▶ inset), showing m/z 1350–5300. All m/z values shown are average.

Identification of the mutation in the TTR gene by direct DNA sequence analysis

DNA was isolated from the peripheral blood leukocytes of the patient. The region containing residues 16–35 corresponds to a portion of exon 2 in the TTR gene. Therefore, the coding sequence of exon 2 was amplified by the polymerase chain reaction (PCR) using exon-2-specific oligonucleotide primers. The resulting PCR product was subsequently sequenced in both directions using the same primers. This direct DNA sequence analysis of exon 2 showed a TTC → TGC transversion in the codon at position 33 (Fig. 6 ▶), consistent with the Phe → Cys substitution.

Figure 6.

Direct DNA sequence analysis of exon 2 of the TTR gene from the DNA sample of patient ATTR-00084. Peaks are labeled to indicate the nucleotide bases C (○), T (▵), A (□), and G (*).

Identification of the S-cysteinylation of the novel Phe → Cys substitution at position 33 by MS/MS analysis

An aliquot of the Lys-C digest of the combined HPLC fractions (peaks a–c) was analyzed by MS/MS analysis using an ESI quadrupole/orthogonal acceleration time-of-flight (TOF) mass spectrometer to obtain structural information of the peptide containing the modification. In a product ion mass spectrum, the mass difference between sequential fragment ions in each b- and y-fragment ion series corresponds to the mass of an amino acid residue (Biemann 1988). The [M+3H]³⁺ m/z 741.74 ion of the S-cysteinylated peptide-containing residues 16–35 was isolated and fragmented using collision-induced dissociation (CID) to obtain sequence information. The product ion mass spectrum is shown in Figure 7 ▶. Enough sequence coverage was obtained to show that there is a Phe → Cys substitution at position 33 and that the Cys33 in the primary sequence is S-cysteinylated. The y₂ and y₃ fragment ions were detected at m/z 303.22 and 525.23, respectively. The mass difference is 222.01 u. The monoisotopic mass of a Cys residue is 103.01 Da (versus 147.07 Da for a Phe residue). S-Cysteinylation would produce an additional monoisotopic mass change of +119.00 Da. Taken together, the total monoisotopic mass of an S-cysteinylated Cys residue is 222.01 Da. The mass difference between the y₂ and y₃ fragment ions corresponds to the total mass obtained from the S-cysteinylation of the Phe → Cys substitution at position 33. This MS/MS analysis unambiguously identifies the S-cysteinylation of the novel Phe → Cys substitution at position 33.

Figure 7.

The ESI CID product ion mass spectrum of the [M+3H]³⁺ ion of the S-cysteinylated Phe33Cys variant peptide at m/z 741.74 from the Lys-C digest of an aliquot of the TTR-related components from the combined HPLC fractions containing peaks a–c (see Fig. 3A ▶ inset). All m/z values shown are monoisotopic.

Discussion

A novel TTR variant Phe33Cys was identified in a middle-aged woman of Polish ancestry living in the United States. The clinical features of the disease were consistent with those of other variant TTRs and included vitreous opacities, cardiomyopathy, mild sensory neuropathy, and renal disease. The family history confirmed inheritance of the disease in two generations. In addition, we report extensive modification of the Cys33 residue similar to those observed at the normally occurring Cys10 residue.

Identification of S-sulfonation and S-thiolation of the novel Phe33Cys variant TTR

The peaks containing the Phe33Cys variant in the ESI mass spectrum, shown in Figure 3B ▶, could now be assigned. This novel ATTR Phe33Cys variant has two potential sites for S-sulfonation and S-thiolation: one at the normal Cys10 position and the other at the novel Cys33 site. The peak observed at 13,836 Da is +75 Da higher in mass than the peak observed at 13,761 Da for the unmodified wild-type TTR (Fig. 3A ▶); this peak contained the Phe33Cys variant with S-cysteinyl conjugation at Cys10 or at Cys33 (M_cal = 13,837 Da). The +75-Da mass shift corresponded to the Phe → Cys substitution (−44 Da) at position 33 and the S-cysteinylation (+119 Da) of Cys10 or Cys33. Similarly, the peak at 13,797 Da contained the Phe33Cys variant with S-sulfonation at Cys10 or Cys33 (M_cal = 13,796 Da). S-Sulfonation (+80 Da) of the Phe33Cys variant [Phe → Cys substitution (−44 Da)] at Cys10 or Cys33 would give a mass shift of +36 Da, which agrees with the observed 36-Da mass shift between 13,761 Da and 13,797 Da.

The peaks at 13,916 Da, 13,955 Da, 14,012 Da, and 14,141 Da (Fig. 3B ▶) contained S-sulfonation and S-thiolation at both the Cys10 and Cys33 residues. S-Cysteinylation of both the Cys10 and Cys33 residues in the Phe33Cys variant TTR would produce the peak observed at 13,955 Da (M_cal = 13,956 Da). The peak at 13,916 Da was a composite of two possible conjugated situations of the Phe33Cys variant: S-sulfonation at Cys10 and S-cysteinylation at Cys33 or vice versa (M_cal = 13,916 Da). Similarly, the peak at 14,012 Da contained a composite of two components: S-cysteinylglycine at Cys10 and S-cysteinylation at Cys33 or vice versa (M_cal = 14,013 Da). Likewise, the peak at 14,141 Da contained a composite of two components: S-glutathionylation at Cys10 and S-cysteinylation at Cys33 or vice versa (M_cal = 14,142 Da).

Formation of S-sulfonation and S-thiolation of TTR

It is not known whether posttranslational modification of TTR occurs intracellularly or extracellularly. However, in plasma, the reduced, oxidized, and protein-bound forms of homocysteine, cysteine, cysteinylglycine, and glutathione interact by redox and disulfide exchange reactions (Halliwell and Gutteridge 1990; Ueland et al. 1996). These interactions may play an important role in the extracellular antioxidant defense system (Halliwell and Gutteridge 1990; Ueland et al. 1996). Because TTR is an extracellular protein, the formation of S-thiolated TTR may be part of these disulfide exchange reactions. In contrast, the formation of the S-sulfonated TTR involves sulfitolysis of the S-thiolated TTR. Sulfitolysis of disulfide bonds in proteins produces S-sulfonated proteins (Cole 1967). The reaction involves the nucleophilic attack of the sulfite ions on disulfide bonds in proteins (Cecil and Wake 1962). For example, S-sulfonation of albumin and fibronectin is well documented (Gregory and Gunnison 1984). Figure 8 ▶ shows reactions that can lead to S-sulfonation of TTR. The human body is constantly exposed to sulfur dioxide (a major air pollutant) and sulfite (antimicrobial and antioxidant agents in foods and beverages). Reaction A in Figure 8 ▶ shows the equilibrium that exists between sulfur dioxide, bisulfite, and sulfite ions. Reactions B and C in Figure 8 ▶ show that S-sulfonation of TTR involves the cleavage of the disulfide bond in the S-thiolated TTR species by either bisulfite or sulfite ions, respectively, releasing cysteine, cysteinylglycine, and glutathione. Because the pK_a2 of sulfurous acid is 7.2 (Tartar and Garretson 1941), at physiological pH (7.4), the bisulfite ions dissociate and thus their concentration is lower than the concentration of the sulfite ions. In addition, kinetic studies have shown that the rate of reaction of low molecular weight disulfides with bisulfite ions is negligible in comparison to that of the sulfite ions (Cecil and McPhee 1955). Thus, formation of S-sulfonated TTR predominantly results from the reaction between S-thiolated TTR and sulfite ions (Fig. 8 ▶, reaction C).

Figure 8.

S-Sulfonation of TTR. (A) Equilibrium of sulfur dioxide, bisulfite, and sulfite ions. Reaction of bisulfite (B) and sulfite (C) ions with S-thiolated TTR.

Sulfitolysis and protein oxidation

Sulfitolysis may disrupt the plasma redox thiol status and affect the clearance of reactive oxygen species, allowing them to oxidize proteins. Sulfitolysis of bovine serum albumin (BSA) and the introduction of the negative charge by the S-sulfonated group have been shown to increase the hydrodynamic volume of BSA (Malhotra and Sahal 1996). In addition, sulfitolysis unfolds BSA by altering its helical content (Malhotra and Sahal 1996). Studies of the formation and clearance of plasma S-sulfonated proteins in rabbits, rat, and rhesus monkey exposed to sulfur dioxide and sulfite show the persistence of S-sulfonated proteins in plasma. S-Sulfonation has been shown to extend the half-life of the proteins (Gunnison and Palmes 1973, 1978).

Sulfitolysis of S-thiolated TTR to generate the S-sulfonated TTR may affect the protein in a similar way. For example, by reversed-phase HPLC analysis, the S-sulfonated TTR seems to be more hydrophobic than the S-cysteinylated TTR (Fig. 3A ▶, inset). The S-cysteinylated TTR eluted in peak a at 16.4 min (52.6% acetonitrile), whereas the S-sulfonated TTR eluted in peak b at 17.1 min (53.6% acetonitrile). In addition, the negative charge of the S-sulfonated TTR may affect the stability of the TTR tetramer, allowing it to dissociate. Introduction of the additional Cys residue at position 33 and its subsequent modification provide a potential doubling of the capacity for sulfonolysis.

Posttranslational modifications and TTR amyloidogenesis

The link between amino acid substitutions in TTR and ATTR is well established. Certain amino acid substitutions destabilize the tetramer and cause it to dissociate into monomeric subunits that undergo conformational changes to intermediates that self-associate into amyloid fibrils (Lai et al. 1996; Lashuel et al. 1998; Quintas et al. 1999). However, these studies were done using recombinant TTR and thus, do not take posttranslational modification of the protein into account. In plasma, TTR is extensively modified with thiol adducts (Fig. 3 ▶). Typically, the unmodified TTR only accounts for ~5%–15% of the total TTR circulating in plasma. The other 85%–95% is posttranslationally modified in the form of S-sulfonation and S-thiolation (S-cysteinylation, S-cysteinylglycine, and S-glutathionylation). These forms of TTR have been detected by MS (Kishikawa et al. 1996, 1999a; Théberge et al. 1999) and IEF in urea gradients (Altland et al. 1999). Thus, in plasma, TTR exists as a mixed tetramer (unmodified and posttranslationally modified forms). The microheterogeneity of TTR has been shown to affect the quaternary structure of TTR and its ability to bind to thyroxine (Pettersson et al. 1989).

In vitro studies have shown that the S-cysteinylated Val30Met TTR (S-cysteinylation at Cys10) has a greater propensity to form amyloid fibrils than either the unmodified Val30Met TTR, the unmodified wild-type TTR, or the S-cysteinylated wild-type TTR at pH 5.5 (Ando et al. 1999). A study using IEF in urea gradients under equilibrium conditions has indicated that the S-sulfonated form of TTR may have a stabilizing effect on the TTR tetramers by increasing the tetramer-to-monomer ratio (Altland and Winter 1999). However, in vitro studies also show that the S-sulfonated TTR is the most amyloidogenic form of TTR (Kishikawa et al. 1999b). The S-sulfonated group adds a negative charge to each TTR monomer or four negative charges to the TTR tetramer. Although it is not fully understood how net molecular charge can affect amyloidogenicity in proteins, studies using synthetic peptide models have indicated that the total net charge of the molecule, in addition to the conformational state of the molecule, can contribute to amyloid fibril formation (Lopez De La Paz et al. 2002).

Posttranslational modifications and oxidative stress

Formation of S-sulfonated proteins may disrupt the redox thiol status in plasma, allowing excessive reactive oxygen species to oxidize proteins. The level of oxidized proteins increases with age, possibly due to the increase of the generation of reactive oxygen species (Stadtman 1992; Berlett and Stadtman 1997). For instance, the level of the protein carbonyl content and protein hydrophobicity has been shown to increase with age (Chao et al. 1997). In addition, the protein carbonyl content in the brain of patients with Alzheimer is greater than that in the brain of age-matched control individuals (Smith et al. 1991). Oxidative stress and a high level of protein carbonyl content have been observed in amyloid tissue of ATTR Val30Met patients (Ando et al. 1997). Furthermore, oxidative stress is known to induce amyloid fibril formation in the rat (Tajiri et al. 2002). Increases in the ratio of posttranslationally modified Val30Met TTR relative to unmodified Val30Met TTR have been reported in ATTR patients (Suhr et al. 1998). In this study, the higher percentage of posttranslationally modified Val30Met TTR found in the Japanese patients may be correlated to the lower average age of onset in these patients (45 years old) compared to the Swedish patients (56 years old; Suhr et al. 1999).

Protein conformational changes and degradation

Such posttranslational modifications, as discussed above, may alter the structure of TTR. For example, according to the crystal structure of wild-type TTR, the Cys10 residue is located at the edge of β-strand A. Its sulfur atom could form a sulfur-hydrogen bond (NH. . .S) to the main chain amide hydrogen of the Gly57 residue in β-strand D (Blake et al. 1978; Terry et al. 1993). When the Cys10 residue is posttranslationally modified, the sulfur atom of Cys10 is no longer available to form the NH. . .S bond to the main chain amide hydrogen of Gly57. The disruption of this hydrogen bonding process may alter the structure of the TTR, causing it to adopt a different conformation. Likewise, the Cys33 residue is within a structured region of TTR, and its posttranslational modification may interrupt normal folding. Interestingly, only one other Cys substitution (at position 114) has been described by DNA sequence analysis (Ueno et al. 1990). However, no data have been reported on the post-translational modification of Cys114.

Protein conformational changes resulting from amino acid substitution and posttranslational modification may affect the rate of protein degradation. Degradation of extracellular proteins involves pinocytosis and specific receptor-mediated endocytosis. Through these processes, extracellular proteins are taken inside the cell for degradation in lysosomes. The mechanism of TTR cellular uptake and degradation is not well understood. However, cellular uptake of TTR has been shown to be receptor mediated (Divino and Schussler 1990), and the structure of the TTR can affect the receptor recognition (Sousa and Saraiva 2001). For example, the nonamyloidogenic Thr119Met variant has the highest proclivity to enter cells, whereas the amyloidogenic Leu55Pro does not enter cells at all (Sousa and Saraiva 2001). Posttranslational modifications, such as those reported here, in addition to amino acid substitution can affect the structure of the TTR and thus affect its ability to bind to these receptors.

The site of TTR amyloid fibril formation is not known. TTR amyloid fibril formation may take place outside the cell or inside the lysosome. Activated neutrophils can release hypochlorous acid into the extracellular space (Weiss 1989). This process could create an acidic microenvironment, allowing TTR to misfold and self-assemble into amyloid fibrils extracellularly. Alternatively, once TTR is in the lysosome, the rate of amyloid fibril formation could occur faster than its rate of degradation. Build-up of protease-resistant proteins inside the lysosome has been reported (Nixon and Cataldo 1993). In neurons, the rupture of lysosomes may promote cell autolysis with subsequent neurodegeneration (Nixon and Cataldo 1993). The lysosomal enzymes (elastase and cathepsin G) have been found associated with amyloid fibrils isolated from patients (Skinner et al. 1986; Stone et al. 1993). In addition, the lysosomal enzymes cathepsins B and D have been detected in senile plaques from the brains of patients with Alzheimer, but not from the brains of age-matched control individuals. Thus, the amyloid fibrils formed inside the lysosome could be released into extracellular spaces during cell autolysis. Regardless of where the amyloid fibrils form, altering of the protein structure through modifications may affect its ability to bind to cell surface receptors and possibly affect its ability to be degraded by lysosomal enzymes.

Conclusions

In this paper, we report the identification of S-sulfonation and S-thiolation of a novel Phe33Cys variant TTR isolated from a patient diagnosed with ATTR. Current studies directed at elucidating the molecular mechanism of TTR fibrillogenesis do not take posttranslational modifications into account. Many factors may contribute to the amyloidogenesis of TTR because mutations in the TTR gene alone cannot explain the variability of clinical features as well as delayed onset of the disease. Posttranslational modification may be one of these factors. In plasma, the majority of TTR (85%–95% of total TTR) in circulation is posttranslationally modified. The unmodified form only accounts for ~5%–15% of the total TTR. This microheterogeneity creates mixed TTR tetramers, which may have different stability than that of tetramers consisting of only unmodified TTR used in current models. In addition, post-translational modification may change the conformation of TTR, thus affecting its cellular uptake and degradation. Future studies aimed at explaining the mechanism of TTR fibrillogenesis should incorporate the effect of posttranslational modification.

Materials and methods

Case report

A 51-year-old woman (ATTR-00084) of Polish ancestry living in the United States was referred for evaluation after proteinuria prompted a kidney biopsy that was positive for amyloid deposits. Sixteen years before the evaluation, she experienced visual disturbances and had eye surgery for vitreous opacities. She reported symptoms of neuropathy in her hands. She did not have dyspnea on exertion, orthopnea, lightheadedness, or gastrointestinal symptoms. Her family history indicated that her father was diagnosed with amyloidosis and died at age 62 of congestive heart failure. He also had neuropathy in his hands and feet. Two paternal aunts died in their 70s. Both had blindness and had received pacemakers. On physical examination, the patient had a normal blood pressure without orthostasis, had no organomegaly, and had no neurologic deficits.

An electrocardiogram showed normal heart rhythm, normal voltage, and early signs of a conduction abnormality with a borderline first-degree atrioventricular (AV) block. An echocardiogram was consistent with cardiac amyloid disease with a moderate concentric left ventricular thickening and an intraventricular septal measurement of 15 mm (normal 7–11 mm). Electrophysiologic testing of the cardiac conduction system was carried out and showed normal sinus node recovery time and no inducible AV block. Tests of kidney function showed a 24-h urine protein of 1.7 g and a normal serum creatinine of 0.6 mg/dL. An abdominal fat aspiration was positive for amyloid deposits. A bone marrow biopsy was positive for amyloid deposits but showed no sign of a plasma cell dyscrasia. The serum and urine immunofixation electrophoreses were also negative for a monoclonal protein. An IEF study was positive for a mutant TTR, and further characterization of the variant TTR was undertaken using mass spectrometry and direct DNA sequence analysis.

The patient received an orthotopic liver transplant 1 year after her initial evaluation. Physical examination and cardiac studies remain unchanged 1 year after the liver transplant. Clinical data were collected with the approval of the Institutional Review Board at Boston University School of Medicine.

Initial IEF screening analysis

Sera from the patient, a normal control individual, and a patient who had ATTR Val30Met were screened using IEF. Details of our IEF protocol have been previously described (Connors et al. 1998). Typically, 50 μL of serum was electrophoresed on a nondenaturing 7.5% polyacrylamide gel. The portion of the gel containing the TTR was excised and placed on a broad range (pH 4.0–7.0) Immobiline gel (Amersham Pharmacia Biotech, Piscataway, NJ). IEF was carried out under partially denaturing conditions in 2.5-M urea. The TTR bands were visualized with Coomassie Blue stain.

Purification of TTR from the patient’s serum

The TTR was immunoprecipitated from the serum of patient ATTR-00084 as described previously (Kishikawa et al. 1996). The TTR-antibody complex was dissolved in 80:10:10 (v/v/v) water: acetonitrile:acetic acid and passed through a Millipore Microcon YM-100 centrifugal filter to remove the antibody. For further purification, the filtrate was applied to an analytical Vydac C4 reversed-phase HPLC column and eluted at 0.75 mL/min. The gradient was held at 5% acetonitrile for 8 min, then continued at 40%–85% acetonitrile for 30 min, and held at 85% acetonitrile for 5 min before returning to initial conditions (5% acetonitrile).

ESI MS of intact TTR

The molecular mass of intact TTR was determined in the positive ion mode using a Micromass Quattro II ESI triple quadrupole mass spectrometer equipped with a nanospray Z-Spray source (Beverly, MA). The instrument was calibrated using sodium trifluoroacetate ion clusters (Moini et al. 1998). This instrument gives average masses for large biomolecules. After calibration, this instrument was capable of achieving at least 70 ppm mass accuracy for the deconvoluted mass spectrum. The purified TTR sample was dissolved in 50:50:0.5 (v/v/v) of methanol:water:formic acid, and 2 μL was loaded into a nanospray tip. The sample was nanosprayed into the mass spectrometer by increasing the capillary potential slowly from 0 to 1.2 kV until a stable ion current was observed. The cone voltage and ion source temperature were held at 34 V and 82°C, respectively.

MALDI MS of the TTR Lys-C digest

Aliquots of the purified TTR sample from the patient were digested with lysyl endopeptidase Lys-C (Wako BioProducts, Richmond, VA) at an enzyme-to-substrate ratio of 1:100 in 100 mM ammonium bicarbonate (NH₄HCO₃) at pH 8 at 37°C for 17 h. The enzymatic digestion was quenched by drying the reaction mixture in a Savant Instruments SC110 Speedvac concentrator. To confirm the presence of disulfide linkages, an aliquot of the Lys-C digest was treated with 2 mM DTT in 50 mM NH₄HCO₃ at pH 8 for 15 min at 37°C, before drying as described above.

The resulting peptide mixtures were then dissolved in 30:70:0.1 (v/v/v) of acetonitrile:water:TFA, mixed with the MALDI matrix 2,5-dihydroxybenzoic acid (DHB; 10 mg/mL), and analyzed using a Finnigan MAT Vision 2000 MALDI TOF mass spectrometer in the positive ion linear mode with delayed extraction. The instrument was calibrated using a standard peptide mixture (Agilent Technologies, Palo Alto, CA) consisting of oxytocin [M+H]⁺ m/z 1008.2, Arg-8-vasopressin [M+H]⁺ m/z 1085.3, angiotensin I [M+H]⁺ m/z 1282.5, somatostatin [M+H]⁺ m/z 1638.9, chicken atrial natriuretic peptide [M+H]⁺ m/z 3161.7, human recombinant insulin [M+H]⁺ m/z 5808.7, and recombinant hirudin [M+H]⁺ m/z 6964.5. After this external calibration, the instrument was capable of achieving at least 1000 ppm mass accuracy in the linear mode with delayed extraction. In this mode, all the masses observed were average values.

Mass spectrometric sequence analysis

Peptide sequence information from the Lys-C digest of the TTR was obtained using MS/MS analysis with an Applied Biosystems/MDS-SCIEX QSTAR Pulsar i ESI (Foster City, CA) quadrupole/orthogonal acceleration TOF mass spectrometer in the positive ion mode. For MS experiments, the instrument was calibrated using the [M+2H]²⁺ ion (m/z 879.9704) and [M+4H]⁴⁺ ion (m/z 440.4892) of porcine renin substrate tetradecapeptide. For MS/MS experiments, the instrument was calibrated using the b₈ (m/z 1028.5317) and y₂ (m/z 269.1137) fragment ions of the [M+3H]³⁺ ion (m/z 586.9829) of the porcine renin substrate tetradecapeptide. After calibration, this instrument was capable of achieving at least 10 ppm mass accuracy in both the MS and MS/MS modes with a minimum resolution of 1:9000 (fwhm). The isotopic clusters of the multiply charged form [M+nH]ⁿ⁺ of individual peptides were selected for collision-induced dissociation.

Direct DNA sequence analysis

Total genomic DNA was isolated from peripheral blood leukocytes. The coding sequence of exon 2 was amplified using the forward and reverse oligonucleotide primer pair: 5′-TCTTGTT TCGCTCCAGATTTC-3′ (forward), 5′-CAGATGATGTGAGCC TCTCTC-3′ (reverse; Integrated DNA Technologies, Inc, Coralville, IA). PCR was performed through 39 cycles involving denaturation at 94°C for 30 sec, annealing at 61°C for 1 min, and extention at 72°C for 30 sec. The PCR products were separated by electrophoresis on 1.5% agarose gels and visualized by staining with ethidium bromide. Purification was accomplished using QIAquick PCR purification kit (Qiagen Inc, Valencia, CA), and nucleotide sequences in both the forward and reverse directions were obtained using the same amplification oligonucleotides. Dideoxy terminator fluorescent DNA sequencing reactions were carried out using the ABI PRISM BigDye Terminators cycle sequencing kit (Applied Biosystems). The resulting products were purified by ethanol precipitation and electrophoresed on the ABI PRISM 377 DNA sequencer at the Molecular Genetics Core at Boston University School of Medicine.

Abbreviations

• ATTR, familial transthyretin amyloidosis

• CID, collision-induced dissociation

• DHB, 2,5-dihydroxybenzoic acid

• DTT, dithiothreitol

• ESI, electrospray ionization

• fwhm, full width half maximum

• HPLC, high-performance liquid chromatography

• IEF, isoelectric focusing

• M_cal, calculated mass

• MALDI, matrix-assisted laser desorption/ionization

• [M+H]⁺, protonated molecule

• MS, mass spectrometry

• MS/MS, tandem MS

• m/z, mass-to-charge ratio

• ppm, part per million

• RFLP, restriction fragment length polymorphism

• SSCP, single-strand conformation polymorphism

• TFA, trifluoroacetic acid

• TOF, time-of-flight

• TTR, transthyretin

• u, atomic mass unit

• wt, wild type