Hormone affinity and fibril formation of piscine transthyretin: The role of the N-terminal

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Abstract

Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd = 10.6 ± 1.7 nM) and T4 (Kd = 9.8 ± 0.97 nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd = 252.3 ± 15.8 nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.

Introduction

Transthyretin (TTR) is one of several thyroid hormone-binding proteins (THBP) which transport thyroid hormones thyroxine (T4) and triiodothyronine (T3) in the circulation of vertebrates. This protein is synthesized and secreted mainly by the liver and its functionally active form is a tetramer, the central core of which is proposed to transport one or two molecules of thyroid hormones (THs) (Blake et al., 1978, Schreiber and Richardson, 1997, Wojtczak et al., 1996). Subsequently TTR was also identified in the choroid plexus of mammals where it is proposed to play an important role in the transport of T4 across the blood brain barrier (Blay et al., 1993, Chanoine et al., 1992, Hamberger et al., 1990, Harms et al., 1991, Schreiber et al., 1990, Southwell et al., 1993).

The TTR tetramer is a highly stable protein, however, dissociation and misfolding of this protein can lead to its conversion into insoluble amyloid structures that can deposit extracellularly. This process is associated with severe amyloid disease in humans: familial amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SSA) (reviewed in Benson and Uemichi, 1996, Ingenbleek and Young, 1994, Schreiber and Richardson, 1997, Sipe, 1992). Tetramer dissociation seems to be the critical step in misfolding and in vitro studies have shown that the TTR tetramer can form fibrils under acidic conditions at 37 °C (Hammarstrom et al., 2001, Lai et al., 1996).

TTR has now been identified in most vertebrate groups; it is present in placental mammals, Australian and American marsupials, birds, reptiles, amphibians and fish (Dickson et al., 1985, Duan et al., 1991, Duan et al., 1995, Larsson et al., 1985, Mita et al., 1984, Prapunpoj et al., 2002, Prapunpoj et al., 2000, Richardson et al., 1994, Richardson et al., 1997, Santos and Power, 1996, Santos and Power, 1999, Yamauchi et al., 1993, Yamauchi et al., 1999). Moreover, recently a family of transthyretin-like proteins (TLPs) has been identified in bacteria, plants and animals including non-vertebrates and vertebrates (Eneqvist et al., 2003, Hennebry et al., 2006a). It has been suggested that the TTR gene, only present in vertebrates, probably arose as a result of a duplication of the ancestral TLP gene (Hennebry et al., 2006a, Prapunpoj et al., 2000, Richardson, 2002). Existent studies indicate an overall structural conservation between TLP and TTR (Hennebry et al., 2006b, Lundberg et al., 2006); however, no evidence of TH binding ability could be found for TLP (Eneqvist et al., 2003). Such observations have led to the suggestion that TTR is a vertebrate innovation and evolved as a TH binder.

The distribution of TTR in blood, liver and the choroid plexus of different vertebrates are variable, making it difficult to establish a consensual model for the evolution of this gene. For example, TTR is present principally in the liver and to a lesser extent in the brain and other tissue of teleost fish (Power et al., 2000, Santos and Power, 1996, Santos and Power, 1999) but in turtles (Trachemys scripta) it is expressed principally in the choroid plexus (Richardson et al., 1997). Moreover, some metatherian (marsupial) species contain TTR in the blood stream, whereas others do not (Richardson et al., 1994). In addition to differences in tissue distribution of TTR between species, differences in the thyroid hormone-binding properties of the protein have also been observed. The resolution of the crystal structure of human, chicken and rat TTR has revealed that the functional tetramer contains two structurally identical binding sites in the central channel (Blake et al., 1974, Hamilton et al., 1993, Sunde et al., 1996, Wojtczak, 1997, Wojtczak et al., 1992). The amino acids that are thought to be involved in T4 binding in the central channel of TTR appear to have been conserved between mammals and lower vertebrates. However, binding studies of plasma TTR with radiolabeled T4 and T3 have established that mammalian TTR has the highest affinity for T4 while avian TTR preferentially binds T3 (Chang et al., 1999). Similar preferences for T3 were found for this protein in crocodile (Prapunpoj et al., 2002) and amphibians (Prapunpoj et al., 2000, Yamauchi et al., 1993). The molecular basis for the different affinity of mammalian and lower vertebrate TTR for the thyroid hormones remains to be established but it has been proposed that changes in the N-terminus of the protein may be an important factor. This region is the least conserved between species and in birds, reptiles or amphibian it is longer, having three additional amino acids, and is more hydrophobic than mammalian TTR (Aldred et al., 1997). The identification of TTR in teleost fish is relatively recent, the protein was isolated and the N-terminus sequenced from juvenile masu salmon (Onchorhynchus masou) plasma (Yamauchi et al., 1999) and a full-length cDNA for sea bream TTR was isolated from the liver of juvenile sea bream (Santos and Power, 1996, Santos and Power, 1999). Ligand-binding studies with masu salmon TTR revealed that it has three times higher affinity for T3 (Kd = 13.8 nM) compared to T4 (Kd between 40 and 50 nM) (Yamauchi et al., 1999). The crystal structure of sea bream TTR has been determined and showed that the overall topology of sea bream TTR is conserved (Eneqvist et al., 2004, Folli et al., 2003). The thyroid hormone-binding site is also highly conserved, although Ser 117 (human sequence) is substituted by Thr in sea bream TTR (Folli et al., 2003). Taking into consideration structural studies of human TTR in complex with l-thyroxine it seems unlikely that this substitution will affect TH binding (Wojtczak et al., 2001). However, preliminary qualitative binding studies suggest sea bream TTR preferentially binds T3. Despite conservation of TTR structure between sea bream and other vertebrates, the surface potential, most noticeably in the thyroid hormone-binding site is more negative in the sea bream and the N-terminus of the protein is longer (Power et al., 2000).

As a first step to understand the basis for the differing hormone affinities between fish and mammalian TTR, wild type and N-terminal mutant sea bream TTRs were produced. A specific ligand-binding assay was developed and TH:TTR interactions characterised. Also, the consequences of N-terminal mutations for acid-mediated TTR fibril formation were assessed. Such studies may help to elucidate the regions of the molecule which are important for TTR's TH binding preference and provide preliminary evidence about the role of the N-terminus in tetramer stability.

Section snippets

Cloning of sea bream TTRWT and mutants

For production of sbTTRWT a vector pET 24d(+) construct was used (Eneqvist et al., 2004) containing the mature protein encoding gene (lacking the signal sequence of 18 amino acids predicted by SignalP v3.0 WWW Server) (Bendtsen et al., 2004, Nielsen et al., 1997) preceded by a methionine as indicated 1MAPTPTDKHGGSDTRCPL18. Production of the mutant proteins was based on amplification by PCR of the full-length sea bream TTR cDNA in pBluescript SK(+) (Santos and Power, 1999) using primers

Expression and purification of recombinant sbTTRWT and mutants

Sequencing of TTR expression vector constructs confirmed that sbTTRWT and the 2 mutants M6 and M12 were successfully ligated into vectors and were in frame. Optimization studies of recombinant protein expression with sbTTRWT revealed that a 0.2 mM IPTG concentration and 3 h incubation post-induction at 30 °C were appropriate conditions for induction. Expression was carried out in E. coli BL21 DE3pLysS strain and recombinant protein analysed using denaturing SDS-PAGE 15% followed by Western

Evolution of transthyretin binding affinity for THs T3 and T4

Over the last few years, TTR cDNA and amino acid sequences have been described for a number of representatives of different phylogenetic groups. Several studies concerning TTR affinity for the thyroid hormones T3 and T4 seem to indicate that during evolution TTR progressively lost its higher affinity for T3 in lower vertebrates and became a T4 binder in eutherian mammals (reviewed in Power et al., 2000). This evolution is consistent with physiologically advantageous adaptations regarding TH

Acknowledgments

We thank Dr. Jorge Martins for the technical support in the fluorescence measurements.

Work co-financed by POCI 2010 and the European social funds attributed by the Portuguese National Science Foundation (FCT) to project POCTI/CVT/38703/2001, Pluriannual project to CCMAR and a PhD fellowship to IM (SFRH/BD/6091/2001).

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