Effects Of V30A Mutation Of Transthyretin On Familial Amyloidotic Polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is a rare but fetalautosomal-dominant hereditary polyneuropathy which is termed as an amyloiddeposition disease analogous to Alzheimer and Parkinson disease. Clinical pathologyshows deposit of amyloid fibrils in tissue gaps, leading to impairment on peripheraland autonomic nervous system during early stage and organ failure and death at theadvanced stage which may take several to ten plus years. The incidence of FAP ishigh in Portugal, Sweden and Japan. Only sporadic cases have been reported in Chinathat separated across the country.Single point mutations in the transthyretin (TTR) gene are the main cause ofFAP. Almost every single FAP patient harbors a point mutation in the TTR gene. Todate, more than100point mutations in the TTR gene have been reported, with themajority of them closely related to FAP. The most common one is V30M among thesepoint mutations in the TTR gene. The human TTR gene locus has been localized tochromosome18at q12.1. It has four coding exons which encode a protein monomerwith127amino acid residues. Under the physiological condition, TTR is a stablehomologous tetramer and its main function is to transport thyroid hormone andvitamin A. Point mutations in the TTR gene may decrease TTR structural stabilityleading to tetramer dissociation into monomers, which then aggregate into oligomersand subsequent amyloid fibrils.It is currently believed that decline of TTR protein stability is a direct causalfactor of the FAP disease. Both the positions of the point mutations in the TTR geneand the properties of the amino acid substitutions are closely related to TTR proteinstability. The dissociation rate of TTR protein is also an influencing factor determining the formation rate of amyloid fibrils. The tetramer dissociation andmonomer unfolding rate together constitute the rate-limiting step for amyloidformation. These may all directly influence the severity and the onset of FAP.In August2009, a five-generation Chinese FAP family was discovered by theFirst Hospital of Jilin University. Thirty-eight blood samples from the proband, twoother patients, and the rest of the family members had been collected. AutomatedDNA sequencing of the TTR coding region revealed a heterozygous V30A mutation inthe three FAP patients but not in the rest of the family members. Histochemicalanalysis showed serious deposition of amyloid fibrils in a series of different organs ofthe proband. The clinical symptoms confirmed that the positions of TTR amyloiddeposition corresponded with the organ abnormalities and dysfunction, includingautonomic nervous system dysfunction and skeletal muscle damage symptoms such aspoor sensory of the extremities and perception, impairment of autonomic nervoussymptoms, and muscle atrophy of torso and limbs. Therefore, we speculate that thepathogenesis of this five-generation FAP family may be due to the V30A mutation ofthe TTR gene.The V30A mutation is rare and the associated FAP cases scattered all over theworld. The first FAP case harboring the V30A mutation was discovered in1992in aGerman family. It has been rarely reported since then. In our country two cases havebeen reported one as a man carrier in2003and the other as a V30A FAP family in2007. For the reported V30A FAP cases, the researchers only focused on the clinicalfeatures and genetic characteristics of the patients. However, the effects of the V30Amutation on TTR structural stability and amyloid deposition have not been studied.Further, a relationship between the V30A mutant protein and the pathologicalsymptoms of FAP has not been established. In this study, we sought to investigate thestructure stabilities and dissociation kinetics of recombinant wild-type, V30A andV30M TTR proteins to determine the effects of V30A mutation on amyloid formationand neuronal toxicity so as to prove that the V30A mutation is the indeed causalpathological factor for this Chinese FAP family.First, we successfully constructed an E. coli system (pQE30vector and M15 strain) to express and purify the TTR proteins. The original TTR gene sequence issubstituted by “preference codons” of E. coli. The recombinant TTR proteins have asix His-tag at the N-terminal allowing these proteins to bind to Ni-NTA beads forone-step protein purification.4mg TTR proteins could be generated from100mlculture of E. coli. The recombinant TTR proteins maintained tetramer structures atpH7.4determined by non-reducing SDS-PAGE electrophoresis.Next, we evaluated the structure stabilities of the wild-type TTR and the V30Aand V30M mutants in vitro. OD400nm optical density analysis and Thioflavin (ThT)fluorescence detection methods were used to detect amyloid formation of these TTRproteins. Under pH pressure, the V30A and V30M especially the V30A mutationsignificantly promoted the formation of amyloid fibrils. Results from kinetic studiesdemonstrated that the V30A mutant had the fastest amyloid formation rate at pH4.2compared to the wild-type TTR and the V30M variant. Subsequently, we examinedthe effects of TTR ligands Thyroxine (T4) and Flufenamic acid (Flu) on amyloidformation and found that the V30A mutation significantly reduced the ability of T4toprevent amyloid formation, reflected by a significant increase in the half inhibitionrate (IC50). Consistent results were obtained from isothermal titration calorimetryexperiments, showing that the V30A mutation directly reduced Flu binding affinity toTTR protein.Additional detailed comparative analyses of the thermodynamic and kineticstabilities of the quaternary, tertiary and secondary structures between the wild-typeTTR and the two mutant TTRs were performed. Quaternary structure analyses underpH or urea pressure revealed that the V30A mutant tetramers had the fastest rate ofdissociation into dimers and monomers detected by SDS-PAGE and resveratrolfluorescence assays compared to the wild-type. By Trp fluorescence spectrometrydetection, we found that under urea gradient pressure, V30A mutant was the first tounfold and expose intra-molecular tryptophan residues. Circular dichroism analysesrevealed that the V30A mutant was the first to lose its beta sheet structure comparedto the wild-type TTR proteins. The influence of V30M was closed to the previousreports. These results strongly suggest that the V30A mutation accelerates the TTR tetramer dissociation and monomer unfolding. Molecular dynamics simulation resultsshowed that the hydrophilic surface area of V30A and radius of gyration are muchhigher than that of the wild-type and V30M TTR, indicating that the mutation ofV30A loosens the structure of TTR and decreases the overall structure stability.In order to simulate the same condition in vivo as the V30A heterozygouscarrier of FAP patients in this family, we also determined the quaternary and tertiarystructural stabilities of a wild-type-V30A mixture at1:1ratio. Although thewild-type-V30A mixture is more stable than the V30A mutant, it is definitely lessstable than the wild-type TTR.Taken together, our results from both thermodynamic and kinetic studies haveconsistently shown that the V30A mutation decreases the stability of TTR tetramerleading to tetramer dissociation, monomer unfolding, and amyloid fibril formation.Finally, we wanted to determine whether the decreased stability of the TTRV30A mutant is related to neurotoxicity. MTT assay and trypan blue staining wereused to measure in vitro cytotoxicity of the wild-type and mutant TTRs on aneuroblastoma cell line IMR-32. Interestingly, incubation of the V30A mutant withthe IMR-32cells resulted in a significant decrease of viable cells and a significantincrease of cell death compared to that of the wild-type TTR. These results providestrong evidence that the V30A variant TTR is significantly more toxic to the IMR-32neuroblastoma cells than the wild-type TTR, presumably due to its structuralinstability. This was accompanied by significantly increased generation of reactiveoxygen species (ROS), greater induction of the DNA double-strand break biomarkerphospho-H2A.X, cleavage of PARP and Caspase-3, and induction of Beclin-1. Theseresults suggest that treatments of the IMR-32cells with the V30A variant caused celldeath through apoptosis and potentially autophagy presumably through induction ofROS and DNA double-strand breaks.In summary, our results show that the V30A mutation decreases the quaternary,tertiary and secondary structure stabilities of TTR, leading to the formation ofamyloid fibrils and toxicity to neuroblastoma cells potentially through induction ofoxidative stress and DNA damage, leading to apoptosis and autophagy. Our results provide compelling evidence that the V30A mutation maybe the causal factor of theFAP disease in this Chinese family.