Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretin

Abstract

Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70-80 degrees C in neutral to mild alkaline buffers or at 37 degrees C and slightly acidic pH (6-7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis.

Beyond Genetic Factors in Familial Amyloidotic Polyneuropathy: Protein Glycation and the Loss of Fibrinogen's Chaperone Activity

“…Therefore, we analysed the relative amounts of TTR monomers and dimers in FAP and control subjects by SDS-PAGE, as previously used to evaluate the relative amount of TTR dimer to monomer species [44]. Also, the stable sample-specific dimer/monomer ratios, achieved under conditions of partial dimer to monomer transition, reflect the conformational monomer stability relative to the dimeric form [45]. Once a dimer dissociates, the monomers are captured in a complex with SDS and their ability to associate as a dimer is compromised.…”

Serum levels of variants of transthyretin down‐regulation in cholangiocarcinoma

“…Typically, native TTR only accounts for 5$15% of total TTR circulating in plasma. The other 85$95% is posttranslationally modified in the forms of S-sulfonation and S-thiolation [Altland and Winter, 2003]. Importantly, it has been reported that the levels of TTR could decrease in cases of severe liver disease, malnutrition, and acute inflammation [Imanishi, 1981;Marten et al, 1996;Ritchie et al, 1999].…”

“…2A). The differential expressed band at $16 kDa could very possibly be the protein corresponding to peaks observed on chips, for the reason that migration rates for proteins with molecular masses less than 20 kDa may become more dependent on differences of structural features than of masses [Altland and Winter, 2003]. To confirm the doubt, differential expression band at about $16 kDa was excised from the gels for trypsin digestion and identified by ESI-MS/MS.…”

Genetic microheterogeneity of human transthyretin detected by IEF

“…The diagnosis of TTR amyloidosis was verified by clinical symptoms, identification of amyloid in biopsy specimens by appropriate staining (plus immunohistochemical identification of TTR in some cases) and the identification of a TTR mutation by DNA analysis. PAGE followed by IEF in urea gradients or by SDS gradient PAGE was performed as described [3,5]. Gels with IPG ranging from pH 4.2 to 6.8 over distance of 12 cm were used for IEF.…”