Partially denaturing Western blot of Transthyretin in plasmas of V30M mutation carriers

Abstract

Human transthyretin (TTR) is a homotetrameric protein essential for the transport of thyroxine and retinol in plasma of healthy individuals. Point mutations, mainly a substitution of valine to methionine at position 30 (V30M), result in disruption of TTR complexes and release of TTR monomers responsible for building amyloid deposits in the interstice of heart and brain tissues. Denaturing immunoblotanalysis of plasma samples, with either monoclonal antibodies (MAbs) raised against the whole TTR molecule or with monospecific serum directed to the C-terminal track containing amino acid residues 71 to 98 (p71/98), readily identified the 14 Kd monomeric form of TTR in both V30M mutant-carrier and in non-carrier individuals. P71/98 also identified a 55-60 Kd polypeptide (TTRt) corresponding to a fraction of TTR tetramers that either escape disruption by the combined use of SDS and heat treatment and/or refolded during the first stages of PAGE/SDS running. A milder treatment avoiding the heating of plasma samples prior to immunoblot resulted in a notable increase in TTRt and disappearance of monomeric TTR in samples from V30M-negative individuals but not in those from V30M-positive carriers where free TTR monomers are systematically detected by p71/98 serum. Our results suggest that minimizing the TTR complex disruption before PAGE-SDS/ immunoblot analysis could be simple strategy to sort plasmas from V30M-positive and V30M-negative individuals in the clinical laboratory