Transthyretin mutation Leu-55-Pro significantly alters tetramer stability and increases amyloidogenicity.
A recently reported variant of human transthyretin (TTR), Leu-55-Pro, implicated as the causative agent in early-onset familial amyloid polyneuropathy was expressed and characterized, and its denaturation pathway and amyloidogenicity were compared to those of wild-type transthyretin. The overlap-extension polymerase chain reaction (PCR) methodology was used to introduce the Leu-55-Pro mutation into the transthyretin DNA sequence and to construct a new expression system. The Leu-55-Pro variant of transthyretin was expressed with a leader sequence to ensure secretion into the periplasmic space of Escherichia coli. Transthyretin's resistance to sodium dodecyl sulfate- (SDS-) induced denaturation was utilized to measure the quaternary stability as a function of pH employing SDS-polyacrylamide gel electrophoresis (PAGE) in the presence and absence of an amyloid fibril inhibitor, Z 3-14. These studies reveal that the Leu-55-Pro TTR tetramer is significantly less stable than wild-type TTR. This is relevant because we have previously shown that the partial denaturation of transthyretin is sufficient to effect amyloid fibril formation from a denaturation intermediate which may be a structured monomer. The ability of Leu-55-Pro TTR to denature to the amyloidogenic intermediate at pHs where the wild-type protein is stable may explain the variant's propensity to form amyloid fibrils in vitro and in vivo where the wild-type protein remains stable and nonamyloidogenic. Congo red binding, polarized light microscopy, and electron microscopy confirm the characteristic structure of amyloid fibrils produced from Leu-55-Pro TTR in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
McCutchen
S L
SL
Department of Chemistry, Texas A&M University, College Station 77843-3255.
Colon
W
W
Kelly
J W
JW
D000682:Amyloid / Q000096:biosynthesis*; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011228:Prealbumin / Q000235:genetics* / Q000378:metabolism; D011392:Proline / Q000235:genetics*
D000682:Amyloid; D017931:DNA Primers; D011228:Prealbumin; D011392:Proline; D007930:Leucine