Abstract:
: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)
journal_name
Biochemistryjournal_title
Biochemistryauthors
McCutchen SL,Colon W,Kelly JWdoi
10.1021/bi00096a024subject
Has Abstractpub_date
1993-11-16 00:00:00pages
12119-27issue
45eissn
0006-2960issn
1520-4995journal_volume
32pub_type
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