2. Chronic Disease Program, Ottawa Hospital Research Institute, U Ottawa, 725 Parkdale Ave, Ottawa, ON K1Y4E9, Canada.
Background: A number of human conditions collectively known as amyloidosis were found to be associated with self-aggregation of a specific group of proteins. This fibril like protein aggregates deposit in different tissues including the central nervous system leading to amyloid polyneuropathies (AP), systemic senile amyloidosis (SSA), amyloidotic cardiomyopathy (AC) and carpal tunnel syndrome (CTS). The most common protein in this group is human transthyretin (hTTR). Similar to beta amyloid peptide of Alzheimer's Disease, hTTR forms amyloid type fibrils following its proteolytic cleavage at the C-(carboxy) terminus leading to aggregation. hTTR is normally present in blood where it binds with thyroid hormone thyroxine T4 and Retinol Binding Protein-Vitamin A complex and mediates their transport. It is also present in lower amount in cerebrospinal fluid as a major carrier of thyroxine hormone. Deposition of hTTR fibril aggregates is responsible for SSA usually observed in older population with age >60 years which is regulated by its mutant forms. The exact mechanism of this disorder is still unclear. Studies confirmed that C-terminally truncated hTTR undergoes rapid self-aggregation leading to amyloidosis. The site of this cleavage and the protease involved has not been fully characterized although it is proposed to be at Lys48↓Thr49 residues.
Objective: The major goal of this study is to confirm that hTTR is cleaved by a trypsin like enzyme at the position indicated by vertical arrow Ala-Ser-Gly-Lys48↓Thr49-Ser-Glu-Ser and that this cleavage can be blocked by a trypsin-inhibitor.
Methods: A 20 mer peptide comprising the wild type sequence from residue (41-60) of hTTR was synthesized, purified by RP-HPLC and fully characterized by mass spectrometry. Three additional mutant peptides namely Lys48/Ala, Ser52/Pro and Thr49/Ala were also prepared. These mutant peptides were designed because of their pathological implications in SSA syndrome in varying degrees. All peptides were digested in vitro with trypsin under identical condition to examine and compare their cleavage efficiencies.
Results: Our results showed that while the control wild type peptide was fully cleaved at Lys48↓Thr49 by trypsin within an hour the corresponding Lys48/Ala mutant remained completely uncleaved under identical condition. In contrast the Ser52/Pro and Thr49/Ala mutant peptides were cleaved with varying efficiency. These results are consistent with those reported for the physiological full length hTTR proteins. Moreover the above cleavage can be blocked efficiently by a trypsin inhibitor (dimethyl formamide), reported for the first time in this study. Energy minimised 3D modeling study revealed that Lys48/Ala mutant exhibits a unique structural conformation compared to the corresponding wild type control peptide suggesting its potential role in regulating proteolytic cleavage. Overall our data confirmed the key role of a trypsin like enzyme in hTTR proteolysis leading to its truncation which facilitates its self-aggregation leading to amyloidosis. The study also highlighted a potential therapeutic strategy for hTTR associated amyloidosis by targeting this enzyme with specific inhibitors.
Keywords: Human transthyretin (hTTR), hTTR proteolysis, hTTR mutation, trypsin, reverse phase high performance layer chromatography (RP-HPLC), mass spectrum, trypsin inhibitor, trypsin activation, protein aggregation