Transthyretin, a new cryptic protease.
Transthyretin (TTR) is a plasma homotetrameric protein that acts physiologically as a transporter of thyroxine (T(4)) and retinol, in the latter case through binding to retinol-binding protein (RBP). A fraction of plasma TTR is carried in high density lipoproteins by binding to apolipoprotein AI (apoA-I). We further investigated the nature of the TTR-apoA-I interaction and found that TTR from different sources (recombinant and plasmatic) is able to process proteolytically apoA-I, cleaving its C terminus after Phe-225. TTR-mediated proteolysis was inhibited by serine protease inhibitors (phenylmethanesulfonyl fluoride, Pefabloc, diisopropyl fluorophosphate, chymostatin, and N(alpha)-p-tosyl-l-phenylala-nine-chloromethyl ketone), suggesting a chymotrypsin-like activity. A fluorogenic substrate corresponding to an apoA-I fragment encompassing amino acid residues 223-228 (Abz-ESFKVS-EDDnp) was used to characterize the catalytic activity of TTR, including optimum reaction conditions (37 degrees C and pH 6.8) and catalytic constant (K(m) = 29 microm); when complexed with RBP, TTR activity was lost, whereas when complexed with T(4), only a slight decrease was observed. Cell lines expressing TTR were able to degrade Abz-ESFKVS-EDDnp 2-fold more efficiently than control cells lacking TTR expression; this effect was reversed by the presence of RBP in cell culture media, therefore proving a TTR-specific proteolytic activity. TTR can act as a novel plasma cryptic protease and might have a new, potentially important role under physiological and/or pathological conditions.
Liz
Márcia Almeida
MA
Molecular Neurobiology, Instituto de Biologia Molecular e Celular, Rua Campo Alegre 823, 4150-180 Porto, Portugal.
Faro
Carlos José
CJ
Saraiva
Maria João
MJ
Sousa
Mónica Mendes
MM
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011228:Prealbumin / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism
D016632:Apolipoprotein A-I; D010446:Peptide Fragments; D011228:Prealbumin; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D010450:Endopeptidases