Proteolytic activity of Crocodylus porosus transthyretin protease and role of the terminal polypeptide sequences

Abstract

Human transthyretin (TTR), a recently identified protease, participates in the biology of high density lipoprotein and in the nervous system. In the present study, we determined whether TTR from a non-mammal Crocodylus porosus (crocTTR) has proteolytic activity and whether the N-and C-termini of the TTR polypeptide affect the proteolytic activity. The proteolytic activity of crocTTR and three chimeric crocTTRs: xenoN/crocTTR (crocTTR in which the N-terminal sequence was replaced with that of Xenopus laevis TTR), pigC/crocTTR (crocTTR in which the C-terminal sequence was replaced with that of Sus scrofa TTR), and xenoN/pigC/crocTTR (crocTTR in which the N-and C-terminal sequences were replaced with that of X. laevis TTR and S. scrofa TTR, respectively) were studied and compared. Using either casein or apoAI as a substrate, crocTTR had a lower proteolytic activity than human TTR. Replacing the C-terminal sequence of crocTTR increased the activity (casein: 1008 ± 36 pmol/min; apoAI: 231 ± 43 pmol/min), whereas replacing the N-terminal sequence decreased the activity (casein: 299 ± 26 pmol/min; apoAI: 31.5 ± 2.0 pmol/min). The activity of xenoN/pigC/crocTTR (casein: 502 ± 11 pmol/min; apoAI: 371 ± 23 pmol/min) was higher than those of crocTTR or xenoN/crocTTR, but similar to that of pigC/crocTTR. These results are the first to show the proteolytic activity of reptile TTR, and that the activity is changed when the N-and/or C-terminal amino acid sequences of the TTR subunit are changed.

The influence of Zn2+-binding motif sequence on the catalytic activity of transthyretin towards amyloid beta

“…Moreover, a minor band of dimeric TTR with the mass ∼2 folds of monomeric TTR was observed in both mutated TTRs, which also similar to their wild types. This result was in accordance with other TTRs previously reported [9,19]. The dimer of TTR could be seen when TTR was not completely denatured even by heating under reducing condition for 30 min.…”

“…Our recent experiments showed that TTRs from Crocodylus porosus and Gallus gallus also have the proteolytic activity toward Aβ and other TTR substrates although the HXHXE motif does not exist [9,17]. Moreover, changing N-and/or C-terminal region of TTR, to be longer and more hydrophobic, increases its proteolytic activity [9]. From these findings, it raised the hypothesis that the sequence nearby the catalytic site, especially those in the Nand C-terminus of TTR, participate in the proteolytic mechanism of TTRs.…”

“…Although the entire amino acid sequence of crocTTR polypeptide is highly conserved and similar to that of the eutherian TTRs, the Zn 2+ -chelating sequence HXHXE does not exist in the reptile TTR. The addition of a few amino acid residues to the N-and/or C-terminal sequence of C. porosus and human TTR polypeptides resulted in chimeric TTRs with higher and more specificity of the proteolytic activity [9]. Accordingly, we hypothesized not only Zn 2+ -chelating motif sequence HX-HXE but also the sequence nearby particularly those in the N-and C-terminal regions of TTR polypeptide play roles in the proteolytic activity of TTR.…”

“…Predominant changes in TTR structure occur at the N-terminal sequence, and the N-terminal region of the avian and the reptilian are longer and more hydrophobic than that of the eutherian [5,6]. The changes influence on not only the function of TTR as distributors for TH and retinal [7,8], but also its catalytic property [9,10]. Beside functioning as TH and retinal distributors, TTR proteolytic activity towards amyloid beta (Aβ) and a few specific target proteins is evidenced for its important physiological roles in the central and peripheral nervous systems of human [11,12].…”

The hydrophobic C‐terminal sequence of transthyretin affects its catalytic kinetics towards amidated neuropeptide Y

“…Our previous studies demonstrated a connection between the alteration in length and hydropathy of the amino acid sequence of the N‐terminal segment of TTR and the binding affinities for thyroid hormone and RBP . A few changes of amino acid sequence have also been observed in the C‐terminal sequence of TTR and these were demonstrated to lead to an increase in the binding affinity for RBP and catalytic activity towards apolipoprotein A‐I . However, the effect of the changes in the C‐terminal region on the catalytic activity of TTR towards amidated NPY remains unknown.…”