Abstract
The primary structure of a new Kunitz-type protease inhibitor InhVJ from the sea anemone Heteractis crispa (Radianthus macrodactylus) was determined by protein sequencing and cDNA cloning. InhVJ amino acid sequence was shown to share high sequence identity (up to 98%) with the other known Kunitz-type sea anemones sequences. It was determined that the P1 Thr at the reactive site resulted in a decrease of the Ki of InhVJ to trypsin and α-chymotrypsin (7.38 × 10−8 M and 9.93 × 10−7 M, respectively). By structure modeling the functional importance of amino acids at the reactive site as well as at the weak contact site were determined. The significant role of Glu45 for the orientation and stabilization of the InhVJ-trypsin complex was elucidated. We can suggest that there has been an adaptive evolution of the P1 residue at the inhibitor reactive site providing specialization or functional diversification of the paralogs. The appearance of a key so-called P1 Thr residue instead of Lys might lead to refinement of inhibitor specificity in the direction of subfamilies of serine proteases. The absence of Kv channel and TRPV1-receptor modulation activity was confirmed by electrophysiological screening tests.
Highlights
Protein inhibitors regulate the activity of proteases involved in biochemical processes such as blood clotting, complement system, digestion, inflammation, apoptosis and others
It is worth mentioning that the sea anemone Heteractis crispa (Radianthus macrodactylus) produces, besides P1 Lys/Arg inhibitors, atypical polypeptides with the Thr residue at P1 position of its reactive site
In this paper we report the determination of the primary structure and structure-function features of a new inhibitor InhVJ from the sea anemone H. crispa
Summary
Protein inhibitors regulate the activity of proteases involved in biochemical processes such as blood clotting, complement system, digestion, inflammation, apoptosis and others. The most known sea anemone inhibitors belong to the widely studied BPTI/Kunitz inhibitor family [24], except for Kasal-type inhibitors from Anemonia sulcata [13] Their stable molecules consist of 56–60 amino acid residues, six of which are conservatively positioned cysteine residues forming three disulfide bonds. The majority of the sea anemone inhibitors have a positively charged Lys or Arg residue at the P1 position of the reactive site, which is known to be essential for inhibition of serine proteases, such as trypsin and α-chymotrypsin [11,17]. It is worth mentioning that the sea anemone Heteractis crispa (Radianthus macrodactylus) produces, besides P1 Lys/Arg inhibitors, atypical polypeptides with the Thr residue at P1 position of its reactive site. The structures of serine protease complexes were analyzed
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