Abstract
This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar K(i) values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature.
Highlights
Several protein inhibitors of metallocarboxypeptidases have already been described
Isolation and Purification of SmCI—The proteinaceous inhibitor was isolated from the tentacle crown of S. magnifica by both affinity and reverse phase chromatography
We identified and characterized a novel metallocarboxypeptidase inhibitor (SmCI) isolated from the tentacle crown of the marine annelid S. magnifica
Summary
Several protein inhibitors of metallocarboxypeptidases have already been described. Results: We have characterized of a tri-domain inhibitor from Sabellastarte magnifica, recombinant forms and truncated variants. Among the available natural sources of protease inhibitors, one of the most attractive and rather unexplored is the marine fauna, especially invertebrates (including numerous phyla, genera, and species) Several such inhibitors capable of independently inhibiting proteases of different mechanistic classes have been described, in the phyla Cnidaria [17,18,19,20,21,22,23,24,25,26,27], Mollusca (28 –31), and Annelida [32, 33]. We describe the isolation, characterization, cDNA cloning, and sequence analysis of a novel tight-binding metallo-CP inhibitor found in S. magnifica This inhibitor is bifunctional, acting on serine proteases, featuring three BPTI/ Kunitz domains not present in other proteinaceous CP inhibitors. Our findings provide insight into the distinctive features of such molecules in the still quite unexplored world of marine invertebrates as a potential rich and diverse source of new substances of biotechnological interest
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