Specific inhibition of procollagen C-endopeptidase activity by synthetic peptide with conservative sequence found in chordin.
Procollagen C-endopeptidase (BMP-1) and N-endopeptidase (ADAMTS-2) are key enzymes for correct and efficient conversion of fibrillar procollagens to their self assembling monomers. Thus, they have an essential role in building and controlling the quality of extracellular matrices (ECMs). Here, we tested inhibition of activity of the largest variant of BMP-1, a recombinant mammalian tolloid (mTld), in vitro by three synthetic peptides with conservative amino-acid sequences found in chordin using procollagen type I as a substrate. We also verified the specific action of best inhibitory 16 amino-acid peptide in the procollagen type I cleavage assay with the use of ADAMTS-2 (procollagen N-endopeptidase). Subsequently, we determined the critical residues and minimal sequence of six amino acids in the original 16 amino-acid peptide required to maintain the inhibitory potential. Studies on the interactions of 6 and 16 amino acid long peptides with the enzyme revealed their binding to non-catalytic, regulatory domains of mTld; the inhibitory activity was not due to the competition of peptides with the substrate for the enzyme active center, because mTld did not cleave the peptides. However, in the presence of mTld both peptides underwent cyclization by disulfide bond formation. Concluding, we have shown that procollagen C-endopeptidase may be specifically blocked via its non-catalytic domains by synthetic peptide consisting of 6 amino acids in the sequence found in highly conservative region of chordin. Thus, we hypothesize that the 6 amino-acid peptide could be a good candidate for anti-fibrotic drug development.
Department of General and Molecular Biology, Center of Excellence for Research and Teaching of Molecular Biology of Matrix and Nanotechnology, Silesian Medical University in Katowice, Katowice, Poland.
Marta
M
Lesiak
Aleksandra
A
Augusciak-Duma
Anna
A
Szydlo
Ksymena
K
Pruszczynska
Aleksander L
AL
Sieron
D051722:ADAM Proteins / Q000378:metabolism; D000071096:ADAMTS Proteins; D000071121:ADAMTS4 Protein; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D002417:Cattle; D017124:Conserved Sequence; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000494:pharmacology*; D011348:Procollagen N-Endopeptidase / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D055394:Tolloid-Like Metalloproteinases
D019485:Bone Morphogenetic Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C054234:chordin; D045726:Metalloproteases; D055394:Tolloid-Like Metalloproteinases; D051722:ADAM Proteins; D000071096:ADAMTS Proteins; D008666:Metalloendopeptidases; C000609307:ADAMTS2 protein, human; D011348:Procollagen N-Endopeptidase; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1; D000071121:ADAMTS4 Protein