Inhibition of Enzymic Activity of Procollagen C-Endopeptidase Inhibits Angiogenesis

1. Inhibition of Enzymic Activity of Procollagen C-Endopeptidase Inhibits Angiogenesis KCPACG (328 to ?) EAICGG (432 to ?) CGYEKP (510 to ?) VHLDNK (734 to ?) ALEKSANDER L. Sieron,Marta Lesiak,Aleksandra Augusciak, Anna K. Szydlo,Ksymena Urbanek, Martyna Dubrawska Department of General, Molecular Biology and Genetics, Centre of Excellence for Research and Teaching on Molecular Biology of Matrix and Nanotechnology Silesian Medical University in Katowice, e-mail: alsieron@slam.katowice.pl Anna Zborek -Center of Oncology, Maciej Kajor & Dariusz Golka -Department ofPathomorphology Procollagen C-endopeptidase (BMP-1) and procollagen N-endopeptidase (ADAMTS-2) are two enzymes crucial for conversion of fibrillar procollagens to self assembling collagen monomers. Thus, here we tested inhibition of the largest variant of BMP-1, a recombinant mammalian tolloid (mTld) in vitro, on procollagen type I and in angiogenesis, ex vivo in cultured rat aortas, and in chick embryos. Our results revealed that a peptide consisting of 16 residues with amino acid sequence found in non-collagenous substrate for mTld, Chordin inhibited the enzyme activity at micromolar concentrations. Subsequently, we determined amino acids and minimal 6 amino acid sequence required to maintain the inhibition. The peptides inhibited the microvessels formation in cultured rat aortas, blocked blood vessel growth in chick embryos, and slowed cells proliferation in cell cultures. Studies on interactions of inhibitors with the enzyme revealed interactions with regulatory domains of mTld. The interaction of peptide with the enzyme changed its conformation by cycling it through the disulfide bond. Concluding, we were able to block the angiogenesis inhibiting activity of mTld. The results strongly indicate the key role(s) of mTld in formation of new blood vessels and maintenance of their growth. HIPOTHESIS: PEPTIDES TESTED Peptide BLOCKING N-PROPEPTIDE C-PROPEPTIDE YES S1.16 S S YES S1.19 TRIPLEHELICAL DOMAIN S S ADAMTS-2 BMP-1/mTld S1.9 NO S1.16c NO BLOCKERS = Drugs modulating amounts of: S S WEAK AS1.1 S A. collagen: to much of collagen = fibrosis = scars collagen = ECM = angiogenesis = tumor growth AS2.1 WEAK S VERYWEAK AS3.1 • B. active lysyl oxidise : weak collagen cross-linking = fragile bloodvessels • weak elastin cross-linking = fragile blood vessels AS1.11 YES S S BLOCKING OF mTld ACTIVITY BINDING OF PEPTIDES TO mTld FRAGMENTS Blocking of angiogenesis by both peptides S1.16 & AS1.11 was due to inhibition of cell proliferation S1-16 S1-16c S1-16 (16-mer) PEPTIDE [~160mg/ml] S S S S S S Incubation Time at 35oC [min.] with the peptide without mTld without mTld 0 Temperature Denatured mTld 0 without mTld 60 120 11.1 15.1 Peptide AS1.11 (6-mer) [~160mg/ml] S S with mTld 30 without mTld Both peptides S1.16 & AS1.11 inhibited growth of blood vesels in cultured chick embryos 120 with mTld with mTld 120 • FRAGILE BLOOD VESSELS (confirmed) • = • SLOWER TUMOR GROWTH (in progress) 60 with mTld plus BME after incubation 120 with mTld 120 11.1 15.1 Supporting grants to ALS: LTR 03/19/97, 2. KBN – 3P05D03723, and 3. NN-1-008/03