Production of Recombinant Proteins: ADAMTS-2 – in insects cells Sf9; BMP-1/mTLD – in mammalian cells HT1080

Polyclonal Antibodies Against Recombinant Proteins: In New Zeeland rabbits by Bio-Synthesis, Inc (Lewisville, TX, USA); Secondary antibodies conjugated with horseradish

peroxidase were used to visualize the antigens by the ECL method (Amersham)Preparation of Synthetic Peptides: By Cybesyn (USA) and Sigma (Germany)

Enzymatic Activity Assays: 2 units of active mTld or ADAMTS-2 were incubated with about 0.3 mg of procollagen type I. Reaction products were separated in 7.5% SDS-polyacrylamide gels

and assayed following staining and documenting with a digital camera using a software 1D-Kodak and Image Quant v5.2 (GE Healthcare, USA formerly Molecular Dynamics, CA, USA).

Assay of Interactions of the mTld and Its Domains with Peptides: The peptides was incubated with procollagen C-endopeptidase or its -chymotrypsin derived fragments. The

reaction products were analyzed by Western method. Interactions of the peptides with native form of mTld were also assayed by standard reverse phase high pressure liquid chromatography

Inhibition of growth of the blood vessels and cells: Incubation of peptides AS1.11 and S1.16 with cultured chick embryos and three types of mammalian cells (HT1080, HUVEC – endothelial

cells and fibroblasts).

Inhibition of Enzymic Activity of Procollagen C-Endopeptidase Inhibits Angiogenesis

Aleksandra Duma1, Marta Lesiak1, Anna Szydlo1, Maciej Kajor3, Aleksander L. Sieron1. 1Department of General, Molecular Biology and Genetics, Silesian Medical University in Katowice, Centre of Excellence for Research and Teaching of Molecular Biology of Matrix and Nanotechnology, Network CoE BioMedTech Silesia, 2Department of Pathomorphology, Silesian Medical University in Katowice

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 a 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.

Supporting grants: LTR 03/19/97, KBN – 3P05D03723, NN-1-008/03

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

REFERENCES

ACKNOWLEDGEMENTS

VHLDNK (734 to ?)

EAICGG (432 to ?)

KCPACG (328 to ?)

CGYEKP (510 to ?)

FRAGILE BLOOD VESSELS (confirmed)=

SLOWER TUMOR GROWTH (in progress)

CONCLUSIONS

HIPOTHESIS:

BLOCKERS = Drugs modulating amounts of:A. collagen: to much of collagen = fibrosis = scars collagen = ECM = angiogenesis = tumor growthtumor growthB. active lysyl oxidise : weak collagen cross-linking = fragile blood vessels; weak elastin cross-linking = fragile blood vessels

ADAMTS-2

TRIPLEHELICAL DOMAIN

N-PROPEPTIDE C-PROPEPTIDE

BMP-1/mTldBMP-1/mTld

PRO-COLLAGEN

Peptide name(a)

(Corresponding Residues in

Chordin)

Amino Acid Sequence M.W.(b) Soluble in

pH in water

S1.9 (737-745) Ser-Gln-Pro-Val-His-Leu-Pro-Asp-Gln 1245 water neutral

S1.16 (720-735) Gln-Lys-Arg-Thr-Val-Ile-Cys-Asp-Pro-Ile-Val-Cys-Pro-Pro-Leu-Asn 2022 water neutral

S1.16c (720-735) Gln-Lys-Arg-Thr-Val-Ile-Cys-Asp-Pro-Ile-Val-Cys-Pro-Pro-Leu-Asn(c) 2019 water neutral

S1.19 (727-745) Asp-Pro-Ile-Val-Cys-Pro-Pro-Leu-Asn-Cys-Ser-Gln-Pro-Val-His-Leu-Pro-Asp-Gln 2298 water neutral

AS1.1 (720-735) Gln-Lys-Arg-Thr-Val-Ile-Ser-Asp-Pro-Ile-Val-Cys-Pro-Pro-Leu-Asn(d) 2005 water neutral

AS2.1 (720-735) Gln-Lys-Arg-Thr-Val-Ile-Cys-Asp-Pro-Ile-Val-Ser-Pro-Pro-Leu-Asn 2005 water neutral

AS3.1 (720-735) Gln-Lys-Arg-Thr-Val-Ile-Ser-Asp-Pro-Ile-Val-Ser-Pro-Pro-Leu-Asn 1989 water neutral

AS1.11 (726-731) Cys-Asp-Pro-Ile-Val-Cys 874 water neutral

AS1.11c (726-731) Cys-Asp-Pro-Ile-Val-Cys 871 water neutral

Table 1. Sequence of peptides tested for inhibition of procollagen C-endopeptidase activity in mTld.(a) All peptides were conjugated with biotin on their amino-termini. (b) M.W. stands for molecular weight of the peptide.(c) Cys in italic – the cysteines forming internal disulfide bridge.(d) Ser in italic – the residues substituting for Cys.

1.Prockop D.J., Kivirikko K.I., Tuderman L., Guzman N.A. (1979) Biosynthesis of collagen. N. Engl. J. Med. 301:12-23, 75-85.

2.Li, S-W., Sieron, A.L., Fertala, A., Hojima, Y., Arnold, W.V., Prockop, D.J. (1995) The C-proteinase that processes procollagens to collagens is identical to the protein previously

identified as bone morphogenic protein-1. Proc. Natl. Acad. Sci. USA 93:4127-5130.3.Sieron A.L., Tretiakova A., Jameson B.A., Segall M.L., Lund-Katz S., Khan M.T., Li S.W. and

Stöcker W. (2000) Structure and function of procollagen C-proteinase (mTolloid) domains determined by protease digestion, circular dichroism, binding to procollagen type I, and

computer modeling. Biochemistry 39:3231-3239.

Fig 1. Assay of specific inhibitory activity of synthetic peptide inhibitors on procollagen C- and N-endopeptidase.

Fig 2. Interactions of synthetic peptide inhibitors with domains of procollagen C-endopeptidase.

Fig 3. Inhibition of growth of blood vessels in cultured chick embryos. Fig 4. Inhibition of proliferation of the cell as

a percent of control due to action of synthetic peptides

1. The tests conducted in these studies indicate that the minimal fragment required for interaction with the inhibitors is located in the CUB4/5 since it was present in all analyzed tryptic

fragments. It, also, explains the specificity of the inhibitor to just mTld but not ADAMTS-2 or astacin and other MMPs (data not shown).

2. The inhibitory peptide could interfere with the action of the second EGF-like motif possibly binding to the CUB4/5 domain and this way changing its space orientation. Thus, our results

support the hypothesis that the CUB4/5 domains may be responsible in mTld for its substrate specificity.

3. The observation that linear forms of the inhibitory peptides were better then their cyclic variants could be explained by assumption that the site for binding of the inhibitory peptide is

somewhere inside of the CUB4/5 structure or in just one of these two domains, therefore, better accessible for elongated structure and only after reaching the niche the inhibitor changes its

conformation forming the disulfide bond between the two Cys residues stabilizing the interaction. The inhibitory peptide showed strong affinity to the enzyme, as it has been

observed in the assays with denatured mTld that part of the inhibitor pole was converted to its cyclic form. The change of conformation in the presence of denatured enzyme, also, explains

binding of the inhibitory peptides to -chymotrypsin generated peptides electro-blotted following heat denaturation and SDS PAGE separation.

4. The inhibitory action of peptides lacking either of the two cysteines indicates that cysteine is critical for peptide activity at least in assays conducted in vitro.

5. The inhibition of procollagen C-endopeptidase activity could be utilized in regulation of processes that depend on the mTld activity such as ECM building and control of its quality.

6. The inhibition of mTld activity by the inhibitory peptides should also affect cell proliferation in such situations as tumour growth, angiogenesis, and fibrosis.