The proteolytic profile of human cancer procoagulant suggests that it promotes cancer metastasis at the level of activation rather than degradation.
Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.
Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, P.O. Box 77000, Port Elizabeth, 6031, South Africa.
Nalise Low Ah
NL
Kee
Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, P.O. Box 77000, Port Elizabeth, 6031, South Africa.
Jason
J
Krause
Biomedical Biotechnology Research Unit, Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown, 6140, South Africa.|Centre for Chronic Disease, College of Health and Biomedicine, Victoria University, Melbourne, VIC, Australia.
Gregory L
GL
Blatch
Laboratory of Biomolecular Function, Graduate School of Life Sciences, Tohoku University, Sendai, 981-8555, Japan.
Koji
K
Muramoto
Department and Graduate School of Sustainable Resource Sciences, Mie University, 1577 Kurima-Machiyacho, Tsu, 514-8507, Japan.
Kazuo
K
Sakka
Department and Graduate School of Sustainable Resource Sciences, Mie University, 1577 Kurima-Machiyacho, Tsu, 514-8507, Japan.
Makiko
M
Sakka
Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, P.O. Box 77000, Port Elizabeth, 6031, South Africa.
Ryno J
RJ
Naudé
Probiodrug AG, Weinbergweg 22, 06120, Halle, Germany.
Leona
L
Wagner
Probiodrug AG, Weinbergweg 22, 06120, Halle, Germany.
Raik
R
Wolf
Department of Drug Design and Target Validation, Fraunhofer-Institute for cell therapy and immunology, Weinbergweg 22, 06120, Halle, Germany.
Jens-Ulrich
JU
Rahfeld
Department of Drug Design and Target Validation, Fraunhofer-Institute for cell therapy and immunology, Weinbergweg 22, 06120, Halle, Germany.
Hans-Ulrich
HU
Demuth
Department of Pharmaceutical Biochemistry, Medical University of Łódź, ul. Muszynskiego 1, 90151, Lodz, Poland.
Wojciech P
WP
Mielicki
Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, P.O. Box 77000, Port Elizabeth, 6031, South Africa. carminita.frost@nmmu.ac.za.
Carminita L
CL
Frost
D000595:Amino Acid Sequence; D024141:Collagen Type IV / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007797:Laminin / Q000378:metabolism; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D009369:Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D059748:Proteolysis; D013379:Substrate Specificity
D024141:Collagen Type IV; D005353:Fibronectins; D007797:Laminin; D009363:Neoplasm Proteins; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant
CP assay; Extracellular matrix; Factor X; Fibronectin; Kinetics; Peptide substrates