Isolation of Two Plant Proteinases in Latex from Carica candamarcensis Acting as Mitogens for Mammalian Cells

Abstract

In a prior study we showed evidence that latex from Carica candamarcensis contains a protein fraction that stimulates mammalian cell proliferation. In this report we describe the isolation of two proteinases responsible for this effect. Both proteinases (P1, P2) display a relative mass of 23 kDa and following chromatographic purification stimulate proliferation of fibroblastic and epithelial cells. P2 added to L929 fibroblasts at 2.5 nM enhances proliferation by 60 %. We further demonstrate that its cellular effect is linked to an increase in activity of Erk2, a component of the MAP kinase pathway. To our knowledge, this is the first known plant proteinase to exert a proliferative effect in mammalian cells. This novel mitogenic property attributed to a purified cysteine proteinase may explain some of the therapeutic actions attributed to these enzymes.

Key words

Carica candamarcensis - Cariaceae - latex - mitogens - proteinases

References

• 1 Dussourd D E, Eisner T. Vein-cutting behavior: insect counterploy to the latex defense of plants.  Science. 1987;  237 898-901
• 2 Silva L G, Garcia O, Lopes M TP, Salas C E. Changes in protein profile during coagulation of latex from Carica papaya .  Braz J Med Biol Res. 1997;  30 615-9
• 3 Furie B, Furie B C. The molecular base of blood coagulation.  Cell. 1988;  53 505-18
• 4 Moutim V, Silva L G, Lopes M TP, Fernandes G W, Salas C E. Spontaneous processing of peptides during coagulation of latex from Carica papaya .  Plant Sci. 1999;  142 115-21
• 5 Clarck R AF. The molecular and cellular biology of wound repair. Second edition Editor Clarck RAF Plenum Press New York; 1998
• 6 Ahmed S U, Bar-Peled M, Raikhel N V. Cloning and subcellular location of an Arabidopsis receptor-like protein that shares common features with protein-sorting receptors of eukaryotic cells.  Plant Physiol. 1997;  114 325-36
• 7 Komatsu S, Abe K, Karibe H. Epidermal growth factor binding proteins in rice.  Biol Pharm Bull. 1996;  19 905-8
• 8 Kohorn B D, Lane S, Smith T A. An Arabidopsis serine/threonine kinase homologue with an epidermal growth factor repeat selected in yeast selected for its specificity for a thylakoid membrane protein.  Proc Natl Acad Sci. 1992;  89 10 989-92
• 9 Molloy C J, Pawlowski J E, Taylor D S, Turner C E, Weber H, Peluso M. Thrombin receptor activation elicits rapid protein tyrosine phosphorylation and stimulation of the raf-1/MAP kinase pathway preceding delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for an obligate autocrine mechanism promoting cell proliferation induced by G-protein-coupled receptor agonist.  J Clin Invest. 1996;  97 1173-83
• 10 Bravo L M, Hermosilla L, Salas C E. A biochemical comparison between latex from C. candamarcensis and C. papaya .  Braz J Med Biol Res. 1994;  27 2831-42
• 11 Baeza G, Correa D, Salas C E. Proteolytic enzymes in C. candamarcensis .  J Sci Food and Agric. 1990;  51 1-9
• 12 Silva C A, Gomes M TR, Ferreira R S, Rodrigues K CL, do Val C G, Lopes M TP, Mello V J, Salas C E. A mitogenic protein fraction in latex from Carica candamarcensis .  Planta Medica. 2003;  69 1-7
• 13 Gravina de Moraes M, Termignoni C, Salas C E. Biochemical characterization of a new cysteine endopeptidase from Carica candamarcensis L.  Plant Sci. 1994;  102 11-18
• 14 Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.  Anal Biochem. 1976;  72 248-54
• 15 Pereira M T, Lopes M TP, Meira W O, Salas C E. Purification of a cysteine proteinase from Carica candamarcensis L. and cloning of a genomic putative fragment coding for this enzyme.  Protein Express Purif. 2001;  22 249-57
• 16 Mynott T L, Ladhams A, Scarmato P, Engwerda C R. Bromelain from pineapple stems, proteolitically blocks activation of extracellular regulated kinases-2 in T cells.  J Immunol. 1999;  163 2568-75
• 17 Houck J C, Chang C M, Klein G. Isolation of an effective debriding agent from the stems of pineapple plants.  Int J Tissue React. 1983;  5 125-34
• 18 Hewitt H, Whittle S, Lopez S, Bailey E, Weaver S. Topical use of papaya in chronic skin ulcer therapy in Jamaica.  West Indian Med J. 2000;  49 32-3
• 19 Starley I F, Mohammed P, Schneider G, Bickler S W. The treatment of paediatric burns using topical papaya.  Burns. 1999;  25 636-9
• 20 Ford C N, Reinhard E R, Yeh D, Syrek D, De Las Morenas A, Bergman S B, Williams S, Hamori C A. Interim analysis of a prospective, randomized trial of vacuum-assisted closure versus the healthpoint system in the management of pressure ulcers.  Ann Plast Surg. 2002;  49 55-61
• 21 Walreavens V, Jaziri M, van Beeumen J, Schnek A G, Kleinschmidt T, Looze Y. Isolation and preliminary characterization of the cysteine-proteinases from the latex of Carica candamarcensis Hook.  Biol Chem Hoppe-Seyler. 1993;  374 501-6