Tomato cystine-knot miniproteins: purification and new insights into the molecular mechanisms of their anti-angiogenic properties

D Treggiari1, B Molesini1, G Zoccatelli1, M Degan2, T Pandolfini1, P Minuz2. 1Department of Biotechnology, University of Verona, Verona, Italy, 2Department of Medicine, University of Verona, Verona, Italy

In a previous study we identified two tomato cystine-knot miniproteins (TCMP-1 and -2) potentially endowed with biological activities. We described their antiangiogenic properties in vitro, with inhibition of endothelial tube formation and cell migration induced by epidermal growth factor (EGF), associated with inhibition of ERK 1/2 phosphorylation (Cavallini et al. Br J Pharmacol 2011;162:1261-73). TCMPs belong to a large family of cysteine-rich proteins characterized by three intramolecular disulfide bonds forming a structural motif called cystine-knot. These proteins are widespread in eukaryotes and are being characterised for a broad spectrum of therapeutical activities (Kolmar H. Curr Pharm Des 2011;17:4329-36). The present study aims to further characterize and give a mechanistic insight into the anti-angiogenic activity of the cystine-knot miniprotein which is most abundant in mature tomato fruit (TCMP-2). We first set up a method for the extraction and purification of TMCPs from fruits and processed tomato products. Briefly, samples were homogenised, precipitated with 85% ammonium sulphate and dialysed; afterwards preparative FPLC gel filtration chromatography was carried out using agarose and dextran column (HiLoad 16/60 Superdex 75TM). Further purification was performed by reverse phase HPLC using a linear gradient of acetonitrile (30% to 40% in 40 minutes) and C4 columns (Altech Alltima C4-WP). To identify TCMP-2, the isolated protein was subjected to electrophoretic analysis and immunoblotting with primary rabbit polyclonal antibody (PRIMM). The purified TCMP-2 proved to be resistant to gastric peptidases (pepsin 40 μg mL-1 and pancreatin 24 μg mL-1) in simulated in vitro digestion. To evaluate the potential interaction with growth factor receptors we tested purified TCMP-2 (200 nmol L-1) on human umbilical vein endothelial cells (HUVECs) migration on 9.6 cm2 wells using 10 ng mL-1 vascular endothelial growth factor-A (VEGF-A) alone or in combination with 5 ng mL-1 (Mehta VB, Besner GE, Growth Factors 2007;25:253-63). The scratch wound-healing assay (n=15-55) showed that TCMP-2 inhibits by approximately 50% the increase in cell migration induced by VEGF-A (mean difference: -66 cells/field; 95% CI: -108, -25; P<0.01) and by EGF plus VEGF-A (-93 cells/field; 95% CI: -166, -19; P<0.001) (basal: 265±53 cells/field; VEGF-A: 418±79; EGF+VEGF-A: 446±100; Mean±SD; ANOVA: P<0.0001). Furthermore, an expression profile analysis of 85 genes involved in human angiogenesis pathway has been undertaken using the Human Angiogenesis RT2 Profiler PCR Array (SABiosciences). A significant (P<0.05) decrease in the expression of integrin-αV/β3 (i.d.NM_002210) and β2-microglobulin (i.d. NM_004048) (23% and 40%, respectively) was detected in HUVECs cells stimulated with 5 ng mL-1 EGF and treated with 100 nmol L-1 fruit TCMP-2, whereas VEGF-A (i.d. NM_003376) expression was doubled compared to untreated cells (P<0.05). The present results confirm the biological activity of TCMP-2 on endothelial cells. The inhibitory effects of TCMP-2 on cell migration implicates the interaction with EGF and VEGF-A receptors and their downstream signalling systems. This might explain the reduced expression of genes involved in the angiogenic process in EGF-stimulated HUVEC and a possible compensatory increase in VEGF-A transcription. Further investigation is required to evaluate the potential therapeutic implication of our findings.