Lupinus albus Protein Components Inhibit MMP-2 and MMP-9 Gelatinolytic Activity In Vitro and In Vivo

Abstract

Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are regarded as important clinical targets due to their nodal-point role in inflammatory and oncological diseases. Here, we aimed at isolating and characterizing am MMP-2 and-9 inhibitor (MMPI) from Lupinus albus and at assessing its efficacy in vitro and in vivo. The protein was isolated using chromatographic and 2-D electrophoretic procedures and sequenced by using MALDI-TOF TOF and MS/MS analysis. In vitro MMP-2 and 9 inhibitions were determined on colon adenocarcinoma (HT29) cells, as well as by measuring the expression levels of genes related to these enzymes. Inhibitory activities were also confirmed in vivo using a model of experimental TNBS-induced colitis in mice, with oral administrations of 15 mg·kg^-1. After chromatographic and electrophoretic isolation, the L. albus MMP-9 inhibitor was found to comprise a large fragment from δ-conglutin and, to a lower extent, small fragments of β-conglutin. In vitro studies showed that the MMPI successfully inhibited MMP-9 activity in a dose-dependent manner in colon cancer cells, with an IC50 of 10 µg·mL^-1 without impairing gene expression nor cell growth. In vivo studies showed that the MMPI maintained its bioactivities when administered orally and significantly reduced colitis symptoms, along with a very significant inhibition of MMP-2 and -9 activities. Overall, results reveal a novel type of MMPI in lupine that is edible, proteinaceous in nature and soluble in water, and effective in vivo, suggesting a high potential application as a nutraceutical or a functional food in pathologies related to abnormally high MMP-9 activity in the digestive system.

Keywords: Lupinus albus protein; MMP inhibitor; MMP-2; MMP-9; gastrointestinal diseases; gelatinases; nutraceutical.

Figure 1

L. albus protein profile and corresponding MMP-9 inhibitory activity. (A) Size exclusion chromatography (SEC) in a Superdex 75 column of L. albus cotyledon total protein extracts. (B) Protein peaks were collected as fractions 1 to 6 and analyzed for polypeptide composition by SDS-PAGE. (C) MMP-9 proteolytic activity of fractions 1 to 6 obtained by SEC, as quantified by the DQ-gelatin method. Results are expressed in arbitrary units of fluorescence and represent an average of three replicates ± SD. ** p < 0.001 when compared to control.

Figure 2

Characterization of the L. albus MMP-9 inhibitory protein. I: HPLC separation and peak purification of fraction 4 obtained by SEC (Figure 1A): (A) Reverse phase HPLC chromatographic profile of the MMP-9 inhibitory protein fraction 4 previously isolated from L. albus cotyledons by gel filtration. The main peaks obtained were collected as fractions 1 to 4. (B) MMP-9 proteolytic activity of fractions 1 to 4 in A, as quantified by the DQ-gelatin method. The results are expressed in arbitrary units of fluorescence and represent an average of three replicates ± SD. * p < 0.05. II: Electrophoretic analyses: (C) The polypeptide profile of fraction 2 in A was analyzed by denaturing electrophoresis performed under non-reducing (NR) and reducing (R) conditions. Representative image of the polypeptide composition of isolated MMPI from L. albus cotyledons separated by SDS-PAGE. (D) Two-dimensional electrophoretic separation of the isolated MMPI using 2D-GE IPG pH 3 to 6, 7 cm long, followed by SDS-PAGE 17.5% (w/v) acrylamide. III: Mass determination: (E) Size exclusion chromatography of the isolated MMPI (fraction 2 in A) performed under non-denaturing conditions, showing its mass. (F) Intact protein analysis of the isolated MMPI by MALDI TOF MS.

Figure 3

Amino-acid sequence of the L. albus MMPI. Mass spectrometry analysis of the isolated MMPI spots obtained from 2D analysis, AL1, and AL2, respectively, as demonstrated on the top right. The matched peptides are shown in red.

Figure 4

L. albus MMPI effect on MMP-9 and MMP-2 activity, cell proliferation, and gene expression in HT29 cells. (A) Dose-effect of the isolated L. albus MMPI on total gelatinolytic activity. The isolated MMPI was added at concentrations of 100, 50, 10, and 5 µg·mL⁻¹ and gelatinolytic activity was measured by the DQ fluorogenic assay. Gelatinase activities are expressed as percentage of controls. (B) Representative image of the zymographic profiles of specific MMP-9 and MMP-2 activities. White bands are consistent with higher gelatinolytic activities. HT29 cells were exposed to 50 µg·mL⁻¹ of the MMPI, and extracellular extracts were loaded on 12.5% (w/v acrylamide) polyacrylamide gels co-polymerized with 1% (w/v) gelatin. (C) HT29 cell growth after 48-hour exposure to different concentrations of the L. albus MMPI and representative picture of HT29 cells morphology in controls and in the highest MMPI concentration. Cells were grown in the presence of 100, 50, 10, and 5 µg protein mL⁻¹ and stained with MTT. Values are expressed as a percentage of the control. (D) L. albus MMPI influence on MMP-2, MMP-9, and TIMP-1 gene expression. Cells were grown in the presence of 50 µg protein·mL⁻¹ and transcripts were quantified by real-time PCR (RT-qPCR). Relative gene expression values are presented as log²-fold-change values in relation with the control conditions, using as reference gene Beta-actin. Data were normalized in relation to controls and the graphic is expressed as 2^−ΔΔCt values. All values represent the averages of at least three replicate experiments (n = 3) ± SD in each assay. ** p < 0.001 when compared to controls.

Figure 5

Effect of the L. albus MMPI administration on the colon tissue gelatinase activities of MMP-2 and MMP-9 from colitis-induced mice. (A): Representative image of the zymographic profiles of MMP-9 and MMP-2 activities of the colons. Protein extracts of the colon were loaded on 12.5% (w/v acrylamide) polyacrylamide gels co-polymerized with 1% (w/v) gelatin. (B): Densitometric analysis of the gelatinolytic activity of MMP-9 and MMP-2 obtained in the zymographies. Control group (n = 6); Colitis group (n = 10); Colitis + MMPI =colon from animals treated with L. albus MMPI (15 mg·kg⁻¹, n = 9, p.o.). Results are average of at least three replicates. ^# p < 0.05 vs. Colitis; * p < 0.05 vs. Control and ** p < 0.001 vs. Control.