Identification of chlorophyll a-b binding protein AB96 as a novel TGFβ1 neutralizing agent

Abstract

The discovery of compounds and proteins from plants has greatly contributed to modern medicine. Vernonia amygdalina Del. (Compositae) is used by humans and primates for a variety of conditions including parasitic infection. This paper describes the serendipitous discovery that V. amygdalina extract was able to bind to, and functionally inhibit, active TGFβ1. The binding agent was isolated and identified as chlorophyll a-b binding protein AB96. Given that active TGFβ1 contributes to the pathology of many infectious diseases, inhibiting these processes may explain some of the benefits associated with the ingestion of this species. This is the first plant-derived cytokine-neutralizing protein to be described and paves the way for further such discoveries.

Figure 1

VA aqueous extract [VA(aq)] contains an active TGFβ1 neutralizing agent. The effects of VA extract on endogenous (a) and exogenous (b) TGFβ1 levels in the supernatant of monocyte derived dendritic cells. n = 1 for a; n = 3 for b. (c) Unbound (remaining in supernatant after incubation) and bound (liberated after washing, acidification and neutralization) active TGFβ1 concentrations (starting conc.: 2 ng/ml) after incubation with VA(aq) for 2 h at room temperature (RT). Representative of 3 independent assays. Technical triplicates. Mean +/− SEM shown. (d) The unbound and bound IL-10, IL12, latent TGFβ1 and active TGFβ1 concentrations (starting conc.: 4 ng/ml) after incubation with VA(aq) for 2 h at RT. Technical triplicates. Mean +/− SEM shown. (e) The unbound and bound active TGFβ1 concentrations (starting conc.: 2 ng/ml) after incubation with sesquiterpene lactones and VA(aq) for 2 h at RT. Technical triplicates. Mean +/− SEM shown. (f) The levels of active TGFβ1 bound to aqueous extracts (1 mg/ml) from different species of plants [normalized to VA(aq)] when incubated with 2 ng/ml active TGFβ1. Species in order of x axis: (1) Vernonia amygdalina Del. (Compositae). (2) Vernonia arkansana DC. (Compositae). (3) Helianthus annuus L. (Compositae). (4) Aster dumosus Hoffm. (Compositae). (5) Campanula lactiflora M.Bieb. (Campanulaceae). (6) Petroselinum crispum (Mill.) Fuss (Apiaceae). (7) Ocimum basilicum L. (Lamiaceae). (8) Solanum lycopersicum L. (Solanaceae). (9) Spinacia oleracea L. (Amaranthaceae). (10) Rosa sericea Wall. Ex Lindl. (Rosaceae). (11) Vitis vinifera L. (Vitaceae). (12) Dieffenbachia amoena Bull. (unresolved, Araceae) (13) Dracaena marginata Hort. (Asparagaceae). (14) Lilium candidum L. (Liliaceae). Technical triplicates. Mean +/− SEM shown. (g) The effect of VA extract on luciferase activity in a functional active TGFβ1 reporter cell line. Biological triplicates. Mean +/− SEM shown. Representative of 2 independent assays. (h) The levels of TGFβ1 in different fractions eluted from an anion exchange column after loading with indicated factors, using indicated % of 2 M NaCl. Unpaired student’s t-test: *p < 0.05.

Figure 2

Chlorophyll a-b binding protein (CabBP) AB96 binds to and neutralizes active TGFβ1. (a) Analyzed mass spectrometry data showing pulled out proteins when using indicated bait proteins when a threshold of min number of peptides = 2, protein threshold = 20% and peptide threshold = 95%. (b) The chlorophyll a-b binding protein AB96 peptides from Pisum Sativum (Fabaceae), observed by mass spectrometry are highlighted in yellow. Green represents amino acid modifications: M, oxidation and C, carbamidomethyl. Pink represents Magnesium binding sites. (c) Quantitation of chlorophyll a-b binding protein AB96 pulled from VA(aq) using indicated bait protein. (d) Concentration of active TGFβ1 eluted (bound fraction) from different concentrations of chlorophyll a-b binding protein AB96 (CabBP) or Soybean trypsin inhibitor (STI) control after incubation for 2 h at RT. Mean +/− SEM shown. Example of 2 independent experiments (e) The effect of VA(aq), anti-TGFβ1 antibody, CabBP, STI and relevant controls, on luciferase activity in an active TGFβ1 reporter cell line. Biological quintuplets. Mean +/− SEM shown. Unpaired student t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.