Structural Characterization and in vitro Antiproliferative Activity of Non-Specific Lipid Transfer Protein 1 (nsLTP1) from Fennel (Foeniculum vulgare) Seeds

Abstract

Non-specific lipid transfer proteins (nsLTPs) are cationic proteins involved in intracellular lipid shuttling, in growth and reproduction, as well as in defense against pathogenic microbes. Even though the primary and spatial structures of some nsLTPs from different plants indicate their similar features, they exhibit distinct lipid-binding specificities signifying their various biological roles that dictate further structural study. The present study determined the complete amino acid sequence, in silico 3D structure modeling, and in vitro antiproliferative activity of nsLTP1 from fennel (Foeniculum vulgare) seeds. Fennel is a member of the family Umbelliferae (Apiaceae) native to southern Europe and the Mediterranean region. It is used as a spice, medicine and fresh vegetable. Fennel nsLTP1 was purified using gel filtration and reverse-phase high-performance liquid chromatography (RP-HPLC). Its homogeneity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. The purified nsLTP1 was treated with 4-vinyl pyridine, and the modified protein was then digested with trypsin. The complete amino acid sequence of nsLTP1 was established by intact protein sequence up to 28 residues, overlapping tryptic peptides, and cyanogen bromide (CNBr) peptides. Hence, it is confirmed that fennel nsLTP1 is a 9433 Da single polypeptide chain consisting of 91 amino acids with eight conserved cysteines. Moreover, the 3D structure was predicted to have four α-helices interlinked by three loops and a long C-terminal tail. In vitro lipid-binding study and molecular docking studies confirmed that the order of binding efficiency among the four studied fatty acids was linoleic acid>linolenic acid>stearic acid>palmitic acid. In vitro antiproliferative activity of fennel revealed the inhibition in the growth of MCF-7 human breast cancer cells in a dose-dependent manner with an IC50 value of 6.98 µM. The dose-dependent increase in the percentage of apoptotic cells may imply the apoptotic cell killing mechanism. It also modulated the expression of proapoptotic and antiapoptotic genes, which further proposes the induction of apoptosis. Thus, the finding from this study unveils the potential use of fennel nsLTP1 as a breast cancer therapeutic agent, which therefore warrants further molecular studies as well as in vivo animal studies.