The structural characterization and bioactivity assessment of nonspecific lipid transfer protein 1 (nsLTP1) from caraway (Carum carvi) seeds

Abstract

Background: Carum carvi (caraway) of the Apiaceae family has been used in many cultures as a cooking spice and part of the folk medicine. Previous reports primarily focus on the medicinal properties of caraway seed essential oil and the whole seeds extract. However, no effort has been made to study caraway proteins and their potential pharmacological properties, including nonspecific lipid transfer protein (nsLTP), necessitating further research. The current study aimed to characterize nonspecific lipid transfer protein 1 (nsLTP1) from caraway seed, determine its three-dimensional structure, and analyze protein-ligand complex interactions through docking studies. We also evaluated nsLTP1 in vitro cytotoxic effect and antioxidant capacity. Additionally, nsLTP1 thermal- and pH- stability were investigated.

Methods: Caraway nsLTP1 was purified using two-dimensional chromatography. The complete amino acid sequence of nsLTP1 was achieved by intact protein sequence for the first 20 residues and the overlapping digested peptides. The three-dimensional structure was predicted using MODELLER. Autodock Vina software was employed for docking fatty acids against caraway nsLTP1. Assessment of nsLTP1 cytotoxic activity was achieved by MTS assay, and the Trolox equivalent antioxidant capacity (TAC) was determined. Thermal and pH stability of the nsLTP1 was examined by circular dichroism (CD) spectroscopy.

Results: Caraway nsLTP1 is composed of 91 residues and weighs 9652 Da. The three-dimensional structure of caraway nsLTP1 sequence was constructed based on searching known structures in the PDB. We chose nsLTP of Solanum melongena (PDB ID: 5TVI) as the modeling template with the highest identity among all other homologous proteins. Docking linolenic acid with caraway protein showed a maximum binding score of -3.6 kcal/mol. A preliminary screening of caraway nsLTP1 suppressed the proliferation of human breast cancer cell lines MDA-MB-231 and MCF-7 in a dose‑dependent manner with an IC₅₀ value of 52.93 and 44.76 μM, respectively. Also, nsLTP1 (41.4 μM) showed TAC up to 750.4 μM Trolox equivalent. Assessment of nsLTP1 demonstrated high thermal/pH stability.

Conclusion: To the best of our knowledge, this is the first study carried out on nsLTP1 from caraway seeds. We hereby report the sequence of nsLTP1 from caraway seeds and its possible interaction with respective fatty acids using in silico approach. Our data indicated that the protein had anticancer and antioxidant activities and was thermally stable.

Keywords: Antioxidant; Caraway; Cytotoxicity; Molecular modeling; Nonspecific lipid transfer protein; Phylogenetic tree.

Fig. 1

Isolation of caraway proteins. a Fractionation profile of proteins from caraway (Carum carvi) on HiLoad 26/600 Superdex 200 pg column. b Electrophoretic profile by Tris/Tricine SDS-PAGE (10%) of caraway seeds proteins precipitates and gel filtration chromatography fractions. Lane M, standard molecular weight marker; Lane C, crude proteins; and Lane 1–8, eluted gel filtration fractions

Fig. 2

Chromatographic profile and analysis of caraway nsLTP1. a 2D-RP-HPLC elution profile of pooled gel filtration fractions containing caraway nsLTP1 on Aeris 3.6 µm WIDEPORE C4 250 × 4.6 mm column. b MALDI-TOF mass spectra of RP-HPLC purified caraway nsLTP1. c Amino acid sequence of nsLTP from caraway seeds. Solid line represent the N-terminal sequence of intact protein, while arrows represent cleaved peptides sequenced after trypsin and CNBr digestion

Fig. 3

Multiple sequence alignment of caraway nsLTP1 performed through Muscle program. Amino acids in multiple sequences are colored on the basis of conserved residues by Jalview. Scale bar at the top shows the position of amino acids. Left side represents several nsLTPs. The golden bar displays the substitution that occurred due to subtle changes in amino acids. Consensus bar in black color indicates the most common conserved residues of the alignment. Carum carvi sequence was set as a reference for numbering convention

Fig. 4

Neighbor-joining based unrooted phylogenetic tree of nsLTP protein sequences by MEGA11 software. Bootstrap value is present at each node. Diamond shape denotes caraway as a reference sequence

Fig. 5

Three-dimensional structure of caraway nsLTP1 in ribbon style. a Modeled caraway nsLTP1. N and C represent two terminals (basic and acidic end) of protein. 1H, 2H, 3H, and 4H represent four helices present in this protein model, along with their positions. Cysteine residues making disulfide bridges are highlighted with yellow and loops are colored grey. b Superimposed structure of modeled caraway (in pink color) with its template structure 5TVI (in yellow)

Fig. 6

Binding conformations of four fatty acids with caraway nsLTP. a linolenic acid, (b) linoleic acid, (c) stearic acid, and (d) palmitic acid. The binding site of caraway nsLTP structure around 4Å is also labeled with amino acids

Fig. 7

Dose-dependent cytotoxic activity of caraway nsLTP1 after 48 h treatment. Data from three independent experiments are presented with mean and standard deviation (SD). The IC₅₀ was calculated using nonlinear regression analysis; For MDA-MB-231 cells, the value was 52.93 μM, while for MCF-7 cells, it was 44.76 μM

Fig. 8

The total antioxidant capacity of caraway nsLTP1. For 41.4 μM caraway nsLTP1, the total antioxidant capacity was 750.4 ± 6.9 μM, and for 10.4 μM caraway nsLTP1, it was 268.2 μM ± 0.29 Trolox equivalent. Data is represented by Trolox equivalent with mean and standard deviation (SD) highlighted as *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 9

The thermal treatments effect on caraway nsLTP1 secondary structure. Caraway nsLTP1 (A) and Lysozyme (B) CD spectra were collected at 20, 40, 60, 80, and 95 °C and after cooling to 20 °C