Structural and functional characterization of a novel immunomodulatory glycoprotein isolated from ajowan...

Ajowan (Trachyspermum ammi L.) spice has been used in food preparations and also as a traditional medicine in Ayurveda. Although a number of pharmacological activities have been attributed to ajowan, its role in immunomodulation is not known. The main objective of the present study is to examine the macromolecular immunomodulatory components. Macrophage activation was studied by nitric oxide (NO) release, phagocytosis and secretion of pro-inflammatory cytokines as the markers. Ethanol precipitate (fractional) of ajowan aqueous extract was subjected to conventional chromatography (Q Sepharose followed by Bio-Gel P-100). One of the proteins (30.7 kDa; ajowan glycoprotein or Agp) showed effective mitogenic activity towards splenocytes. Agp is a O-linked glycoprotein with the glycans contributing to one-third of the molecular mass. It has a high content of glutamic acid, serine, aspartic acid and proline whereas galactose (45.7%), arabinose (34.5%), glucose (7%), mannose (5%) and xylose (4%) are the constituent sugars. Secondary structure analysis indicated that Agp contains 79% α-helices and 21% random coil. Internal sequencing of the tryptic peptides did not show homology with the existing proteins in the database (BLAST). Agp at 1 μg/mL induced proliferation of B-cell enriched murine splenocytes and activated macrophages in releasing NO and promoted phagocytosis (p < 0.01). RAW 264.7 cells produced pro-inflammatory cytokines (IL-12, TNF-α and IFN-γ) at 1 μg/mL Agp (p < 0.01). Deproteinized Agp (dpAgp) failed to elicit activation of murine immune cells, whereas deglycosylated Agp (20 kDa; dgAgp) showed compromised efficiency. This is the first report of an immunomodulatory protein from ajowan.