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Structural and functional characterization of a novel immunomodulatory glycoprotein isolated from ajowan (Trachyspermum ammi L.)

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Abstract

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.

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Abbreviations

Agp:

Ajowan glycoprotein

dpAgp:

Deproteinized Agp or Pronase-treated Agp

dgAgp:

Deglycosylated Agp

AGP:

Arabinogalactan protein

ALC:

Average local confidence

MLN:

Myeloid lymph node

NO:

Nitric oxide

NAC:

Nylon wool adherent cells

NNAC:

Nylon wool non-adherent cells

PEC:

Peritoneal exudate cells

TI:

T- cell independent

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Acknowledgements

We wish to thank Prof. Ram Rajasekharan, Director, CSIR–CFTRI, Mysuru for his support and constant encouragement. Our sincere thanks to Dr. Sridevi A. Singh and Dr. Govindaraju K, Department of Protein Chemistry and Technology, CSIR-CFTRI for help and advice in carrying out CD studies and amino acid analysis, respectively. One of the authors (RRS) would like to thank the Council of Scientific and Industrial Research, New Delhi, India for the grant of Research Fellowship.

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  1. Department of Biochemistry, CSIR–Central Food Technological Research Institute (CFTRI), KRS Road, Mysore, Karnataka, 570020, India

    R. R. Shruthi, Y. P. Venkatesh & G. Muralikrishna

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  1. R. R. Shruthi
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  2. Y. P. Venkatesh
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  3. G. Muralikrishna
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Correspondence to G. Muralikrishna.

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Following the approval from the Institutional Animal Ethical Committee (IAEC), all experimental procedures involving the handling and caring of animals have been carried out in accordance with the defined ethical guidelines.

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Shruthi, R.R., Venkatesh, Y.P. & Muralikrishna, G. Structural and functional characterization of a novel immunomodulatory glycoprotein isolated from ajowan (Trachyspermum ammi L.). Glycoconj J 34, 499–514 (2017). https://doi.org/10.1007/s10719-017-9771-x

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