title: Comparative evaluation of T11 target structure and its deglycosylated derivative nullifies the importance of glycan moieties in immunotherapeutic efficacy.
abstract: Sheep red blood cell (SRBC), a non-specific biological response modifier that has long been used as a classical antigen, has been shown to exert an immunomodulatory and anti-tumor activities in experimental animals. The active component of SRBC, which is responsible for such effects, was found to be a cell surface acidic glycoprotein molecule, known as T11 target structure (T11TS). In the present study, T11TS was isolated and purified to homogeneity using a five-step protocol involving isolation of sheep erythrocyte membrane from packed cell volume, 20% ammonium sulfate cut of the crude membrane proteins mixture, immunoaffinity purification using mouse anti-sheep CD58 mAb (L180/1) tagged matrix, preparative gel electrophoresis, and gel electroelution process. Finally, the purity and identity of the proteins were confirmed by the matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis. The in silico glycosylation site analysis showed that the extracellular domain contained three N-glycosylation sites (N-12, N-62, and N-111) and one O-glycosylation site (T-107). However, the experimental analysis negated the presence of O-linked glycan moieties on T11TS. To investigate the role of glycan moieties in the current immunotherapeutic regime, T11TS and its deglycosylated form (dT11TS) were administered intraperitoneally (i.p.) in N-ethyl-N-nitrosourea-induced immune-compromised mice at 0.4 mg/kg body weight. It was observed that both the forms of T11TS could activate the compromised immune status of mice by augmenting immune receptor expression (CD2, CD25, CD8, and CD11b), T-helper 1 shift of cytokine network, enhanced cytotoxicity, and phagocytosis activity. Therefore, the results nullify the active involvement of the N-linked glycan moieties in immunotherapeutic efficacy of T11TS.
authors.affiliation: Department of Laboratory Medicine, Calcutta School of Tropical Medicine, Kolkata, West Bengal, India.
authors.forename: Sirshendu
authors.initials: S
authors.lastname: Chatterjee
authors.forename: Sagar
authors.initials: S
authors.lastname: Acharya
authors.forename: Pankaj
authors.initials: P
authors.lastname: Kumar
authors.forename: Ananya
authors.initials: A
authors.lastname: Chatterjee
authors.forename: Suhnrita
authors.initials: S
authors.lastname: Chaudhuri
authors.forename: Anirban
authors.initials: A
authors.lastname: Ghosh
authors.forename: Swapna
authors.initials: S
authors.lastname: Chaudhuri
mesh_terms: D000818:Animals; D018801:CD2 Antigens / Q000737:chemistry* / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology*; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D007167:Immunotherapy / Q000379:methods*; D008264:Macrophages / Q000166:cytology; D051379:Mice; D010587:Phagocytosis; D011134:Polysaccharides / Q000737:chemistry*; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins / Q000737:chemistry; D012756:Sheep; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D018801:CD2 Antigens; D006023:Glycoproteins; D011134:Polysaccharides; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins