Virtual Screening and Binding Analysis of Potential CD58 Inhibitors in Colorectal Cancer (CRC).

Human cell surface receptor CD58, also known as lymphocyte function-associated antigen 3 (LFA-3), plays a critical role in the early stages of immune response through interacting with CD2. Recent research identified CD58 as a surface marker of colorectal cancer (CRC), which can upregulate the Wnt pathway and promote self-renewal of colorectal tumor-initiating cells (CT-ICs) by degradation of Dickkopf 3. In addition, it was also shown that knockdown of CD58 significantly impaired tumor growth. In this study, we developed a structure-based virtual screening pipeline using Autodock Vina and binding analysis and identified a group of small molecular compounds having the potential to bind with CD58. Five of them significantly inhibited the growth of the SW620 cell line in the following in vitro studies. Their proposed binding models were further verified by molecular dynamics (MD) simulations, and some pharmaceutically relevant chemical and physical properties were predicted. The hits described in this work may be considered interesting leads or structures for the development of new and more efficient CD58 inhibitors.

Cis interactions between CD2 and its ligands on T cells are required for T cell activation.

CD2 is largely described to promote T cell activation when engaged by its ligands, CD48 in mice and CD58 in humans, that are present on antigen-presenting cells (APCs). However, both CD48 and CD58 are also expressed on T cells. By generating new knockout mouse strains lacking CD2 or CD48 in the C57BL/6 background, we determined that whereas CD2 was necessary on T cells for T cell activation, its ligand CD48 was not required on APCs. Rather, CD48 was also needed on T cells. One exception was during cytotoxicity, which required CD48 on T cells and APCs. Fluorescence resonance energy transfer (FRET) studies in nonimmune cells provided evidence that cis interactions between CD2 and CD48 existed within individual cells. CD2-CD48 interactions on T cells enabled more robust T cell receptor (TCR) signals, including protein tyrosine phosphorylation. Using T cells from a CD2 knock-in mouse in which a tag was inserted at the carboxyl terminus of CD2, mass spectrometry analyses revealed that the role of CD2 in T cell activation correlated with its ability to interact with components of the TCR complex and the protein tyrosine kinase Lck. CD2-CD58 provided a similar function in human T cells. Thus, our data imply that T cell-intrinsic cis interactions of CD2 with its ligands are required for TCR signaling and T cell activation. Interactions with ligands on APCs contribute during cytotoxicity.

Structure-based identification of inhibitors disrupting the CD2-CD58 interactions.

The immune system has very intricate mechanisms of fighting against the invading infections which are accomplished by a sequential event of molecular interactions in the body. One of the crucial phenomena in this process is the recognition of T-cells by the antigen-presenting cells (APCs), which is initiated by the rapid interaction between both cell surface receptors, i.e., CD2 located on T-cells and CD58 located on APCs. Under various pathological conditions, which involve undesired immune response, inhibiting the CD2-CD58 interactions becomes a therapeutically relevant opportunity. Herein we present an extensive work to identify novel inhibiting agents of the CD2-CD58 interactions. Classical molecular dynamics (MD) simulations of the CD2-CD58 complex highlighted a series of crucial CD58 residues responsible for the interactions with CD2. Based on such results, a pharmacophore map, complementary to the CD2-binding site of CD58, was created and employed for virtual screening of ~ 300,000 available compounds. On the ~ 6000 compounds filtered from pharmacophore mapping, ADME screening leads to ~ 350 molecules. Molecular docking was then performed on these molecules, and fifteen compounds emerged with significant binding energy (< - 50 kcal/mol) for CD58. Finally, short MD simulations were performed in triplicate on each complex (i) to provide a microscopic view of the ligand binding and (ii) to rule out possibly weak binders of CD58 from the identified hits. At last, we suggest eight compounds for in vitro testing that were identified as promising hits to bind CD58 with a high binding affinity.

Modulation of co-stimulatory signal from CD2-CD58 proteins by a grafted peptide.

Peptides were designed to inhibit the protein-protein interaction of CD2 and CD58 to modulate the immune response. This work involved the design and synthesis of eight different peptides by replacing each amino acid residue in peptide 6 with alanine as well as grafting the peptide to the sunflower trypsin-inhibitor framework. From the alanine scanning studies, mutation at position 2 of the peptide was shown to result in increased potency to inhibit cell adhesion interactions. The most potent peptide from the alanine scanning was further studied for its detailed three-dimensional structure and binding to CD58 protein using surface plasmon resonance and flow cytometry. This peptide was used to graft to the sunflower trypsin inhibitor to improve the stability of the peptide. The grafted peptide, SFTI-a1, was further studied for its potency as well as its thermal, chemical, and enzymatic stability. The grafted peptide exhibited improved activity compared to our previously grafted peptide and was stable against thermal and enzymatic degradation.

Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM.

The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell-cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.

Investigating cyclic peptides inhibiting CD2-CD58 interactions through molecular dynamics and molecular docking methods.

The CD2-CD58 protein-protein interaction is known to favor the recognition of antigen presenting cells by T cells. The structural, energetics, and dynamical properties of three known cyclic CD58 ligands, named P6, P7, and RTD-c, are studied through molecular dynamics (MD) simulations and molecular docking calculations. The ligands are built so as to mimic the C and F ß-strands of protein CD2, connected via turn inducers. The MD analyses focus on the location of the ligands with respect to the experimental binding site and on the direct and water-mediated hydrogen bonds (H bonds) they form with CD58. Ligand P6, with a sequence close to the experimental ß-strands of CD2, presents characteristics that explain its higher experimental affinity, e.g., the lower mobility and flexibility at the CD58 surface, and the larger number and occurrence frequency of ligand-CD58 H bonds. For the two other ligands, the structural modifications lead to changes in the binding pattern with CD58 and its dynamics. In parallel, a large set of molecular docking calculations, carried out with various search spaces and docking algorithms, are compared to provide a consensus view of the preferred ligand binding modes. The analysis of the ligand side chain locations yields results that are consistent with the CD2-CD58 crystal structure and suggests various binding modes of the experimentally identified hot spot of the ligands, i.e., Tyr86. P6 is shown to form a number of contacts that are also present in the experimental CD2-CD58 structure.

Costimulatory Function of Cd58/Cd2 Interaction in Adaptive Humoral Immunity in a Zebrafish Model.

CD58 and CD2 have long been known as a pair of reciprocal adhesion molecules involved in the immune modulations of CD8+ T and NK-mediated cellular immunity in humans and several other mammals. However, the functional roles of CD58 and CD2 in CD4+ T-mediated adaptive humoral immunity remain poorly defined. Moreover, the current functional observations of CD58 and CD2 were mainly acquired from in vitro assays, and in vivo investigation is greatly limited due to the absence of a Cd58 homology in murine models. In this study, we identified cd58 and cd2 homologs from the model species zebrafish (Danio rerio). These two molecules share conserved structural features to their mammalian counterparts. Functionally, cd58 and cd2 were significantly upregulated on antigen-presenting cells and Cd4+ T cells upon antigen stimulation. Blockade or knockdown of Cd58 and Cd2 dramatically impaired the activation of antigen-specific Cd4+ T and mIgM+ B cells, followed by the inhibition of antibody production and host defense against bacterial infections. These results indicate that CD58/CD2 interaction was required for the full activation of CD4+ T-mediated adaptive humoral immunity. The interaction of Cd58 with Cd2 was confirmed by co-immunoprecipitation and functional competitive assays by introducing a soluble Cd2 protein. This study highlights a new costimulatory mechanism underlying the regulatory network of adaptive immunity and makes zebrafish an attractive model organism for the investigation of CD58/CD2-mediated immunology and disorders. It also provides a cross-species understanding of the evolutionary history of costimulatory signals from fish to mammals as a whole.

Residues Comprising the Enhanced Aromatic Sequon Influence Protein N-Glycosylation Efficiency.

N-Glycosylation is an important co- and/or post-translational modification that occurs on the vast majority of the one-third of the mammalian proteome that traverses the cellular secretory pathway, regulating glycoprotein folding and functions. Previous studies on the sequence requirements for N-glycosylation have yielded the Asn-X-Ser/Thr (NXS/T) sequon and the enhanced aromatic sequons (Phe-X-Asn-X-Thr and Phe-X-X-Asn-X-Thr), which can be efficiently N-glycosylated. To further investigate the influence of sequence variation on N-glycosylation efficiency in the context of a five-residue enhanced aromatic sequon, we used the human CD2 adhesion domain (hCD2ad) to screen the i-2, i-1, i+1, and i+2 residues flanking Asn at the i position. We found that aromatic residues, especially Trp, and sulfur-containing residues at the i-2 position improved N-glycosylation efficiency, while positively charged residues such as Arg suppressed N-glycosylation. Thiol, hydroxyl, and aliphatic-based side chains at the i-1 position had higher N-glycosylation efficiency, and Cys, in particular, compensated for the negative effect of Arg at the i-2 position. Small residues and Ser at the i+1 position increased the likelihood of N-glycosylation, and Thr is better than Ser at the i+2 position. We devised an algorithm for prediction of N-glycosylation efficiency using the SAS software, employing the 120 sequences studied as a training set. We then introduced the optimized-enhanced aromatic sequons into other glycoproteins and observed an enhancement in N-glycan occupancy that was further supported by modeling the high-affinity interaction between the optimized sequence on hCD2ad and a human oligosaccharyltransferase (OST) subunit. The findings in this study provide useful information for enhancing or suppressing N-glycosylation at a site of interest and valuable data for a better understanding of OST-catalyzed N-glycosylation.

Molecular characterization and expression of CD2 in Nile tilapia (Oreochromis niloticus) in response to Streptococcus agalactiae stimulus.

The cluster of differentiation 2 (CD2), functioning as a cell adhesion and costimulatory molecule, plays a crucial role in T-cell activation. In this paper, the CD2 gene of Nile tilapia, Oreochromis niloticus (designated as On-CD2) was cloned and its expression pattern under the stimulation of Streptococcus agalactiae was investigated. Sequence analysis showed On-CD2 protein consists of two extracellular Ig-like domains, a transmembrane region, and a long proline-rich cytoplasmic tail, which is a hallmark of CD2, and several important structural characteristics required for T-cell activation were detected in the deduced amino acid sequence of On-CD2. In healthy tilapia, the On-CD2 transcripts were mainly detected in the head kidney, spleen, blood and thymus. Moreover, there was a clear time-dependent expression pattern of On-CD2 after immunized by formalin-inactivated S. agalactiae and the expression reached the highest level at 12 h in the brain and head kidney, 48 h in the spleen, and 72 h in the thymus, respectively. This is the first report on the expression of CD2 induced by bacteria vaccination in teleosts. These findings indicated that On-CD2 may play an important role in the immune response to intracellular bacteria in Nile tilapia.

Glycosylation Modulates Human CD2-CD58 Adhesion via Conformational Adjustment.

Human CD2 is a transmembrane cell surface glycoprotein found on T lymphocytes and natural killer cells and plays important roles in immune recognition. The interaction between human CD2 and its counter receptor CD58 facilitates surface adhesion between helper T lymphocytes and antigen presenting cells as well as between cytolytic effectors and target cells. In this study, the molecular effect of glycosylation of CD2 on the structure and dynamics of the CD2-CD58 adhesion complex were examined via MD simulation to help understand the fundamental mechanism of glycosylation that controls CD2-CD58 adhesion. The present result and detailed analysis revealed that the binding interaction of human CD2-CD58 is dominated by three hot spots that form a binding triangle whose topology is critical for stable binding of CD2-CD58. Our study found that the conformation of human CD2, represented by the topology of this binding triangle, is significantly adjusted and steered by glycosylation toward a particular conformation that energetically stabilizes the CD2-CD58 complex. Thus, the fundamental mechanism of glycosylation of human CD2 is to promote CD2-CD58 binding by conformational adjustment of CD2. The current result and explanation are in excellent agreement with previous experiments and help elucidate the dynamical mechanism of glycosylation of human CD2.

Linking 3D and 2D binding kinetics of membrane proteins by multiscale simulations.

Membrane proteins are among the most functionally important proteins in cells. Unlike soluble proteins, they only possess two translational degrees of freedom on cell surfaces, and experience significant constraints on their rotations. As a result, it is currently challenging to characterize the in situ binding of membrane proteins. Using the membrane receptors CD2 and CD58 as a testing system, we developed a multiscale simulation framework to study the differences of protein binding kinetics between 3D and 2D environments. The association and dissociation processes were implemented by a coarse-grained Monte-Carlo algorithm, while the dynamic properties of proteins diffusing on lipid bilayer were captured from all-atom molecular dynamic simulations. Our simulations show that molecular diffusion, linker flexibility and membrane fluctuations are important factors in adjusting binding kinetics. Moreover, by calibrating simulation parameters to the measurements of 3D binding, we derived the 2D binding constant which is quantitatively consistent with the experimental data, indicating that the method is able to capture the difference between 3D and 2D binding environments. Finally, we found that the 2D dissociation between CD2 and CD58 is about 100-fold slower than the 3D dissociation. In summary, our simulation framework offered a generic approach to study binding mechanisms of membrane proteins.

Label-free measurements of the diffusivity of molecules in lipid membranes.

An important and characteristic property of a cell membrane is the lateral mobility of protein molecules in the lipid bilayer. This has conventionally been measured by labeling the molecules with fluorescent markers and monitoring their mobility by different fluorescence-based techniques. However, adding the label to the studied molecule may affect the system, so it is an assumption in almost all experiments that the measured mobility of the biomolecule with its label is the same as that of the unlabeled molecule. However, this assumption is rarely tested due to a lack of suitable methods. In this work, a new technique to perform label-free diffusivity measurements is developed and used to measure the effect of the label for two common protein-lipid systems: 1) streptavidin (SA) coupled to a supported lipid bilayer (SLB) through biotinylated lipids and 2) the extracellular part of the T-cell adhesion protein CD2, coupled to an SLB through histidine tags to nickel-chelating lipids. A measurable (≈12%) decrease in diffusivity is found for both labeled proteins, even though the molecular mass of the label is almost 100 times smaller than those of the proteins (≈50 kDa). The results illustrate the importance of being able to study different biophysical properties of cell membranes and their mimics without relying on fluorescent labels, especially if fluorescent labeling is difficult or is expected to affect the nature of the intermolecular interactions being studied.

Multimeric and differential binding of CIN85/CD2AP with two atypical proline-rich sequences from CD2 and Cbl-b*.

The CD2AP (CD2-associated protein) and CIN85 (Cbl-interacting protein of 85 kDa) adaptor proteins each employ three Src homology 3 (SH3) domains to cluster protein partners and ensure efficient signal transduction and down-regulation of tyrosine kinase receptors. Using NMR, isothermal titration calorimetry and small-angle X-ray scattering methods, we have characterized several binding modes of the N-terminal SH3 domain (SH3A) of CD2AP and CIN85 with two natural atypical proline-rich regions in CD2 (cluster of differentiation 2) and Cbl-b (Casitas B-lineage lymphoma), and compared these data with previous studies and published crystal structures. Our experiments show that the CD2AP-SH3A domain forms a type II dimer with CD2 and both type I and type II dimeric complexes with Cbl-b. Like CD2AP, the CIN85-SH3A domain forms a type II complex with CD2, but a trimeric complex with Cbl-b, whereby the type I and II interactions take place at the same time. Together, these results explain how multiple interactions among similar SH3 domains and ligands produce a high degree of diversity in tyrosine kinase, cell adhesion or T-cell signaling pathways.

High sensitive gold-nanoparticle based lateral flow Immunodevice for Cd2+ detection in drinking waters.

In this work for first time a lateral flow immunosensor device (LFID) for Cd(2+) determination in drinking and tap waters using the Cd-EDTA-BSA-AuNP conjugate as signal producer tool is introduced. The principle of working is based on competitive reaction between the Cd-EDTA-BSA-AuNP conjugate deposited on the conjugation pad strip and the Cd-EDTA complex formed in the analysis sample for the same binding sites of the 2A81G5 monoclonal antibody, specific to Cd-EDTA but not Cd(2+) free, which is immobilized onto the test line. The device has a large response range within 0.4-2000ppb, being the linear response between 0.4 and 10ppb. The quantification and detection limits of 0.4 and 0.1ppb, respectively, represent the lowest ones reported so far for paper based metal sensors. The obtained detection limit is 50 times lower than the maximum contamination level required for drinking water. Here we also show a new option for increasing the sensibility in the LFDs with competitive format, through the decreasing in concentrations of the Cd-EDTA-BSA-AuNP conjugate deposited in the conjugation strip and the mAbs deposited in the test and control zones until to reach optimized concentrations. It is an important result take into account that the increase in sensibility is one of the challenges in the field of LFD sensors, where are focused many of the ongoing researches. In addition, a specificity study of the device for several metal interferences, where potential metal interferences are masked with the use of the EDTA and OVA optimized concentrations, is presented too.

Immunosuppression by co-stimulatory molecules: inhibition of CD2-CD48/CD58 interaction by peptides from CD2 to suppress progression of collagen-induced arthritis in mice.

Targeting co-stimulatory molecules to modulate the immune response has been shown to have useful therapeutic effects for autoimmune diseases. Among the co-stimulatory molecules, CD2 and CD58 are very important in the early stages of generation of an immune response. Our goal was to utilize CD2-derived peptides to modulate protein-protein interactions between CD2 and CD58, thereby modulating the immune response. Several peptides were designed based on the structure of the CD58-binding domain of CD2 protein. Among the CD2-derived peptides, peptide 6 from the F and C ß-strand region of CD2 protein exhibited inhibition of cell-cell adhesion in the nanomolar concentration range. Peptide 6 was evaluated for its ability to bind to CD58 in Caco-2 cells and to CD48 in T cells from rodents. A molecular model was proposed for binding a peptide to CD58 and CD48 using docking studies. Furthermore, in vivo studies were carried out to evaluate the therapeutic ability of the peptide to modulate the immune response in the collagen-induced arthritis (CIA) mouse model. In vivo studies indicated that peptide 6 was able to suppress the progression of CIA. Evaluation of the antigenicity of peptides in CIA and transgenic animal models indicated that this peptide is not immunogenic.

Exploring the molecular mechanism of stabilization of the adhesion domains of human CD2 by N-glycosylation.

N-Glycosylation is one of the most common cotranslational and post-translational modifications occurring in protein biosynthesis and plays a critical role in protein folding and structural diversification. Molecular dynamics studies of two benchmark systems, the NH(2)-terminal human CD2 adhesion domain (HsCD2ad), and the NH(2)-terminal rat CD2 adhesion domain (RnCD2ad) were carried out to investigate the energetic and dynamic effect of N-glycosylation on protein's stability. Our study revealed that N-glycosylation of HsCD2ad at the type I ß-bulge turn strengthens the relevant hydrogen bonds, in particular, the hydrogen bond between Asn(65)OD1-Thr(67)HG1. Dynamic cross correlation map analysis showed that nonglycosylated HsCD2ad has strong anticorrelated motions, whereas glycosylated HsCD2ad largely destroyed this anticorrelated motion. As a result, N-glycosylation energetically and dynamically stabilizes HsCD2ad. In contrast, N-glycosylation of RnCD2ad does not display observable effect on protein's stabilization. The current theoretical result is in excellent agreement with the recent thermodynamic experiment of Culyba et al. and indicates that enthalpy and entropy may both contribute to the stabilization of human CD2 by N-glycosylation.

N-glycosylation of enhanced aromatic sequons to increase glycoprotein stability.

N-glycosylation can increase the rate of protein folding, enhance thermodynamic stability, and slow protein unfolding; however, the molecular basis for these effects is incompletely understood. Without clear engineering guidelines, attempts to use N-glycosylation as an approach for stabilizing proteins have resulted in unpredictable energetic consequences. Here, we review the recent development of three "enhanced aromatic sequons," which appear to facilitate stabilizing native-state interactions between Phe, Asn-GlcNAc and Thr when placed in an appropriate reverse turn context. It has proven to be straightforward to engineer a stabilizing enhanced aromatic sequon into glycosylation-naïve proteins that have not evolved to optimize specific protein-carbohydrate interactions. Incorporating these enhanced aromatic sequons into appropriate reverse turn types within proteins should enhance the well-known pharmacokinetic benefits of N-glycosylation-based stabilization by lowering the population of protease-susceptible unfolded and aggregation-prone misfolded states, thereby making such proteins more useful in research and pharmaceutical applications.

A structural basis for the biochemical behavior of activation-induced deoxycytidine deaminase class-switch recombination-defective hyper-IgM-2 mutants.

Hyper-IgM syndrome type 2 stems from mutations in activation-induced deoxycytidine deaminase (AID) that abolish immunoglobulin class-switch recombination, causing an accumulation of IgM and absence of IgG, IgA, and IgE isotypes. Although hyper-IgM syndrome type 2 is rare, the 23 missense mutations identified in humans span almost the entire gene for AID resulting in a recessive phenotype. Using high resolution x-ray structures for Apo3G-CD2 as a surrogate for AID, we identify three classes of missense mutants as follows: catalysis (class I), substrate interaction (class II), and structural integrity (class III). Each mutant was expressed and purified from insect cells and compared biochemically to wild type (WT) AID. Four point mutants retained catalytic activity at 1/3rd to 1/200th the level of WT AID. These "active" point mutants mimic the behavior of WT AID for motif recognition specificity, deamination spectra, and high deamination processivity. We constructed a series of C-terminal deletion mutants (class IV) that retain catalytic activity and processivity for deletions ≤18 amino acids, with ΔC(10) and ΔC(15) having 2-3-fold higher specific activities than WT AID. Deleting 19 C-terminal amino acids inactivates AID. WT AID and active and inactive point mutants bind cooperatively to single-stranded DNA (Hill coefficients ∼1.7-3.2) with microscopic dissociation constant values (K(A)) ranging between 10 and 250 nm. Active C-terminal deletion mutants bind single-stranded DNA noncooperatively with K(A) values similar to wild type AID. A structural analysis is presented that shows how localized defects in different regions of AID can contribute to loss of catalytic function.

Thermodynamic impact of embedded water molecules in the unfolding of human CD2BP2-GYF domain.

GYF domains are small polyproline-recognition modules adopting a structural arrangement consisting of a single α-helix packed against a small ß-sheet. Although most families of proline-rich recognition modules have been extensively characterized in terms of function, structure, or conformational flexibility, little is known about GYF domain functionality and folding. We have undertaken the thermodynamic characterization of the unfolding of CD2BP2-GYF domain by combining differential scanning calorimetry and circular dichroism under different pH conditions. The experimental data can be well-described in terms of a two-state equilibrium, although an unusually high heat capacity of the native state reflects a considerable conformational flexibility and dynamics of CD2BP2-GYF domain. In addition, the normalized thermodynamic parameters of unfolding (enthalpy, entropy and heat capacity) are roughly a factor of two greater than expected. In contrast, stability curves reveal an ordinary unfolding behavior of CD2BP2-GYF domain in terms of Gibbs energies, incurring thus unusually strong enthalpy-entropy compensation. This phenomenon, previously described as "thermodynamic homeostasis", has been associated in different examples to the contribution of occluded water (solvent) molecules into the protein structure. By means of CASTp server, we have found seven cavities/pockets scattered throughout of the CD2BP2-GYF structure, each able to harbor at least one water molecule. This structural feature provides rationalization for the atypical enthalpy values observed for CD2BP2-GYF because each water molecule is able to organize an extra amount of hydrogen bonds in the native state. In addition, these bound waters increase the vibrational entropy of the protein, which could also be responsible for an increase in protein flexibility and may thus fully explain the homeostatic behavior experimentally observed.

Comparative evaluation of T11 target structure and its deglycosylated derivative nullifies the importance of glycan moieties in immunotherapeutic efficacy.

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.