Figure 3. The binding of FAg to TLR4 is mediated by carbohydrate residues.

Binding of AgW to human peripheral monocytes is inhibited by penta and hexa Chitooligosaccharides. (A) Human PBMCs were incubated with biotinylated AgW in the presence or absence of chitosugars viz; chitobiose, chitotriose, chitotetrose, chitopentaose and chtitohexaose followed by staining with Streptavidin FITC and CD14⁺ cells were analysed by FACS. % mean ± SEM of six experiments are shown. * P<0.001, versus AgW binding without chitosugars (Student's t-test). (B) Binding of AgW to TLR4 is inhibited by chtx. ELISA plates were coated with AgW. After blocking with skimmed milk-PBS, human PBMC lysate were pre incubated with different concentrations of chtx for 30 minutes at 37°C and were then transferred to AgW coated plates. Cell lysates without pre incubation with chtx was used as control. The plates were thoroughly washed and were incubated with rabbit anti-human TLR4 and its binding was detected by using peroxidase conjugated anti-rabbit IgG. The enzyme activity was measured using OPD as substrate. % mean ± SEM of two experiments are shown * P<0.001, versus cell lysates without preincubated with chtx (Student's t-test). (C) In silico analysis between chtx and TLR4. Three dimensional molecular docking between chtx and extra cellular domain of mouse TLR4 was performed using patch dock software demonstrating the interaction of chtx to the internal pocket of extra cellular domain of TLR4.