Figure 2.

Cellular DC-SIGN binds strongly to both viable mycobacteria and the mycobacterial component ManLAM through its primary binding site. (a) K562-DC-SIGN transfectants express high levels of DC-SIGN but lack expression of the other reported ManLAM receptors MR, CD11b, and CD11c. Transfectants were generated as described previously (reference 12). Open histograms represent the isotype controls, and filled histograms indicate the specific antibody staining. (b) DC-SIGN, expressed by K562 transfectants, binds strongly to intact M. bovis BCG and the mycobacterial component ManLAM but not to M. smegmatis and AraLAM. The adhesion of cells to the LAM glycans was determined using the fluorescent bead adhesion assay. Binding to viable mycobacteria was determined by measuring the binding of K562 transfectants to FITC-conjugated mycobacteria (MOI 20) using flow cytometry. Specificity was determined by measuring binding in the presence of blocking antibodies against DC-SIGN. Standard deviation for the fluorescent bead adhesion assay and the mycobacteria binding assay was <5 and <2%, respectively. One representative experiment out of three is shown. (c) The Val³⁵¹ amino acid residue is not essential for the interaction of DC-SIGN with M. bovis BCG and ManLAM, similar to HIV-1 gp120, whereas it is essential for ICAM-3 binding. Binding to the V351G DC-SIGN mutant expressed by K562 cells was measured as described for panel b. Specificity was determined by measuring binding in the presence of blocking antibodies against DC-SIGN, mannan or EGTA. Standard deviation <5% (fluorescent bead adhesion assay) and <2% (mycobacteria binding assay). One representative experiment out of three is shown.