FIGURE 2.

Lys-299 and Lys 313 are extended binding sites for the recognition of sulfated glycans. A, WT and mutant forms of Langerin-Fc expression vectors were transfected into HEK293T cells, and 1 ml of the resulting culture supernatants was incubated with 10 μl of Protein A-Sepharose. The gels were then incubated with SDS sample buffer, and the eluted fractions were run on SDS-PAGE and blotted with peroxidase-labeled goat anti-human IgG (H+L). B, culture supernatants of WT and mutant forms of Langerin-Fc were precomplexed with Cy3-labeled anti-human Fcγ and analyzed by glycoconjugate microarray. Binding of WT and mutant forms of Langerin-Fc to invertase, 6′-sulfo-LacNAc, and 6-sulfo-GlcNAc are shown relative to WT.