Construction, structure, and verification of dodecameric form of soluble CD83. (A) A dodecameric form of soluble CD83 (dodCD83) was constructed by fusing the CD83 extracellular portion, Fcγ portion of human IgG1, and human IgA tailpiece. The primary structure of dodCD83 is shown as a monomeric form. (B) The predicted structure of dodCD83. (C) With or without reducing treatment by dithiothreitol, purified dodCD83 protein was subjected to SDS-PAGE, transferred to a PVDF membrane, and blotted with goat anti-human Ig (H+L) Ab. (D) Purified dodCD83 (100 μg) was loaded onto a size-exclusion HPLC column and fractionated based on size. Purified human IgM, IgG, or gel filtration standard was also run and used as a reference protein. (E) CHO/CD83 cells were preincubated with graded amounts of dodCD83 or control protein and subsequently stained with PE-labeled anti-CD83 mAb. (F) T-cell leukemic lines Jurkat and HPB-ALL and monocytic cell line U937 cells were analyzed for CD83L expression using dimeric and dodecameric of soluble CD83. Cells were incubated with either dodCD83 (open curve) or control dodecameric protein (filled curve), followed by incubation with PE-conjugated secondary antibody.
