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PD-L1 is a member of the B7 protein family, most of whose members so far were identified as dimers in a solution and crystalline state, either complexed or uncomplexed with their ligand(s). The binding of PD-L1 with its receptor PD-1 (CD279) delivers an inhibitory signal regulating the T cell function. Simultaneously with the Garboczi group, we successfully solved another structure of human PD-L1 (hPD-L1). Our protein crystallized in the space group of C222(1) with two hPD-L1 molecules per asymmetric unit. After comparison of reported B7 structures, we have found some intrinsic factors involved in the interaction of these two molecules. Based on these results, we tend to believe this uncomplexed hPD-L1 structure demonstrated its potential dimeric state in solution, although it could just be an evolutionary relic, too weak to be detected under present technology, or still a functional unit deserved our attentions.
Antigens, CD ; chemistry ; immunology ; B7-H1 Antigen ; Crystallography, X-Ray ; Evolution, Molecular ; Humans ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Secondary ; T-Lymphocytes ; chemistry ; immunology

8.Comparison of immunomagnetic beads and hespan precipitation for isolation of mononuclear cells from umbilical cord blood.

Min LI ; Shu-Ying FAN ; Guang-Hui SUN ; Pei-Ru XU ; Aerziguli TURERXUN

Chinese Journal of Contemporary Pediatrics 2009;11(9):757-760

9.Detection of homodimer formation of CD99 through extracelluar domain using bimolecular fluorescence complementation analysis.

Gowoon CHOI ; Sang Wook LEE ; Kyoung Cheon JUNG ; Eun Young CHOI

Experimental & Molecular Medicine 2007;39(6):746-755

Although various functions of CD99 have been reported, such as apoptosis and homotypic aggregation of thymocyte and transendothelial migration of immune cells, biochemical/molecular natures of CD99 are still elusive. Using mouse CD99 gene, we show that CD99 forms homodimer through its extracellular domain. Expression of mouse CD99 is up-regulated on T cells after CD3-mediated activation, like the case for human CD99. The potential of CD99 to form homodimer was tested with a recently developed bimoleular fluorescence complementation analysis (BiFC). In BiFC analysis, the dimerization-induced fluorescence was strong near the perinuclear region and was faded at the cell membrane. However, surface expression of CD99 was still detected by flow cytometry, suggesting that CD99 either in monomer form or in association with other molecules exists on the cell surface. In BiFC analysis using CD99 mutants with its extracellular, transmembrane, or cytosolic domains changed to corresponding human CD4 domains, the mutant replaced with human CD4-extracellular domain did not produce fluorescence. Purified soluble CD99-Fc fusion proteins bound to CD99-Fc immobilized onto the gold sensor chip in surface plasmon resonance analysis, confirming that the extracellular domain was responsible for dimer formation. Intracytoplasmic staining for CD99 expression in the thymocytes and mature T cells showed that most of the cells, even the cells with low surface level of CD99, contained the molecule inside the cell. Our results suggest that majority of CD99 homodimers may exit in the cell and be exported to the cell surface, dissociating from each other, after a certain regulatory signal is delivered.
Animals ; Antigens, CD/chemistry/*isolation & purification ; Cell Adhesion Molecules/chemistry/*isolation & purification ; Flow Cytometry ; *Fluorescence ; Luminescent Measurements/*methods ; Mice ; Molecular Biology/*methods ; T-Lymphocytes/immunology