title: A Salmonella typhi OmpC fusion protein expressing the CD154 Trp140-Ser149 amino acid strand binds CD40 and activates a lymphoma B-cell line.
abstract: CD154 is a type II glycoprotein member of the tumour necrosis factor (TNF) ligand family, which is expressed mainly on the surface of activated T lymphocytes. The interaction with its receptor CD40, plays a central role in the control of several functions of the immune system. Structural models based on the homology of CD154 with TNF and lymphotoxin indicate that binding to CD40 involves three regions surrounding amino acids K143, R203 and Q220, and that strands W140-S149 and S198-A210 are critical for such interactions. Also, it has been reported that two recombinant CD154 fragments, including amino acid residues Y45-L261 or E108-L261 are biologically active, whereas other polypeptides, including S149-L261, are not. Therefore, we decided to construct a fusion protein inserting the W140-S149 amino acid strand (WAEKGYYTMS) in an external loop of the outer membrane protein C (OmpC) from Salmonella enterica serovar Typhi and assess its ability to bind CD40 and activate B cells. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis demonstrated that the chimeric OmpC-gp39 protein conserved its ability to form trimers. Binding to CD40 was established by three variants of enzyme-linked immunosorbent assay, a direct binding assay by coating plates with a recombinant CD40-Fc protein and through two competition assays between OmpC-gp39 and recombinant CD154 or soluble CD40-Fc. Flow cytometry analysis demonstrated that OmpC-gp39 increased the expression levels of major histocompatibility complex II, CD23, and CD80, in Raji human B-cell lymphoma similarly to an antibody against CD40. These results further support that the CD154/CD40 interaction is similar to the TNF/TNF receptor. This is the first report of a bacterial fusion protein containing a small amino acid strand form a ligand that is able to activate its cognate receptor.
mesh_terms: D000595:Amino Acid Sequence; D002051:Burkitt Lymphoma / Q000276:immunology* / Q000378:metabolism; D019013:CD40 Antigens / Q000378:metabolism*; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D018272:Porins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012485:Salmonella typhi / Q000276:immunology*; D014407:Tumor Cells, Cultured
chemical_list: D019013:CD40 Antigens; C113724:OmpC protein; D018272:Porins; D011993:Recombinant Fusion Proteins