Construction and expression of a soluble form of human CD30 ligand with functional activity.
CD30 engagement in human lymphoid cells induces pleiotropic cellular responses that affect cellular viability and proliferation, cytokine production, and nuclear factor kappaB (NF-kappaB) nuclear translocation. Studies examining the molecular basis for this pleiotropism thus far have relied on the use of antibodies and cells transfected with CD30L to trigger CD30, two methods of receptor induction that present important limitations: antibodies are not physiological receptor-triggering molecules and CD30L transfectants induce high background intracellular signaling in the cells under study. We have generated and expressed a functional soluble human CD30L molecule (sCD30L/CD8alpha) comprised of the extracellular domain of human CD30L fused to the extracellular domain of the human CD8alpha chain. Immunoprecipitation and Western blot analysis of sCD30L/CD8alpha revealed the existence of at least two forms of sCD30L/CD8alpha, which exhibited molecular sizes consistent with the existence of monomeric and trimeric forms of the molecule. Binding analyses performed using a soluble CD30 fusion protein (sCD30/gamma1) confirmed the ability of sCD30L/CD8alpha to bind to CD30. Functionally, immobilized sCD30L/CD8alpha-induced cell death in the CD30-expressing lines Karpas-299 and HDLM-2 and reduced proliferative levels in Karpas-299; these effects were inhibitable by the addition of sCD30/gamma1. These studies demonstrate the utility of sCD30L/CD8alpha in characterizing the normal function of CD30L and CD30 and indicate the natural ability of soluble forms of CD30L to trimerize.
Powell
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Program in Molecular Biology, Loyola University School of Medicine, Maywood, Illinois 60153, USA.
Li
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Jäck
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Ellis
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D015153:Blotting, Western; D053285:CD30 Ligand; D016827:CD8 Antigens / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017730:Ki-1 Antigen / Q000378:metabolism; D015448:Leukemia, B-Cell; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012995:Solubility; D014162:Transfection; D014407:Tumor Cells, Cultured
D053285:CD30 Ligand; D016827:CD8 Antigens; D017730:Ki-1 Antigen; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C505774:TNFSF8 protein, human