The B-chain of mistletoe lectin I efficiently stimulates calcium signaling in human Jurkat T-cells.
Mistletoe lectin I (ML I), a heterodimeric disulfide-linked type II ribosome inactivating protein, exhibits immunomodulatory potency in stimulating the cytokine release in vitro and in vivo. However, data concerning early activation events in T-cells induced by ML I and its A and B chain preceding cytokine secretion and the receptors involved are of limited availability. Here we show by flow cytometric measurements that human T-lymphoblastoid Jurkat cells express surface glycoprotein receptors for ML I. One of which is shown to be the CD2 antigen involved in a variety of T-cell signaling events. The lectin induces in Jurkat T-cells an increase of the cytosolic calcium concentration ([Ca(2+)](i)) consisting of both, the transient release of Ca(2+) from internal stores and a sustained influx of extracellular Ca(2+). Studies with isolated A- and B-chains provided evidence that the lectin-induced increase in [Ca(2+)](i) is mediated by ML IB. The ML I and ML IB stimulated cellular calcium responses are inhibited by saccharidic competitors. In transiently transfected E6.1 cells ML IB stimulated the expression of the luciferase reporter construct pNFAT-TA-Luc that is activated through the nuclear factor of activated T-cells (NFAT). The ML IB stimulated expression of the reporter luciferase (Luc) is completely inhibited by cyclosporin A (0.2 microM) and by FK 506 at 0.05 microM. Pretreatment of Jurkat E6.1 cells with 1-deoxymannojirimycin (dMJ), an inhibitor of cis-Golgi alpha-mannosidase I, strongly reduced cell binding of ML IB-FITC and the ML IB induced calcium response. Benzyl-alpha-GalNAc, an inhibitor of O-linked glycosylation, has slightly decreasing effects in ML IB-FITC binding and was without effects on the lectin stimulated increase in [Ca(2+)](i). Inhibition of the lectin induced calcium responses by cholera toxin and by inhibitors of protein kinases as well as the absence of calcium responses in CD3- and CD45- Jurkat T-cell clones suggest that ML IB has the potency to induce early T-cell activation events.
Walzel
H
H
Institute of Medical Biochemistry and Molecular Biology, University of Rostock, Schillingalle 70, D-18057 Rostock, Germany.
Blach
M
M
Neels
P
P
Schulz
U
U
Wollenhaupt
K
K
Brock
J
J
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D001212:Asialoglycoproteins / Q000494:pharmacology; D001667:Binding, Competitive; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000276:immunology*; D002772:Cholera Toxin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D060748:Fetuins; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D037102:Lectins / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000032:analysis; D014784:Mistletoe / Q000276:immunology*; D037121:Plant Lectins; D028321:Plant Preparations; D010940:Plant Proteins; D047428:Protein Kinase Inhibitors; D054790:Ribosome Inactivating Proteins, Type 2; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism* / Q000502:physiology; D014118:Toxins, Biological / Q000494:pharmacology*; D000509:alpha-Fetoproteins / Q000494:pharmacology
D000276:Adjuvants, Immunologic; D001212:Asialoglycoproteins; D004791:Enzyme Inhibitors; D060748:Fetuins; D037102:Lectins; D008562:Membrane Glycoproteins; D037121:Plant Lectins; D028321:Plant Preparations; D010940:Plant Proteins; D047428:Protein Kinase Inhibitors; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; D000509:alpha-Fetoproteins; C017029:asialofetuin; C488862:mistletoe lectin I; C009674:ribosome inactivating protein, Viscum; D002772:Cholera Toxin; D002118:Calcium