CD20 is an antigen expressed on normal and malignant human B cells that is thought to function as a receptor during B cell activation. Here we report the isolation of a CD20‐specific cDNA clone from a lambda gt11 library using a polyclonal antiserum raised against purified CD20 antigen. Additional cDNA clones were then isolated from a lambda gt10 library. Alignment of the sequences of overlapping lambda clones reveal a single consensus sequence except for a divergence that preceded the first methionine within the open reading frame. Normal B cells and B cell lines contain a prominent 2.6 kb mRNA and a lower level of a 3.3 kb mRNA. An oligonucleotide derived from one of the divergent sequences hybridized to the 3.3 kb mRNA only, indicating that the two mRNA species are derived from an alternative splicing mechanism. The predicted amino acid sequence of CD20 reveals three major hydrophobic regions of approximately 53, 25 and 20 amino acids. CD20 lacks an NH2‐terminal signal peptide and contains a highly charged COOH‐terminal domain. Although CD20 is immunoprecipitated as a doublet of 33 and 35 kd proteins from B cells, in vitro translation of CD20 cDNA produced a single 33 kd protein that was specifically immunoprecipitated with monoclonal CD20 antibodies. CD20 was strongly phosphorylated on resting B cells after CDw40 stimulation, suggesting that CD20 may be functionally regulated by a protein kinase(s).
Previous studies have shown that binding of interleukin 1 (IL-1) to its receptor and intracellular processing of the IL-i/IL-i receptor complex appear to be different in Band T-lymphocyte cell lines. In this study we used a B-lymphoid cell line, 70Z/3, and T-lymphoid cell line, EL-4 6.1 C10, to explore further the differences that exist between IL-1 receptors on cells of B and T lineage. We show that a monoclonal antibody against the IL-1 receptor on EL-4 cells does not bind to the IL-1 receptor on 70Z/3 cells. This rinding suggests that there are structural differences in the extracellular domains of the IL-1 receptors on the two cell lines. Furthermore, affinity crosslinking showed that the molecular mass of the IL-1 receptor on EL-4 is 87 kDa, whereas that of 70Z/3 is significantly lower (66 kDa). Activation of phospholipid/Ca2+-dependent protein kinase, protein kinase C, by phorbol 12-myristate 13-acetate (PMA) greatly reduced the number ofIL-1 binding sites on 70Z/3. But, in sharp contrast, PMA had no effect on surface IL-1 receptor expression on EL-4 cells despite having an equally potent effect in activating protein kinase C. The different effects of protein kinase C suggest that the cytoplasmic domains of the IL-1 receptors in 70Z/3 and EL-4 may also be different. Lastly, a probe containing the entire coding region of the murine T-cell IL-1 receptor hybridized under high stringency conditions with mRNA from EL-4 cells but not with mRNA from 70Z/3 cells. Taken together, the observations made in this study suggest that major structural differences exist between the IL-1 receptors on B and T lymphocytes.Interleukin 1 (IL-1) is a polypeptide hormone produced by various cell types. IL-1 initiates a diverse range of biological effects by binding to specific surface receptors (for review, see ref. 1). The pre-B-lymphocyte cell line 70Z/3 and the T-lymphoma cell line EL-4 have proven to be useful systems to study the biological effects of IL-1 (2-5). 70Z/3 cells have receptors whose binding properties are significantly different from the IL-1 receptors (IL-lRs) on EL-4 cells (5-7). After binding to its receptor IL-1 is internalized and quickly degraded by 70Z/3 but not by 6,8). In this study we set out to determine what other differences might exist between the IL-iRs in the two cell lines. MATERIALS AND METHODSCell Lines. Murine pre-B-lymphoid cell line 70Z/3 and murine thymoma EL-4 6.1 C10 were grown in complete RPMI 1640 medium supplemented with 5% fetal calf serum, 2 mM glutamine, 50 ,uM mercaptoethanol, penicillin (100 units/ml), and streptomycin (0.01%) at 370C in a 5% C02/ 95% air atmosphere.Reagents. Recombinant human IL-la was produced in Escherichia coli as described (9). IL-la was labeled with Na251I using a chloramine-T method as described (10). The crosslinking agent dithiobis(succinimidylpropionate) was purchased from Pierce. Dibutyl phthalate and bisphthalate oils were obtained from Kodak.Preparation and Radiolabeling of Anti-EL4R Antibody. The monoclonal anti-IL-lR antibody (M5) was gener...
The electron paramagnetic resonance (EPR) and Mössbauer properties of native horseradish peroxidase have been compared with those of a synthetic derivative of the enzyme in which a mesohemin residue replaces the natural iron protoporphyrin IX heme prosthetic group. The oxyferryl pi cation radical intermediate, compound I, has been formed from both the native and synthetic enzyme, and the magnetic properties of both intermediates have been examined. The optical absorption characteristics of compound I prepared from mesoheme-substituted horseradish peroxidase are different from those of the compound I prepared from native enzyme [DiNello, R. K., & Dolphin, D. (1981) J. Biol. Chem. 256, 6903-6912]. By analogy to model-compound studies, it has been suggested that these optical absorption differences are due to the formation of an A2u and an A1u pi cation radical species, respectively. However, the EPR and Mössbauer properties of the native and synthetic enzyme and of their oxidized intermediates are quite similar, if not identical, and the data favor an A2u radical for both compounds I.
Programmed cell death (PCD), or apoptosis, is characterized by several morphologic alterations and eventual cleavage of nuclear DNA into oligonucleo-some-length fragments. We defined a human B cell line, Ramos, that responds with PCD following ligation of surface IgM. Of the DNA in Ramos cells 3%-10% was fragmented as early as 4 h after IgM ligation. Propidium iodide staining demonstrated that 20%-40% of Ramos cells became apoptotic by 18 h and further established that cells transiting into the S phase of the cell cycle were susceptible to PCD. Addition of several agents to the Ramos cells abrogated anti-IgM-induced PCD, including the phorbol 12-myristate 13-acetate (PMA). In contrast to the effect of PMA, the 4 alpha PMA isomer of PMA neither activated protein kinase C (PKC) nor rescued the cells from anti-IgM-induced PCD, confirming a role for PKC in negating apoptosis. To explore the effect of physiologic signals on anti-IgM-induced PCD, antibodies against the CD20 or CD40 molecules were added in concert with anti-IgM. Both CD20 and CD40 synergize with anti-IgM to augment proliferation but neither molecule activates PKC in Ramos cells. Both anti-CD20 and anti-CD40 reduced the number of cells undergoing anti-IgM-induced PCD. Unlike the effect of anti-CD40, addition of anti-CD20 to anti-IgM-stimulated cells negated PCD only in a subset of cells. Maximal rescue occurred following the addition of anti-CD40 and occurred by 4 h and at least up to 20 h of culture. These data show that (a) PCD can be initiated in B cells entering the S phase of the cell cycle, (b) PCD can be triggered by engagement of surface IgM in the absence of ancillary signals or PKC activation, and (c) rescue from PCD can occur by several mechanisms, either PKC dependent or PKC independent.
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