Light (L) chains that edit anti-DNA heavy (H) chains rescue B-cell development by suppressing DNA binding. However, exceptional editor L chains allow B cells to reach splenic compartments even though their B-cell receptors remain autoreactive. Such incompletely edited B cells express multireactive antibodies that accumulate in the Golgi and are released as insoluble, amyloid-like immune complexes. Here, we examine examples of incomplete editing from the analysis of variable to joining (VJ) gene junction of the variable (Vλx) editor L chain. When paired with the anti-DNA heavy chain, VH56R, the Vλx variants yield antibodies with differing specificities, including glycosaminoglycan reactivity. Our results implicate these specificities in the evasion of receptor editing through intracellular sequestration of IgM and the release of insoluble IgM complexes. Our findings can be extrapolated to human L chains and have implications for understanding a latent component of the Ig repertoire that could exert pathogenic and protective functions.
B-Lymphocytes/metabolism , Glycosaminoglycans/metabolism , Golgi Apparatus/metabolism , Immunoglobulin M/metabolism , Models, Molecular , Protein Conformation , Amino Acid Sequence , B-Lymphocytes/immunology , Base Sequence , Fluorescent Antibody Technique , Glycosaminoglycans/immunology , Humans , Hybridomas , Immunoassay , Immunoglobulin M/immunology , Immunoglobulin Variable Region/genetics , Luminescent Measurements , Microscopy, Confocal , Molecular Sequence Data , X-Ray Diffraction
To investigate the manner in which B cells with lambda light (L) chains undergo receptor editing, we have studied hybridoma panels from 56R/kappa-deleted (kdel) mice. 56R/kdel mice only produce four L chains (lambda1, lambda2, lambda3, and lambdaX). They also have a simplified heavy (H) chain repertoire: All B cells start out with a 56R anti-DNA H chain. A few frankly autoreactive 56R lambda1 cells appear to escape into the periphery, but the majority of the peripheral B cell repertoire in 56R/kdel is made up of B cells expressing the 56R H chain with the lambdaX L chain. Surprisingly, 56R lambdaX B cells are multireactive, binding to a variety of self and nonself antigens, including dsDNA (albeit at reduced affinity compared with the other lambda L chains). Another significant population in the 56R/kdel mouse consists of allelically included B cells that express lambdaX along with another L chain. The multireactivity of both 56R lambdaX and 56R lambdaX/lambda1 receptors could contribute to autoimmunity if these B cells were to become activated. Also found among 56R/kdel hybridomas are clones that have inactivated the H chain and secrete only L chains. These clones may represent products of exhaustive rearrangement. Multireactivity, allelic inclusion, and L chain secretion are three consequences of editing at the lambda locus that may predispose toward the development of autoimmunity.
Autoimmunity/genetics , Gene Rearrangement , Immunoglobulin Light Chains/chemistry , Receptors, Antigen, B-Cell/immunology , Alleles , Animals , Autoantibodies/chemistry , B-Lymphocytes/metabolism , Genotype , Hybridomas/metabolism , Immunoglobulin Light Chains/metabolism , Mice , Mice, Transgenic , Models, Molecular , Receptors, Antigen, B-Cell/metabolism , Sequence Analysis, DNA
BACKGROUND & OBJECTIVES: Normal epithelial or endothelial cells can undergo anoikis, a type of apoptosis, when they are detached from their extracellular matrices (ECM), while most tumor cells derived from epithelial or endothelial tissues lose this feature. Most of the studies indicate that anoikis resistance of tumor cells is closely related to abnormal signal transductioin. The aim of this research was to screen out the tumor cell lines that are anoikis resistant, then to investigate the relationship between the protein tyrosine phosphorylation of signaling molecules and anoikis resistance feature of tumor cells. METHODS: Anoikis resistance of breast tumor cell lines MCF-7, Bcap-37, and MDA-MB-231 was determined by DNA ladder assay, flow cytometry analysis, and soft agar assay upon suspending culture. A normal epithelial cell line MDCK (Madin-Darby canine kidney) was taken as a control. The inhibitory effect of genistein, a general protein tyrosine kinase inhibitor, on anoikis resistance of tumor cells was analyzed at the same time. The differences of total protein tyrosine phosphorylation levels between suspended and attached cultural conditions of three breast cancer cell lines were examined by Western blot analysis. RESULTS: All the three tumor cell lines were distinctly anoikis resistant. Further study showed that these features could be suppressed by genistein. The results of Western blot analysis showed that the general level of tyrosine phosphorylation in suspended MDCK cells was decreased compared with that in attached cells, while tyrosine phosphorylation level of some proteins increased (more than 3.5-6.5 folds) aberrantly in suspended tumor cells. CONCLUSION: All the three breast tumor cells in this study are anoikis resistant respectively to some extent, and aberrant tyrosine phosphorylation of signaling proteins may play a role in the process of anoikis resistance of these tumor cells.
Anoikis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Tyrosine/metabolism , Animals , Cell Line, Tumor , Dogs , Female , Genistein/pharmacology , Humans , Phosphorylation
G protein-coupled receptor kinase-2 (GRK),also known as beta1-adrenergic receptor kinase(beta-ARK1), plays an important role in agonist-induced desensitization of the beta-adrenergic receptors. Activation of protein kinase C (PKC) is able to stimulate phosphorylation and activation of GRKs and induce desensitization of G protein-coupled receptor. However, detail mechanism of interaction between PKC and GRK2 and the effect of GRK2 on activity of PKC remain unknown. Pleckstrin homology (PH) domain is a kind of functionally domain containing about 120 amino acids, which exists on many protein molecules that involve in cellular signal transduction. A PH domain located in GRK2 residue 548 to 660 may play a significant role in mediating interaction between PKC and GRK2. In present study, we revealed that PKC could associate with PH domain of GRK2 in pull-down assay in vitro. Co-immunoprecipitation displayed binding of PKC to GRK2 in intact Jurkat cells after prolonged stimulation of epinephrine. Assay of PKC beta1 kinase activity indicated that the binding of the PH domain of GRK2 to PKC beta 1 could down-regulate activity of PKC beta 1 kinase. Thus, GRK2 may play a negative feedback regulatory role on PKCbeta1 activity in interaction between GRK2 and PKCbeta 1.
Blood Proteins/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation, Enzymologic/genetics , Phosphoproteins/chemistry , Protein Kinase C/genetics , Repressor Proteins/metabolism , Trans-Activators/metabolism , Agammaglobulinaemia Tyrosine Kinase , Animals , COS Cells , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Dogs , Epinephrine/pharmacology , Humans , Jurkat Cells , Peptides/metabolism , Protein Binding/genetics , Protein Kinase C/metabolism , Protein Kinase C beta , Protein Structure, Tertiary/genetics , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/metabolism , Sequence Homology, Amino Acid , U937 Cells , beta-Adrenergic Receptor Kinases
The gene encoding the Ig-like domain of tyrosine protein kinase receptor EphB2 was cloned into the expressing vector pET28a. Under induction with IPTG, the positive strain expressed the fusion protein with a hexahistidine tail on the N-terminal. The protein was purified under denaturing conditions using metal chelate chromatography. The purity was up to 94%. The purified-protein-coated ELISA plate was used as target to screen recombinant phages able to bind onto it, and after three rounds of affinity screening, 19 phages that could bind specifically with EphB2 were isolated from a random phage-displayed seven-peptide library. The peptide sequences of the positive phage clones were analyzed.
