EBV/C3d receptor (CR2) interacts by its intracytoplasmic carboxy-terminal domain and two distinct binding sites with the p53 anti-oncoprotein and the p68 calcium-binding protein.

EBV/C3d receptor (CR2) interacts with the p53 anti-oncoprotein expressed in the human B lymphoma cells, Raji but not in normal B cells, and with the p68 calcium-binding protein, expressed in normal B lymphocytes but not in transformed B lymphocytes. To characterize the CR2 domain interacting with these two intracellular proteins, we synthesized a 34-amino acid peptide, pep34, corresponding to its intracytoplasmic carboxy-terminal domain and analyzed its binding and antigenic properties. Binding of 125I-labeled p53 or 125I-labeled p68 on immobilized pep34 was specific, additive, and totally inhibited by unlabeled p53 or p68, respectively, but not by unlabeled p68 or p53, respectively. Antigenic properties of pep34 were analyzed by immunizing rabbits with particle-bound pep34. Polyclonal anti-pep34 Ab carried anti-CR2 specificities that recognized only the intracellular domain of CR2. In addition, anti-pep34 Ab also carried anti-p53 or anti-p68 specificities. Anti-p53 or anti-p68 specificities were not due to putative common structural or conformational antigenic determinants between the pep34 synthetic peptide and the p68 or p53 proteins. These anti-p53 and anti-p68 specificities were identified as anti-idiotypic anti-CR2 Ab mimicking either p53 or p68 binding sites of CR2. These data clearly establish that despite its short length, the intracytoplasmic C-terminal tail of CR2 is involved in direct protein-protein interactions with the two intracellular regulatory proteins, p53 and p68. An additional feature of these data is the demonstration that particle-bound pep34 triggered "in vivo" anti-Id Ab restricted to either p53 or p68 specificities.     

Expression of CR2 (the C3dg/EBV receptor, CD21) on normal human peripheral blood T lymphocytes.

The expression of CR2 (the C3dg/EBV receptor, CD21) on normal human T lymphocytes was investigated using purified peripheral blood T cells and indirect immunofluorescence with biotinylated anti-CR2 mAb and streptavidin-phycoerythrin. Thirty to 40% of normal peripheral blood T lymphocytes expressed CR2 Ag. The cells expressed three nonoverlapping epitopes of CR2. The specificity of the staining for CR2 epitopes was demonstrated by the ability of unlabeled anti-CR2 mAb but not of anti-CR1 mAb of the same isotype to compete for the binding of biotinylated anti-CR2 mAb to T cells. The intensity of staining of T lymphocytes with anti-CR2 mAb was approximately 10-fold lower than that of peripheral blood B cells. CR2 was immunoprecipitated from purified T lymphocytes as a single protein of apparent Mr 145,000. The presence of CR2 on normal human T lymphocytes suggests that the receptor may modulate the function of T cells in the immune response and the susceptibility of the cells to infection by lymphocytotropic viruses.     

Activation of the EBV/C3d receptor (CR2, CD21) on human B lymphocyte surface triggers tyrosine phosphorylation of the 95-kDa nucleolin and its interaction with phosphatidylinositol 3 kinase

We previously demonstrated that CR2 activation on human B lymphocyte surface triggered tyrosine phosphorylation of a p95 component and its interaction with p85 subunit of phosphatidylinositol 3' (PI 3) kinase. Despite identical molecular mass of 95 kDa, this tyrosine phosphorylated p95 molecule was not CD19, the proto-oncogene Vav, or the adaptator Gab1. To identify this tyrosine phosphorylated p95 component, we first purified it by affinity chromatography on anti-phosphotyrosine mAb covalently linked to Sepharose 4B, followed by polyacrylamide gel electrophoresis. Then, the isolated 95-kDa tyrosine phosphorylated band was submitted to amino acid analysis by mass spectrometry; the two different isolated peptides were characterized by amino acid sequences 100% identical with two different domains of nucleolin, localized between aa 411--420 and 611--624. Anti-nucleolin mAb was used to confirm the antigenic properties of this p95 component. Functional studies demonstrated that CR2 activation induced, within a brief span of 2 min, tyrosine phosphorylation of nucleolin and its interaction with Src homology 2 domains of the p85 subunit of PI 3 kinase and of 3BP2 and Grb2, but not with Src homology 2 domains of Fyn and Gap. These properties of nucleolin were identical with those of the p95 previously described and induced by CR2 activation. Furthermore, tyrosine phosphorylation of nucleolin was also induced in normal B lymphocytes by CR2 activation but neither by CD19 nor BCR activation. These data support that tyrosine phosphorylation of nucleolin and its interaction with PI 3 kinase p85 subunit constitute one of the earlier steps in the specific intracellular signaling pathway of CR2.     

Expression, molecular association, and functions of C3 complement receptors CR1 (CD35) and CR2 (CD21) on the human T cell line HPB-ALL.

We have investigated the expression, molecular association, ligand binding properties, and ability to transduce intracellular signals of CR1 and CR2 C3 receptors on cells of the human HPB-ALL T cell line. CR1 and CR2 on HPB-ALL cells bound polymeric C3b and C3dg and several anti-CR1 and anti-CR2 mAb recognizing different epitopes of the receptors on normal peripheral blood cells. Immunoprecipitated CR1 and CR2 exhibited similar m.w. to those of the receptors on normal peripheral blood T and B lymphocytes. CR1 and CR2 were partially associated in the form of CR1/CR2 complexes in the cell membrane as assessed by the ability of the receptors to cocap and cointernalize and to form a detergent-sensitive complex upon immunoprecipitation analysis. Triggering of CR2 with mAb OKB7 that recognizes an epitope associated with the ligand binding site of the receptor induced an increase in intracellular free calcium concentration in HPB-ALL cells. The signal provided by mAb OKB7 did not synergize with that triggered by anti-CD3 mAb UCHT1. Triggering of CR1 did not result in changes in intracellular free calcium concentration. Our observations have significance for the biology of normal human T cells because the majority of peripheral blood T cells that express CR1 also expressed CR2 and because a change in (Ca2+)i was induced by mAb OKB7 in purified normal T cells. These functions may be relevant for the regulatory role of C3 fragments on the immune response to T-dependent Ag and for the penetration into T cells of lymphocytotropic viruses.     

Identification of an epitope in the major envelope protein of Epstein-Barr virus that mediates viral binding to the B lymphocyte EBV receptor (CR2)

The Epstein-Barr virus gp350/220 envelope protein mediates virus attachment to the EBV/C3dg receptor (CR2) of human B lymphocytes. Synthetic peptides corresponding to two regions in gp350/220, which have a similar amino acid sequence with the complement C3dg protein, were used to identify a receptor binding epitope. A peptide corresponding to the N terminus of gp350/220, EDPGFFNVE, bound to purified CR2 and to CR2 positive but not CR2 negative B and T lymphoblastoid cell lines. Soluble monomeric gp350/220 peptide blocked CR2 binding to immobilized EBV, while multimeric forms of the N-terminal gp350/220 peptide conjugated to albumin efficiently blocked recombinant gp350/220 and C3dg binding to B cells as well as EBV-induced B cell proliferation and transformation. These studies indicate that the N-terminal region of gp350/220 plays a crucial role in mediating the earliest stages of EBV infection of B cells and provides a molecular basis for the restricted host cell EBV tropism.     

Isolation and characterization of a novel 68,000-Mr Ca2+-binding protein of lymphocyte plasma membrane.

A 68 000-Mr protein is a major component of a Nonidet P-40-insoluble fraction of lymphocyte plasma membrane prepared from human B lymphoblastoid cells ( BRI 8) and pig mesenteric lymph nodes. The association of the protein with the detergent-insoluble complex depends on free Ca2+ concentrations of greater than 10 microM. The human and pig 68 000-Mr proteins were purified and appear to be homologous on the basis of amino acid composition and peptide mapping. The protein is monomeric, has pI 5.8 and a single high-affinity Ca2+-binding site (KD 1.2 microM). The results are discussed in terms of the possible role of the 68 000-Mr protein as an intracellular Ca2+ receptor in lymphocytes.     

gp 140, the C3d/EBV receptor (CR2), is phosphorylated upon in vitro activation of human peripheral B lymphocytes

gp 140, the C3d/EBV receptor (CR2), is a specific marker of human B lymphocytes. Very recent data suggest that CR2 is a membrane site involved in early B cell activation. These properties of CR2 led us to analyze the molecular events associated with gp 140. We analyzed whether in some conditions of B lymphocyte activation, CR2 could be phosphorylated. We have found that when highly enriched peripheral B cells were cultured for 48 h with anti-mu Ab and/or SAC, in order to provide an optimal activating signal, phosphorylation of the CR2 was induced.     

Activated human B lymphocytes express cyclooxygenase-2 and cyclooxygenase inhibitors attenuate antibody production

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases and target cyclooxygenases 1 and 2 (Cox-1, Cox-2) that are responsible for PG production. Newer Cox-2-selective drugs have been heavily prescribed to quench inflammation. Little is known about whether or not these drugs influence human B lymphocytes and their ability to produce Ab. We report herein that activated human B cells not only highly express Cox-2 and produce PGs, but that the NSAID indomethacin and Cox-2-selective drugs profoundly inhibit the ability of human B cells to produce IgG and IgM in vitro. Human blood B cells highly express Cox-2 mRNA and protein and produce PGs after activation with CD40L, pansorbin, or CD40L plus BCR engagement. Cox-2 is also highly expressed by human tonsil B cells, as shown by immunohistochemistry. Cox-inhibiting drugs modestly affect purified B cell proliferation but profoundly reduce Ab production. The ability of whole blood to produce IgM and IgG following stimulation is also strongly inhibited. In support that Cox-2 plays a seminal role in B lymphocyte Ab production, Cox-2 knockout mice have 64% less IgM and 35% less IgG than normal littermate controls. These findings support that NSAIDs and the new Cox-2-selective drugs have an unsuspected target, the B cell, and attenuate Ab production in humans. Use of NSAIDs may therefore influence autoantibody production in autoimmune diseases and may dampen humoral immunity in response to antigenic challenge/vaccination.     

Studies on the subcellular localization of the membrane-bound fraction of intestinal calcium-binding protein.

Sorbitol density gradient centrifugation applied to intestinal mucosa homogenates resulted in a complete separation of soluble calcium-binding protein from the bound fraction of calcium-binding protein, providing further documentation of the bound pool of calcium-binding protein. The peak of the bound calcium-binding protein was not associated with the major peaks of any of the markers used, but was associated with minor peaks of alkaline phosphatase, RNA, and glucose-6-phosphatase. Lack of association of bound calcium-binding protein with (Na+ + K+)-ATPase indicated that the bound calcium-binding protein is not on the basolateral membrane. Differential centrifugation fractionation indicated that the bound calcium-binding protein is not associated with nuclei or mitochondria. The bound calcium-binding protein also could not be detected in partially purified brush borders. Exclusion of the brush border and basolateral membranes as the location of the bound calcium-binding protein suggests an intracellular locale.     

Role of endogenous brain-derived neurotrophic factor and sortilin in B cell survival

Brain-derived neurotrophic factor (BDNF), a major neuronal growth factor, is also known to exert an antiapoptotic effect in myeloma cells. Whereas BDNF secretion was described in B lymphocytes, the ability of B cells to produce sortilin, its transport protein, was not previously reported. We studied BDNF production and the expression of its receptors, tyrosine protein kinase receptor B and p75 neurotrophin receptor in the human pre-B, mature, and plasmacytic malignant B cell lines under normal and stress culture conditions (serum deprivation, Fas activation, or their combination). BDNF secretion was enhanced by serum deprivation and exerted an antiapoptotic effect, as demonstrated by neutralization experiments with antagonistic Ab. The precursor form, pro-BDNF, also secreted by B cells, decreases under stress conditions in contrast to BDNF production. Stress conditions induced the membranous expression of p75 neurotrophin receptor and tyrosine protein kinase receptor B, maximal in mature B cells, contrasting with the sequestration of both receptors in normal culture. By blocking Ab and small interfering RNA, we evidenced that BDNF production and its survival function are depending on sortilin, a protein regulating neurotrophin transport in neurons, which was not previously described in B cells. Therefore, in mature B cell lines, an autocrine BDNF production is up-regulated by stress culture conditions and exerts a modulation of apoptosis through the sortilin pathway. This could be of importance to elucidate certain drug resistances of malignant B cells. In addition, primary B lymphocytes contained sortilin and produced BDNF after mitogenic activation, which suggests that sortilin and BDNF might be implicated in the survival and activation of normal B cells also.     

Analysis of the human immune repertoire using human monoclonal antibodies

The investigations of the human immune response to cancer and other diseases have been hampered by the difficulty in determining the specificity of low-titered antibodies in serum, and the inability to define the specificity of individual lymphocytes. In order to study these issues, we developed the hybridoma technology so that human monoclonal antibodies (hM Ab) could be reliably and reproducibly obtained. Using a variety of fusion partners of both mouse and human origin, a large number of human immunoglobulin-secreting hybrids have been generated. We have found that 5 to 10% of the hybridomas produced secrete hM Ab reactive with antigens (Ag) expressed by human cells. Specificity analysis and cellular localization studies of the Ag have been performed for a large number of these hM Ab, and several general points have emerged from our study: (A) A significant proportion of the evaluable B-cell repertoire is directed to the production of antibodies reactive with Ags expressed by human cells. (B) The great majority of these Ags have an intracellular location, and are broadly distributed, being expressed by both normal and malignant cells. (C) Intracellular and cell surface differentiation Ags and other Ags with restricted distribution have been defined by hM Ab, including a series of cell surface and intracellular Ags not detected on normal cells. (D) The relationship of these findings to cancer is unclear as hM Ab showing distinctive distributions have been generated from the lymphocytes of normal individuals as well as tumor-bearing patients. (E) hM Ab with distributions distinct from any known mouse monoclonal antibodies (mM Ab) have been obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 16 amino acid synthetic peptide derived from human C3d triggers proliferation and specific tyrosine phosphorylation of transformed CR2-positive human lymphocytes and of normal resting B lymphocytes.

We demonstrate herein that p16, a 16 amino acid synthetic peptide derived from human C3d, which carried LYNVEA sequence of C3d reacting with CR2 and C3d present in trypsin-cleaved C3, triggered "in vitro" and "in vivo" phosphorylations and "in vitro" proliferation of human B lymphocytes, depending on the stage of cell differentiation. Indeed, p16 and C3dT induced "in vivo" tyrosine phosphorylation of pp105 and "in vitro" proliferation only of CR2-positive and not of CR2-negative cell lines. In addition, p16 and C3dT also induced "in vivo" tyrosine phosphorylation of pp100 and "in vitro" proliferation of only small dense resting B lymphocytes and not other B lymphocyte subpopulations nor T lymphocytes. These data suggest that induction of pp100 and pp105 phosphorylation by p16 and C3dT could represent an early event associated with expression of CR2 in the regulation of human B lymphocyte proliferation.     

Monoclonal and anti-idiotypic anti-EBV/C3d receptor antibodies detect two binding sites, one for EBV and one for C3d on glycoprotein 140, the EBV/C3dR, expressed on human B lymphocytes

Glycoprotein (gp) 140, the EBV/C3dR of B lymphocytes, is a membrane site involved in human cell regulation. To analyze the specificities of the binding sites for EBV and for C3d on the gp 140 molecule, two distinct approaches were used. First, anti-EBV/C3dR mAb were prepared against highly purified EBV/C3dR. Nine anti-EBV/C3dR mAb were obtained. Four of these anti-EBV/C3dR mAb inhibited C3d binding but not EBV binding on gp 140, whereas four others exerted an inverse effect. These differences could not be due to differences in isotype, antibody concentration, affinity constant, and number of molecules bound on cell surface, as these parameters were identical for the nine used mAb. Second, polyclonal anti-idiotypic antibodies (Ab2) were prepared against F(ab)'2 fragments of polyclonal anti-EBV/C3dR (Ab1). Ab2 recognized the variable portion of Ab1 as controlled by immunoblotting experiments. Ab2, which did not react with the cell surface, inhibited Ab1 binding on Raji cells. Ab2 mimicked the EBV/C3dR by its properties to bind to particle-bound C3d and EBV, preventing their binding on Raji cell surface. C3d binding specificities contained in Ab2 were isolated by affinity chromatography on C3b/C3bi-Sepharose. These specificities, being the internal image of C3d binding site of EBV/C3dR, reacted with Ab1 and inhibited particle-bound C3d binding on Raji cells but did not react with EBV. Taken together, these data support strongly that gp 140, the EBV/C3dR, carried two distinct binding sites, one for EBV and one for C3d.     

Primary structure of the human, membrane-associated Ca2+-binding protein p68 a novel member of a protein family.

cDNA clones encoding human 'p68', a membrane-associated Ca2+-binding protein, were isolated from a lambda gt11 expression library of the human T-leukaemia cell line J6, by using a rabbit antiserum against denatured purified lymphocyte p68, and from a liver cDNA library by using 32P-labelled p68 cDNA fragments. The amino acid sequence of p68, deduced from the sequences of overlapping cDNA clones, is described. The results show that p68 is closely related to a family of proteins which includes intracellular substrates of the EGF receptor and pp60src tyrosine kinases. The p68 amino acid sequence is internally repetitive, being constructed from eight repeats of varying lengths, each of which contains a highly conserved sequence. Multiple copies of the latter sequence are also present in the related proteins p36, lipocortin I and protein II. We discuss how the common structural features of these proteins may reflect common functions and, furthermore, how the eight repeat structure of p68 may have evolved. The sequences of independent cDNAs suggest that alternatively-spliced mRNAs could encode different p68 protein species. This suggestion is consistent with the observation that purified p68 migrates as a closely-spaced doublet when analysed by SDS-PAGE.     

A subfamily of RNA-binding DEAD-box proteins acts as an estrogen receptor alpha coactivator through the N-terminal activation domain (AF-1) with an RNA coactivator, SRA

One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identified a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor alpha (hER alpha) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC-1/TIF2 family protein and the hER alpha A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1) and SRA were co-immunoprecipitated with estrogen-bound hER alpha in MCF7 cells and in partially purified complexes associated with hER alpha from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hER alpha and TIF2 in the nucleus. The presence of p72/p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen-induced transactivation of hER alpha. These findings indicate that p72/p68 acts as an ER subtype-selective coactivator through ER alpha AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with SRA and the SRC-1/TIF2 family proteins.     

NMR solution structure of complement-like repeat CR8 from the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein is a member of the low density lipoprotein receptor family and contains clusters of cysteine-rich complement-like repeats of about 42 residues that are present in all members of this family of receptors. These clusters are thought to be the principal binding sites for protein ligands. We have expressed one complement-like repeat, CR8, from the cluster in lipoprotein receptor-related protein that binds certain proteinase inhibitor-proteinase complexes and used three-dimensional NMR on the 13C/15N-labeled protein to determine the structure in solution of the calcium-bound form. The structure is very similar in overall fold to repeat 5 from the low density lipoprotein receptor (LB5), with backbone root mean square deviation of 1.5 A. The calcium-binding site also appears to be homologous, with four carboxyl and two backbone carbonyl ligands. However, differences in primary structure are such that equivalent surfaces that might represent the binding interfaces are very different from one another, indicating that different domains will have very different ligand specificities.     

Characterization of the baboon erythrocyte C3b-binding protein

E from primates demonstrate type 1 CR (CR1) with binding specificities for C3b and C4b. In the present study we characterized the E C3b-binding protein of baboons. We showed that three out of four mouse mAb and one polyclonal antiserum, raised against human E CR1, cross-reacted with baboon E. In addition, one anti-human CR1 mAb (1B4) and a polyclonal anti-human CR1 inhibited the binding of C3b opsonized immune complexes to baboon E. Finally, a mAb to human CR1 (E11) recognized epitopes on E of a variety of nonhuman primates, including baboons. SDS-PAGE analysis of biochemically purified baboon E membrane fractions reactive with E11 demonstrated a 65-kDa protein as a major component. Affinity absorption and elution experiments verified this protein to be E11 reactive as well as a C3b binding protein. E surface radiolabeling, followed by C3i affinity purification, confirmed that this 65-kDa protein is the only C3b-binding protein present on the baboon E membrane. We postulate that the baboon E 65-kDa protein is the equivalent of the human E CR1. In addition, there appear to be antigenic similarities between the baboon E 65-kDa protein and the human E CR1.     

Cellular distribution of p68, a new calcium-binding protein from lymphocytes.

A Ca2+-binding protein of mol. wt. 68 000 ( p68 ) is a major component of a Nonidet P-40 insoluble fraction of human and pig lymphocyte plasma membrane. An affinity-purified rabbit antibody has been produced against p68 and used to study its cellular distribution. The antibody stained fixed and permeabilised human B lymphoblastoid cells, peripheral blood lymphocytes and sections of human tonsil. Whole cells, however, were not stained, indicating that the protein was not represented at the cell surface. This assignment was consistent with the detection of p68 in immunoprecipitates from biosynthetically- but not surface-labelled cells. It is concluded that p68 is located on the cytoplasmic face of the plasma membrane. Subcellular fractionation experiments confirmed that p68 was largely membrane-bound in lymphocytes, although a small soluble fraction (approximately 10% of the total) was detected. Sub-fractionation of lymphocyte membranes revealed that p68 was associated not only with the plasma membrane but also with other endomembrane systems. As judged by immunoprecipitation, p68 was present in a variety of cultured cell lines of both lymphoid and non-lymphoid origin. p68 demonstrated a diffuse distribution in fixed and permeabilised fibroblasts which did not correspond to the distribution of either microfilaments or intermediate filaments. However, in detergent-extracted cells the protein was localised in a lamina-like network. A similar immunofluorescent staining pattern has recently been observed for spectrin-related proteins in the detergent-resistant cytoskeleton of fibroblasts. It is suggested that p68 is part of a sub-membranous cytoskeletal complex not only in lymphocytes but also in other cell types.