c3d receptor (CR2) in the human burkitt b lymphoma cell, raji

Molecular immunology, Vol. 29, No. 9, pp.

0161-5890/92 $5.00 fO.00 0 1992 Pergamon Press Ltd

f.113--l 120, 1992

Printed in Great Britain.

VIRUS/C3d NUCLEAR LOCALIZATION OF THE EPSTEIN-BARR RECEPTOR (CR2) IN THE HUMAN BURKITT B LYMPHOMA CELL, RAJI” ALINE GAuFI%E,t ANNIE VIRON,~ MONIQUE BAREL,~ JACQUES HERMANN,? EDMOND PUVION$ and RAYMOND FRADE~§ ~Immunochimie des Regulations Cellulaires et des Interactions Virales, INSERM U.354, H6pital Saint-Antoine, 75012 Paris, France and $Institut de Recherches Scientifiques sur le Cancer, UPR 272 CNRS, BP8, 94801 Villejuif Cedex, France (First received 20 November 1991; ucce~ted in revisedform 20 Ju~u~ry 1992) Abstract-Epstein-Barr virus/C3d receptor (CR2) is a glycoprotein of mol. wt 140,000 expressed on the surface of Raji cells. We previously isolated phosphorylated CR2 from purified Raji cell nuclei. We have analyzed the nuclear localization of CR2 by electron microscope immunochemistry of thin sections of Raji cells and we have compared the binding properties of CR2 expressed on purified plasma membranes or nuclei. Anti-CR2 mAb immunogold labeling of thin sections of Raji cells identified CR2 at the nuclear surface and also within the nucleus. Nuclear envelope associated CR2 was localized mainly at nuclear pores. Within the nucleus, CR2 was associated with ribonucleoprotein (RNP) interchromatin fibrils. This labeling was preserved in nuclear matrix preparations. CR2 expressed on the surfaces of purified nuclei or on the ceil surface interacted with soluble and particIe-bound C3bi/C3d. Monoclonal anti-CR2 antibodies, which recognized extracellular domains of CR2, reacted differently with CR2 depending on its subcellular localization. The presence of CR2 in nuclei may be due to translocation of the cell surface CR2 and/or the presence of two distinct intracellular pathways for mature CR2.

INTRODUCTION

The receptor for the Epstein-Barr virus (EBV) and for the C3d fragment of C3, the human third component of the complement, is a glycoprotein of mol. wt 140,000 (EBV/C3dR, CD21 or CR2) (Bare1 et al., 1981; Frade et al., 1985b; Frade, 1990). The presence of distinct binding sites on CR2 for EBV and C3d was demonstrated by using either anti-CR2 mAb and polyclonal anti-idiotypic anti-CR2 antibodies (Bare1 et al., 1988) or deletion mutant glycoproteins and synthetic peptides of gp350/220, the viral capsid protein which mediates EBV binding to CR2 (Tanner et al., 1988). These data have

been recently confirmed using human-mouse chimeras of CR2 (Molina et al., 1991). CR2 is involved in human B lymphocyte activation (Frade, 1986). Indeed, crosslinking of CR2 at the cell surface by specific extracellular ligands increased B l~phocyte proliferation in synergy with T-cell factors (Frade et al., 198%; Melchers et al., 1985; Nemerow et al., 1985; Masucci et al., 1987). CR2 was phosphorylated during “‘in vitro” activation of human B lymphocytes (Bare1 et al., 1986; Changelian and Fearon, 1986). Analysis of the intracellular role of CR2 in human B lymphocytes was performed by analyzing the intracellular components which interacted directly with this receptor and by studying its subcellular distribution. We have recently shown that CR2 interacts specifically with two distinct intracellular components, depending on the normal or transfo~ed state of human B lymphocytes. Indeed, CR2 reacted with the p.53 antioncoprotein expressed in the human B lymphoma cell line Raji, but not in normal B lymphocytes (Bare1 et al., 1989). In addition, CR2 reacted in human tonsil B lymphocytes with ~68, a calcium-binding protein, not detected in Raji cells (Bare1 et al., 1991). The presence of CR2 in Raji cell nuclei was suggested from analysis of subcellular localization of phosphorylated CR2 (Delcayre et al., 1987). CR2 isolated from nuclei interacted with a nuclear p 120 ribonucleoprotein, ~120 RNP. “In vitro” phosphorylation of ~120 RNP depended on the presence of CRZassociated kinases (Delcayre et al., 1987). No biochemical differences were

*This work was supported by Institut National de la Sante et de la Recherche Medicale (INSERM), Association pour la Recherche contre le Cancer (ARC), Fondation de France, Fondation de la Recherche Medicale, Association de Recherche sur la Polyarthrite and Valmon Inc. gAddress correspondence and reprint requests to: Raymond Frade, Immunochimie des Regulations Cellulaires et des Interactions Virales, INSERM U.354, Hopital SaintAntoine, 75012 Paris, France. Abbreviations: CR2, Complement receptor type 2; C3d, cleavage fragment of third complement component; C3bi/C3d, mixture of C3 fragments generated by trypsin treatment of C3; RNP, ribonucleoprotein; SDS-PAGE, polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate; NP-40, Nonidet-P40; PMSF, phenylmethylsulfonyl-fluo~de; M,, apparent molecular weight; mAb, monoclonal antibody. “,MM BY--F 1113

ALINE GAUFFREet al.

1114

detected by gel electrophoresis analysis between whole or fragmented CR2 isolated from plasma membranes or purified nuclei (Delcayre, 1989). To further study the properties of CR2 present in Raji nuclei, we analyzed the subcellular and subnuclear distribution of CR2 by electron microscope immunochemistry on thin sections of Raji cells. In addition, we analyzed the binding properties of CR2 expressed an Raji cell surfaces or in purified nuclei with the extracellular ligands human C3bi/C3d and anti-CR2 mAb. MATERIALS

Human

AND METHODS

cells

Human cell lines used were: Raji, a Burkitt B lymphoma, CR2 positive cell line and CEM, a T lymphoma CR2 negative cell line. Cells were grown in RPM1 1640 supplemented with 10% heat-inactivated fetal calf serum (Flow Labs, Rockville, MD, U.S.A.), 2 mM L-glutamine, 100 U/ml penicillin and 85 PM streptomycin, at 37°C in a 5% CO, incubator. Subcellular

,fractionation

of cells

Plasma membranes were prepared by differential centrifugation according to the method of McKee1 and Jarett (1970) as modified by Delcayre et al. (1987). Nuclei were purified following the method of Blobel and gradient of Potter (1966) on a discontinuous 0.55 : 1 : 1.6 M sucrose. Nuclear

matrix preparation

Nuclear matrix preparations were made as described by Berezney and Coffey (1977). Briefly, purified nuclei were incubated in 20 mM Tris-HCl (pH 7.5), 5 mM MgCl,, 250 mM sucrose, containing 0.1% BSA, then successively treated in this buffer with 1Opg DNAse I, 2 M NaCl, 1% Triton X100, 50 pg DNAse I and 50 pg RNAse A. After each treament, the nuclear particles were washed twice in the same buffer. The residual nuclear particles were retained as nuclear matrix. Binding of soluble fractions

or particle-bound

C3bilC3d

to cell

After preincubation for 30 min with either polyclonal anti-CR2 antibodies (Frade et al., 1984) or the IgG fraction of non-immune serum, cells or purified nuclei were incubated in barbital buffer (pH 7.5), 50 mM NaCl, 2 mM CaCl,, 3 mM MgCl,, 250 mM sucrose, 0.1% BSA with 106cpm of ‘251-C3bi/C3d labeled as previously described (Bare1 et al., 1988). After 60 min incubation at 4°C samples were layered on 200 ~1 butylphthalate-nonylphthalate (4: 1, Kodak, Rochester) and centrifuged for 5 min at 7000g. The pelleted fractions were counted for radioactivity. Specific binding of particlebound C3bi/C3d to cells was measured by rosette assay, as previously described (Frade et al., 1984). Binding qf monoclonal

antibodies

to cell fractions

Cells or purified nuclei were incubated in barbital buffer (pH 7.5) 50 mM NaCl, 2 mM CaCl,, 3 mM

MgCl,, 250 mM sucrose with the same amount of either anti-transferrin receptor mAb (Becton-Dickinson, Sunnyvale, CA, U.S.A.) or with anti-CR2 mAb for 45 min at 4°C. After extensive washes, samples were successively incubated with the IgG fraction of rabbit anti mouse IgG (Dako, Denmark) and with 1O”cpm of ‘“SI-protein A. Immunocytochemistry For standard electron microscope immunochemistry, Raji cells or nuclear matrix preparations were fixed at 4°C for 1 hr with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.3). After centrifugation, the pellets were dehydrated in methanol prior to Lowicryl K4M embedding (Carlemalm et al., 1982). Ultrathin sections were floated successively on drops of distilled water, PBS and 5% BSA. They were then incubated for 1 hr with 1: 10 dilution of antibodies in PBS, subsequently washed for 15 min in PBS and incubated for 30 min with a 1: 10 dilution in PBS of goat anti-mouse IgG conjugated to gold particles, 10 nm dia (Janssen Pharmaceutics Beerse, Belgium). After a final PBS wash, the grids were rinsed with a jet of distilled water and air-dried. Staining of grids was carried out either with uranyl acetate (10 min) and lead citrate (30 set) or with the EDTA regressive staining method. In this method, thin sections were first overstained with uranium and then treated with EDTA which removed the metal more effectively from desoxibonucleoprotein than from RNP, thereby visualizing preferentially the RNP components (Bernhard, 1969; Puvion et al., 1984). Control samples were prepared in the same way except that normal mouse serum or an irrelevant mouse anti-transferrin receptor mAb was used instead of the anti-CR2 mAb. RESULTS

The localization of CR2 in Raji nuclei was analyzed by electron microscope immunochemistry on thin sections of Raji cells embedded in lowicryl, a polymer which preserves the integrity of nuclei structures but not that of plasma membranes, therefore preventing the labeling of the latter. The subcellular localization of CR2 was analyzed using monoclonal anti-CR2 antibodies (mAbs) prepared against highly purified receptor (Bare1 et al., 1988) and detected by goat anti-mouse IgG conjugated to gold particles. Three anti-CR2 mAbs, MR.l, MR.2 and MR.4 (Bare1 et al., 1988), gave positive gold staining in the nuclei and cytoplasm of Raji thin sections (Fig. 1). In the cytoplasm, anti-CR2 immunogold labeling was mainly detected in areas containing polyribosomes. Small clusters of immunogold particles were observed in contact with the nuclear envelope, especially near nuclear pores. The clusters were distributed in nuclear and cytoplasmic areas lining the nuclear pore channel [Fig. l(b,c)]. In addition, positive staining was detected in the intranuclear compartment. In the control, thin sections were not stained by a mouse mAb, recognizing the transferrin receptor expressed on the cell surface [Fig. l(a)]. When the EDTA regressive staining method

Nuclear

localization

of CR2

Fig. 1. Immunogold labeling of Lowicryl embedded Raji cells using anti-CR2 mAb. (a) Thin sections were labeled using anti-transferrin receptor mAb as control and few gold particles are seen (arrowheads). (b, c) Thin sections were labeled with anti-CR2 mAb. Identical results were obtained with MR.], MR.2 or MR.3. Gold particles are distributed over both nucleus (N) and cytoplasm (CY). The nucleolus (NU) and the ergastoplasm (ER) are not significantly labeled. Arrows indicate a selective accumulation of labeling at the nuclear envelope. Few gold particles are present at the plasma membranes (PM) (arrow heads). Staining: uranyl acetate and lead citrate. (a) x 30,000. (b) x 30,000. (c) x 60,000.

1115

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ALINE GAUFFRE et al.

was used on thin sections, anti-CR2 mAb immunogold labeling was co-localized on RNP interchromatin fibrils, whilst nucleoli were not labeled (Fig. 2). The sub-nuclear localization of CR2 was analyzed by preparing nuclear matrix (Berezney and Coffey, 1977). In these conditions, 85% of the total nuclear proteins and 55% of ‘Z51-labeled components of the nuclear surface were extracted. In nuclear matrix preparations, immunolabeling by anti-CR2 mAb was observed on the internal matrix despite RNAse digestion (Fig. 3). This suggested that CR2 is part of the matricial proteins

belonging to the salt resistant complexes of hnRNP (Gallinaro et al., 1983). The residual nucleoli were not labeled. Perinuclear labeling observed in Fig. 2 was abolished after the above treatments of nuclei (Fig. 3). The binding properties of CR2 present on highly purified nuclei of Raji cells were analyzed using specific extracellular ligands as soluble and particle-bound C3bi/C3d, which expressed the C3d site (Frade et al., 1984; Bare1 et al., 1988). Soluble ‘2SI-C3bi/C3d and particle-bound C3bi/C3d bind specifically to Raji purified nuclei as well as on cell surfaces (Table l), since

Fig. 2. Immunogold labeling of Lowicryl embedded Raji cells using anti-CR2 mAb. RNP were preferentially visualized by the EDTA regressive staining method. (a) Thin sections were incubated with anti-transferrin receptor mAb used as control and very few gold particles were seen. (b) Thin sections were incubated with anti-CR2 mAb. Identical results were obtained with MR.1, MR.2 or MR.3. The nucleolus (NU) and bleached condensed chromatin (CH) are not labeled; gold particles (arrows) are observed within the nucleus over the RNP fibrils (CY, cytoplasm). (a) x 30,000. (b) x 30,000.

Nuclear

localization

of CR2

1117

Fig. 3. Nuclear matrix following RNAse digestion. (a) Thin sections were incubated with anti-transferrin receptor mAb used as control and few gold particles were seen. (b) Thin sections were incubated with anti-CR2 mAb. Identical results were obtained with MR.], MR.2 or MR.3. Gold particles are observed over the fibrillar network of residual internal matrix (arrows). The residual nucleolus (NU) is not labeled (CY, residual cytoplasm). Arrowheads show the lamina (L). (a) x 40,000. (b) x 40,000.

Table 1. Binding of specific extracellular ligands of CR2 to cells and highly purified nuclei

Fraction Raji cells Raji nuclei CEM cells CEM nuclei “IgG fraction

Preincubated with NIS IgG anti-CR2 NIS IgG anti-CR2 NIS IgG anti-CR2 NIS IgG anti-CR2

iz51-C3bi/C3d bound ( x lo-’ cpm) 28 * 1 7fl 15f 1 8+1 4*1 4+1 6+1 6+1

Ab Ab Ab Ab

of non-immune

serum.

C3bi/C3d rosettes W) 50 8 45 13 8 8 10 10

they were inhibited by polyclonal anti-CR2 antibodies (Frade et al., 1984). No specific binding was measured on nuclei purified from CEM, a CR2 negative T cell line (Delcayre er al., 1987). Additionally, specific binding of 10 anti-CR2 mAbs, MR.1 to MR.9, prepared against solubilized CR2 (Bare1 et al., 1988) and OKB-7, all interacting with the extracellular domain of CR2, were compared on Raji cells and purified Raji nuclei (Fig. 4). These anti-CR2 mAbs interacted with both cell surfaces and nuclei of Raji cells, whilst an anti-transferrin receptor (anti-TrR), used as a control, interacted with the Raji cell surface but not with Raji nuclei. Among the antiCR2 mAbs used, the highest binding to Raji cells was measured with OKB-7, MR.2 and MR.8, and to Raji

ALINE GAUFFRE et

1118

OK&7

MR.4

MR.6

MR.5

MR.8

al.

MR.1

MR.7

MR.6

MR.2

MR.7

MR.6

MI4.P

mAb

OKB-7

MR.4

MR.6

MR.6

MR.6

MR.1

MR.3

antl.TrR

mAb

Fig. 4. Comparison

of the specific binding of anti-CR2 mAbs to Raji cells and to purified Raji nuclei. Raji cells (A) or highly purified nuclei of Raji (B) were incubated for 45 min at 4°C with anti-CR2 mAb or anti-transferrin receptor mAb. Bound IgG was detected by incubation with rabbit anti-mouse IgG and with IOhcpm of “‘I-protein A, successively. Each point represents the mean of triplicate dete~inations from five separate experiments. Nonspecific binding was measured on cells and nuclei using an anti-C3d mAb and cpm were substracted from each value. nuclei with MR.1, MR.2 and MR.8. Thus, OKB-7 and

MR. I presented significantly different reactivities with CR2 expressed on different subcellular fractions. All other mAb presented similar patterns of binding to Raji cells and nuclei. DISCUSSION We have analyzed the nuclear localization of CR2 by electron microscope immunochemistry of thin sections

of the human B lymphoma Raji cells and the binding properties of CR2 present in highly purified Raji nuclei. Electron microscope immunochemistry of thin sections of Raji cells was performed using anti-CR2 mAb, prepared against highly purified receptor (Bare1 et al., 1988). OKB-7, a monoclonal anti-CR2 prepared by others randomly against B cells and helpful in immunoprecipitation studies or cell surface labeling, did not label thin sections of Raji cells. It has already been demonstrated by the Authors and others that anti-CR2 mAbs react

Nuclear localization differently with CR2, depending on the cells or the experimental conditions used. Anti-CR2 mAbs, prepared against highly purified receptor enabled detection of CR2 in the cytoplasm, at the nuclear surface and also at the intranuclear level. Anti-CR2 labeling of the cytoplasm was mainly detected in areas containing polyribosomes. This labeling was more likely due to the presence of CR2 precursor forms of mol. wts 110,000 and 130,000 in the cytoplasm (Weis and Fearon, 1985) and of 130,O~ in low-density microsome fraction, which contains polyribosomes (Delcayre et al., 1987). CR2 localized at the nuclear surface was easily visualized on the nuclear envelope, predominantly on both sides of nuclear pores. CR2 was not associated with the lamina. The pattern of anti-CR2 labeling was similar to that described by others using monoclonal antibodies prepared against a group of pore complex glycoproteins (Davis et a!., 1986, 1987; Snow et al., 1987). It has been suggested that glycoproteins localized in the nuclear pore complex may have a role in nucleocytoplasmic transport of macromolecules such as proteins and RNAs (Davis et al., 1986, 1987; Snow et al., 1987). Gold particles associated with subcellular structures were quantified as 20, 30 and 250 particles per pm2 in nucleus, cytoplasm and nuclear pores, respectively. At the intranuclear level, CR2 was associated with RNP interchromatin fibrils. This labeling was preserved after nuclear matrix preparation by Triton Xl00 and RNAse treatment. The “in situ” intranuclear localization of CR2 on RNP interchromatin fibrils is in good correlation with the “in vitro” propensity of CR2 to interact with a nuclear ribonucleoprotein, ~120 RNP (Delcayre et al., 1987). Indeed, we previously showed that CR2 solubilized and immunoprecipitated from purified Raji nuclei interacted with nuclear ~120 RNP (Delcayre et ai., 1987). Localization of CR2 on RNP interchromatin fibrils also correlates with the property of CR2 to interact directly with the anti-oncoprotein ~53 (Bare1 et ai., 1989). Indeed, ~53 anti-oncoprotein has been localized by electron microscopy on RNP interchromatin fibrils of transformed cell nuclei, even in the absence of large T antigen (Caron de Fromentel et al., 1986). CR2 present on the surfaces of purified nuclei interacted with soluble or particle-bound C3d-like fragments and with monoclonal antibodies which react with the extracellular domain of CR2 (Bare1 et al., 1988; Ahearn and Fearon, 1989), thus suggesting that nuclear CR2 was in a functional conformational state. Preliminary comparison of the molecular weights of (1) whole CR2 molecules purified on C3bi/C3d-sepharose; (2) partially deglycosylated forms obtained by treatment of rZSI-CR2 molecules with neuramidase and mixed glycosidases; (3) CR2-fragments generated by V8-proteinase; and (4) phosphopeptides generated by trypsin, showed no significant differences between CR2 molecules isolated from plasma membranes or purified nuclei (Delcayre, 1989). However. among the panel of anti-CR2 mAbs

1119

of CR2

used, two of them, 0KB7 and MR. 1, reacted differently with the receptor depending on its localization on the cell or nucleus surfaces. This could be due to differences in conformation, environment or small biochemical differences not detectable by gel electrophoresis, between CR2 molecules localized on cell or nucleus surfaces. The role of CR2 in Raji cell nuclei remains unknown. However, nuclear localization of CR2 could be due to at least two main (but not exclusive) mechanisms. (1) Translocation to the nucleus of complexes formed at the cell surface between CR2 and its extra~ellular ligands, such as gp350/220, the capsid protein of EBV, or human C3d. Indeed, CR2 mediates internalization of EBV into normal B cells and B lymphoblastoid cells (Nemerow and Cooper, 1984) and of purified recombinant gp350/220 into B lymphocytes (Tanner et al., 1987). In these conditions, CR2 would have the role of an intranuclear carrier for its ligands. (2) The presence of two distinct intracellular pathways for mature CR2, some CR2 molecuies being expressed on the cell surface and others in the nucleus. In this latter localized in the RNP interchromatin

hypothesis, CR2 fibrils of the nu-

cleus may act as a regulatory factor through its interactions either with the pl20RNP which could be involved as nuclear ribonucleoproteins in packaging, enzymatic processing and transport of pre-mRNA (Gerace and Burke, 1988) or with the ~53 anti-oncoprotein involved in the regulation of tumor progression (Levine et al., 1991). Acknowledgement-The authors would like to thank Gerard Drevet for technical assistance. REFERENCES Aheam J. M. and Fearon D. T. (1989) Structure and function of complement receptors, CR1 (CD35) and CR2 (CD21). Adv. Immun. 46, 183-219. Bare1 M., Charriaut C. and Frade R. (1981) Isolation and characterization of a C3d receptor-like molecule from membranes of a human B lymphoblastoid cell line (Raji). FEDS Lett. 136, 111-114. Bare1 M., Fiandino A., Delcayre A., Lyamani F. and Frade R. (1988) Monocional and anti-idiotypi~ anti-EBV/C3d receptor antibodies detect two binding sites, one for EBV and one for C3d on gpl40, the CR2 expressed on human B lymphocytes. J. fmmun. 141, 1590-1595. Bare1 M., Fiandino A., Lyamani F. and Frade R. (1989) Epstein-Barr virus/C3d receptor (EBV/C3dR) reacts with ~53, a cellular anti oncogene-encoded membrane phosphoprotein: detection by polyclonal anti-idiotypic antiEBV/C3dR antibodies (Ab2). Proc. natn. Acad. Sci. U.S.A. 86, 10,05410,058. Bare1 M., Gauffre A., Lyamani F., Fiandino-Tire1 A., Hermann J. and Frade R. (1991) Intracellular interaction of Epstein-Barr virus receptor with ~68, a calcium-binding protein, present in normal and not in transformed B lymphocytes. J. Zmmun. 147, 12861291. Bare1 M., Vazquez A., Charriaut C., Aufredou M. T., Galanaud P. and Frade R. (1986) Gp140, the C3d/EBV receptor (CR2) is phosphorylated upon in r+tro activation of human peripheral B lymphocytes. FEBS Left. 197, 353-356.

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