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International audience; Cathepsin L, a cysteine protease, is overexpressed in human tumor cells and plays a major role in melanoma progression. Our aim was herein to identify molecular mechanisms, which contribute to its overexpression. We found that cathepsin L protein expression correlated with mRNA level in tumor cells. Therefore, we focused on mechanisms involved in cathepsin L mRNA regulation. CpG island was localized in the 5' region of cathepsin L gene that encompassed regulatory regions identified as essential for promoter activity. CpG dinucleotides, not methylated in any melanoma cells analysed, were methylated in a B lymphoma cell line, which poorly express cathepsin L. Our data demonstrate that in lymphoma cells, cathepsin L silencing was methylation-dependent. Furthermore, gene amplification was involved in cathepsin L overexpression in one melanoma cell line, while transcriptional mechanisms but not mRNA stability are responsible of cathepsin L overexpression in others melanoma cells. In addition, NF-Y, Sp1, Sp2 and Sp3 transcription factors, essential to basal cathepsin L transcription, are not directly involved in overexpression. Thus, our data provides the first demonstration that cathepsin L expression in tumor cells is under the control of distinct molecular mechanisms.

RB18A (TRAP220/DRIP205) is a cofactor of transcription. We herein demonstrated that RB18A downregulated p53 and upregulated MDM2 promoters. These RB18A regulations, not modified by p53wt expression, were inhibited by mutant p53 (p53mut) expression, which directly interacts with RB18A D5 domain. In addition, RB18A via its D4 domain, also interacts directly and specifically with MDM2 protein inhibiting p53mut degradation. Altogether, these mechanisms contribute to maintain a high level of p53mut expression in tumor proliferating cells. Therefore, RB18A plays a central role to control p53wt and p53mut protein content and functions in cells through a loop of regulation, which involves MDM2.

We previously demonstrated that CR2 activation on human B lymphocyte surface specifically triggered tyrosine phosphorylation of the 95-kDa nucleolin, this leading to its binding on SH2 domains of p85 sub-unit of PI 3-kinase and to activation of this enzyme. The specificity of CR2 pathway was clearly demonstrated as neither CD19 nor BCR could induce tyrosine phosphorylation of nucleolin in normal B lymphocytes. These data led us to investigate herein additional molecular events, which were triggered by CR2 activation, upstream and downstream to PI 3-kinase activation. Upstream, we demonstrated that pp60src, a tyrosine kinase of the src family, was involved in tyrosine phosphorylation of nucleolin, while syk tyrosine kinase was not. We also demonstrated a direct protein-protein interaction of pp60src with nucleolin in a CR2-dependent and CD19-independent pathway. Downstream, we demonstrated that CR2 activation also triggered Akt and GSK3 enzyme activation, this pathway being under the control of pp60src tyrosine kinase activation. These regulatory functions of activated CR2 were specific as independent of syk tyrosine kinase and of CD19 and BCR activation. Thus, CR2 activation recruits a specific mechanism to activate PI 3-kinase and its subsequent pathways, this mechanism being different to those recruited by CD19 and BCR.

We previously demonstrated that RB18A, a member of TRAP220/DRIP205/PBP family, in vivo acted as a cofactor of transcription by differently regulating p53wt transactivating activity on physiological promoters. Using p53-negative cells transfected with different constructs, we herein demonstrated that RB18A down-regulated p53wt-dependent apoptosis. This biological regulation was due to a specific diminution of p53wt protein level, as level of p53mut and GAPDH proteins was not modified. This p53wt diminution was dependent on proteasome activity, as inhibited by MG-132 inhibitor. This specific p53wt degradation was correlated with an increase in expression of MDM2, which promoted p53wt degradation into proteasome. RB18A up-regulated MDM2 expression by activating MDM2 promoter, even in absence of p53wt. Altogether, these data emphasized that RB18A could regulate p53wt function not only by direct interaction between both proteins, but also by up-regulating promoter activity of MDM2, a p53-regulating partner.

Human C3 is a multipotent molecule which participates to different events involved in immune response as complement activation, antigen presentation, cell–cell interactions and cell proliferation. Thus, proteinases which cleave C3 may modify C3-dependent cellular functions. This led us to identify two membrane-associated proteinases which cleave human C3: (a) A p57 serine proteinase expressed on human erythrocyte membranes—This p57 proteinase shared antigenic determinants with ankyrine and may be involved in clearance of immune complexes; (b) A p41 cysteine proteinase, which shares antigenic determinants, amino-acid sequence and specific activity with procathepsin-L—This p41 C3-cleaving cyteine proteinase is also involved in tumorigenic and metastatic properties of human melanoma in nude mice. Indeed, pretreatment of highly tumorigenic and metastatic melanoma cells with anti-p39 Ab totally abolished their tumorigenicity and significantly decreased the number of experimental lung metastases in nude mice. Furthermore, overexpression of procathepsin-L in nonmetastatic melanoma cells increased their tumorigenicity and switched their phenotype to highly metastatic cells in nude mice. Altogether, these data support that expression and secretion of procathepsin-L, which cleaves human C3, might be one of the multiple mechanisms by which tumor cells escape the immune surveillance.

CR2 extracellular domain is constituted of 15 or 16 Short Consensus Repeats (SCR), with additional SCR 11 localized between SCRs 10 and 12. We amplified Raji cDNA library, with specific primers where SCR 11 is localized. This generated a new fragment of 643 bp (16b SCR), in addition to the two expected transcripts of 489 (15 SCR) and 667 (16a SCR) bp. Sequencing these three fragments and the corresponding genomic DNA, demonstrated the presence of a 24 bp deletion in 16b SCR, without change of open reading frame and that this 24 bp region was flanked by two splicing acceptor sites. This supported a new alternative splicing of CR2, with generation of a third distinct mRNA. This third transcript was expressed in human CR2 positive T cells, normal or transformed B cells and EBV negative B cell lines. The 24 bp deletion corresponds to a proline-rich region, which may influence CR2 conformation and more likely have consequences on CR2 extra and intracellular interactions.

Immunological screening with the anti-p53 moAb, PAb1801 of a cDNA expression library, prepared from human B lymphoma cells, led us to identify a new human 205 kDa protein called RB18A for 'Recognized By PAb1801 moAntibody'. Immunoblotting or immunoprecipitation of fusion protein or in vitro translated protein, respectively, demonstrated that RB18A protein was recognized by several anti-p53 moAb reacting with the N or C-terminal domains of p53. Full length sequence of RB18A cDNA and computer analysis demonstrated that despite common antigenic determinants between RB18A and p53 proteins, nucleotide and deduced protein sequences did not reveal any significant homologies. RB18A mRNA was detected in all tissues tested except in kidney. In addition, RB18A protein shared identical functions with p53 protein: binding to DNA or to p53 and self-oligomerization. Furthermore, RB18A regulated p53 specific binding on his DNA consensus binding site. These functions were associated to the C-terminal domain of RB18A protein and more specifically to the PAb421 binding site present in this domain. The activation by RB18A of p53 binding on DNA was induced through an unstable interaction between both proteins. Altogether, our data demonstrated that RB18A protein shares antigenic and functional properties with p53 and regulated p53 functions.

A comparative study of tyrosine phosphorylation was performed on peripheral blood lymphocytes from systemic lupus erythematosus (SLE) patients and from healthy donors. Freshly isolated SLE lymphocytes presented an elevated tyrosine phosphorylation level when compared to healthy donors lymphocytes (p = 0.005). Among all phosphorylated proteins, those called p120, p110, p80 and p55-p60 were more phosphorylated. The level of tyrosine phosphorylation of p120 and p110 proteins discriminated significantly (p = 0.0048, respectively, p = 0.02) between SLE patients and healthy donors. Lymphocytes form SLE patients and healthy donors were then stimulated by cross-linking T cell antigens (CD3, CD4, CD8) to further distinguish the signal transduction between normal and pathologic lymphocytes. No statistical differences in the tyrosine phosphorylation pattern, following CD4 or CD8 cross-linking, were observed between SLE patients and healthy donors lymphocytes. CD3 cross-linking induced an effect on tyrosine phosphorylation different in SLE patients versus healthy donors lymphocytes. Thus, the lymphocytes of SLE patients were refractile in anti-CD3 stimulation in comparison with the healthy donors lymphocytes. Chi-square analysis demonstrated that a significantly larger number of healthy donors responded to anti-CD3 stimulation compared to SLE patients (p = 0.03). The high frequency of tyrosine phosphorylation of p110 and p80 proteins, following CD3 stimulation, in normal versus SLE lymphocytes, suggested that these proteins could be involved in abnormal signal transduction in SLE cells.

Epstein-Barr virus/C3d receptor (CR2, CD21) interacts with three intracellular proteins: the p53 anti-oncoprotein expressed in human B lymphoma cells, the p68 calcium binding protein expressed in normal B lymphocytes and the nuclear p120 ribonucleoprotein (RNP). We previously demonstrated that p53 and p68 interacted with the intracytoplasmic carboxy-terminal domain of CR2. To analyse the amino acid sequence of CR2 binding sites for p53 and p68, we synthesized different peptides whose sequences were derived from this carboxy-terminal domain. Thus, CR2 bound to p53 and p68 through two distinct binding sites localized on the N-terminal and on the central part of its carboxy-terminal domain, characterized by the amino acid sequences of KHRERNYYTD and KEAFHLEARE, respectively. CR2 site reacting with the nuclear p120RNP was determined using either anti-CR2 mAb directed against its extracellular domain or pep34, pep14/SCR3 and pep14/SCR4, synthetic peptides whose sequences corresponded to the intracellular 34 amino acid domain or to sites of the extracellular domain of CR2, respectively. Data support that CR2 interacts with p120RNP through the DEGYRLQGPPSSRC amino acid sequence of its extracellular SCR4 domain. Furthermore, phosphorylation of CR2 inhibits its interaction with the nuclear p120RNP. Binding of CR2, through its intracellular and extracellular domains, with the p53 oncoprotein and p120RNP, respectively, and the co-localization of these three proteins on nuclear interchromatin fibrils, suggest that CR2 could act as a crosslinker between these two nuclear proteins to regulate their functions.

Human erythrocytes express a p57 membrane serine proteinase which cleaves C3, the third component of complement. Amino acid analysis of the first 20 N-terminal residues of the purified p57 proteinase demonstrated 100% homology with residues 910-929 of erythrocyte ankyrin, a sequence localized in its Mr = 62 kDa fragment. Thus, we analyzed whether ankyrin could generate the p57 C3-cleaving activity. We demonstrate herein that: 1) anti-ankyrin antibodies react with purified p57 proteinase; 2) anti-p57 proteinase antibodies react with purified ankyrin but not with spectrin, another cytoskeleton component; 3) while purified ankyrin did not carry any detectable proteinase activity, limited proteolysis of ankyrin by immobilized chymotrypsin generated this C3-cleaving serine proteinase. Spectrin did not generate any C3-cleaving activity. This is the first demonstration that erythrocyte ankyrin could generate a proteinase activity, which in addition, cleaves specifically human C3.

The switch of human melanoma cell phenotype from non to highly tumorigenic and metastatic is triggered by the increase of procathepsin L secretion, which modifies the tumour microenvironment. The aim of the present study was to identify components involved in the regulation of procathepsin L secretion in melanoma cells. We focused on Rab family members, i.e. Rab3A, Rab4A, Rab4B, Rab5A, Rab8A, Rab11A, Rab27A and Rab33A, which are involved in distinct regulatory pathways. From analysis of mRNA and protein expression of these Rab components and their knockdown by specific siRNAs (small interfering RNAs) it emerged that Rab4A protein is involved in the regulation of procathepsin L secretion. This result was strengthened as procathepsin L secretion was either inhibited by expression of a Rab4A dominant-negative mutant or increased by overexpression of the wild-type Rab4A. Rab4A regulation: (i) discriminates between procathepsin L secretion and expression of intracellular cathepsin L forms; (ii) did not modify other Rab proteins and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression, or IL-8 (interleukin-8) and MMP-2 (matrix metalloproteinase-2) secretion; and (iii) was still efficient during unglycosylated procathepsin L secretion. Thus down- or up-regulation of Rab4A expression or Rab4A function triggered inhibition or increase of procathepsin L secretion respectively. Furthermore, Rab4A regulation, by modifying procathepsin L secretion, switches the tumorigenic phenotype of human melanoma cells in nude mice.

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 cell surface interacted with soluble and particle-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.

International audience; The RB18A/MED1 human gene, also named TRAP220, DRIP205 and PBP, encodes for a single 205 kDa component, which interacts with nuclear receptors and transcription factors. RB18A/MED1 chromosome localization on locus 17q12-q21.1 suggests its involvement in human cancers. We herein analyzed RB18A/MED1 expression in human melanoma cell lines. We found that RB18A/MED1 is either highly or weakly expressed in melanoma cells, depending on their respectively non or highly-tumorigenic phenotype. We therefore investigated the possible existence of a relationship between the RB18A/MED1 expression level and melanoma cell phenotype. For this purpose, we down-regulated RB18A/MED1 expression by transfecting melanoma cells with a RB18A/MED1 small interfering RNA (siRNA), specific to the 3'-untranslated region of native RB18A/MED1 RNA, already demonstrated to inhibit specifically RB18A/MED1 protein expression. A nonspecific (scrambled) siRNA was used as control. This RB18A/MED1 siRNA did not modify the expression of cathepsin L forms or lamin A/C, nor the secretion of procathepsin L and MMP2 in transfected cells. Analysis using a microarray membrane with 113 cancer-related genes, western blot and specific tests, demonstrated that RB18A/MED1 knockdown significantly inhibits tissue inhibitor of metalloproteinase-3 expression, and increases uPAR expression, two genes well known to be involved in melanoma cell invasion, through modifications of the tumor microenvironment. Indeed, RB18A/MED1 knockdown in melanoma cells in vitro increased their invasive properties, without modification of cell proliferation. Furthermore, RB18A/MED1 knockdown in vivo switched melanoma phenotype from non to strongly-tumorigenic in nude mice. Our data thus demonstrated for the first time that a decrease of RB18A/MED1 expression in human melanoma cells increases their tumorigenic phenotype.

International audience; We previously demonstrated that the switch from non- to highly tumorigenic phenotype of human melanoma cells is directly related to procathepsin L secretion, which increased cell resistance to complement-mediated cell lysis. Involvement of procathepsin L secretion in tumor growth was clearly demonstrated by three different strategies: (1) inhibition of secreted procathepsin L activity; (2) increase of procathepsin L secretion; and (3) inhibition of procathepsin L secretion. This latter strategy was triggered by intracellular expression of anti-human cathepsin L single-chain variable fragment (ScFv). These previous experiments were performed by processing melanoma cells before their injection into nude mice. We herein designed a new lentiviral vector in which this anti-cathepsin L ScFv was cloned. This lentiviral vector was optimized to allow the highest intracellular expression of anti-cathepsin L ScFv in transduced melanoma cells. In these transduced cells, procathepsin L secretion was strongly inhibited. In addition, injection of this anti-cathepsin L ScFv lentiviral vector into tumors already induced in nude mice inhibited tumor growth and associated angiogenesis. This is the first report to demonstrate that targeting procathepsin L secretion with anti-cathepsin L ScFv lentiviral construct constitutes a new gene therapy in the challenge to inhibit the growth of tumors induced by human melanoma cells.

Overexpression of cathepsin L, a cysteine protease, and consequently procathepsin L secretion switch the phenotype of human melanoma cells to highly tumorigenic and strongly metastatic. This led us to identify the DNA regulatory sequences involved in the regulation of cathepsin L expression in highly metastatic human melanoma cells. The results of the present study demonstrated the presence of regulatory sequences in the 3′ region downstream of the cathepsin L gene and in the 3′- and 5′-flanking regions of GC/CCAAT sites of its promoter. In addition, we established that the 5′-UTR (untranslated region) was the most important region for cathepsin L expression. This 5′-UTR integrated an alternative promoter and sequences involved in post-transcriptional regulation. Transfection experiments of bicistronic reporter vectors and RNAs demonstrated that the cathepsin L 5′-UTR contained a functional IRES (internal ribosome entry site). This complete IRES was present only in one of the three splice variants, which differed in their 5′-UTR. Then, we analysed cathepsin L expression in this human melanoma cell line grown under hypoxia. We demonstrated that under moderate hypoxic conditions (1% O2) intracellular expression of cathepsin L was up-regulated. Hypoxia significantly increased only the expression of the transcript which contains the complete IRES, but inhibited promoter activity. These results suggest that the presence of an IRES allowed cathepsin L mRNA translation to be efficient under hypoxic conditions. Altogether, our results indicated that in vivo a tumour hypoxic environment up-regulates cathepsin L expression which promotes tumour progression.

International audience; It is well established that CD21 activation on human B cell surface triggers B cell proliferation. We previously demonstrated that CD21 activation also triggers tyrosine phosphorylation of two components, p95 and p120, both interacting with SH2 domains of the p85 subunit of PI 3-kinase. We successively identified p95 as the nucleolin and the first signal transduction pathway specifically triggered by CD21 activation, i.e.: pp60Src activation, tyrosine phosphorylation of p95 nucleolin, its interaction with SH2 domains of p85 subunit and PI 3-kinase activation, followed by AKT-GSK-3 activations. We herein identified the p120 component as the protooncoprotein Cbl and the first steps associated to its activation. First, CD21 activation triggered Cbl tyrosine phosphorylation, which required c-Src kinase but not PI 3-kinase or Syk kinase activities. Involvement of Src kinase in this step was supported by inhibition of Cbl phosphorylation and its interactions with other components when cells were either preincubated with specific Src inhibitor or transfected with dominant-negative c-Src form. Second, once tyrosine phosphorylated, Cbl interacts with SH2 domains of p85 subunit, SH2 domains of Crk-L and with tyrosine phosphorylated Syk kinase. The third and unexpected feature was to found that, at the contrary of BCR or of CD19 (herein also analyzed for the first time), CD21 activation triggers dissociation of Cbl-Vav complex. Thus, these results provide the first molecular basis of a new signal transduction pathway specifically triggered by CD21 activation.

We demonstrated previously that the switch from nonmetastatic to highly metastatic phenotype of human melanoma cells is directly related to secretion of procathepsin L form. This cysteine proteinase was identified on the basis of its property to cleave human C3, the third component of complement. In an attempt to control procathepsin L secretion, we have recently generated an anti-cathepsin L single chain variable fragment (ScFv) from an anti-cathepsin L monoclonal antibody generated against recombinant cathepsin L. We herein selected clones stably transfected with this anti-cathepsin L ScFv and analyzed them for changes in tumor growth and metastasis. We show that in stably transfected clones, anti-cathepsin L ScFv strongly inhibited the secretion of procathepsin L without modifying the intracellular amount or processing pattern of cathepsin L forms. Confocal analysis demonstrated colocalization of endogenous cathepsin L and anti-cathepsin L ScFv. In addition, expression of this ScFv strongly inhibited generation of tumor and metastasis by these human melanoma clones in nude mice. In vivo, the anti-cathepsin L ScFv-transfected cells produced tumors with decreased vascularization (angiogenesis) concomitant with increased apoptosis of tumor cells. Matrigel assay also demonstrated that melanoma invasiveness was completely abolished. Thus, this is the first demonstration that anti-cathepsin L ScFv could be used to inhibit the tumorigenic and metastatic phenotype of human melanoma, depending on procathepsin L secretion, and could therefore be used as a molecular tool in a therapeutic cellular approach.

International audience; We previously demonstrated that increase of procathepsin L secretion by human melanoma cells strongly increased their tumourigenicity and switched their phenotype from low to highly metastatic. Thus, we herein analysed whether it was possible to inhibit procathepsin L secretion using anti-cathepsin L ScFv. For this purpose, we produced different forms of fusion cathepsin L in prokaryotic or eukaryotic expression systems. An anti-cathepsin L monoclonal antibody (mAb), named 3D8, was isolated from mice immunized with purified procathepsin L-His. This 3D8 mAb interacted with an epitope localized on the 156—197 amino acid sequence of cathepsin L and recognized recombinant or native forms of cathepsin L synthesized by human melanoma cells. An active anti-cathepsin L ScFv was generated and characterized from 3D8 mAb heavy and light variable chains. Then, human melanoma cells were transiently co-transfected with 3D8 ScFv and cathepsin L cDNAs. Data demonstrated that increase of 3D8 ScFv expression in human melanoma cells totally inhibited procathepsin L secretion and induced accumulation of intracellular procathepsin L. Our results constitute the first demonstration that anti-cathepsin L ScFv could be used in human melanoma cells to inhibit procathepsin L secretion. This ScFv represents a new molecular tool to explore cell therapy of human melanomas.

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.

The major neutralizing epitope (MNE) for the Epstein-Barr virus (EBV) is present on its envelope glycoprotein gp350/220 (hereafter referred to as gp350) in close proximity to the virus-receptor (CR2) binding site and is recognized by the neutralizing murine monoclonal antibody (mAb) 72A1. We studied the reactivities of 72A1 and another anti-gp350 mAb 2L10 (which does not neutralize EBV) with gp350 expressed on three different lymphoid cell lines (Raji, CEM.NKr and BJA-B). Our results indicate that gp350 expressed on the surface of CR2-positive cells interacts with the viral receptor and that this interaction masks the major EBV-neutralizing epitope. The interaction was reversible and the masked epitope was revealed on incubation with an excess of anti-CR2 mAb OKB7. Gp350-expressing CEM-NKr cells with intact MNE exhibited significantly higher (P ≤ 0.05) lysis in gp350-specific antibody-dependent cellular cytotoxic assays compared with its Raji counterpart. The present results may have important implications for the use of soluble viral receptors as therapeutic agents in acute and chronic EBV and other viral infections (e.g., HIV-1). J. Leukoc. Biol. 64: 192–197; 1998.

Human erythrocytes express at the membrane level a p57 serine proteinase which cleaves C3, the third component of complement. We demonstrated herein that human serum carries an inhibitory activity against this p57 membrane proteinase. Purification allowed to identify this inhibitor as the α 1-proteinase inhibitor ( α 1-PI) on the basis of its molecular weight, antigenicity and amino acid sequence identity. Data demonstrated that α 1-PI is the unique and strong serum inhibitor of the p57 proteinase activity: inhibition studies showed that α 1-PI inhibited p57 proteinase activity with a k ass value of 10 5 M −1 s −1 . Inhibition of p57 proteinase by α 1-PI was due to formation of a SDS-stable complex between both components. We suggest that inhibition of the membrane p57 proteinase activity by serum α 1-PI may be involved in the regulation of C3 fragment generation and/or in clearance in liver of C3b bearing immune complexes by erythrocyte-CR1.

We recently demonstrated that DM-4, a human melanoma cell line highly metastatic in nude mice, expressed a p41 C3-cleaving proteinase. This p41 proteinase is a cysteine proteinase, associated to cell surface and involved in tumorigenicity and metastatic properties of these tumor cells. We demonstrate herein that DM-4 cells also secrete the p41 proteinase. In addition, analysis of cellular components which reacted with the p41 proteinase led us to demonstrate that DM-4 cells synthesized and secreted human C3. Secreted C3 is cleaved by the secreted p41 proteinase and a C3dg-like fragment is generated. This is the first demonstration that a human melanoma cell line co-expresses and co-secretes human C3 and a C3-cleaving cysteine proteinase, antigenically related to procathepsin L.

We previously identified, on normal or tumour cells, two membrane proteinases, p57 and p65, that cleave human C3, the third component of complement, thus regulating C3′s biological properties. Whereas p57 was purified from human erythrocytes, p65 was identified using polyclonal anti-p57 antibodies on a human melanoma cell line resistant to complement lysis. Analysis of cell distribution of C3-cleaving proteinases established that DSm, a murine melanoma cell line, expressed a C3-cleaving proteinase distinct from p57 and p65 proteinases. Thus we purified the C3-cleaving proteinase solubilized from membranes of DSm cells. The purified proteinase, termed ‘p39’ on the basis of its molecular mass of 39 kDa, was identified, using specific proteinase inhibitors, as a cysteine proteinase. Anti-p39 antibodies, prepared against highly purified p39, localized the p39 C3-cleaving proteinase mainly at the cell surface and demonstrated that p39 is also secreted. Anti-p39 antibodies inhibited solubilized C3-cleaving activity. Preincubation of DSm cells with anti-p39 F(ab′)2 fragments increased up to 60% complement cell susceptibility. Amino acid analysis of N-terminal and three other regions of p39 demonstrated that this C3-cleaving proteinase carries 100% identity within four regions of procathepsin L. This is the first demonstration that a melanoma cell line expresses on its surface and secretes a p39 C3-cleaving cysteine proteinase that shares sequence identities with procathepsin L. Thus the p39 cysteine proteinase represents a new member of the C3-cleaving proteinase family associated with, and/or expressed on, the cell surface.

CR2 is involved in regulation of human B lymphocyte proliferation by interacting, through distinct domains, with extracellular, cell surface or intracellular components. Contribution of CR2 intracytoplasmic domain in CR2 regulatory functions remains unclear. Thus, we used pep34, a 34 amino acid synthetic peptide whose sequence corresponds to CR2 intracytoplasmic domain. Pep34 was incorporated into B lymphocytes which were then activated by EBV or C3d through CR2. Our data demonstrate that pep34 inhibits 100% B lymphocyte proliferation triggered by EBV or C3d. Irrelevant peptide had no effect. When B lymphocyte proliferation was triggered by a multipotent B cell activator as SAC, pep34 did not exert any inhibitory effect. Our data demonstrate that pep34 inhibits B lymphocyte proliferation only when lymphocytes are triggered through CR2. Thus, this strongly supports that despite its short length. CR2 intracytoplasmic domain participates to regulatory functions of this receptor.

We previously demonstrated that human C3d or pep16, a 16-amino acid synthetic peptide derived from human C3d, induced in vivo and in vitro tyrosine phosphorylation of pp105, an intracellular component found only in human cells that express CR2 at their surface. To determine the contribution of CR2 molecules to this enzymatic regulation, we first analyzed whether activation of CR2 by other extracellular CR2 ligands could trigger such regulation in cell extracts. Subsequently, we used cell extracts of either CR2-positive cells depleted in CR2 molecules by absorption with anti-CR2 antibodies or CR2-negative cells transfected with CR2 cDNA. We demonstrate here that pp105 phosphorylation was induced when CR2 was activated by C3d and pep16 as well as by gp350, the Epstein-Barr virus capsid protein or OKB7, an anti-CR2 monoclonal antibody (mAb). HB5, another anti-CR2 mAb, which did not activate B lymphocytes through CR2, did not induce pp105 phosphorylation. Thus, C3d, pep16, gp350, and OKB7 presented similar properties in activating CR2 to trigger pp105 phosphorylation and in regulating B lymphocyte proliferation, while HB-5 had no effect on either assays. Furthermore, our data demonstrate that the presence of CR2 activated by its extracellular ligands regulates pp105 phosphorylation through two distinct pathways: one which also requires the presence of non-activated CD19, and one which is independent of CD19. The involvement of CD19 in the first pathway was not due to the formation of putative CR2-CD19 complexes. Both pathways were TAPA-1 independent. This is the first demonstration that activated CR2 molecules can play a regulatory role in enzymatic function, such as phosphorylation, despite the absence of CD19 and TAPA-1.

We originally isolated gp140, the receptor of human C3 (the third component of complement) from Raji, a human B lymphoma cell line (1981). gp140 is the C3d receptor (1983, 1985a) and also the Epstein-Barr virus receptor (1985b). Activated EBV/C3dR regulates B lymphocyte proliferation (1985c). EBV interaction with EBV/C3dR is the first step of B lymphocyte transformation (1985b). Activated EBV/C3dR interacts through its intracytoplasmic domain, with the p53 oncoprotein (1989, 1992, 1995) or a p68 Cabp (1991, 1992, 1995). We identified the two first signaling pathways specifically triggered by activated EBV/C3dR. The first pathway, independent of CD19 and BCR activation, is characterized by: 1) activation of pp60src kinase to tyrosine phosphorylate nucleolin; 2) tyrosine phosphorylated nucleolin interacts with the SH2 domains of the p85 subunit of PI-3 kinase, activating this latter; 3) activated PI 3-kinase recrutes the AKT-GSK3 pathway (1999, 2001, 2003). The second pathway is characterized by: 1) Cbl phosphorylation; 2) interactions of phosphorylated Cbl with Crk-L, Syk and the p85 subunit of PI-3-kinase; 3) dissociation of phosphorylated Cbl from Vav (2006). This dissociation, opposed to the Cbl-Vav association triggered by CD19 or BCR, emphasizes the specificity of EBV/C3dR signaling pathways. We herein identified the functional relationship between these two pathways.

A monoclonal antibody was raised against p57, a serine proteinase, characterized by an apparent molecular weight of 57 kDa, and purified from human erythrocyte membranes. P57 proteinase cleaves the human third component of complement, C3. The antibody selected, MP1, of IgG2a isotype, precipitated specifically the p57 antigen which carried the C3/C3b-cleaving activity present in membrane crude extract of human erythrocytes. P57 proteinase eluted from MP1-sepharose was inhibited by 5 x 10(-4) M PMSF, enhanced by 0.5% SDS and generated C3 fragments identical to those generated by membrane crude extract of human erythrocytes. All these properties were identical to those of the p57 previously purified by biochemical procedures. In addition, 5000 binding sites were detected on cell surface. This MP1 monoclonal antibody will be helpful to analyse the role of p57 in human erythrocytes.

We present herein the first evidence that human C3 and, with a higher efficiency, trypsin-cleaved C3 enhanced in vitro phosphorylation of a cellular component, characterized by an apparent molecular weight of 105 kDa, pp105, present in the human B lymphoma cells, Raji. This regulatory activity was associated with C3d fragment generated in trypsin-cleaved C3. A 16 amino-acid peptide, carrying the LYNVEA sequence of C3d reacting with the C3d receptor (CR2), was synthetized. P16 enhanced, in a dose-dependent curve between 0.3 to 10 μM, in vitro phosphorylation of pp105, as well as C3d fragments present in trypsin-cleaved C3. A fibrinogen-related synthetic peptide of 15 amino acids, used as control, had no effect on pp105 phosphorylation. P16 and trypsin-cleaved C3 regulate pp105 phosphorylation through identical pathways. Thus, p16 represents the 16 amino-acid sequence of C3 which regulated in vitro phosphorylation of pp105.

We originally isolated gp140, the receptor of human C3 (the third component of complement) of human B lymphocytes (1981). gp140 is the C3d receptor (1985a) and also the Epstein-Barr virus receptor (1985b). EBV/C3dR regulates proliferation (1985c) and transformation (1985b) of human B lymphocytes. Thus, we analyzed intracellular events triggered by activated EBV/C3dR. EBV/C3dR activation triggered its intracellular interactions with a p120 nuclear RNP (1987) and the p53 oncoproteine (1995). Then, we first identified two signaling pathways specifically triggered by activated EBV/C3dR. The first pathway is characterized by PI-3 kinase activation: upstream PI-3 kinase, pp60src kinase is activated to tyrosine phosphorylate nucleolin. Then, tyrosine phosphorylated nucleolin interacts with SH2 domains of the p85 subunit of PI-3 kinase, activating this latter;downstream PI 3-kinase, AKT-GSK3 pathway is activated. This pathway is independent of BCR and CD19 activations (1999, 2001, 2003). The second pathway is characterized by: Cbl phosphorylation;Cbl interactions with Crk-L, Syk and the p85 subunit of PI-3-kinase;dissociation of the Cbl-Vav complex (2006). This dissociation, in opposite to Cbl-Vav association triggered by BCR and CD19, emphasizes the specificity of EBV/C3dR signaling. We herein identified the functional relationship between both pathways. This work is supported by INSERM

In a defined medium in which transferrin (3 micrograms/ml) was the only source of exogenous proteins, Raji cells of the human pre-B lymphoblastoid cell line died within 48 h after forming polykaryons. The simple addition of purified C3 at a concentration equal to or higher than 3 micrograms/ml allowed Raji cells to divide. This preliminary report provides a defined system for studying the mitogenic effect of human C3 or C3 fragments upon proliferation of human B-cells lines.

During the "respiratory adaptation" of Bacillus coagulans, it was possible to dissociate the kinetics of cytochrome a and a3 synthesis with carbon monoxide. The synthesis of cytochrome a3 is preferentially repressed when the pH of the incubation medium is pH 6.5 instead of pH 5.5. However, though the total synthesis of tetrapyrrole compounds is the same at both pH values, the excretion of coproporphyrin III is much increased at pH 6.5. Bacillus coagulans, sensitive to the "glucose effect", shows the "pH effect" only in the presence of high glucose concentrations. The repression of the oxidase complex synthesis by a slight increase of the extracellular pH appears directly related to the increase of the extracellular coproporphyrin III.

gp140, previously identified as a 140-kDa C3b-binding membrane glycoprotein present on Raji cell surface, was shown to be the C3dg/C3d receptor of B lymphocytes (CR2). Specific polyclonal anti-gpl40, prepared by immunizing rabbits with this highly purified C3 receptor, blocked Raji cell rosettes with EC3b, EC3bi, EC3dg and EC3d, and also blocked normal lymphocyte rosettes with EC3dg and EC3d without affecting CR1 or CR3 activity. Moreover, a monoclonal anti-C3 (C3b/#130), described by others as reacting with the d region highly expressed on ECSbi, EC3dg and EC3d and poorly exposed on EC3b, completely inhibited EC3bi, EC3dg and EC3d rosettes with Raji cells, but had no effect on EC3b rosettes. Treatment of Raji cells with rabbit anti-gp140 blocked the uptake of three 125I-labeled monoclonal antibodies anti-B2, HB-5 and OKB7 reported to react with C3d-binding proteins, indicating that each of these monoclonal antibodies recognizes epitopes present on gp140. The neutrophil C3dg receptor was examined to determine its relationship to lymphocyte CR2. While neutrophil rosettes with EC3d were undetectable, a specificity for C3d was suggested by the inhibition of EC3dg rosettes by fluid phase C3d-complexes bearing no detectable C3dg. However, such neutrophil EC3dg and EC3bi rosettes were not inhibited by rabbit anti-gp140 nor an excess of anti-CRl, anti-CR2, and anti-CR3. In addition, neutrophils did not bind 125I-labeled anti-gp140, anti-B2, or HB-5. Thus, the neutrophil C3dg receptor is distinct from gp140, the lymphocyte CR2, and should be designated CR4.

gp140, the C3d/EBV receptor (CR2), previously isolated and characterized from human B lymphocytes, was identified on human platelets: (a) by measuring the specific binding of either polyclonal anti-gp140 IgG and monoclonal anti-C3d/EBVR antibodies, as OKB-7 and HB-5, or human C3d; (b) by isolating gp140 from solubilized platelet components with polyclonal anti-gp140 IgG or monoclonal OKB-7, using immunoprecipitation and electro-immunoblotting assays; (c) by inducing specific activation of human platelets. Cross-linking of this receptor by polyclonal anti-gp140 IgG induced aggregation of human platelets and stimulated ATP release. Absence of lactate dehydrogenase release and inhibition by EDTA and prostacyclin of anti-gp140-induced aggregation, support strongly active aggregation and absence of lysis. Platelet aggregation by anti-gp140 required metabolic activities and was modulated by fibrinogen, paf-acether or thrombin. OKB-7 triggered human platelet aggregation when cross-linked by anti-mouse second-step antibodies. In the same way, platelet activation by C3d fragment was detected, in presence of fibrinogen, only when C3d was cross-linked on the cell surface by anti-C3d F(ab′)2 fragments.

Purified human native third component of complement, C3, was found to inhibit in vitro natural killer (NK) cell cytotoxicity in both mouse and human systems. The effect was dose and time dependent, a 50% inhibition being reached with 190 nM C3 (35 micrograms/ml) added during the NK assay or after a 30-min preincubation of the effector cells with this C3 concentration. C3 was shown to act at the effector-cell population level because pretreatment of the target cells did not modify the NK lysis. The inhibition was not due to general cytotoxicity nor to cell agglutination. Moreover, another in vitro cytotoxicity system (represented by alloreactive cytotoxic lymphocytes) was not affected by purified C3. Structural analysis of the active part of the C3 molecule shows that the C3-induced inhibition is supported by the C3a fragment. Release of carboxyl-terminal arginine residue by carboxypeptidase B, converting C3a into des-Arg77-C3a, did not alter the inhibitory effect displayed by this fragment. These results suggest that C3a may play an important role in the regulation of NK activity.

The C3d receptor is a specific marker of B lymphocytes. Recently we have shown that C3d receptor activity is carried by a gp 140 membrane antigen. A polyclonal antibody has been prepared by immunizing a rabbit with highly purified gp 140 molecule isolated from membranes of the human B lymphoblastoid cell line Raji and its high specificity was previously demonstrated. We tested the effect of this antibody to the C3d receptor on the B cell proliferative response. Purified B cells from human blood were induced to proliferate by a B cell growth factor (BCGF)-containing partially purified supernatant from activated T cells. The anti-C3d receptor F(ab')2 enhanced the BCGF-dependent B cell proliferation. This effect was dose dependent, was observed in the presence of different concentrations of BCGF and did not correspond to a change in the time course of the response. The anti-C3d receptor F(ab')2 had no mitogenic effect in the absence of T cell supernatant. In contrast the undigested anti-C3d receptor IgG suppressed the BCGF-dependent B cell proliferation. These results emphasize the potentialities of anti-gp 140 F(ab')2 to explore the involvement of the C3d receptor in the regulation of B cell response to T cell products.

The aim of this work was to detect Fc gamma receptors on human lymphoblastoid cells using a solid phase radioimmunoassay (RIA), specific to human IgG. RIA was performed by coupling rabbit anti-human IgG (AHIgG) to acrylamide beads to make immunobeads. The specificity and sensitivity of binding of human [125I]IgG to immunobeads permitted detection of as little as 10(-10) M unlabeled human IgG or aggregated human IgG (AHIgG) in competitive assay. The cells were incubated with an AHIgG concentration (3 x 10(-10) M) giving 25% inhibition in the RIA; unbound AHIgG concentration was then measured in the cell supernatants using RIA. Results show that Fc gamma receptors could be detected at 20 degrees C or at 37 degrees C (but not at 4 degrees C) on the four B cell lines tested. At whatever temperature of incubation, Fc gamma receptors were not detected on three T cell lines nor on two 'non T-non B' cell lines. This method allows screening of a large number of Fc gamma receptor positive cells. It is also useful for detecting Fc gamma receptors in membrane preparations or in detergent extracts of human B cell membranes.

Analysis of the interaction of human C3 fragments with human B lymphoma cell line, led us to isolate gp 140, the C3 receptor of Raji cells. Rabbit anti-gp 140 was prepared against this highly purified receptor. Using these polyclonal antibodies, it was found that: (a) gp 140 is the C3d receptor (CR2) which reacts with the C3d site expressed on C3d, C3dg, C3bi and at a less extent on C3b. Gp 140 is a specific marker of human B lymphocytes; (b) gp 140 is also the Epstein-Barr virus receptor (EBVR); (c) CR2 is a membrane site involved in B-cell regulation; and (d) the C3d/C3dg receptor (CR2) of human B lymphocytes is distinct to the C3dg receptor (CR4) of human neutrophils.

Specific membrane receptors for breakdown products of the third component of human complement, C3, are expressed in several mammalian cells. At present, 5 receptors are described, i.e., C3a, C3b, C3d, iC3b and H altered C3b receptors [I]. Biochemical and molecular analysis of 2 of these, namely C3b and C3d receptors, have been approached. ture medium associated with a 72 000 Mr glycoprotein [9], whereas attempts to purify C3b receptors from its membranes have been unsuccessful [lo]. This is the first report of the solubilization from the membrane of the human B lymphoblastoid cell line, Raji, of a 140 OOOMr glycoprotein carrying a C3b binding activity. Some properties of this membrane component were analyzed.

gp140, the EB/C3d receptor (EBV/C3dR; CR2), is a membrane site involved in human B cell regulation. Cross-linking of this receptor on the cell surface by its specific ligands led to the enhancement of B cell proliferation in synergy with T cell factors. In vitro activation of human peripheral B lymphocytes by cross-linking membrane immunoglobulins with anti-mu antibody induced EBV/C3dR phosphorylation. These studies were pursued by analyzing cell-free phosphorylation of EBV/C3dR isolated from Raji cell fractions, and immobilized on OKB7, a monoclonal anti-EBV/C3dR antibody. Three EBV/C3dR-related antigens which could be cell-free phosphorylated were detected: gp140, the EBV/C3dR, p130 and p120. gp140, the mature form of EBV/C3dR, was isolated from plasma membrane and from purified nuclei. p130 was identified as an intracellular intermediate of EBV/C3dR glycosylation, localized in low-density microsomes. Phosphoamino acid analysis of EBV/C3dR allowed the detection of phosphotyrosine and phosphoserine residues. These data suggest that EBV/C3dR could carry an autophosphorylation activity and could be associated to serine kinases. Using polyclonal anti-p120 antibody and anti-120 kDa nuclear ribonucleoprotein monoclonal antibody (mAb), p120 was identified as a nuclear ribonucleoprotein antigenically not related to EBV/C3dR. Detection of p120 on EBV/C3dR, immobilized on OKB7, was due to interactions between both antigens, instead of anti-EBV/C3dR mAb cross-reactivity with p120. Cell-free phosphorylation of p120 was under the control of EBV/C3dR. However, it is not yet established whether other nuclear or membrane components were involved in the control of p120 cell-free phosphorylation by EBV/C3dR. From the data presented herein, we propose that phosphorylation of a 120-kDa nuclear ribonucleoprotein by EBV/C3dR-associated kinases could represent a crucial step in in vivo regulation of human B cell activation.

Gp140 is the Epstein-Barr virus receptor and the C3d receptor (EBVR/C3dR) of human B lymphocytes. Recently, we have shown that cross-linking of EBVR/C3dR on cell surface by polyclonal anti-gp140 induced B cell activation, in presence of T cell factors. Immunoregulatory abnormalities of EBV-induced B cell activation have been demonstrated in rheumatoid arthritis (RA) patients. These data prompted us to analyze the putative presence of anti-EBVR/C3dR autoantibodies in human sera. The IgG fractions from eleven RA and 10 normal sera were tested for their ability to: (a) bind to Raji cell surface; (b) inhibit the binding to cell surface of 3 anti-EBVR/C3dR monoclonal antibodies (mAb), which recognized different epitopes on gp140; (c) inhibit the binding of particle-bound C3d and (d) react with 1% Nonidet-P40-solubilized gp140 from Raji cell membranes, in immunoblotting assays. Three RA sera carry anti- EBVR/C3dR autoantibodies which react with gp140 expressed on Raji cell surface or its solubilized form. The purification of monomeric IgG fraction of selected RA sera ruled out involvement of immune complexes carrying C3 molecules, which could interfere in these assays. One of these 3 RA sera was able to inhibit the binding to cell surface of anti-EBVR/C3dR mAb and particle-bound C3d. However, the 2 other RA sera, found positive by immunoblotting, did not inhibit particle-bound C3d and presented differences in their inhibitory effect on anti-EBVR/C3dR mAb binding to Raji cell surface. These data allow us to demonstrate differences which exist in the properties of anti-EBVR/C3dR autoantibodies. These autoantibodies were not detected in all the normal and other RA sera. Anti-EBVR/C3dR autoantibodies could play a role “in vivo” in B lymphocyte activation of RA patients.