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Investigation of the ligand binding site of the C5a receptor
60 COMPARATIVE STUDY OF THE INTRACELLULAR EVOLUTION OF C3b FREE OR BOUND TO ANTIGEN I N U937 C E L L S U S E D AS A N T I G E N P R E S E N T I N G CELLS. Christian L. V1LLIERS, Catherine REY-MILLET, Marie-Bernadette VILLIERS, Isabelle KERBLAT and Maurice G. COLOMB Laboratoire d'Inununochimie, DBMS/ICH, INSERM U238, CENG, Grenoble, France. The covalent binding of C3b to various antigens modifies their handling by antigen presenting cells at various levels such as internalisation and proteolytic processing. Uptake and internalisation of free C3b, free tetanus toxin (TT) and of in vitro preformed C3b-TT complexes were studied using U937 as antigen presenting cells : binding of radiolabelled TT to the cells was increased by eight fold when experiments were carried out with covalent complexes, whereas the interaction of C3b with the cells was not modified by its covalent binding to the antigen. Intracellular proteolysis of C3b and C3b-TT complexes were studied using subcellular tractions purified on percoll gradient after U937 cells cracking. In the presence of endosomal or lysosomal proteases, free or bound C3b was proteolysed into C3c and C3dg-like fragments upon two hours incubation at pH 5.0, whereas only 50 % of the ester bond in C3b-TT complexes were disrupted. The major protease involved in this proteolysis was identified as Cathepsin B, from inhibition studies and from comparison with proteolysates obtained with purified cathepsin B. These results show : -1- that C3b is progressively proteolysed inside U937 cells -2- that in spite of this cleavage, C3 fragments remain largely bound to TT via the stable ester link. Thus, C3b or C3 residual fragments bound to "IT are likely to control TT proteolysis in antigen presenting cells. How this influences class I1 loading with TT peptides is under investigation.
COVALENT BINDING OF C3b TO TETANUS TOXIN: INFLUENCE ON UPTAKE/INTERNALIZATION OF ANTIGEN BY S P E C I F I C A N D N O N - S P E C I F I C B CELLS. M.-B. VILLIERS, C.L. VILLIERS, M.R. JACQUIER-SARLIN, F.M. GABERT, A.M. JOURNET and M. G. COLOMB. Laboratoire d'lmmunochimie, DBMS/ICH, INSERM U238, CENG, Grenoble, France. Complement C3 is a thioester containing protein which was described as playing a role in cell-mediated immune response: opsonization of antigens (Ag) with C3b involves covalent binding to Ag and subsequent non-covalent binding of C3b-Ag complexes to cellular C3b receptors. Previous results demonstrated that C3b-binding on Tetanus Toxin (TT) reduced by a factor 100 the amount of Ag needed for specific T cells proliferation. Several parameters are implied in such an effect, among them a delay of the proteolysis of TT when complexed with C3b as described before. As the mechanism of Ag uptake by Ag presenting cells could be different for TT alone (membrane IgG=mIgG) or complexed (CR1, CR2),and thus, could lead to a different way of presentation, we analysed the uptake/internalization of Tr-C3b complexes (preformed in vitro) by specific (4.2) and non specific (BL15) lymphoblastoid B cells. The kinetics of incorporation of radiolabelled TT and Tr-C3b are similar in both cells althought uptake of 'IT alone by BL15 is very low. On the contrary, C3b binding on TT leads to the internalization of Ag in almost the same amount in 4.2 and BL15. Competition assays demonstrate that TT-C3b uptake by specific B ceils is largely through C3b receptors and less through mIgG. Purification of subcellular fractions using percoll gradients allowed us to analyse the distribution of internalized TT and Tr-C3b in endosomes and lysosomes. The overall results show that, after 2h incubation of TI" or TT-C3b at 37°C with 4.2 and BL15, the distribution of Ag does not differ significantly, except in the case of 177 alone and BL15, reflecting a lower uptake of Ag by B cells in the absence of specific mlgG. These results point out the importance of complement C3 fragments and their receptors in the uptake of C3b-Ag even in case of specific B cells.
INVESTIGATION OF THE LIGAND BINDING SITE OF THE CSa RECEPTOR T. Vogt, S. Zahn, T. Hebell, B. Fischer, and O. GOtze Department of Immunology, Georg-August-University, Kreuzbergring 57, D-37075 GtRtingen, Germany Receptors for the chemotactic factors C5a and formyI-Met-LeuPhe (fMLP) are necessary for the migration of myeloid cells to sites of inflammation along gradients of these chemoattractants which also stimulate cytotoxic responses in receptor-bearing cells. The receptors for C5a (CSaR) and for fMLP (MLPR) are 35% identical in amino acid sequence. They belong to the family of G-protein coupled receptors whose members are characterized by their seven membrane-spanning domains. To determine the binding site of the C5aR for human C5a, chimeric receptors have been constructed that contain different parts of the C5aR and the MLPR. Using PCRgenerated DNA that encodes the C5aR and the MLPR, respectively, 3 chimeric molecules were constructed. A) Two MLPR in which the first extracellular domains (EXl) were exchanged against different length EXl segments of the C5aR, B) A construct whose first two extracellular domains (EXl and EX2) consist of C5aR-specific sequences whereas EX3 and EX4 were taken from the MLPR. After transfection of these constructs into COS-7 cells, the membrane expression of the chimeric receptors was verified with polyclonal antibodies and a monoclonal antibody to the first extracellular domain of the C5aR. Ligand-binding studies of the transfected cells using this antibody and human C5a showed that neither the first extracellular domain alone nor the first and second extracellular domains of the C5aR together are sufficient for the binding of C5a Supported by DFG (SFB236, B6)
MOLECULAR BASIS OF HOMOZYGOUS FACTOR I DEFICIENCY. TJ Vyse, RB SimS, BJ Morley, MJ Walport. Rheumatology Unit, RPMS, Hammersmith Hospital, London, UK tMRC Immunochemistry Unit, Oxford, UK Factor I cleaves C3b, thereby preventing the operation of the alternative pathway amplification loop. If Factor I is absent there is consumption of alternative pathway proteins, in particular C3 and factor B. Inherited Factor I deficiency has been described in 15 families and is usually manifest in homozygotes by a susceptibility to pyogenic infection and to immune complex disease. The cDNA sequence of Factor I is known, although the genomic organisation has not been described. Southern blot analysis of normal genomic DNA reveals that the Factor I gene is 25 kilobases in length. Analysis of three homozygous Factor I deficient individuals, two from one pedigree and one from another, suggests that in one pedigree there is a 5.5 kilobase duplication in the vicinity of the gene; in the other there is no gross abnormality. Using a cDNA probe, two cosmids have been isolated that cover the Factor I gene apart from a 2 kilobase gap between them. Restriction maps of these cosmids have been constructed and the intron-exon organisation of the gene will be determined. Restriction fragments towards the 5' end of the gene hybridise to an Alu probe. The presence of Alu repeat sequences within the gene may have led to the duplication found in one of the Factor I deficient pedigrees. The Factor I deficient patients are being investigated by the polymerase chain reaction (PCR) using fibroblasts as a source for RNA since we have shown that normal fibroblasts synthesise sufficient Factor I mRNA to be visualised by Northern blot analysis. The patient with the gene duplication has no Factor I mRNA detectable by Northern blot analysis. However, Factor I mRNA can be amplified by reverse transcriptase-PCR and the products are the same length as obtained using normal fibroblasts. Therefore in this pedigree a very low level of normal-sized Factor I mRNA is being synthesised.
COVALENT BINDING OF C3b TO TETANUS TOXIN: INFLUENCE ON UPTAKE/INTERNALIZATION OF ANTIGEN BY S P E C I F I C A N D N O N - S P E C I F I C B CELLS. M.-B. VILLIERS, C.L. VILLIERS, M.R. JACQUIER-SARLIN, F.M. GABERT, A.M. JOURNET and M. G. COLOMB. Laboratoire d'lmmunochimie, DBMS/ICH, INSERM U238, CENG, Grenoble, France. Complement C3 is a thioester containing protein which was described as playing a role in cell-mediated immune response: opsonization of antigens (Ag) with C3b involves covalent binding to Ag and subsequent non-covalent binding of C3b-Ag complexes to cellular C3b receptors. Previous results demonstrated that C3b-binding on Tetanus Toxin (TT) reduced by a factor 100 the amount of Ag needed for specific T cells proliferation. Several parameters are implied in such an effect, among them a delay of the proteolysis of TT when complexed with C3b as described before. As the mechanism of Ag uptake by Ag presenting cells could be different for TT alone (membrane IgG=mIgG) or complexed (CR1, CR2),and thus, could lead to a different way of presentation, we analysed the uptake/internalization of Tr-C3b complexes (preformed in vitro) by specific (4.2) and non specific (BL15) lymphoblastoid B cells. The kinetics of incorporation of radiolabelled TT and Tr-C3b are similar in both cells althought uptake of 'IT alone by BL15 is very low. On the contrary, C3b binding on TT leads to the internalization of Ag in almost the same amount in 4.2 and BL15. Competition assays demonstrate that TT-C3b uptake by specific B ceils is largely through C3b receptors and less through mIgG. Purification of subcellular fractions using percoll gradients allowed us to analyse the distribution of internalized TT and Tr-C3b in endosomes and lysosomes. The overall results show that, after 2h incubation of TI" or TT-C3b at 37°C with 4.2 and BL15, the distribution of Ag does not differ significantly, except in the case of 177 alone and BL15, reflecting a lower uptake of Ag by B cells in the absence of specific mlgG. These results point out the importance of complement C3 fragments and their receptors in the uptake of C3b-Ag even in case of specific B cells.
INVESTIGATION OF THE LIGAND BINDING SITE OF THE CSa RECEPTOR T. Vogt, S. Zahn, T. Hebell, B. Fischer, and O. GOtze Department of Immunology, Georg-August-University, Kreuzbergring 57, D-37075 GtRtingen, Germany Receptors for the chemotactic factors C5a and formyI-Met-LeuPhe (fMLP) are necessary for the migration of myeloid cells to sites of inflammation along gradients of these chemoattractants which also stimulate cytotoxic responses in receptor-bearing cells. The receptors for C5a (CSaR) and for fMLP (MLPR) are 35% identical in amino acid sequence. They belong to the family of G-protein coupled receptors whose members are characterized by their seven membrane-spanning domains. To determine the binding site of the C5aR for human C5a, chimeric receptors have been constructed that contain different parts of the C5aR and the MLPR. Using PCRgenerated DNA that encodes the C5aR and the MLPR, respectively, 3 chimeric molecules were constructed. A) Two MLPR in which the first extracellular domains (EXl) were exchanged against different length EXl segments of the C5aR, B) A construct whose first two extracellular domains (EXl and EX2) consist of C5aR-specific sequences whereas EX3 and EX4 were taken from the MLPR. After transfection of these constructs into COS-7 cells, the membrane expression of the chimeric receptors was verified with polyclonal antibodies and a monoclonal antibody to the first extracellular domain of the C5aR. Ligand-binding studies of the transfected cells using this antibody and human C5a showed that neither the first extracellular domain alone nor the first and second extracellular domains of the C5aR together are sufficient for the binding of C5a Supported by DFG (SFB236, B6)
MOLECULAR BASIS OF HOMOZYGOUS FACTOR I DEFICIENCY. TJ Vyse, RB SimS, BJ Morley, MJ Walport. Rheumatology Unit, RPMS, Hammersmith Hospital, London, UK tMRC Immunochemistry Unit, Oxford, UK Factor I cleaves C3b, thereby preventing the operation of the alternative pathway amplification loop. If Factor I is absent there is consumption of alternative pathway proteins, in particular C3 and factor B. Inherited Factor I deficiency has been described in 15 families and is usually manifest in homozygotes by a susceptibility to pyogenic infection and to immune complex disease. The cDNA sequence of Factor I is known, although the genomic organisation has not been described. Southern blot analysis of normal genomic DNA reveals that the Factor I gene is 25 kilobases in length. Analysis of three homozygous Factor I deficient individuals, two from one pedigree and one from another, suggests that in one pedigree there is a 5.5 kilobase duplication in the vicinity of the gene; in the other there is no gross abnormality. Using a cDNA probe, two cosmids have been isolated that cover the Factor I gene apart from a 2 kilobase gap between them. Restriction maps of these cosmids have been constructed and the intron-exon organisation of the gene will be determined. Restriction fragments towards the 5' end of the gene hybridise to an Alu probe. The presence of Alu repeat sequences within the gene may have led to the duplication found in one of the Factor I deficient pedigrees. The Factor I deficient patients are being investigated by the polymerase chain reaction (PCR) using fibroblasts as a source for RNA since we have shown that normal fibroblasts synthesise sufficient Factor I mRNA to be visualised by Northern blot analysis. The patient with the gene duplication has no Factor I mRNA detectable by Northern blot analysis. However, Factor I mRNA can be amplified by reverse transcriptase-PCR and the products are the same length as obtained using normal fibroblasts. Therefore in this pedigree a very low level of normal-sized Factor I mRNA is being synthesised.