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Definition of an extended MHC class II-peptide binding motif for the autoimmune disease-associated Lewis rat RT1.BL molecule
I P.1.05.14 I Definition of an extended
MHC class II-Delltide
moufforthe autoimmune ~ ~ disease-associated Lewis ratRTI .BLmolecule
-
bindhlg
MC. Grosfeld-Bkllemeyer ’ I. JooHen ** M. van der Kraan 1 M.H.M. Wauben q 1Institute of Mectious Diseases & Immunol~, Faculty of Veterinary Medicine, Utrecht University. The Netherlands, 2 T~spiantation-Serology Lab., University Hospital Niimegen, The Netherlands 1
I
Introductton: Several approaches towards immunotherapy of T cell mediated diseases focus at the level of the TCR-peptfde-MHC complex. For an efficient design of MHC binding peptides, the molecular basis of MHC-peptkie interactions needs to be elucidated. The Lewis rat, extremely susceptible to experimental autoimmune diseases, provides a good model to study autoimmune diseases and peptfde-mediated therapy. In this rat most autotmmune associated CD4+ T cell responses are MHC class II, RTI .BL, restricted. Matarfals and Methods: The characteristics of RTI .BL-pepttde interactions were explored. A series of substitution anatogues of two T cell epitopes were examined in a direct peptide-MHC binding assay. Moreover, peptides were designed in which putative MHC anchor residues were specifically deleted or introduced in a polyalanine pepttde. This has led to the definition of a FtT1.BL peptide binding mottt. Results: The motif includes two clusters of residues important for MHC binding. At the N-terminus positions 1 and 3 prefer a small hydrophilic uncharged or hydrophobic anchor residue, while at position 7 a bulky hydrophilic, at position 8 a small or hydrophobic anchor and at position 9 a negatively charged residue is prefered. Conclusion: The RT1.BL peptide binding motif is the first peptide-MHC binding motif to be reported in the rat.
P.1.0515
Diploid expression of HLA class I and class II molecules on spermatozoa and their cyclic inverse correlation with inhlbln concentration
J.M. Martfn-Villa, I. Luque, N. Martfnez-Cuiles, A. Core& JR. Regueiro, M. Tim&r, A. Amaiz-Villena. Departamento de Immunologia, Hospital 12 de Octubre, Univetsidad Complutense, Madrid, Spain A diploid expression of class I and class II HLA antigens has been found in purified spermatozoa by using double fluorescence labelling cytofluorometry and relevant monoclonal antibodies; this expression has been confirmed for the first time by the analysis of specific HfA mRNA and metabolic “S labelling followed by immunopracipkation, which demonstrates an active ongoing translation of HLA proteins in germinal cells. Long-living mRNA coming from diploid germinal cells may be translated to HLA molecules in spermatozoa. This translation is controlled (or at least inversely correlated) by a testicular honone (inhibin) in a cyclic fashion. Remarkably, serum levels of inhibin, synthesized by Leydig and Sertoli cells, follow a 12-13 days cycle, with a peak level at day 8; this is probably controlled by FSH (not cyclic in males) and other testicular and/or unknown hormones. Peak levels of inhibin concur with the lower density and percentage of spermatozoa expressing both HLA class I and II molecules (close to 3% by cytofluorometty); lowest levels of inhibin coincide with the highest numbers (3540%) of spermatozoa positive for both HfA molecules and a higher surface density. These observations could put to an end a disconcerttng and long-lasting controversy on the expression/non-expression of HLA antigens on spenatozoa. The possibflfty that HLA-bearing spermatozoa are more capacitated for fertilization than those that do not bear HLA, and the implications of our results on male fertility control are also discussed.
1P.1.05.16 1 The role of biologically active ligands In the structure and function of the human alpha-fetoprotein I.V. Dudich, L.N. Semenkova, E.I. Dudich. institute of Engineering Immunology, Lyubuchanx Moscow Region, Russia Introduction: Alpha-fetoprotein (AFP) is a 89 kDa oncodevelopmental protein with a vast spectrum of the immunosuppressive and cell growth regulative activities. AFP is known to bind with the high affinity different biologically active ligands, such as fatty acids, steroid hormones, growth factors, which could effectively modulate its functional activity. The present study was designed to define the effects of the native ligands on the structural and functional charactenstics of human AFP. MatedaIs and Methods: Ligand-free defatted AFP (cAFP) was obtained by charcoal/O.1 M HCI treatment. Arachkfonic acid and estradiol were used as exogenous ligands for defatted AFP to recover tts native structure. Char&e&tics of the secondary and tertiary structure of AFP molecule were determined by circular dichrolsm (CD) and dfferential scanning microcalorfmetry techniques (DSM). Comparative functional activity of cAFP and AFP was determined by the assessment of cytostatic response of HepG2 cells to these compounds, as was measured by [3H]-thymidine incorporation assay.
Reaub It was demonstrated that thermodynamic parameters of AFP molecule, such as enthalpy of denaturatton and temperature of the denaturation transition, which are the characterfstics of the molecular tertiary structure, critically depended on the ligand content of the AFP mdecule. These changes of the thermodynamic parameters are reversible. The thermodynamic parameters of cAFP molecule completely recovered after addition of the arachidonic acid and estradiol. The changes of the thenodynamic parameters of the AFP molecule after removal of its ligands shows, that certain conformational changes in the tertiary structure of the molecule occurred. The secondary structure of AFP, as was shown by the comparison of the CD spectra of defatted cAFP and AFP, did not depend on the presence of ligands. It was also demonstrated that the concentration-depended equilibrium AFP dimer formation significantly changes the tertiary structure of the monomeres composed these dimer wmplexes. The growth regulative activity of cAFP and AFP was shown to be practically independent on the presence of ligands. The growth inhibition of the HepG2 cell proliferation induced by AFP was shown to be 20% more intensive comparatively to cAFP. Conclusfons: The tertiary structure of AFP molecule was shown to depend significantly on its ligand content. In spite of the significant difference in the tertiary structure of cAFP and AFP both compounds showed not very pronounced differences in the growth regulative activity.
1P.1.05.17 1 T cell activity and MHC II binding capacity of main-chain modified peptides J. Cotton, M. He&, S. Pouvelle, G. Mourier, B. Maillere. CEA-DBpartement d’lngdnierie et dEtudes des Protdines, CE Saclay, France An abundant network of hydrogen bonds between the peptide backbone and conserved residues of the MHC II molecules plays a major interacting role and contributes to the ability of the MHC II molecules to bind a wide range of peptides. As a result, we observed that retro-inveno analogs of T cell peptfdes in which the amide bond was reversed while the side chains retained a correct orientation, did not show any binding capacity. Neither did they elicit T cells nor produce specific antibodies. However, we also demonstrated that punctual modification into the peptfde backbone did not necessarily lead to inactive peptides. These observations have been made using the pepttde 24-38, a snake toxin fragment which binds to I-Ed and stimulates specific T cells. We have systematically replaced the CC-NH linkage in the central region of the 24-38 peptide by reduced w[CHs-NH] and N-methytated ~[CC-NMe] peptkie bonds in order to assess the contribution of respectively the carbonyl and the NH group. As revealed by I-Ed binding assays, pseudopeptides bound wfth various efficacy. Depending on its location into the pepttde sequence, the introduced modiication made the corresponding peptide less potent or unable to compete with the reference peptide or in contrary did not affect its binding capacity. Weak binders to I-Ed generally stimulated a 24-38 T cell hybridoma with low, ff any, efficiency. However, among the good binders, some were as T cell stimulating as the native peptide. Interestingly, others did not yield to T cell stimulation suggesting they formed differently recognized complexes as compared to the 24-38 peptide. These results confirm the major binding contribution artsing from the peptide backbone. They also show that particular positions in the peptide tolerated N-methylation or reduction without dramatic effects on binding and thus may be used to produce agonist or antagonist peptides with increasing stability toward proteases.
P.1.05.18
Defective iron homeostasis in @2-microglobulin knockout mice recapitulates hereditarv hemochromatosis in man
M. Santos1,4, M.W. Schilham’, L.H.P.M. Rademakers2, J.J.M. Marx3, M. de Sousa4, H. Clevers ’ . ’ Department of Immundogy, Eijkman-Wnkler Institute, 3584 CX Utrecht, The Netherlands, *Department of Internal Medicine, Eijkman-Whkler Institute, 3584 CX Utrecht, The Netherlands, 3Department of Patholog)! University Hospital Utrecht, 3584 CX Utrecht, The Netherlands, 4Molecular Patholw and Immunot~ Abel Salazar institute for the Biomedical Sciences, 4400 Porto, Portugal Previously, hepatic iron overload resembling that in Herediiry Hemachromatosis (HH) has been found in ,¶P-microglobulin knock-out (82 m-/-) mice. We have now characterized iron metabolism in 82 m-j- mice. The mutant mice fail to limit the transfer of iron from mucosal cells into the plasma. Transfenin saturation is abnormally high. Pathologic iron depositions occur predominantly in liver parenchymal cells. Reconstitution with normal hematopoietic cells redistributes the iron from parenchymal to Kupffer cells, but does not correct the mucosal defect. We conclude that 1) iron metabolism is defective in the gut mucosa as well as the liver of j32 m-j- mice, and 2) a 82 m-dependent gene product is involved in iron homeostasis. Recently, a novel gene of the MHC class I family, HLA-H, has been found to be mutated in a large proportion of HH patients. Our data provide functional support for the proposed causative role of HLA-H mutations in HH.
MHC class II-Delltide
moufforthe autoimmune ~ ~ disease-associated Lewis ratRTI .BLmolecule
-
bindhlg
MC. Grosfeld-Bkllemeyer ’ I. JooHen ** M. van der Kraan 1 M.H.M. Wauben q 1Institute of Mectious Diseases & Immunol~, Faculty of Veterinary Medicine, Utrecht University. The Netherlands, 2 T~spiantation-Serology Lab., University Hospital Niimegen, The Netherlands 1
I
Introductton: Several approaches towards immunotherapy of T cell mediated diseases focus at the level of the TCR-peptfde-MHC complex. For an efficient design of MHC binding peptides, the molecular basis of MHC-peptkie interactions needs to be elucidated. The Lewis rat, extremely susceptible to experimental autoimmune diseases, provides a good model to study autoimmune diseases and peptfde-mediated therapy. In this rat most autotmmune associated CD4+ T cell responses are MHC class II, RTI .BL, restricted. Matarfals and Methods: The characteristics of RTI .BL-pepttde interactions were explored. A series of substitution anatogues of two T cell epitopes were examined in a direct peptide-MHC binding assay. Moreover, peptides were designed in which putative MHC anchor residues were specifically deleted or introduced in a polyalanine pepttde. This has led to the definition of a FtT1.BL peptide binding mottt. Results: The motif includes two clusters of residues important for MHC binding. At the N-terminus positions 1 and 3 prefer a small hydrophilic uncharged or hydrophobic anchor residue, while at position 7 a bulky hydrophilic, at position 8 a small or hydrophobic anchor and at position 9 a negatively charged residue is prefered. Conclusion: The RT1.BL peptide binding motif is the first peptide-MHC binding motif to be reported in the rat.
P.1.0515
Diploid expression of HLA class I and class II molecules on spermatozoa and their cyclic inverse correlation with inhlbln concentration
J.M. Martfn-Villa, I. Luque, N. Martfnez-Cuiles, A. Core& JR. Regueiro, M. Tim&r, A. Amaiz-Villena. Departamento de Immunologia, Hospital 12 de Octubre, Univetsidad Complutense, Madrid, Spain A diploid expression of class I and class II HLA antigens has been found in purified spermatozoa by using double fluorescence labelling cytofluorometry and relevant monoclonal antibodies; this expression has been confirmed for the first time by the analysis of specific HfA mRNA and metabolic “S labelling followed by immunopracipkation, which demonstrates an active ongoing translation of HLA proteins in germinal cells. Long-living mRNA coming from diploid germinal cells may be translated to HLA molecules in spermatozoa. This translation is controlled (or at least inversely correlated) by a testicular honone (inhibin) in a cyclic fashion. Remarkably, serum levels of inhibin, synthesized by Leydig and Sertoli cells, follow a 12-13 days cycle, with a peak level at day 8; this is probably controlled by FSH (not cyclic in males) and other testicular and/or unknown hormones. Peak levels of inhibin concur with the lower density and percentage of spermatozoa expressing both HLA class I and II molecules (close to 3% by cytofluorometty); lowest levels of inhibin coincide with the highest numbers (3540%) of spermatozoa positive for both HfA molecules and a higher surface density. These observations could put to an end a disconcerttng and long-lasting controversy on the expression/non-expression of HLA antigens on spenatozoa. The possibflfty that HLA-bearing spermatozoa are more capacitated for fertilization than those that do not bear HLA, and the implications of our results on male fertility control are also discussed.
1P.1.05.16 1 The role of biologically active ligands In the structure and function of the human alpha-fetoprotein I.V. Dudich, L.N. Semenkova, E.I. Dudich. institute of Engineering Immunology, Lyubuchanx Moscow Region, Russia Introduction: Alpha-fetoprotein (AFP) is a 89 kDa oncodevelopmental protein with a vast spectrum of the immunosuppressive and cell growth regulative activities. AFP is known to bind with the high affinity different biologically active ligands, such as fatty acids, steroid hormones, growth factors, which could effectively modulate its functional activity. The present study was designed to define the effects of the native ligands on the structural and functional charactenstics of human AFP. MatedaIs and Methods: Ligand-free defatted AFP (cAFP) was obtained by charcoal/O.1 M HCI treatment. Arachkfonic acid and estradiol were used as exogenous ligands for defatted AFP to recover tts native structure. Char&e&tics of the secondary and tertiary structure of AFP molecule were determined by circular dichrolsm (CD) and dfferential scanning microcalorfmetry techniques (DSM). Comparative functional activity of cAFP and AFP was determined by the assessment of cytostatic response of HepG2 cells to these compounds, as was measured by [3H]-thymidine incorporation assay.
Reaub It was demonstrated that thermodynamic parameters of AFP molecule, such as enthalpy of denaturatton and temperature of the denaturation transition, which are the characterfstics of the molecular tertiary structure, critically depended on the ligand content of the AFP mdecule. These changes of the thermodynamic parameters are reversible. The thermodynamic parameters of cAFP molecule completely recovered after addition of the arachidonic acid and estradiol. The changes of the thenodynamic parameters of the AFP molecule after removal of its ligands shows, that certain conformational changes in the tertiary structure of the molecule occurred. The secondary structure of AFP, as was shown by the comparison of the CD spectra of defatted cAFP and AFP, did not depend on the presence of ligands. It was also demonstrated that the concentration-depended equilibrium AFP dimer formation significantly changes the tertiary structure of the monomeres composed these dimer wmplexes. The growth regulative activity of cAFP and AFP was shown to be practically independent on the presence of ligands. The growth inhibition of the HepG2 cell proliferation induced by AFP was shown to be 20% more intensive comparatively to cAFP. Conclusfons: The tertiary structure of AFP molecule was shown to depend significantly on its ligand content. In spite of the significant difference in the tertiary structure of cAFP and AFP both compounds showed not very pronounced differences in the growth regulative activity.
1P.1.05.17 1 T cell activity and MHC II binding capacity of main-chain modified peptides J. Cotton, M. He&, S. Pouvelle, G. Mourier, B. Maillere. CEA-DBpartement d’lngdnierie et dEtudes des Protdines, CE Saclay, France An abundant network of hydrogen bonds between the peptide backbone and conserved residues of the MHC II molecules plays a major interacting role and contributes to the ability of the MHC II molecules to bind a wide range of peptides. As a result, we observed that retro-inveno analogs of T cell peptfdes in which the amide bond was reversed while the side chains retained a correct orientation, did not show any binding capacity. Neither did they elicit T cells nor produce specific antibodies. However, we also demonstrated that punctual modification into the peptfde backbone did not necessarily lead to inactive peptides. These observations have been made using the pepttde 24-38, a snake toxin fragment which binds to I-Ed and stimulates specific T cells. We have systematically replaced the CC-NH linkage in the central region of the 24-38 peptide by reduced w[CHs-NH] and N-methytated ~[CC-NMe] peptkie bonds in order to assess the contribution of respectively the carbonyl and the NH group. As revealed by I-Ed binding assays, pseudopeptides bound wfth various efficacy. Depending on its location into the pepttde sequence, the introduced modiication made the corresponding peptide less potent or unable to compete with the reference peptide or in contrary did not affect its binding capacity. Weak binders to I-Ed generally stimulated a 24-38 T cell hybridoma with low, ff any, efficiency. However, among the good binders, some were as T cell stimulating as the native peptide. Interestingly, others did not yield to T cell stimulation suggesting they formed differently recognized complexes as compared to the 24-38 peptide. These results confirm the major binding contribution artsing from the peptide backbone. They also show that particular positions in the peptide tolerated N-methylation or reduction without dramatic effects on binding and thus may be used to produce agonist or antagonist peptides with increasing stability toward proteases.
P.1.05.18
Defective iron homeostasis in @2-microglobulin knockout mice recapitulates hereditarv hemochromatosis in man
M. Santos1,4, M.W. Schilham’, L.H.P.M. Rademakers2, J.J.M. Marx3, M. de Sousa4, H. Clevers ’ . ’ Department of Immundogy, Eijkman-Wnkler Institute, 3584 CX Utrecht, The Netherlands, *Department of Internal Medicine, Eijkman-Whkler Institute, 3584 CX Utrecht, The Netherlands, 3Department of Patholog)! University Hospital Utrecht, 3584 CX Utrecht, The Netherlands, 4Molecular Patholw and Immunot~ Abel Salazar institute for the Biomedical Sciences, 4400 Porto, Portugal Previously, hepatic iron overload resembling that in Herediiry Hemachromatosis (HH) has been found in ,¶P-microglobulin knock-out (82 m-/-) mice. We have now characterized iron metabolism in 82 m-j- mice. The mutant mice fail to limit the transfer of iron from mucosal cells into the plasma. Transfenin saturation is abnormally high. Pathologic iron depositions occur predominantly in liver parenchymal cells. Reconstitution with normal hematopoietic cells redistributes the iron from parenchymal to Kupffer cells, but does not correct the mucosal defect. We conclude that 1) iron metabolism is defective in the gut mucosa as well as the liver of j32 m-j- mice, and 2) a 82 m-dependent gene product is involved in iron homeostasis. Recently, a novel gene of the MHC class I family, HLA-H, has been found to be mutated in a large proportion of HH patients. Our data provide functional support for the proposed causative role of HLA-H mutations in HH.