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3.2-07 Dissociation and reassociation of Ia-peptide complexes
3.2 -07
DISSOCIATION AND REASSOCIATION OF Ia-PEPTIDE COMPLEXES. Jonathan M. Lee and Tania H. Watts, Department of Immunology, University of Toronto, Toronto, Ontario, Canada. MHC Class II molecules are peptide binding proteins involved in antigen presentation to T cells. A popular model of antigen presentation proposes that Class 11 molecules are endocytosed from the surface of an antigen presenting cell (APC), and that subsequent acidification of the endosome releases the peptides bound to the Class II molecules, freeing their peptide binding site for other peptides. An opposing view is that only newly synthesized MHC Class II molecules bind peptides. In order to gain further insight into presentation, we been antigen have investigating the ability of purified Ia to sequentially bind, release, and rebind peptide. We have used T-cell activation and fluoresencc binding assays to study the interaction between the peptide Ovalbumin 323-339 (OVA323-339) and the murine Class II molecule I-Ad. Our results suggest that Ia-peptide complexes are much more stable than previously reported: measureable dissociation occurs only at pH~4 and at pH 3.0 the complex has a half life of approximately 5h at 37OC. Although the OVA323-339/I-Ad complex is very stable, it is possible to almost completely dissociate the OVA323-339/I-Ad complex at low pH and at neutral pH rebind OVA323-339 to the uncomplexed l-Ad. This suggests that acid dissociation dots not destroy the I-Ad molecule, and that intracellular dissociation and reassociation of peptide-Ia complexes could possibly occur. However the stability of the peptide/Ia complex indicates that this dissociation could not be readily explained by a brief transit through a mildly acidic (pH 5) endosomal compartment, rather, o prolonged residence in an acidic compartment or active cellular processes would bc rcquircd.
4.1-01
HUMAN TROPHOBLAST EXPRESSES A NON CLASSICAL HLA CLASS I MOLECULE. Shirley A.Ellis and Andrew J.McMichael, Institute of Molecular Medicine,Oxford, U.K. It has previously been demonstrated that extravillous trophoblast from normal human placenta expresses an HLA class I molecule with some unusual characeristics (in ths absence of HLA A,B or C),such as small size and limited polymorphism.We have isolated an HLA class I cDNA clone from a library derived from a choriocarcinoma cell line, BeWo,which appears to express the same molecule.The nucleo. tide sequence which we have obtained represents the produc, of a class I locus other than A,B or C,which we predict would give rise to a molecule with the characteristics described. We have used the polymerase chain reaction to demonstrate the presence of similar class I sequences in cDNA from normal trophoblast.These sequences differ from one another only at the nucleotide level. Expression of MHC antigens on these fetally derived cells is of particular interest because of their unique position at the maternal-fetal interface.It may be essential for this tissue to express a non polymorphic HLA class I molecule because of its putative role in r'rotectinq the fetus from the maternal immune system.
DISSOCIATION AND REASSOCIATION OF Ia-PEPTIDE COMPLEXES. Jonathan M. Lee and Tania H. Watts, Department of Immunology, University of Toronto, Toronto, Ontario, Canada. MHC Class II molecules are peptide binding proteins involved in antigen presentation to T cells. A popular model of antigen presentation proposes that Class 11 molecules are endocytosed from the surface of an antigen presenting cell (APC), and that subsequent acidification of the endosome releases the peptides bound to the Class II molecules, freeing their peptide binding site for other peptides. An opposing view is that only newly synthesized MHC Class II molecules bind peptides. In order to gain further insight into presentation, we been antigen have investigating the ability of purified Ia to sequentially bind, release, and rebind peptide. We have used T-cell activation and fluoresencc binding assays to study the interaction between the peptide Ovalbumin 323-339 (OVA323-339) and the murine Class II molecule I-Ad. Our results suggest that Ia-peptide complexes are much more stable than previously reported: measureable dissociation occurs only at pH~4 and at pH 3.0 the complex has a half life of approximately 5h at 37OC. Although the OVA323-339/I-Ad complex is very stable, it is possible to almost completely dissociate the OVA323-339/I-Ad complex at low pH and at neutral pH rebind OVA323-339 to the uncomplexed l-Ad. This suggests that acid dissociation dots not destroy the I-Ad molecule, and that intracellular dissociation and reassociation of peptide-Ia complexes could possibly occur. However the stability of the peptide/Ia complex indicates that this dissociation could not be readily explained by a brief transit through a mildly acidic (pH 5) endosomal compartment, rather, o prolonged residence in an acidic compartment or active cellular processes would bc rcquircd.
4.1-01
HUMAN TROPHOBLAST EXPRESSES A NON CLASSICAL HLA CLASS I MOLECULE. Shirley A.Ellis and Andrew J.McMichael, Institute of Molecular Medicine,Oxford, U.K. It has previously been demonstrated that extravillous trophoblast from normal human placenta expresses an HLA class I molecule with some unusual characeristics (in ths absence of HLA A,B or C),such as small size and limited polymorphism.We have isolated an HLA class I cDNA clone from a library derived from a choriocarcinoma cell line, BeWo,which appears to express the same molecule.The nucleo. tide sequence which we have obtained represents the produc, of a class I locus other than A,B or C,which we predict would give rise to a molecule with the characteristics described. We have used the polymerase chain reaction to demonstrate the presence of similar class I sequences in cDNA from normal trophoblast.These sequences differ from one another only at the nucleotide level. Expression of MHC antigens on these fetally derived cells is of particular interest because of their unique position at the maternal-fetal interface.It may be essential for this tissue to express a non polymorphic HLA class I molecule because of its putative role in r'rotectinq the fetus from the maternal immune system.