Most cellular interactions essential for the development of an immune response involve the membrane glycoproteins encoded in the major histocompatibility gene complex. The products of the I region, the class II histocompatibility molecules (Ia molecules), are essential for accessory cells such as macrophages to present polypeptide antigens to helper T cells. This interaction, antigen presentation, is needed for T-cell recognition of the antigen and its consequent activation. How the Ia molecules regulate the immune response during antigen presentation is not known, although it is commonly thought to result from their association with the presented antigen. Recent studies, including the elucidation of the structure of the T-cell receptor, favour recognition of a single structure, an antigen-Ia complex. Here we report attempts to determine whether purified Ia glycoproteins have an affinity for polypeptide antigens presented by intact cells in an Ia-restricted manner. We first identified the epitope of a peptide antigen involved in presentation. Several laboratories have shown that globular proteins are altered (processed) in intracellular vesicles of the antigen-presenting cell before antigen presentation. A major component of the T-cell response is directed toward determinants found in the unfolded or denatured molecule, and our laboratory has shown that the determinant of the hen-egg lysozyme protein (HEL), presented in H-2k mice to T cells, is a sequence of only 10 amino acids. This portion resides in an area of the native molecule partially buried inside the molecule, in a beta-sheet conformation. To be presented, intact or native HEL must first be processed in acidic intracellular vesicles. Having isolated the peptide responsible for T-cell recognition of HEL, we sought a physical association of this peptide with purified, detergent-solubilized I-Ak molecules from B-hybridoma cells. We have found such an association, which may explain the role of the Ia glycoproteins in cellular interactions.

We examined the direct binding of a hen egg white lysozyme peptide, , to membrane I-Ak (pro-tein encoded in the A locus of the I region) molecules in the presence of detergent. A number of synthetic peptide derivatives, which did not stimulate our T-cell reactive hybridomas, competed for the binding of HEL(46-61) to I-Ak and also inhibited the functional presentation ofHEL(46-61). Inhibitors included a peptide lacking a tyrosine at position 53 and a peptide corresponding to the autologous lysozyme peptide. Presentation was examined with cells or with supported planar phospholipid membranes bearing only I-Ak and HEL(46-61).Other peptides that did not compete for the binding did not inhibit functional presentation. We concluded that the binding of an immunogenic peptide to I-A is critical for presentation, that the I-A molecule does not discriminate between autologous and foreign related determinants but does recognize structurally different peptides. Our evidence suggests that our immunogenic peptide bears noncontiguous amino acids critical for contact I-A binding interspersed with amino acids critical for interaction with T cells.We have demonstrated that a class II histocompatibility protein, an I-region associated (Ia) molecule, directly binds to a defined synthetic immunogenic peptide derived from hen egg white lysozyme (HEL) (1). We measured the binding of a fluorescently labeled immunogenic HEL peptide, 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-HEL (46-61), to the I-Ak molecule (protein encoded in the A locus ofthe Iregion), in detergent, by equilibrium dialysis. The peptide HEL-(46-61) contains the immunodominant epitope recognized by mice of the k haplotype (2). The binding was shown to be specific, with a Kd of approximately 2 ,uM. Furthermore, the binding was haplotyje specific, in that NBD-HEL(46-61) did not bind to the I-A molecule, a product of a nonresponder allele, when measured under identical conditions. These results may provide necessary insights to understand how Ia molecules can influence T-cell activation in an antigen specific manner.We have now addressed the question of whether synthetic peptide derivatives ofthe immunogenic peptide, generated by a series of amino acid deletions, substitutions, or truncations, can compete with NBD-HEL(46-61) for binding to I-Ak using our established binding assay system (1). At the same time we have studied the effect of some of these same peptides on the functional presentation of MATERIALS AND METHODS Purification of I-Ak. I-Ak was affinity purified on a large scale using an affinity matrix, the anti-I-Ak monoclonal antibody 10-2.16 coupled to CNBr-activated Sepharose 4B from plasma membranes derived from~-1011 TA3 hybridoma cells as described (3). Briefly, a plasma membrane-enriched fraction was isolated, washed in high salt (0.5 M NaCl, 10 mM EDTA), lysed in 0.5% Triton X-100 containing 15 mM triethanolamine, 0.2 mM phenylmethylsulfonyl fluoride, leupeptin at 10 gg/ml at pH 8.0, and passed through the anti-I-Ak affinity column. I-Ak was eluted wi...

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The interactions of palmitoyl-alpha-bungarotoxin (PBGT) with dipalmitoylphosphatidylcholine (DPPC) bilayers have been studied by using high-sensitivity differential scanning calorimetry together with steady-state and time-resolved phosphorescence and fluorescence spectroscopy. The incorporation of PBGT into large single lamellar vesicles causes a decrease in the phospholipid phase transition temperature (Tm), a broadening of the heat capacity function, and a decrease in the enthalpy change associated with the phospholipid gel to liquid-crystalline transition. Analysis of the dependence of this decreased enthalpy change on the protein/lipid molar ratio indicates that each PBGT molecule exhibits a localized effect upon the bilayer, preventing approximately six lipid molecules from participating in the lipid phase transition. Additional calorimetric experiments indicate that binding to acetylcholine receptor enriched membranes causes a small increase in the Tm of the PBGT/DPPC vesicles. Steady-state fluorescence depolarization measurements employing 1,6-diphenyl-1,3,5-hexatriene (DPH) indicate that the association of PBGT with the phospholipid bilayer decreases the apparent order of the bulk lipid below Tm while increasing the order above Tm. These results have been further supported by rotational mobility measurements of erythrosin-labeled PBGT associated with giant (about 2-micron) unilamellar vesicles composed of dielaidoylphosphatidylcholine or dioleoylphosphatidylcholine using the time-dependent decay of delayed fluorescence/phosphorescence emission anisotropy. Rotational correlation times in the submillisecond time scale (about 30 microseconds) indicate that the protein is highly mobile in the fluid phase and that below Tm the rotational mobility is only slightly restricted.(ABSTRACT TRUNCATED AT 250 WORDS)