Mechanisms and consequences of peptide selection by the I-Ak class II molecule.

Important quantitative parameters can be utilized to define the selection and the immunogenicity of protein antigens precisely at a biochemical and a cellular level. Here we describe a naturally processed family of peptides comprising the dominant hen egg white lysozyme epitope, its major contribution to surface I-Ak molecules, the primary and auxiliary peptide anchors involved in its selection, and its display of T-cell receptor contacts. In addition, we explore the importance of the processing events that lead to the generation of residues flanking the minimal core epitope, the quantification of T-cell responses directed toward the epitope, and the ability of the dominant epitope to form two unique conformations within the binding groove. Lastly, we address the relationship between this dominant and a minor lysozyme epitope.

Hindering auxiliary anchors are potent modulators of peptide binding and selection by I-Ak class II molecules.

Selection of particular antigen-derived peptides by class II MHC molecules determines the population of complexes represented on the antigen-presenting cell surface and available for T cell receptor engagement. This discriminating selection process results from unique interactions between the spectrum of peptides generated during antigen processing and the MHC molecules. Here, we examined the selection of peptides by the class II MHC, I-A(k). Our results indicate that although peptide primary anchors are key in MHC binding, auxiliary anchors are a powerful regulatory component in the selection of peptides by I-A(k). Study of the segments surrounding the dominant hen egg white lysozome(48-61) epitope demonstrates that auxiliary anchors also are involved in determining the binding register of I-A(k) along an extended peptide. In addition, we found that unique combinations of auxiliary anchors can act in concert to modulate the binding of peptides to I-A(k).

Structural basis of peptide binding and presentation by the type I diabetes-associated MHC class II molecule of NOD mice.

We have determined the crystal structure of I-Ag7, an integral component in murine type I diabetes development. Several features distinguish I-Ag7 from other non-autoimmune-associated MHC class II molecules, including novel peptide and heterodimer pairing interactions. The binding groove of I-Ag7 is unusual at both terminal ends, with a potentially solvent-exposed channel at the base of the P1 pocket and a widened entrance to the P9 pocket. Peptide binding studies with variants of the hen egg lysozyme I-Ag7 epitope HEL(11-25) support a comprehensive structure-based I-Ag7 binding motif. Residues critical for T cell recognition were investigated with a panel of HEL(11-25)-restricted clones, which uncovered P1 anchor-dependent structural variations. These results establish a framework for future experiments directed at understanding the role of I-Ag7 in autoimmunity.

Temperature-sensitive yeast GPI anchoring mutants gpi2 and gpi3 are defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol. Cloning of the GPI2 gene.

To identify genes required for the synthesis of glycosyl phosphatidylinositol (GPI) membrane anchors in yeast, we devised a way to isolate GPI anchoring mutants in which colonies are screened for defects in [3H]-inositol incorporation into protein. The gpi1 mutant, identified in this way, is temperature sensitive for growth and defective in vitro in the synthesis of GlcNAc-phosphatidylinositol, the first intermediate in GPI biosynthesis (Leidich, S. D., Drapp, D. A., and Orlean, P. (1994) J. Biol. Chem. 269, 10193-10196). We report the isolation of two more conditionally lethal mutants, gpi2 and gpi3, which, like gpi1, have a temperature-sensitive defect in the incorporation of [3H]inositol into protein and which lack in vitro GlcNAc-phosphatidylinositol synthetic activity. Haploid Saccharomyces cerevisiae strains containing any pairwise combination of the gpi1, gpi2, and gpi3 mutations are inviable. The GPI2 gene, cloned by complementation of the gpi2 mutant's temperature sensitivity, encodes a hydrophobic 269-amino acid protein that resembles no other gene product known to participate in GPI assembly. Gene disruption experiments show that GPI2 is required for vegetative growth. Overexpression of the GPI2 gene causes partial suppression of the gpi1 mutant's temperature sensitivity, a result that suggests that the Gpi1 and Gpi2 proteins interact with one another in vivo. The gpi3 mutant is defective in the SPT14 gene, which encodes a yeast protein similar to the product of the mammalian PIG-A gene, which complements a GlcNAc-phosphatidylinositol synthesis-defective human cell line. In yeast, at least three gene products are required for the first step in GPI synthesis, as is the case in mammalian cells, and utilization of several different proteins at this stage is therefore likely to be a general characteristic of the GPI synthetic pathway.