Class I major histocompatibility complex molecules present antigenic peptides to cytotoxic T lymphocytes, which leads to the elimination of virus infected cells. Class I molecules are heterotrimers consisting of a heavy chain, a light chain termed beta2-microglobulin, and a peptide ligand. Assembly of class I molecules begins in the endoplasmic reticulum where the heavy chain associates with beta2-microglobulin, and the heavy chain-beta2-microglobulin heterodimers enter a peptide loading complex where class I molecules acquire peptides. During the biogenesis of class I molecules, ERp57, a thiol oxidoreductase, associates with free class I heavy chains and, at a later stage, with the peptide loading complex. In this thesis, I show for the first time that ERp57 participates in oxidative folding of the heavy chain. Depletion of ERp57 by RNAi delayed heavy chain disulfide bond formation and slowed folding of the heavy chain alpha3 domain. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no such effect. Since ERp57 associates with the lectin-chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, I examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells, suggesting that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin-chaperones. ERp57 also forms a mixed disulfide with tapasin within the peptide loading complex and I found that the formation of this mixed disulfide is independent of its interaction with calnexin and calreticulin. I also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide-binding site in substrate recognition. Finally, by inactivating the redox active sites of ERp57, I demonstrate that its enzymatic activity is dispensable in stabilizing the loading complex and in supporting efficient peptide loading. Thus, ERp57 plays a structural rather than catalytic role within the peptide loading complex.

Genomic organization of the major histocompatibility complex (MHC) can profoundly influence gene function and multigene family evolution. Situated at the interface of individual genetic variation and the adaptive immune response, MHC class I and II loci are intensively studied for disease associations and used as markers of adaptive genetic variation in evolutionary ecology research. Genomic sequence of MHC-containing cosmid clones from the red-billed gull (Larus scopulinus, Charadriiformes: shorebirds, gulls, and allies) was obtained for comparative analysis of avian MHC evolution. MHCI polymorphism was further investigated using cDNA library screening and locus-specific genotyping protocols. This first information regarding MHC organization and MHCI variation in charadriiforms suggests a complex evolutionary history to MHC architecture in birds. Duplication of MHCIIα loci in tandem MHCIIα/β pairs and their proximity to MHC-region gene COL11A2 are similar to arrangements in nonavian vertebrates, and contrast with the “minimal essential” MHC of the chicken (Gallus gallus, Galliformes: gamebirds). MHCI–TAP2 organization is shared with Galloanserae (gamebirds + waterfowl), as is a proposed major classical function for this MHCI gene. In contrast, the placement of MHCI genes adjacent to sequence from chromosomes 3, 5, and 22 of the chicken and zebra finch (Taeniopygia guttata, Passeriformes: perching birds) indicates interchromosomal rearrangements in birds and the possible genomic dispersal of nonclassical MHCI genes in the red-billed gull. Screening for avian malaria, genetic parentage tests, and field data from red-billed gulls at Kaikoura Peninsula, New Zealand were combined with MHCI genotypes to investigate relationships with disease and reproduction. Plasmodium infection was confirmed in red-billed gulls, and breeding condition was negatively associated with malarial infection and positively related to variation at the putative major MHCI locus. A low rate of extrapair paternity was identified across thirteen breeding seasons. Partners without extrapair young (EPY) had greater MHCI dissimilarity than was expected by chance, whereas lower individual MHCI variation and elevated hatching failure existed for pairs with EPY. In addition to contributing to studies of MHC evolution, sexual selection, and disease dynamics in the New Zealand avifauna, this research will facilitate studies of MHC genes in related charadriiforms, many of which are of conservation concern.

Genomic organization of the major histocompatibility complex (MHC) can profoundly influence gene function and multigene family evolution. Situated at the interface of individual genetic variation and the adaptive immune response, MHC class I and II loci are intensively studied for disease associations and used as markers of adaptive genetic variation in evolutionary ecology research. Genomic sequence of MHC-containing cosmid clones from the red-billed gull (Larus scopulinus, Charadriiformes: shorebirds, gulls, and allies) was obtained for comparative analysis of avian MHC evolution. MHCI polymorphism was further investigated using cDNA library screening and locus-specific genotyping protocols. This first information regarding MHC organization and MHCI variation in charadriiforms suggests a complex evolutionary history to MHC architecture in birds. Duplication of MHCIIα loci in tandem MHCIIα/β pairs and their proximity to MHC-region gene COL11A2 are similar to arrangements in nonavian vertebrates, and contrast with the “minimal essential” MHC of the chicken (Gallus gallus, Galliformes: gamebirds). MHCI–TAP2 organization is shared with Galloanserae (gamebirds + waterfowl), as is a proposed major classical function for this MHCI gene. In contrast, the placement of MHCI genes adjacent to sequence from chromosomes 3, 5, and 22 of the chicken and zebra finch (Taeniopygia guttata, Passeriformes: perching birds) indicates interchromosomal rearrangements in birds and the possible genomic dispersal of nonclassical MHCI genes in the red-billed gull. Screening for avian malaria, genetic parentage tests, and field data from red-billed gulls at Kaikoura Peninsula, New Zealand were combined with MHCI genotypes to investigate relationships with disease and reproduction. Plasmodium infection was confirmed in red-billed gulls, and breeding condition was negatively associated with malarial infection and positively related to variation at the putative major MHCI locus. A low rate of extrapair paternity was identified across thirteen breeding seasons. Partners without extrapair young (EPY) had greater MHCI dissimilarity than was expected by chance, whereas lower individual MHCI variation and elevated hatching failure existed for pairs with EPY. In addition to contributing to studies of MHC evolution, sexual selection, and disease dynamics in the New Zealand avifauna, this research will facilitate studies of MHC genes in related charadriiforms, many of which are of conservation concern.

Class I major histocompatibility complex molecules present antigenic peptides to cytotoxic T lymphocytes, which leads to the elimination of virus infected cells. Class I molecules are heterotrimers consisting of a heavy chain, a light chain termed beta2-microglobulin, and a peptide ligand. Assembly of class I molecules begins in the endoplasmic reticulum where the heavy chain associates with beta2-microglobulin, and the heavy chain-beta2-microglobulin heterodimers enter a peptide loading complex where class I molecules acquire peptides. During the biogenesis of class I molecules, ERp57, a thiol oxidoreductase, associates with free class I heavy chains and, at a later stage, with the peptide loading complex. In this thesis, I show for the first time that ERp57 participates in oxidative folding of the heavy chain. Depletion of ERp57 by RNAi delayed heavy chain disulfide bond formation and slowed folding of the heavy chain alpha3 domain. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no such effect. Since ERp57 associates with the lectin-chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, I examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells, suggesting that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin-chaperones. ERp57 also forms a mixed disulfide with tapasin within the peptide loading complex and I found that the formation of this mixed disulfide is independent of its interaction with calnexin and calreticulin. I also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide-binding site in substrate recognition. Finally, by inactivating the redox active sites of ERp57, I demonstrate that its enzymatic activity is dispensable in stabilizing the loading complex and in supporting efficient peptide loading. Thus, ERp57 plays a structural rather than catalytic role within the peptide loading complex.

β2-microglobulin (β2m) exists free of the major histocompatibility complex class I (MHC I) receptor in many bodily fluids. The amount of protein present in these fluids has been found to be a useful prognostic marker for various diseases but outside of its practical value not much is known about this form of β2m. In fish, soluble β2m has not been studied at all. Another unknown in fish is the effects that plants lectins might have on naturally carnivorous species in aquaculture. These plant proteins which bind to specific sugar groups found on cells have been shown to have a multitude of gastrointestinal and immune effects in mammals and can be found in the plant products being fed to carnivorous, cultured fish making them possible toxicants. The two studies of this thesis set out to pioneer knowledge on these subjects using rainbow trout as a model. The first investigation inspected the various body fluids of these fish for their free β2m content. Soluble β2m was found to be present in the plasma, the seminal fluid, ovarian fluid, and the mucus of the skin and intestines. This distribution shows that β2m could indeed make a good biomarker, not only for disease but also for pheromone release and alludes to some possible functions of soluble β2m while opening the way for future research on this form of the protein. The second study looked at the effects of lectins on the gut of rainbow trout by treating RTgutGC, an intestinal epithelial cell line derived from trout, with plant lectins from wheat (WGA) and soybean (SBA), among others. This study found WGA to be a potent inducer of morphological and cytotoxic effects in these cells while other lectins and plant factors were not. WGA was also observed to effect the expression of β2m and the α-chain of the MHC I receptor. This work suggests WGA ingested by trout through the wheat in their diet might be causing them harm and should be studied further. It is also interesting that both studies related β2m to the intestines of trout. This could allow soluble β2m to serves as a marker of WGA’s effect or for WGA to aid in the study of free β2m.

Cattle are vitally important to American agriculture industry, generating over 24.6 billion pounds of beef (by carcass weight), and 79.5 billion dollars in 2005, and over 27 billion dollars in milk sales in 2004. As of July 2006, the U.S. beef and dairy industry is comprised of 104.5 million head of cattle, 32.4 million of which were processed in 2005. The health of the animals has always been an important concern for breeders, as healthy animals grow faster and are more likely to reach market weight. Animals that exhibit natural resistance to disease do not require chemicals to stimulate normal weight gain, and are less prone to disease related wasting. The major histocompatibility complex (MHC) is a collection of genes, many of which function in antigen processing and presentation. The bovine MHC (BoLA) differs from typical mammalian MHCs in that the class II region was disrupted by a chromosomal inversion into two subregions, designated BoLA IIa and BoLA IIb. BoLA IIb was transposed to a position near the centromere on bovine chromosome 23,while BoLA IIa retains its position in BoLA. Comparative sequence analysis of BoLA IIb with the human MHC revealed the location of the region containing the proximal inversion breakpoint. Gene content, order and orientation of BoLA IIb are consistent with the single inversion hypothesis when compared to the corresponding region of the human class II MHC (HLA class II). BoLA IIb spans approximately 450 kb. The genomic sequence of BoLA IIb was used to detect sequence variation through comparison to other bovine sequences, including data from the bovine genome project, and two regions in the BAC scaffold used to develop the BoLA IIb sequence. Analysis of the bovine genome project sequence revealed a total of 10,408 mismatching bases, 30 out of 231 polymorphic microsatellites, and 15 sequences corresponding to the validated SNP panel generated by the bovine genome sequencing project. The two overlapping regions in the BoLA IIb BAC scaffold were found to have 888 polymorphisms, including a total of 6 out of 42 polymorphic microsatellites indicating that each BAC derived from a different chromosome.

The major histocompatibility complex region (MHC) is a multi gene family present in all jawed vertebrates, with a fundamental role in vertebrate immunity. More than two decades of studies have resulted in the characterization of over a dozen MHC regions, and models of evolution explaining that the MHC has gradually increased in size and gene content since its origins without addressing their genomic context or the environmental selective forces. Furthermore, a compelling reconstruction of the evolutionary history of the MHC has been hampered due to phylogenetic gaps and the absence of comparative phylogenetic methods applied to comparative genomics. Here I reconstruct 320 MY of MHC evolution using 42 amniote genomes using improved gene annotations, genomic alignments and phylogenetic algorithms to reconstruct the evolution of the MHC at three levels of phylogenetic resolution. The first one describes 25 MY of evolution of the primate MHC using eight Human and four non-Human primate MHC haplotypes. Results suggests that highly dense gene segments have a strikingly conserved gene organization, and six conserved and highly rearranging segments overlap genes that are most commonly associated to disease. Phylogenomic analysis implies that the MHC has remained stable in gene content and size, with significantly increased duplication rates in the primate ancestors. The second one describes 280 MY of MHC evolution through the first characterization of reptilian MHC region, which combines mammalian, reptilian, Bird and amphibian characteristics, which favors the hypothesis of the existence of a primordial MHC in which natural killer receptors, CD1 and lectin genes co-exist. The Anolis MHC expands our understanding of the origins of the exceptionally small Bird MHC regions and provides further information about the organization and size of the ancestral amniote MHC. The third one compares 42 amniote MHC regions and map gene duplications and losses to further evaluate the mode and tempo of the evolution of the region. Comparative phylogenetic methods imply that the genomic and environmental factors affect the diversification of MHC during 320 My of evolution.

No description available.

<p>Nearly thirty percent of all newly synthesized polypeptides are targeted for rapid proteasome-mediated degradation. These rapidly degraded polypeptides (RDPs) are the primary source of antigenic substrates for the major histocompatibility complex (MHC) class I presentation pathway, allowing for the immunosurveillance of newly synthesized proteins by cytotoxic T lymphocytes. Despite the recognized role of RDPs in MHC class I presentation, it remains unclear what molecular characteristics distinguish RDPs from their more stable counterparts. It has been proposed that premature translational termination products may constitute a form of RDP; indeed, in prokaryotes translational drop-off products are normal by-products of protein synthesis and are subsequently rapidly degraded. </p><p>To study the cellular fate of premature termination products, the antibiotic puromycin was used to modulate prematurely terminated polypeptide production in human cells. At low concentrations, puromycin doubled the fraction of rapidly degraded polypeptides, with enhanced degradation predominantly affecting small polypeptides, consistent with rapid degradation of truncated translation products. Immunoprecipitation experiments using anti-puromycin antisera demonstrated that the majority of peptidyl-puromycins are rapidly degraded in a proteasome-dependent manner. Low concentrations of puromycin increased the recovery of cell surface MHC class I-peptide complexes, indicating that prematurely terminated polypeptides can be processed for presentation via the MHC I pathway. In the continued presence of puromycin, MHC I export to the cell surface was inhibited, coincident with the accumulation of polyubiquitinated proteins. The time- and dose-dependent effects of puromycin suggest that the pool of peptidyl-puromycin adducts differ in their targeting to various proteolytic pathways which, in turn, differ in the efficiency with which they access the MHC class I presentation machinery. These studies highlight the diversity of cellular proteolytic pathways necessary for the metabolism and immunosurveillance of prematurely terminated polypeptides which are, by their nature, highly heterogeneous.</p> / Dissertation

β2-microglobulin (β2m) exists free of the major histocompatibility complex class I (MHC I) receptor in many bodily fluids. The amount of protein present in these fluids has been found to be a useful prognostic marker for various diseases but outside of its practical value not much is known about this form of β2m. In fish, soluble β2m has not been studied at all. Another unknown in fish is the effects that plants lectins might have on naturally carnivorous species in aquaculture. These plant proteins which bind to specific sugar groups found on cells have been shown to have a multitude of gastrointestinal and immune effects in mammals and can be found in the plant products being fed to carnivorous, cultured fish making them possible toxicants. The two studies of this thesis set out to pioneer knowledge on these subjects using rainbow trout as a model. The first investigation inspected the various body fluids of these fish for their free β2m content. Soluble β2m was found to be present in the plasma, the seminal fluid, ovarian fluid, and the mucus of the skin and intestines. This distribution shows that β2m could indeed make a good biomarker, not only for disease but also for pheromone release and alludes to some possible functions of soluble β2m while opening the way for future research on this form of the protein. The second study looked at the effects of lectins on the gut of rainbow trout by treating RTgutGC, an intestinal epithelial cell line derived from trout, with plant lectins from wheat (WGA) and soybean (SBA), among others. This study found WGA to be a potent inducer of morphological and cytotoxic effects in these cells while other lectins and plant factors were not. WGA was also observed to effect the expression of β2m and the α-chain of the MHC I receptor. This work suggests WGA ingested by trout through the wheat in their diet might be causing them harm and should be studied further. It is also interesting that both studies related β2m to the intestines of trout. This could allow soluble β2m to serves as a marker of WGA’s effect or for WGA to aid in the study of free β2m.