Melbourne Veterinary School - Theses

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Type: PhD thesis × Author: Gill, Jeffrey William. ×

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    The expression of T19 proteins on CD+ T cells
    Gill, Jeffrey William. (University of Melbourne, 2001)
    The subject of this thesis was the structure and expression of T19 proteins on CD4+ T cells. These molecules possess extracellular SRCR (scavenger receptor cysteine rich) domains appended to a characteristic hinge, transmembrane anchor and unique cytoplasmic domains and were once considered restricted to y? T cells. However, work from this laboratory, prior to this thesis, had demonstrated the expression of T19 transcripts outside the y? lineage. Specifically, one of the lineages that expressed T19 transcripts were CD4+ T lymphocytes sourced from efferent prescapular lymph. What had not yet been demonstrated was whether T19 transcripts were expressed in CD4+ T cells from other tissues, whether T19 molecules were expressed in a tissue specific manner, indicating a role in lymphocyte migration, or indeed, whether the T19 transcripts in efferent prescapular CD4+ T cells were even translated. Moreover, previous work had relied upon RT-PCR technology that may have skewed or biased the range of T19 molecules identified. The early part of this thesis was concerned with the cloning and characterisation of cDNAs generated from T19 transcripts purified from within CD4+ lymphocytes collected from efferent prescapular and efferent mesenteric lymphatics. These cloning experiments were conducted using a molecular probe that corresponded to the unique hinge, transmembrane anchor and cytoplasmic domains of the T19 multigene family. This probe was used to screen cDNA libraries that were constructed in order to obtain a more representative view of the repertoire of T19 molecules expressed in both efferent prescapular and efferent mesenteric CD4+ T cells than was possible using RT-PCR technology. A separate, non-overlapping probe, corresponding to the extracellular SRCR domains of the T19 multigene family was then employed to confirm the identity of any clonally purified putative T19 cDNAs. These experiments indicated that T19 genes were indeed expressed in efferent mesenteric CD4+ T cells, although at a ten-fold lower level than in efferent prescapular CD4+ T cells. This was the first direct evidence of tissue specific differences in T19 expression within a purified T cell subset. The sequence data from these T19 cDNAs indicated that there was an unexpectedly large repertoire of T19 transcripts expressed in these tissues. No clonal redundancy was identified either within or between anatomical sites. Thus, while less definitive than was hoped, the data supported an hypothesis of site-specificity in T19-isoform expression. The sequence data also identified that many of the T19 transcripts within CD4+ T cells coded for putatively soluble polypeptides. This latter finding was also unexpected. Furthermore, the data indicated that a large gene family of closely related proteins were expressed in these tissues, rather than a smaller number of alternately spliced genes. Selected cDNAs were genetically engineered for prokaryotic expression, with the specific aim of employing the resultant recombinant proteins as immunogens. Accordingly, a chimaeric protein was constructed that possessed three extracellular SRCR domains appended to a bacterial maltose binding protein (MBP). The MBP enabled the successful purification of the recombinant protein by affinity chromatography. One of these recombinants, EP4a'k, was particularly amenable to protein expression. Following confirmation of the recombinant construct's primary structure by MALDI-TOF mass spectrometric analysis, EP4a'k was used directly as a molecular probe in an attempt to identify its cognate in vivo ligand and to elicit antisera in rabbits. While the antisera displayed activity towards the T19-derived SRCR domains of EP4a'k, it did not bind to detectable populations of T lymphocytes. Nor did it appear to bind to any soluble T19 isoforms in cell-free lymph. These results indicate that either the native protein corresponding to EP4a'k was expressed at levels too low to detect with the techniques employed in these studies, or that EP4a'k had not elicited antibodies which bound its native counterpart. EP4a'k itself did not bind to detectable populations of T lymphocytes indicating that either its cognate ligand was expressed at levels too low to detect with the techniques employed in these studies, or that EP4a'k did not bind this ligand. In the final experimental chapter of this thesis, the recently available Celera databases were accessed in a series of in silico cloning experiments. Contrary to expectations and to previous, but unpublished, data T19 genes were not found in either human or mouse genomes. However, although the data in mouse was limiting due to its non-contiguous nature, structurally and possibly functionally analogous molecules were identified within the human genome. These findings suggest, for the first time, that the T19 multigene family is an Artiodactyl invention and that related molecules perform their function within other mammals. In the final discussion of this thesis, the aforementioned data was discussed in light of previously available data in the context of a role for T19 in the site-specific functions of T cells and the evolution of a diverse range of functionally related proteins within mammals.
  • Item
    No Preview Available
    The expression of T19 proteins on CD+ T cells
    Gill, Jeffrey William. (University of Melbourne, 2001)
    The subject of this thesis was the structure and expression of T19 proteins on CD4+ T cells. These molecules possess extracellular SRCR (scavenger receptor cysteine rich) domains appended to a characteristic hinge, transmembrane anchor and unique cytoplasmic domains and were once considered restricted to y? T cells. However, work from this laboratory, prior to this thesis, had demonstrated the expression of T19 transcripts outside the y? lineage. Specifically, one of the lineages that expressed T19 transcripts were CD4+ T lymphocytes sourced from efferent prescapular lymph. What had not yet been demonstrated was whether T19 transcripts were expressed in CD4+ T cells from other tissues, whether T19 molecules were expressed in a tissue specific manner, indicating a role in lymphocyte migration, or indeed, whether the T19 transcripts in efferent prescapular CD4+ T cells were even translated. Moreover, previous work had relied upon RT-PCR technology that may have skewed or biased the range of T19 molecules identified. The early part of this thesis was concerned with the cloning and characterisation of cDNAs generated from T19 transcripts purified from within CD4+ lymphocytes collected from efferent prescapular and efferent mesenteric lymphatics. These cloning experiments were conducted using a molecular probe that corresponded to the unique hinge, transmembrane anchor and cytoplasmic domains of the T19 multigene family. This probe was used to screen cDNA libraries that were constructed in order to obtain a more representative view of the repertoire of T19 molecules expressed in both efferent prescapular and efferent mesenteric CD4+ T cells than was possible using RT-PCR technology. A separate, non-overlapping probe, corresponding to the extracellular SRCR domains of the T19 multigene family was then employed to confirm the identity of any clonally purified putative T19 cDNAs. These experiments indicated that T19 genes were indeed expressed in efferent mesenteric CD4+ T cells, although at a ten-fold lower level than in efferent prescapular CD4+ T cells. This was the first direct evidence of tissue specific differences in T19 expression within a purified T cell subset. The sequence data from these T19 cDNAs indicated that there was an unexpectedly large repertoire of T19 transcripts expressed in these tissues. No clonal redundancy was identified either within or between anatomical sites. Thus, while less definitive than was hoped, the data supported an hypothesis of site-specificity in T19-isoform expression. The sequence data also identified that many of the T19 transcripts within CD4+ T cells coded for putatively soluble polypeptides. This latter finding was also unexpected. Furthermore, the data indicated that a large gene family of closely related proteins were expressed in these tissues, rather than a smaller number of alternately spliced genes. Selected cDNAs were genetically engineered for prokaryotic expression, with the specific aim of employing the resultant recombinant proteins as immunogens. Accordingly, a chimaeric protein was constructed that possessed three extracellular SRCR domains appended to a bacterial maltose binding protein (MBP). The MBP enabled the successful purification of the recombinant protein by affinity chromatography. One of these recombinants, EP4a'k, was particularly amenable to protein expression. Following confirmation of the recombinant construct's primary structure by MALDI-TOF mass spectrometric analysis, EP4a'k was used directly as a molecular probe in an attempt to identify its cognate in vivo ligand and to elicit antisera in rabbits. While the antisera displayed activity towards the T19-derived SRCR domains of EP4a'k, it did not bind to detectable populations of T lymphocytes. Nor did it appear to bind to any soluble T19 isoforms in cell-free lymph. These results indicate that either the native protein corresponding to EP4a'k was expressed at levels too low to detect with the techniques employed in these studies, or that EP4a'k had not elicited antibodies which bound its native counterpart. EP4a'k itself did not bind to detectable populations of T lymphocytes indicating that either its cognate ligand was expressed at levels too low to detect with the techniques employed in these studies, or that EP4a'k did not bind this ligand. In the final experimental chapter of this thesis, the recently available Celera databases were accessed in a series of in silico cloning experiments. Contrary to expectations and to previous, but unpublished, data T19 genes were not found in either human or mouse genomes. However, although the data in mouse was limiting due to its non-contiguous nature, structurally and possibly functionally analogous molecules were identified within the human genome. These findings suggest, for the first time, that the T19 multigene family is an Artiodactyl invention and that related molecules perform their function within other mammals. In the final discussion of this thesis, the aforementioned data was discussed in light of previously available data in the context of a role for T19 in the site-specific functions of T cells and the evolution of a diverse range of functionally related proteins within mammals.