Characterization of a high-affinity, highly selective tryptophan transport system in the human macrophage and the effects of overexpression of tryptophanyl t-RNA synthetase on Jurkat proliferation

Hdl Handle:
http://hdl.handle.net/10675.2/575211
Title:
Characterization of a high-affinity, highly selective tryptophan transport system in the human macrophage and the effects of overexpression of tryptophanyl t-RNA synthetase on Jurkat proliferation
Authors:
Seymour, Robert L.
Abstract:
Suppression of T cell activation by macrophages/dendritic cells via tryptophan degradation has been shown to play an important role in immunotolerance. Tryptophan degradation is carried out by the enzyme indolamine-2,3-dioxegenase (IDO). This model raises many questions. This study addresses two of these questions. First, how does the macrophage gain access to and degrade tryptophan to a level below 50 nM in culture medium? This is achieved despite the fact that the known high affinity tryptophan transport systems accept other amino acids and have Km values for tryptophan ranging fi-om 10-100 pM. In this study we show that the macrophage possesses a high-affinity, highly selective, and Na-independent tryptophan transport system with a Km for tryptophan of about 300 nM. This would allow the macrophage to have effective access to tryptophan at concentrations in the nanomolar range. We also show T cells do not possess this transport system. Second, how does the T cell sense the level of intracellular free tryptophan? It has been shown in the past that if T cells are stimulated in medium containing less than SOOnM tryptophan that they attempt to activate but arrest in mid-Gl of the cell cycle. The enzyme tryptophanyl t-RNA S5mthetase (WRS) charges tRNA*'^ with tryptophan. This enzjmie has two protein isoforms, with one having a non-canonical N-terminal kinase domain. WRS is also upregulated by interferon gamma (IFNy). These characteristics put WRS in a position to be an intracellular free tryptophan sensor. Here we show that transient transfection of the T cell line, Jurkat, with cDNA encoding the kinase-containing isoform of WRS inhibits proliferation. In addition, to the above we have ereated a subline of Jurkat, which stably arrests in the absence of tryptophan. We also show that this new subline is resistant to the drug G418 but is sensitive to hygromycin. When treated with rapamycin the Jurkat sub line will stably arrest in the presence or absence of tryptophan. Rapamycin is a known immunosuppressive agent, which inhibits T cell proliferation. This leads to the speculation of a possible link between the signaling pathways involved in tryptophan sensing and those involved in the effects of rapamycin.
Affiliation:
Department of Pediatrics
Issue Date:
Apr-2004
URI:
http://hdl.handle.net/10675.2/575211
Additional Links:
http://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/305096653?accountid=12365
Type:
Dissertation
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorSeymour, Robert L.en
dc.date.accessioned2015-08-19T02:45:57Zen
dc.date.available2015-08-19T02:45:57Zen
dc.date.issued2004-04en
dc.identifier.urihttp://hdl.handle.net/10675.2/575211en
dc.description.abstractSuppression of T cell activation by macrophages/dendritic cells via tryptophan degradation has been shown to play an important role in immunotolerance. Tryptophan degradation is carried out by the enzyme indolamine-2,3-dioxegenase (IDO). This model raises many questions. This study addresses two of these questions. First, how does the macrophage gain access to and degrade tryptophan to a level below 50 nM in culture medium? This is achieved despite the fact that the known high affinity tryptophan transport systems accept other amino acids and have Km values for tryptophan ranging fi-om 10-100 pM. In this study we show that the macrophage possesses a high-affinity, highly selective, and Na-independent tryptophan transport system with a Km for tryptophan of about 300 nM. This would allow the macrophage to have effective access to tryptophan at concentrations in the nanomolar range. We also show T cells do not possess this transport system. Second, how does the T cell sense the level of intracellular free tryptophan? It has been shown in the past that if T cells are stimulated in medium containing less than SOOnM tryptophan that they attempt to activate but arrest in mid-Gl of the cell cycle. The enzyme tryptophanyl t-RNA S5mthetase (WRS) charges tRNA*'^ with tryptophan. This enzjmie has two protein isoforms, with one having a non-canonical N-terminal kinase domain. WRS is also upregulated by interferon gamma (IFNy). These characteristics put WRS in a position to be an intracellular free tryptophan sensor. Here we show that transient transfection of the T cell line, Jurkat, with cDNA encoding the kinase-containing isoform of WRS inhibits proliferation. In addition, to the above we have ereated a subline of Jurkat, which stably arrests in the absence of tryptophan. We also show that this new subline is resistant to the drug G418 but is sensitive to hygromycin. When treated with rapamycin the Jurkat sub line will stably arrest in the presence or absence of tryptophan. Rapamycin is a known immunosuppressive agent, which inhibits T cell proliferation. This leads to the speculation of a possible link between the signaling pathways involved in tryptophan sensing and those involved in the effects of rapamycin.en
dc.relation.urlhttp://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/305096653?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectTryptophanyl t-RNA Synthetaseen
dc.subjectTryptophanen
dc.subjectTransporteren
dc.subjectindoleamine 2,3 dioxygenaseen
dc.subjectT Cellen
dc.subjectMacrophageen
dc.subjectJurkaten
dc.subjectG418en
dc.subjectRapamycinen
dc.subjectMammalian target of Rapamycinen
dc.titleCharacterization of a high-affinity, highly selective tryptophan transport system in the human macrophage and the effects of overexpression of tryptophanyl t-RNA synthetase on Jurkat proliferationen
dc.typeDissertationen
dc.contributor.departmentDepartment of Pediatricsen
dc.description.advisorMunn, Daviden
dc.description.committeeGanapathy, Vadivel; Ignatowicz, Leszek; Iwashima, Makio; Leibach, Frederick; Mellor, Andrewen
dc.description.degreeDoctor of Philosophy (Ph.D.)en
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