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Functional analysis of GPI-anchored and truncated forms of HLA-A2.1

by Huang, Jui-Han

Abstract (Summary)
GPI-anchors are post-translational modifications attached to a variety of cell surface molecules. A unique property of GPI-anchored polypeptides, which differs from transmembrane proteins, is the ability to spontaneously incorporate into biological membranes. This property allows for the direct alteration of cell surface phenotype without genetic manipulation. The human class I major histocompatibility complex (MHC) molecule, HLA-A2.1, has been correlated with protective immune responses in a number of diseases, including hepatitis B virus-induced chronic active hepatitis and malignant melanoma. Hence, a GPI-modified class I molecule could have immunotherapeutic utility. Toward this end, two artificial, chimeric HLA-A2.1:decay accelerating factor (DAF) DNA sequences were produced. Both chimeric sequences encoded the GPI-modification signals from the 3? end of DAF, but differed in the lengths of DAF sequence encoded. Both constructs were expressed in human cell lines. Serological analyses suggested overall molecular similarity between the HLA-A2.1:DAF and native HLA-A2.1 molecules. HLA-A2.1-alloantigen presentation to HLA-A2.1-specific T cell lines and clones was demonstrated for the shorter polypeptide, HLA-A2.1:GPI-S, but not for the longer molecule, HLA-A2.1: GPI-L. In contrast, both molecules effectively presented a peptide from hepatitis B virus surface antigen (HBV env335-343) to peptide-specific, HLA-A2.1-restricted T cell clones. The HLA-A2.1:GPI-S was expressed in insect cells and immunoaffinity purified. These insect cell-derived HLA-A2.1:GPI-S molecules differ from those derived from human cells because they lack bound peptides. As a result, antigenic peptide association is facilitated. The in vitro assembled complexes could be incorporated into cell surfaces and thereby sensitize target cells to cytolysis by effector T cells. The CD8? T cell co-receptor molecule binds to the ?3 domain of class I MHC molecules. To probe this interaction in a cell-free system, the ?3 domain of the HLA-A2.1 molecule was isolated by recombinant DNA methodology and expressed in E. coli. These ?3 domain polypeptides were shown to bind to a number of human CD8? polypeptide derivatives.
Bibliographical Information:

Advisor:

School:Case Western Reserve University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:functional analysis gpi anchored truncated forms hla a2 1

ISBN:

Date of Publication:01/01/1994

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