Functional analysis of avian class I (BFIV) glycoproteins by epitope tagging and mutagenesis in vitro

Authors

  • Janet Elizabeth Fulton,

    1. USDA-Agricultural Research Service, Avian Disease and Oncology Laboratory, East Lansing, USA
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  • Eileen Lenore Thacker,

    1. USDA-Agricultural Research Service, Avian Disease and Oncology Laboratory, East Lansing, USA
    Current affiliation:
    1. Iowa State University, Veterinary and Reserach Institute, 1802 Elwood Drive, Ames, A 50011-1250, USA
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  • Larry Dean Bacon,

    1. USDA-Agricultural Research Service, Avian Disease and Oncology Laboratory, East Lansing, USA
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  • Henry Donald Hunt

    Corresponding author
    1. USDA-Agricultural Research Service, Avian Disease and Oncology Laboratory, East Lansing, USA
    • USDA-Agricultural Research Service, Avian Disease and Oncology Laboratory, 3606 East Mount Hope Road, East Lansing, MI 48823, USA (Fax: +1-517-337-6776)
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Abstract

Similarities between the physical structures of avian and mammalian major histocompatibility complex (MHC) class I glycoproteins have been proposed based on comparative alignment of their amino acid sequences. To investigate the physical structure of the chicken class I glycoprotein, we cloned the cDNA representing the BFIV locus of the B21 haplotype. A unique, chimeric class I glycoprotein was constructed by incorporating an epitope tag (FLAG) at the N terminus. Monoclonal antibodies to the FLAG epitope served to monitor cellsurface expression for functional analysis of the BFIV21 class I glycoprotein. The chimeric class I glycoprotein was expressed in target cells using an avian leukosis virus (ALV)-derived retrovirus vector (RCASBP). The presence of the FLAG epitope did not interfere with either alloantibody recognition or cytotoxic T lymphocyte interaction. Functional analysis employing site-directed mutagenesis identified BF amino acid residues forming serologic epitopes as well as residues important in antigen presentation to ALV-induced cytotoxic T lymphocytes. BF residues 78 and 81, corresponding to HLA 79 and 82, form an antibody epitope with a slight effect on ALV antigen presentation, consistent with their predicted orientation based on the HLA-A2 crystal structure. Alignment of the BFIV21 sequence with previously published BFIV sequences revealed polymorphisms at position 34 (HLA 34), a monomorphic residue in HLA and H-2. Residue 34 is located in pocket B and is predicted to contact the main-chain carbon of peptides bound in HLA-A2. A site-directed substitution in BFIV residue 34 dramatically alters ALV antigen presentation by the BFIV21 class I glycoprotein. These data indicate that the physical molecular structure of the chicken MHC class I glycoprotein is similar to HLA.

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