Functional analysis of avian class I (BFIV) glycoproteins by epitope tagging and mutagenesis in vitro.
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 cell-surface 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 residues 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.
Fulton
J E
JE
USDA-Agricultural Research Service, Avian Disease and Oncology Laboratory, East Lansing, MI 48823, USA.
Thacker
E L
EL
Bacon
L D
LD
Hunt
H D
HD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000276:immunology*; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D018604:Epitope Mapping; D005805:Genes, MHC Class I; D015395:Histocompatibility Antigens Class I / Q000737:chemistry*; D007111:Immunity, Cellular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D017931:DNA Primers; D018076:DNA, Complementary; D015395:Histocompatibility Antigens Class I; D010455:Peptides