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

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 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.

Studies on the association ofBF1/BF2 gene expression patterns with traits of genetic resistance to Marek's disease in chickens

“…HLA-A and -B molecules are the dominant class I molecules in presenting antigen to cytotoxic T lymphocytes (CTL) as part of the adaptive immune response (Littaua et al, 1991) while HLA-C molecules have been shown to be recognised by CTL induced in only a few viral Food and Agricultural Immunology 225 infections (Littaua et al, 1991). Site-directed mutagenesis demonstrated that a residue critical for binding peptide in mammalian class I molecules was also important for peptide specificity of BF2 molecules (Fulton, Thacker, & Bacon, 1995). BF2 molecules have been described as highly effective in presenting antigen to MHCrestricted, antigen-specific CTL .…”

Comparative genomics of the poultry major histocompatibility complex

“…The physical structure of BF2 in the B21 haplotype ( BF2 * 21 ) is similar to HLA‐A2 (Fulton et al . 1995; Yan et al .…”

“…The predominantly expressed BF2 locus, known as B‐FIV , B‐F major or BF alpha1 , is located between the TAP2 and C4 loci (Fig. 1; Fulton et al . 1995; Kaufman et al .…”

“…The predominantly expressed BF2 locus, known as B-FIV, B-F major or BF alpha1, is located between the TAP2 and C4 loci (Fig. 1;Fulton et al 1995;Kaufman et al 1999a,b). The B complex hybridization data initially resulted in the mistaken inclusion of two additional loci (B-FII and B-FIII) on the original map (Guillemot et al 1988;Kaufman et al 1999a;Miller et al 2004).…”

Mass spectral data for 64 eluted peptides and structural modeling define peptide binding preferences for class I alleles in two chicken MHC-B haplotypes associated with opposite responses to Marek's disease

“…Following verification of the sequences, the constructs were introduced into the avian retroviral vector RCASBP(A) (Petropoulos and Hughes 1991) and transfected into DF1 cells. Transfected DF1 cell culture supernatants were used to infect the RP9 cells and these were in turn selected by flow cytometry for uniform surface expression of the FLAG epitope as previously described (Fulton et al 1995). …”