Assembly and function of the major histocompatibility complex (MHC) I peptide-loading complex are conserved across higher vertebrates

Abstract

Antigen presentation to cytotoxic T lymphocytes via major histocompatibility complex class I (MHC I) molecules depends on the heterodimeric transporter associated with antigen processing (TAP). For efficient antigen supply to MHC I molecules in the ER, TAP assembles a macromolecular peptide-loading complex (PLC) by recruiting tapasin. In evolution, TAP appeared together with effector cells of adaptive immunity at the transition from jawless to jawed vertebrates and diversified further within the jawed vertebrates. Here, we compared TAP function and interaction with tapasin of a range of species within two classes of jawed vertebrates. We found that avian and mammalian TAP1 and TAP2 form heterodimeric complexes across taxa. Moreover, the extra N-terminal domain TMD0 of mammalian TAP1 and TAP2 as well as avian TAP2 recruits tapasin. Strikingly, however, only TAP1 and TAP2 from the same taxon can form a functional heterodimeric translocation complex. These data demonstrate that the dimerization interface between TAP1 and TAP2 and the tapasin docking sites for PLC assembly are conserved in evolution, whereas elements of antigen translocation diverged later in evolution and are thus taxon specific.

Keywords: ABC Transporter; Antigen Processing; Immunology; Major Histocompatibility Complex (MHC); Membrane Protein; Transporter.

FIGURE 1.

Expression screening of TAP subunits and assembly of a PLC. A, core TAP is shown as a homology model based on the x-ray structures of Sav1866 (PDB 2HYD) and an NDB dimer of rat TAP1 (PDB 2IXE) (13, 35). The interaction with tapasin (PDB 3F8U) (36) and the interaction scaffold TMD0 are indicated. B, TAP1 subunits are fused C-terminally to a C3 cleavage site, mVenus, a C8-tag, and a His₁₀-tag, TAP2 subunits are fused C-terminally to a C3 cleavage site, mCerulean, a myc-tag, and the streptavidin-binding peptide (SBP).

FIGURE 2.

Avian and mammalian TAP1 and TAP2 are expressed in human cells. HEK293T cells were cotransfected with TAP1 and TAP2 from different species. A, expression levels of TAP1 and TAP2 were monitored by flow cytometry. The dot blots represent transfections with TAP1 (left), TAP2 (middle) and TAP1/TAP2 (right) from different species. The fluorescence gates of mVenus and mCerulean are shown as gray lines, and numbers represent gate populations in %. A. platyrhynchos TAP1 lacking its correct 3′-region did not express. To analyze the expression and function of TAP2 from A. platyrhynchos, it was coexpressed with G. gallus TAP1. B, expression levels of TAP1 (blue), TAP2 (yellow-orange), as well as TAP1/TAP2(black) are summarized. C, expression of TAP1-mVenus and TAP2-mCerulean was analyzed by SDS-PAGE in-gel fluorescence (TAP1: yellow and TAP2: blue) and immunoblotting. White signals indicate a spectral overlap of the YFP and the CFP channel. TAP1 and TAP2 were detected with a C8- and myc-antibody, respectively.

FIGURE 3.

Avian and mammalian TAP complexes interact with tapasin. Immunodetection of human tapasin after tandem-affinity purification of avian and mammalian TAP complexes expressed in HEK293T. Expression of TAP1 is colored in yellow, that of TAP2 in blue. A white signal indicates a spectral overlap of the YFP and the CFP channel. Tapasin was detected by the antibody 7F6. The input after solubilization (A) and the tandem-affinity purified PLC (B) are shown.

FIGURE 4.

All mammalian TAP1 and TAP2 restore MHC I surface expression in TAP-deficient cells. TAP1-deficient BRE169 (A) or TAP2-deficient STF169 cells (B) were transfected with mammalian TAP1 or TAP2, respectively. Cells were gated on the expression of TAP1-mVenus (BRE169) or TAP2-mCerulean (STF169), respectively, and the MHC I surface expression is shown. MHC I were stained with a mouse antibody W6/32 and a PerCP-Cy5.5-coupled anti-mouse secondary antibody. H. sapiens (black), S. scrofa (red), C. familiaris (orange), B. taurus (green), M. musculus (blue), and R. norvegicus (pink), mock transfections are shown in gray. After cotransfection of BRE169 (C) or STF169 cells (D) with TAP1/TAP2, double positive cells were gated by the fluorescence of TAP1-mVenus and TAP2-mCerulean, and MHC I surface expression is shown. E, the restoration of MHC I surface expression in TAP1- and TAP2-deficient cells by the corresponding TAP1 (white bars) and TAP2 subunits (black bars). Mean fluorescence intensities (MFI%) and the S.D. are provided from two independent experiments.

FIGURE 5.

Avian TAP1/TAP2 restore MHC I peptide loading and surface expression. After transfections of TAP1-deficient BRE169 (A) or TAP2-deficient STF169 cells (B) with avian TAP1 or TAP2, respectively, the cells were gated on the expression of TAP1-mVenus (BRE169) or TAP1-mVenus (STF169). MHC I was stained with a mouse antibody W6/32 and an APC-coupled anti-mouse secondary antibody. Colors are: H. sapiens (black), A. platyrhynchos (red), C. coturnix (orange), G. gallus (green), and M. gallopavo (blue). TAP1-deficient BRE169 (C) or TAP2-deficient STF169 cells (D) were cotransfected with TAP1/TAP2 of the same species. TAP1-deficient BRE169 were cotransfected with TAP1 from C. coturnix (E), G. gallus (F), or M. gallopavo (G) in combination with TAP2 from A. platyrhynchos (red), C. coturnix (orange), G. gallus (green), and M. gallopavo (blue), respectively. Double positive cells were gated by the fluorescence of TAP1-mVenus and TAP2-mCerulean, and the MHC I surface expression is shown. H, ATP-dependent peptide transport in TAP1-deficient BRE169 cells transfected with TAP1-mCherry or combinations of TAP1-mCherry and TAP2-mCerulean. Cells transfected with mCherry (mock) are used as control. Transport is normalized to human TAP1 and the S.D. is provided from three independent experiments.