Modulation of the antigenic peptide transporter TAP by recombinant antibodies binding to the last five residues of TAP1.
The transporter associated with antigen processing (TAP) plays a pivotal role in the major histocompatibility complex (MHC) class I mediated immune response against infected or malignantly transformed cells. It belongs to the ATP-binding cassette (ABC) superfamily and consists of TAP1 (ABCB2) and TAP2 (ABCB3), each of which possesses a transmembrane and a nucleotide-binding domain (NBD). Here we describe the generation of recombinant Fv and Fab antibody fragments to human TAP from a hybridoma cell line expressing the TAP1-specific monoclonal antibody mAb148.3. The epitope of the antibody was mapped to the very last five C-terminal amino acid residues of TAP1 on solid-supported peptide arrays. The recombinant antibody fragments were heterologously expressed in Escherichia coli and purified to homogeneity from periplasmic extracts by affinity chromatography. The monoclonal and recombinant antibodies bind with nanomolar affinity to the last five C-terminal amino acid residues of TAP1 as demonstrated by ELISA and surface plasmon resonance. Strikingly, the recombinant antibody fragments confer thermal stability to the heterodimeric TAP complex. At the same time TAP is arrested in a peptide transport incompetent conformation, although ATP and peptide binding to TAP are not affected. Based on our results we suggest that the C terminus of TAP1 modulates TAP function presumably as part of the dimer interface of the NBDs.
Plewnia
Gabriele
G
Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 9, D-69438 Frankfurt a. M., Germany.
Schulze
Katrin
K
Hunte
Carola
C
Tampé
Robert
R
Koch
Joachim
J
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology*; D003001:Cloning, Molecular; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D006825:Hybridomas; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D011994:Recombinant Proteins; C073119:TAP1 protein, human