The outermost N-terminal region of tapasin facilitates folding of major histocompatibility complex class I
Research output: Contribution to journal › Journal article › Research › peer-review
Tapasin (Tpn) is an ER chaperone that is uniquely dedicated to MHC-I biosynthesis. It binds MHC-I molecules, integrates them into peptide-loading complexes, and exerts quality control of the bound peptides; only when an "optimal peptide" is bound will the MHC-I be released and exported to the cell surface for presentation to T cells. The exact mechanisms of Tpn quality control and the criteria for being an optimal peptide are still unknown. Here, we have generated a recombinant fragment of human Tpn, Tpn(1-87) (representing the 87 N-terminal and ER-luminal amino acids of the mature Tpn protein). Using a biochemical peptide-MHC-I-binding assay, recombinant Tpn(1-87) was found to specifically facilitate peptide-dependent folding of HLA-A*0201. Furthermore, we used Tpn(1-87) to generate a monoclonal antibody, alphaTpn(1-87)/80, specific for natural human Tpn and capable of cellular staining of ER localized Tpn. Using overlapping peptides, the epitope of alphaTpn(1-87)/80 was located to Tpn(40-44), which maps to a surface-exposed loop on the Tpn structure. Together, these results demonstrate that the N-terminal region of Tpn can be recombinantly expressed and adopt a structure, which at least partially resembles that of WT Tpn, and that this region of Tpn features chaperone activity facilitating peptide binding of MHC-I.
Original language | English |
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Journal | European Journal of Immunology |
Volume | 39 |
Issue number | 10 |
Pages (from-to) | 2682-94 |
Number of pages | 13 |
ISSN | 0014-2980 |
DOIs | |
Publication status | Published - 2009 |
Bibliographical note
Keywords: Amino Acid Sequence; Amino Acid Substitution; Antibodies, Monoclonal; Antibody Specificity; Binding, Competitive; Chromatography, Affinity; Endoplasmic Reticulum; Epitope Mapping; Escherichia coli; Escherichia coli Proteins; HLA-A Antigens; Heat-Shock Proteins; Histocompatibility Antigens Class I; Humans; Immunoprecipitation; Membrane Transport Proteins; Peptides; Protein Array Analysis; Protein Binding; Protein Denaturation; Protein Folding; Recombinant Fusion Proteins; beta 2-Microglobulin
ID: 20294663