The outermost N-terminal region of tapasin facilitates folding of major histocompatibility complex class I.
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.
Roder
Gustav
G
Department of Experimental Immunology, Institute of International Health, Immunology and Microbiology, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark. groder@sund.ku.dk
Geironson
Linda
L
Darabi
Anna
A
Harndahl
Mikkel
M
Schafer-Nielsen
Claus
C
Skjødt
Karsten
K
Buus
Søren
S
Paulsson
Kajsa
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology; D002846:Chromatography, Affinity; D004721:Endoplasmic Reticulum / Q000378:metabolism; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D015234:HLA-A Antigens / Q000378:metabolism; D015789:HLA-A2 Antigen; D006360:Heat-Shock Proteins / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D026901:Membrane Transport Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D040081:Protein Array Analysis; D011485:Protein Binding / Q000276:immunology; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D029968:Escherichia coli Proteins; C489400:GrpE protein, E coli; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D006360:Heat-Shock Proteins; D015395:Histocompatibility Antigens Class I; D026901:Membrane Transport Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin; C107762:tapasin