The outermost N-terminal region of tapasin facilitates folding of major histocompatibility complex class I

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The outermost N-terminal region of tapasin facilitates folding of major histocompatibility complex class I. / Roder, Gustav; Geironson Ulfsson, Linda; Darabi, Anna; Harndahl, Mikkel; Schafer-Nielsen, Claus; Skjodt, Karsten; Buus, Soren; Paulsson, Kajsa M.

In: European Journal of Immunology, Vol. 39, No. 10, 2009, p. 2682-2694.

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Roder, Gustav ; Geironson Ulfsson, Linda ; Darabi, Anna ; Harndahl, Mikkel ; Schafer-Nielsen, Claus ; Skjodt, Karsten ; Buus, Soren ; Paulsson, Kajsa M. / The outermost N-terminal region of tapasin facilitates folding of major histocompatibility complex class I. In: European Journal of Immunology. 2009 ; Vol. 39, No. 10. pp. 2682-2694.

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TY - JOUR

T1 - The outermost N-terminal region of tapasin facilitates folding of major histocompatibility complex class I

AU - Roder, Gustav

AU - Geironson Ulfsson, Linda

AU - Darabi, Anna

AU - Harndahl, Mikkel

AU - Schafer-Nielsen, Claus

AU - Skjodt, Karsten

AU - Buus, Soren

AU - Paulsson, Kajsa M

PY - 2009

Y1 - 2009

N2 - 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, alpha Tpn(1-87/80), specific for natural human Tpn and capable of cellular staining of ER localized Tpn. Using overlapping peptides, the epitope of alpha Tpn(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.

AB - 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, alpha Tpn(1-87/80), specific for natural human Tpn and capable of cellular staining of ER localized Tpn. Using overlapping peptides, the epitope of alpha Tpn(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.

KW - Antibodies

KW - Antigen processing

KW - MHC-I

U2 - 10.1002/eji.200939364

DO - 10.1002/eji.200939364

M3 - Article

VL - 39

SP - 2682

EP - 2694

JO - European Journal of Immunology

JF - European Journal of Immunology

SN - 1521-4141

IS - 10

ER -