0 20 40 60 80 100 0 100 200 300 400 500 600 700 0 500 1000 1500 2000 2500 3000 %B mAU B A C 0 100 200 300 400 500 600 700 800 900 1000 0 25 50 75 100 125 150 175 mAU Anion Exchange Chromatography (AEX) Size Exclusion Chromatography (SEC) Volume [mL] Volume [mL] *4 1 23 1 *2 D AEX pool Fermentation EAX Peak *4 SEC Peak *2 SEC Peak 1 Marker No IPTG IPTG - 3 hrs Urea Dissolved s e l p m a S n o i t c a r F s e l p m a S n o i t c a r F SEC pool 66 kDa 45 kDa 36 kDa 29 kDa 24 kDa 20 kDa Tpn 1-87 construct: GrpE-Fxa-Tpn 1-87 construct: Length : M W : pI : 87 amino acids 9313.7 Da 8.0 Length : M W : pI : 288 amino acids 31563.1 Da 5.0 Start End 1 N C 87 Tpn 1-87 s-s 7 71 d n E t r a t S FXa IEGR 1 -5 -201 C N 87 Tpn 1-87 GrpE s-s 7 71 RVNKGTGVK - HLA-A*0201 RVNKGTGVK - HLA-A*0201-T134K RIYSHIAPY - HLA-A*0201-T134K RIYSHIAPY - HLA-A*0201 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 0 50 100 150 200 250 300 350 400 Ctr l Grp E GrpE- FXa- Tpn 1-87 Peptide [nM] % Folded MHC- I 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 0 50 100 150 200 250 300 350 400 Ctr l Grp E GrpE- FXa- Tpn 1-87 Peptide [nM] % Folded MHC- I 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 0 50 100 150 200 250 300 350 400 Ctr l Grp E GrpE- FXa- Tpn 1-87 Peptide [nM] % Folded MHC- I 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 0 50 100 150 200 250 300 350 400 Ctr l Grp E GrpE- FXa- Tpn 1-87 Peptide [nM] % Folded MHC-I A C αTpn 1-87 /4 αTpn 1-87 /19 αTpn 1-87 /44 αTpn 1-87 /80 anti-Crt Marker αGrpE/86 Load Marker RT W1 W3 E1 E2 E3 E4 E5 W2 Elution Wash IP Sample: LCL-721.221A2 αTpn 1-87 /4 IP Fractions WB Sample: WB Sample: WB Sample: LCL-721.221A2 LCL-721.220A2 GrpE-FXa-Tpn 1-87 GrpE-FXa-Tpn 1-87 72 kDa 56 kDa 43 kDa 56 kDa 43 kDa 56 kDa 43 kDa 72 kDa 56 kDa 43 kDa calreticulin wt-tapasin wt-tapasin wt-tapasin calreticulin IP mAb: αTpn 1-87 /80 IP mAb: LCL-721.221A2 LCL-721.220A2 DAPI ERp57 αTpn 1-87 /80 Merged B The First 87 Amino Acids of tapasin Facilitates Folding of Peptide : HLA-A*0201 Complexes Natively Folded Wild-type tapasin Has a Surface Exposed Loop Spanning Residues 40-44 C B A αTpn 1-87 /80: EPPPRPDLDPEL LDPELYLSVHDP LDPEL 0 10 20 30 40 50 60 70 80 90 100 110 120 Peptide Position in Tpn 1-87 Sequence PeptideChip Signal [AU ] 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 0 1000 2000 3000 4000 5000 6000 7000 Peptide-23310 [nM] LOCI CpS Abstract Findings 2 # n o i s u l c n o C 1 # n o i s u l c n o C Mice were immunized with GrpE-FXa-Tpn 1-87 and monoclonal antibodies were gen- erated. In Western blot, the antibodies recognized wild-type tapasin, and only in tapa- sin positive cells (panel a). Both the ERp57 and αTpn 1-87 /80 antibodies stained areas around the nucleus in the tapasin positive LCL-721.221A2 cells (panel b). Of all the monoclonal antibodies that could recognize wild-type tapasin in Western blot, only one, αTpn 1-87 /80, could affinity purify wild-type, natively folded tapasin (panel c). A peptide microarray, consisting of sliding truncations of 12-mer peptides sliding along the GrpE-FXa-Tpn 1-87 sequence with an overlap of 11 amino acids.,was incu- bated with αTpn 1-87 /80 monoclonal antibody, washed, stained with a Cy3 fluoro- chrome labeled secondary goat anti-mouse-IgG antiserum. The sliding truncations re- vealed a single strong signal peak centered on the sequence LDPEL corresponding to human tapasin residues 40-44 (panel a). This region was mapped (loop shown in yellow) onto the protein structure of tapasin (panel b). The αTpn 1-87 /80 interacting peptide (LDPELYLSVHD), was synthesized and could in- hibit the recognition of GrpE-FXa-Tpn 1-87 by αTpn 1-87 /80 in a biochemical assay. (panel c). The IC 50 value of this inhibition was 3 nM suggesting a very strong binding between αTpn 1-87 /80 and GrpE-FXa-Tpn 1-87 . In conclusion, αTpn 1-87 /80 recognizes a surface exposed, linear epitope consisting of RPDLDPELYLS with a LDPEL core sequence. Production of Recombinant Tpn 1-87 ; The First 87 Amino Acids of tapasin Tpn 1-87 Facilitates Folding of Peptide : HLA-A*0201 Complexes Wild-type tapasin Has a Surface Exposed Loop Spanning Residues 40-44 The Monoclonal Antibody, αTpn 1-87 /80, Recogizes Wild-Type tapasin The Outermost N-Terminal Region of tapasin Facilitates Folding of Major The Outermost N-Terminal Region of tapasin Facilitates Folding of Major Histocompatibility Complex Class I Histocompatibility Complex Class I Gustav Roder, Linda Geironson, Anna Darabi, Mikkel Harndahl, Claus Schafer-Nielsen, Karsten Skjødt, Søren Buus, Kajsa Paulsson Gustav Roder, Linda Geironson, Anna Darabi, Mikkel Harndahl, Claus Schafer-Nielsen, Karsten Skjødt, Søren Buus, Kajsa Paulsson Universities of Copenhagen, Lund, and Southern Denmark, and Schafer-N Universities of Copenhagen, Lund, and Southern Denmark, and Schafer-N Tapasin is an ER chaperone that 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 tapasin quality control and the criteria for being an optimal peptide are still unknown. Here, we have generated a recombinant fragment of human tapasin, Tpn 1-87 (representing the 87 N-terminal and ER-luminal amino acids of the mature tapasin protein), and found that Tpn 1-87 spe- cifically facilitates peptide dependent folding of HLA-A*0201. Furthermore, we used Tpn 1-87 to generate a monoclonal antibody, αTpn 1-87 /80,, and its epitope was located to tapasin 40-44, which maps to a surface-exposed loop on the tapasin structure. The GrpE-FXa-Tpn 1-87 fusion protein (panel a) was expressed in E. coli BL21(DE3). GrpE-FXa-Tpn 1-87 was purified by column chromatography in urea-containing buf- fers; first by anion exchange chromatography (panels b,d) and then by size exclusion chromatography (panels c,d). To examine whether GrpE-FXa-Tpn 1-87 affected folding of HLA-A*0201, folded pep- tide : HLA-A*0201 complexes were measured using a biochemical binding assay, with or without GrpE-FXa-Tpn 1-87 . Two HLA-A*0201 binding peptides were examined. GrpE-FXa-Tpn 1-87 increased the amount of folded HLA-A*0201, but no effect of GrpE on the folding of HLA-A*0201 was observed indicating that Tpn 1-87 is solely respon- sible for facilitating peptide binding. Furthermore, a tapasin insentive mutant HLA-I allele, HLA-A*0201-T134K, was also insensitive to Tpn 1-87 . We conclude that Tpn 1-87 is the active part of GrpE-FXa-Tpn 1-87 affecting the folding of HLA-A*0201 and suggest that the specificity of this interaction resembles that of wild-type, full-length tapasin. Peptide #1 HLA-A*0201 HLA-A*0201- T134K Peptide #2