0

20

40

60

80

100

0100200300400500600700

0 500 1000 1500 2000 2500 3000

%B

mAU

B

A

C

0100200300400500600700800900

1000

0 25 50 75 100 125 150 175

mAU

Anion Exchange Chromatography (AEX) Size Exclusion Chromatography (SEC)

Volume [mL] Volume [mL]

*4

1 2 3

1 *2

D

AEX pool

Fermentation EAX Peak *4 SEC Peak *2SEC Peak 1

Marker

No IPTG

IPTG - 3 hr

s

Urea D

issolv

ed

selpmaSnoitcarFselpmaSnoitcarF

SEC pool

66 kDa

45 kDa36 kDa

29 kDa24 kDa

20 kDa

Tpn1-87 construct:

GrpE-Fxa-Tpn1-87 construct:

Length :MW :

pI :

87 amino acids9313.7 Da8.0

Length :MW :

pI :

288 amino acids31563.1 Da5.0

Start End1

N C

87Tpn1-87

s-s7 71

dnEtratS

FXaIEGR

1-5-201

CN

87Tpn1-87GrpE

s-s7 71

RVNKGTGVK - HLA-A*0201

RVNKGTGVK - HLA-A*0201-T134K RIYSHIAPY - HLA-A*0201-T134K

RIYSHIAPY - HLA-A*0201

10-2

10-1

100

101

102

103

104

1050

50100150200250300350400 Ctrl

GrpEGrpE-FXa-Tpn1-87

Peptide [nM]

% F

olde

d M

HC-I

10-2

10-1

100

101

102

103

104

1050

50100150200250300350400 Ctrl

GrpEGrpE-FXa-Tpn1-87

Peptide [nM]

% F

olde

d M

HC-I

10-2

10-1

100

101

102

103

104

1050

50100150200250300350400 Ctrl

GrpEGrpE-FXa-Tpn1-87

Peptide [nM]

% F

olde

d M

HC-I

10-2

10-1

100

101

102

103

104

1050

50100150200250300350400 Ctrl

GrpEGrpE-FXa-Tpn1-87

Peptide [nM]

% F

olde

d M

HC-I

A C

αTpn 1-8

7/4

αTpn 1-8

7/19

αTpn 1-8

7/44

αTpn 1-8

7/80

anti-C

rt

Marker

αGrpE/86

Load

Marker

RT W1 W3 E1 E2 E3 E4 E5W2

ElutionWash

IP Sample: LCL-721.221A2

αTpn1-87/4

IP FractionsWB Sample:

WB Sample:

WB Sample:

LCL-721.221A2

LCL-721.220A2

GrpE-FXa-Tpn1-87

GrpE-FXa-Tpn1-87

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:

αTpn1-87/80IP mAb:

LCL-

721.

221A

2LC

L-72

1.22

0A2

DAPI ERp57 αTpn1-87/80 MergedB

The First 87 Amino Acids of tapasin FacilitatesFolding of Peptide : HLA-A*0201 Complexes

Natively Folded Wild-type tapasin Has a SurfaceExposed Loop Spanning Residues 40-44

CBA

αTpn1-87/80:EPPPRPDLDPEL

LDPELYLSVHDP LDPEL

0102030405060708090

100110120

Peptide Position in Tpn1-87 Sequence

Pept

ideC

hip

Sign

al [A

U]

10-3 10-2 10-1 100 101 102 1030

1000

2000

3000

4000

5000

6000

7000

Peptide-23310 [nM]

LOCI

CpS

Abstract

Findings

2#noisulcnoC1#noisulcnoC

Mice were immunized with GrpE-FXa-Tpn1-87 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 αTpn1-87/80 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, αTpn1-87/80, 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-Tpn1-87 sequence with an overlap of 11 amino acids.,was incu-bated with αTpn1-87/80 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 αTpn1-87/80 interacting peptide (LDPELYLSVHD), was synthesized and could in-hibit the recognition of GrpE-FXa-Tpn1-87 by αTpn1-87/80 in a biochemical assay. (panel c). The IC50 value of this inhibition was 3 nM suggesting a very strong binding between αTpn1-87/80 and GrpE-FXa-Tpn1-87.

In conclusion, αTpn1-87/80 recognizes a surface exposed, linear epitope consisting of RPDLDPELYLS with a LDPEL core sequence.

Production of Recombinant Tpn1-87; The First 87 Amino Acids of tapasin

Tpn1-87 Facilitates Folding of Peptide : HLA-A*0201 Complexes Wild-type tapasin Has a Surface Exposed Loop Spanning Residues 40-44

The Monoclonal Antibody, αTpn1-87/80, Recogizes Wild-Type tapasin

The Outermost N-Terminal Region of tapasin Facilitates Folding of MajorThe Outermost N-Terminal Region of tapasin Facilitates Folding of MajorHistocompatibility Complex Class IHistocompatibility Complex Class IGustav Roder, Linda Geironson, Anna Darabi, Mikkel Harndahl, Claus Schafer-Nielsen, Karsten Skjødt, Søren Buus, Kajsa PaulssonGustav Roder, Linda Geironson, Anna Darabi, Mikkel Harndahl, Claus Schafer-Nielsen, Karsten Skjødt, Søren Buus, Kajsa PaulssonUniversities of Copenhagen, Lund, and Southern Denmark, and Schafer-NUniversities 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, Tpn1-87 (representing the 87 N-terminal and ER-luminal amino acids of the mature tapasin protein), and found that Tpn1-87 spe-

cifically facilitates peptide dependent folding of HLA-A*0201. Furthermore, we used Tpn1-87 to generate a monoclonal antibody, αTpn1-87/80,, and its epitope was located to tapasin 40-44, which

maps to a surface-exposed loop on the tapasin structure.

The GrpE-FXa-Tpn1-87 fusion protein (panel a) was expressed in E. coli BL21(DE3). GrpE-FXa-Tpn1-87 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-Tpn1-87 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-Tpn1-87. Two HLA-A*0201 binding peptides were examined.GrpE-FXa-Tpn1-87 increased the amount of folded HLA-A*0201, but no effect of GrpE on the folding of HLA-A*0201 was observed indicating that Tpn1-87 is solely respon-sible for facilitating peptide binding. Furthermore, a tapasin insentive mutant HLA-I allele, HLA-A*0201-T134K, was also insensitive to Tpn1-87.

We conclude that Tpn1-87 is the active part of GrpE-FXa-Tpn1-87 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