A poster representing Paper-I from Gustav\'s PhD thesis

1. The Outermost N-Terminal Region of tapasin Facilitates Folding of Major
Histocompatibility Complex Class I
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
Abstract
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.
Findings
The First 87 Amino Acids of tapasin Facilitates Natively Folded Wild-type tapasin Has a Surface
Folding of Peptide : HLA-A*0201 Complexes Exposed Loop Spanning Residues 40-44
Production of Recombinant Tpn1-87; The First 87 Amino Acids of tapasin The Monoclonal Antibody, αTpn1-87/80, Recogizes Wild-Type tapasin
A A C
7 71
pn /19
pn 7 /44
0
s-s Length : 87 amino acids
pn 4
/8
αT 1-87 /
αT 1-87
87
N C MW : 9313.7 Da
rt
αT 1-8
r
1-
ke
C
Tpn1-87 construct:
pn
Tpn1-87
ti-
ar
pI : 8.0 IP Sample: LCL-721.221A2
αT
an
M
1 87
Start End
WB Sample: LCL-721.221A2 IP Fractions
FXa 72 kDa
7 71
IEGR s-s Length : 288 amino acids calreticulin
N C MW : 31563.1 Da Wash Elution
GrpE-Fxa-Tpn1-87 construct: GrpE Tpn1-87 56 kDa
r
ke
pI : 5.0
ad
ar
wt-tapasin
1
2
3
T
E1 E2 E3 E4 E5
Lo
-201 -5 1 87
W
W
W
M
R
Start End 43 kDa
IP mAb: αTpn1-87/4
B C 56 kDa
Anion Exchange Chromatography (AEX) Size Exclusion Chromatography (SEC) WB Sample: LCL-721.220A2
700 72 kDa wt-tapasin
100
600 *4 1000 1 *2 43 kDa
80 900 calreticulin
500 800 56 kDa
700
mAU
400 60 600 IP mAb: αTpn1-87/80
mAU
%B
500 43 kDa
300 1 2 3 40 400 56 kDa
200 300 wt-tapasin
20 200 WB Sample: GrpE-FXa-Tpn1-87
100
100 43 kDa
0 0 0 GrpE-FXa-Tpn1-87
0 500 1000 1500 2000 2500 3000 0 25 50 75 100 125 150 175
Volume [mL] Volume [mL]
D
Fermentation
d
EAX Peak *4 SEC Peak 1 SEC Peak *2 B
s lve DAPI ERp57 αTpn1-87/80 Merged αGrpE/86
hr o
TG - 3 iss
er
LCL-721.221A2
D
a rk o IP TG ea
M N IP Ur Fraction Samples Fraction Samples
66 kDa
45 kDa
LCL-721.220A2
36 kDa
29 kDa
24 kDa
20 kDa
AEX pool SEC pool
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-
The GrpE-FXa-Tpn1-87 fusion protein (panel a) was expressed in E. coli BL21(DE3). sin positive cells (panel a). Both the ERp57 and αTpn1-87/80 antibodies stained areas
GrpE-FXa-Tpn1-87 was purified by column chromatography in urea-containing buf- around the nucleus in the tapasin positive LCL-721.221A2 cells (panel b). Of all the
fers; first by anion exchange chromatography (panels b,d) and then by size exclusion monoclonal antibodies that could recognize wild-type tapasin in Western blot, only
chromatography (panels c,d). one, αTpn1-87/80, could affinity purify wild-type, natively folded tapasin (panel c).
Tpn1-87 Facilitates Folding of Peptide : HLA-A*0201 Complexes Wild-type tapasin Has a Surface Exposed Loop Spanning Residues 40-44
Peptide #1 Peptide #2
RVNKGTGVK - HLA-A*0201 RIYSHIAPY - HLA-A*0201 A B C
400 Ctrl 400 Ctrl 120
7000
% Folded MHC-I
% Folded MHC-I
350 350 110
GrpE GrpE
PeptideChip Signal [AU]
100 6000
300 GrpE- 300 GrpE- 90
5000
LOCI CpS
250 250
HLA-A*0201 200
FXa-
Tpn1-87 200
FXa-
Tpn1-87
80
70
4000
60
150 150 50 3000
100 100 40
30 2000
50 50
20 1000
0 -2 0 -2 -1 0 1 2 3 4 5 10
-1 0 1 2 3 4 5
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 0 0
Peptide [nM] 10-3 10-2 10-1 100 101 102 103
Peptide [nM] Peptide Position in Tpn1-87 Sequence
Peptide-23310 [nM]
αTpn1-87/80:
EPPPRPDLDPEL
LDPEL
RVNKGTGVK - HLA-A*0201-T134K RIYSHIAPY - HLA-A*0201-T134K LDPELYLSVHDP
400 400
% Folded MHC-I
Ctrl Ctrl
350
% Folded MHC-I
GrpE 350 GrpE
300 GrpE- 300 GrpE-
HLA-A*0201- 250 FXa- 250 FXa-
200 Tpn1-87
T134K 150
200
150
Tpn1-87
100 100
50 50
0 -2 -1 0 1 2 3 4 5 0 -2 -1 0 1 2 3 4 5
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
Peptide [nM] Peptide [nM] 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
To examine whether GrpE-FXa-Tpn1-87 affected folding of HLA-A*0201, folded pep- human tapasin residues 40-44 (panel a). This region was mapped (loop shown in
tide : HLA-A*0201 complexes were measured using a biochemical binding assay, with yellow) onto the protein structure of tapasin (panel b).
or without GrpE-FXa-Tpn1-87. Two HLA-A*0201 binding peptides were examined. The αTpn1-87/80 interacting peptide (LDPELYLSVHD), was synthesized and could in-
GrpE-FXa-Tpn1-87 increased the amount of folded HLA-A*0201, but no effect of GrpE hibit the recognition of GrpE-FXa-Tpn1-87 by αTpn1-87/80 in a biochemical assay.
on the folding of HLA-A*0201 was observed indicating that Tpn1-87 is solely respon- (panel c). The IC50 value of this inhibition was 3 nM suggesting a very strong binding
sible for facilitating peptide binding. Furthermore, a tapasin insentive mutant HLA-I between αTpn1-87/80 and GrpE-FXa-Tpn1-87.
allele, HLA-A*0201-T134K, was also insensitive to Tpn1-87.
Conclusion #1 Conclusion #2
We conclude that Tpn1-87 is the active part of GrpE-FXa-Tpn1-87 affecting the folding In conclusion, αTpn1-87/80 recognizes a surface exposed, linear epitope consisting of
of HLA-A*0201 and suggest that the specificity of this interaction resembles that of RPDLDPELYLS with a LDPEL core sequence.
wild-type, full-length tapasin.