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Class I pathway
Prediction of proteasomal cleavage and
TAP binidng
Can Keşmir,
TBB, Utrecht University, NL &
CBS, BioCentrum, DTU
Outline
• MHC class I epitopes
– Antigen processing
• Proteasome
– Specificity and Polymorphism
– Prediction methods
• TAP
– Binding motif
• Evolution
• Immune escape
Peptide generation in the class I pathway
Proteasomal cleavage
• ~20%
of all peptide bonds are cleaved
• Average peptide length 8-9 amino acids
• Not all peptide bonds are equally likely
cleaved
• Cleavage more likely after
hydrophobic than after hydrophilic
amino acids
Proteasome specificity
• Low polymorphism
– Constitutive & Immunoproteasome
• Evolutionary conserved
• Stochastic and low
specificity
– Only 70-80% of the
cleavage sites are
reproduced in repeated
experiments
Proteasome evolution (b1 unit)
Human (Hs) - Human
Drosophila (Dm) - Fly
Bos Taurus (Bota) - Cow
Oncorhynchus mykiss (Om) - Fish
…
Constitutive
Immuno- and Constitutive proteasome
specificity
Immuno
Constitutive
P1 P1’
...LVGPTPVNIIGRNMLTQL..
Predicting proteasomal cleavage
• NetChop
– Neural network based method
• PaProc
– Partially non-linear method (a neural network
without hidden neurons????)
• SMM (stabilized matrix method)
• FragPredict
– Based on a statistical analysis of cleavagedetermining amino acid motifs present around
the scissile bond (i.e. also weight matrix like)
NetChop20S-3.0
In vitro digest data from the constitutive proteasome
Toes et al., J.exp.med. 2001
NetChop 3.0 Cterm (MHC ligands)
• NetChop-3.0 C-term
– Trained on class I
epitopes
– Most epitopes are
generated by the
immunoproteasome
– Predicts the
processing specificity
LDFVRFMGVMSSCNNPA
LVQEKYLEYRQVPDSDP
RTQDENPVVHFFKNIVT
TPLIPLTIFVGENTGVP
LVPVEPDKVEEATEGEN
YMLDLQPETTDLYCYEQ
PVESMETTMRSPVFTDN
ISEYRHYCYSLYGTTLE
AAVDAGMAMAGQSPVLR
QPKKVKRRLFETRELTD
LGEFYNQMMVKAGLNDD
GYGGRASDYKSAHKGLK
KTKDIVNGLRSVQTFAD
LVGFLLLKYRAREPVTK
SVDPKNYPKKKMEKRFV
SSSSTPLLYPSLALPAP
FLYGALLLAEGFYTTGA
Prediction performance
TP
Sens 
AP
TN
Spec 
AN
TP  TN  FN  FP
CC 
PP  AN  AP  PN
FP
TP
AP
AN
Sens
Aroc=0.8
Aroc=0.5
1 - spec
Predicting proteasomal cleavage
NetChop-3.0
Sens
Spec
CC PCC Aroc
NetChop20S--3.0
CC
1
0.8
0.6
0.4
0.2
0
-0.2
-0.4
Performance
Performance
1
0.5
0
CC
PCC
Aroc
PAProCI
0.12
0.1
Netchop20S
0.41
NetChop20S-3.0
0.48
0.13
0.56
0.48
0.82
0.55
0.85
tC
Ne
op
3.0
2.0
p
ho
tch
Ne
CI
o
Pr
PA
ct
di
re
gP
a
Fr
• Relative
FragPredict
poor predictive performance
–For MHC prediction CC~0.92 and AUC~0.95
Proteasome specificity
What does TAP do?
TAP affinity prediction
• Transporter Associated
with antigen Processing
• Binds peptides 9-18 long
• Binding determined mostly
by N1-3 and C terminal
amino acids
TAP binding motif matrix
A low matrix entry corresponds to an amino acid
well suited for TAP binding
Peters et el., 2003. JI, 171: 1741.
Predicting TAP affinity
9 meric peptides
>9 meric
ILRGTSFVYV
-0.11 + 0.09 - 0.42 - 0.3 = -0.74
Peters et el., 2003. JI, 171: 1741.
Proteasome, TAP and MHC co-evolution
• Antigen processing and
presentation is highly
ineffective
• Only 1 in 200 peptides will
bind a given MHC complex
• If proteasome and TAP do
not effectively produce
MHC restricted peptides,
antigen processing would be
a severe bottleneck for
antigen recognition
Co-evolution of Proteasome, TAP and MHC
• CP-P1: Constitutive
proteasome specificity at
P1 position
• TAP-9: TAP motif at P9
position
• MHC-9: Average MHC
motif at P9
Co-evolution of Proteasome, TAP and MHC
• IP-P1: Immuno proteasome
specificity at P1 position
• CP-P1: Constitutive
proteasome specificity at P1
position
• TAP-9: TAP motif at P9
position
• MHC-9: Average MHC motif
at P9
Co-evolution (continued)
Kesmir et al. Immunogenetics, 2003, 55:437
What is going on at the N terminal?
Epitope identification
TAP precursor
A2 Epitope FLDGNEMTL
FLDGNEMTL 2.0100
KFLDGNEMTL -2.5300
RKFLDGNEMTL -3.7400
TRKFLDGNEMTL -2.4400
Proteasomal cleavage
STRKFLDGNEMTL...
0.0101 0.6483 0.9955 0.9984 0.4299 0.2261 0.0103 0.0265 0.0099 0.0099 0.9590 0.4670 0.9989
N terminal trimming
>50% need 2-3 amino acids N
terminal trimming
TAP and proteasome independent
presentation
• CTL epitopes are presented
• Other important players
at the cell surface on TAP
in the class I pathway
deficient cell lines
– Signal peptides
•Some CTL epitopes have
– Sec61
very poor TAP binding
affinity
– Diffusion
• Dominant CTL epitopes can
– Proteases
have very poor C terminal
cleavage signal
• Many CTL epitope have
strong internal cleavage sites
Immune escape
• Pathogens evolve under strong selection
pressure to avoid CTL recognition
• Generate point mutations or
insertions/deletions to disturb
– Peptide binding to MHC
– CTL recognition
• Only involve the antigentic peptide region
– Antigen processing
• Can involve peptide flanking region
Immune escape via antigen processing
HIV-1 Nef epitope VPLRPMTY (Milicic et al. JI, 2005, 4618)
IP
IP
CP
Summary
• The most important players (MHC, TAP and proteasome) in the
MHC class I pathway have co evolved to a share a common C
terminal pathway specificity
• We can predict (up to a degree) proteasomal cleavage
• TAP binding motif characterized in a weight matrix
– Binding mostly determined by the N1-3 and C terminal amino acids
• Proteasome produces and TAP transports precursor T cell epitopes
of length 8-13 amino acids
• Epitope trimming in the ER by several amino peptidases (ERAP)
• We still do not understand everything
– Many more important players are involved in the class I path way