Class I pathway Prediction of proteasomal cleavage and TAP binidng
description
Transcript of Class I pathway Prediction of proteasomal cleavage and TAP binidng
Class I pathwayPrediction of proteasomal cleavage and
TAP binidng
Morten Nielsen,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 & Immuno-
proteasome
• Evolutionary conserved• Stochastic and low specificity
– Only 70-80% of the cleavage sites are reproduced in repeated experiments
Proteasome evolution (1 unit)
Constitutive
Immuno
Human (Hs) - HumanDrosophila (Dm) - Fly
Bos Taurus (Bota) - CowOncorhynchus mykiss (Om) - Fish
…
Immuno- and Constitutive proteasome specificity
...LVGPTPVNIIGRNMLTQL..
P1 P1’
Immuno Constitutive
• NetChop– Neural network based method
• PaProc– Weight matrix based method
• FragPredict– Based on a statistical analysis of cleavage-
determining amino acid motifs present around the scissile bond• i.e. also weight matrix like
Predicting proteasomal cleavage
NetChop 3.0 Cterm (MHC ligands)
LDFVRFMGVMSSCNNPA LVQEKYLEYRQVPDSDP RTQDENPVVHFFKNIVT TPLIPLTIFVGENTGVP LVPVEPDKVEEATEGEN YMLDLQPETTDLYCYEQ PVESMETTMRSPVFTDN ISEYRHYCYSLYGTTLE AAVDAGMAMAGQSPVLR QPKKVKRRLFETRELTD LGEFYNQMMVKAGLNDD GYGGRASDYKSAHKGLK KTKDIVNGLRSVQTFAD LVGFLLLKYRAREPVTK SVDPKNYPKKKMEKRFV SSSSTPLLYPSLALPAP FLYGALLLAEGFYTTGA
• NetChop-3.0 C-term– Trained on class I
epitopes– Most epitopes are
generated by the immuno proteasome
– Predicts the immuno proteasome specificity
NetChop20S-3.0In vitro digest data from the constitutive proteasome
Toes et al., J.exp.med. 2001
Prediction performance
€
Sens =TP
AP
Spec =TN
AN
CC =TP ⋅TN − FN ⋅FPPP ⋅AN ⋅AP ⋅PN
TPFP
APAN
Aroc=0.5
Aroc=0.8
1 - spec
Sen
s
Predicting proteasomal cleavage
-0.4-0.2
00.20.40.60.8
1
Performance
FragPredictPAProCI Netchop2.0NetChop3.0
Sens Spec CC
0
0.5
1
Performance
CC PCC Aroc
CC 0.12 0.1 0.41 0.48
PCC 0.13 0.48 0.55
Aroc 0.56 0.82 0.85
FragPredict PAProCI Netchop20S NetChop20S-3.0
NetChop20S-3.0
NetChop-3.0
• Relative poor predictive performance–For MHC prediction CC~0.92 and AUC~0.95
Proteasome specificity
• NetChop is the best available cleavage method– www.cbs.dtu.dk/services/NetChop-3.0
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
Peters et el., 2003. JI, 171: 1741.
A low matrix entry corresponds to an amino acid well suited for TAP binding
TAP affinity prediction
Predicting TAP affinity
9 meric peptides >9 meric
Peters et el., 2003. JI, 171: 1741.
ILRGTSFVYV-0.11 + 0.09 - 0.42 - 0.3 = -0.74
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
More evolution
Constitutive proteasome!!!
What is going on at the N terminal?
S T R K F L D G N E M T L . . .
Epitope identification
TAP precursor A2 Epitope FLDGNEMTL
FLDGNEMTL 2.0100 KFLDGNEMTL -2.5300
RKFLDGNEMTL -3.7400 TRKFLDGNEMTL -2.4400
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
Proteasomal cleavage
N terminal trimming
>50% need 2-3 amino acids N terminal trimming
TAP and proteasome independent presentation
• CTL epitopes are presented at the cell surface on TAP deficient cell lines•Some CTL epitopes have very poor TAP binding affinity• Dominate CTL epitopes can have very poor C terminal cleavage signal • Many CTL epitope have strong internal cleavage sites
• Other important players in the class I pathway
– Signal peptides– Sec61– Diffusion– Proteases
• Mette will tell you more
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
189 S N 0.38 SSWDFITV190 S S 0.59 SSWDFITV191 W S 0.92 SSWDFITV192 D N 0.23 SSWDFITV193 F S 0.87 SSWDFITV194 I S 0.84 SSWDFITV195 T N 0.27 SSWDFITV196 V S 0.96 SSWDFITV197 N S 0.82 SSWDFITV
189 S N 0.38 SSWDFITV 190 S S 0.59 SSWDFITV 191 W S 0.92 SSWDFITV 192 D N 0.23 SSWDFITV 193 F S 0.87 SSWDFITV 194 I S 0.83 SSWDFITV 195 T N 0.13 SSWDFITV 196 V S 0.92 SSWDFITV 197 D S 0.97 SSWDFITV
Moloney murine leukemia virus (MuLV) epitope SSWDFITV
Processed right and recognized by CTL
Processed as SSWDFITVD and has wrong C terminal for MHC binding, not recognized by CTL
Immune escape
Proteasome-mediated digestion analysis of a synthetic 26-mer peptide derived from the Friend sequence shows that cleavage takes place predominantly C-terminal of D, instead of V as is the case for the Moloney MuLV sequence. Therefore, the C terminus of the epitope is not properly generated. Epitope-containing peptide fragments extended with an additional C-terminal D are not efficiently translocated by TAP and do not show significant binding affinity to MHC class I-Kb molecules..
Beekmanet al., JI 2000
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
• Proteasomal cleavage prediction tools exist– NetChop3.0 and NetChop20S-3.0 are among the best
• 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 9-13 amino acids
• Epitope trimming in the ER by several amino peptidases (ERAP)
• We still do not understand every thing– Many more important players are involved in the class I path way