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