Page 188 - Haematologica June

J. Hrdinová et al.
Table 3. Evaluation of ADAMTS13 peptide immunogenicity in silico.
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Binding of peptides to nine common HLA-DR allele families was predicted using the EpiMatrix algorithm developed by EpiVax.An EpiMatrix Score that reflects peptide binding potential to each of the 9 HLA-DR alleles was assigned to each peptide. Peptides with EpiMatrix scores > 5 are predicted to have elevated immunogenic potential; peptides with scores > 10 are predicted to have significant immunogenic potential.In addition,JanusMatrix was used to predict potential cross-reactivity between the peptides and the human proteome, based on conservation of TCR-facing residues. A JanusMatrix Homology Score was assigned to each peptide. Higher scores indicate greater conservation with the human proteome.At the population level,given elevated to high EpiMatrix scores,peptides with low cross-conservation with human proteome (scores < 3) are considered likely to be immunogenic, while peptides with scores > 3 have elevated to high cross-conservation with human proteome and are thus potentially tolerated or tolerogenic.
alleles HLA-DRB1*11 and DQB1*03 in the general popu- lation represents an interesting question that needs further study.
The overall low number of ADAMTS13-derived pep- tides reported may also be due to post-translational mod- ifications of HLA-DR/DQ-presented peptides that inter- fere with their identification. For instance, the presence of a glycan results in a shift in the net mass of the glycan- bearing peptide, impairing its identification by mass spec- trometry. Ten N-glycosylation sites, 8 O-fucosylation sites and 3 C-mannosylation sites have been described for plas- ma-derived ADAMTS13.39,40 As yet, the impact of post- translational modifications such as N-linked glycosylation on MHC-II peptide presentation has not been extensively studied. To our surprise, we also identified a peptide derived from the TSP2 domain of ADAMTS13 (NYSCLDQAR) that has been reported to contain an N- linked glycosylation site.39 A recent publication from our group showed that the asparagine present within this pep- tide contains a bi-antennary complex-glycan.40 As men- tioned earlier, the presence of a glycan interferes with the identification of the glycan-bearing peptide by mass spec- trometry. However, it was previously described that the third position in peptides with consensus sequence Asn-
X-Ser/Thr affects glycosylation efficiency, Thr being asso- ciated with a higher degree of glycosylation compared to Ser.41 It is, therefore, likely that the identified peptide NYSCLDQAR is partially glycosylated, allowing for the identification of the non-glycosylated version in our study. Whether the glycosylated counterpart of this peptide is also presented on HLA molecules remains to be deter- mined. How the presence or absence of an N-linked gly- can at this position modulates peptide/MHC-II/T-cell receptor (TCR) binding represents an interesting question. On one hand, the presence of an N-linked glycan at this position could interfere with the binding of the peptides to HLA-DQ or with the recognition of the HLA-DQ/pep- tide complex by a complementary T-cell receptor (TCR). Interestingly, absence of an O-linked glycan on type II col- lagen has been previously suggested to create a novel T- cell epitope that has been implicated in the development of autoimmune arthritis.42-44 Similarly, CD4+ T cells recog- nizing non-glycosylated forms of ADAMTS13-derived peptides that normally contain a glycan may contribute to the onset of autoimmune TTP. On the other hand, the presence of a glycan can also lead to the formation of a neo-epitope.45 In view of the large number of N-and O- linked glycans on ADAMTS13, it cannot be excluded that
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