Guglielmo Lucchese^1,2, Animesh Amart Sinha³, Darja Kanduc¹

1Department of Biochemistry and Molecular Biology, University of Bari, Italy, of Human Medicine, Michigan State University, Eeast Lansing, Michigan, USA

TABLE OF CONTENTS

1. Abstract

2. Introduction

3. Methods

4. Results

4.1. Single amino acid changes, proteomic similarity, and immune recognition

4.2. Single amino acid changes, proteomic similarity, and immunogenicity/pathogenicity

4.3. Single amino acid changes, proteomic similarity, and therapeutic peptides

5. Discussion

6. Contributions

7. References

1. ABSTRACT

What types of amino acid substitutions are functionally tolerated in an epitope? This question is of importance because the immunogenicity, pathogenicity, and therapeutic potential of a peptide can be determined by a single amino acid change. As an example, a single amino acid change in the encephalitogenic myelin oligodendrocyte glycoprotein WYRSPFSRVV peptide confers the capacity to ameliorate and reverse experimental autoimmune encephalomyelitis. Currently, no rule is available to predict/explain the functional outcomes of amino acid changes. To address this issue, we examined the role of single amino acid changes in immune responses by applying proteomic similarity analyses to available data. We found that the loss or gain of immunological information in a peptide epitope following an amino acid substitution often is related to a gain or loss in the proteomic similarity. Rare, but significant epitopic sequences become immunologically insignificant when an amino acid change makes them common, repeated sequences. This study confirms that low similarity to the host proteome is a major factor in modulating the immune epitope repertoire.

2. INTRODUCTION

The functional importance of even a single amino acid (aa) substitution is evident at both the physiological and pathological levels. For example:

- Single aa changes confer constitutive activation of mTOR, a serine/threonine kinase that regulates a variety of cellular functions such as growth, proliferation, and autophagy and is also overactivated in cancer cells (1).

- One aa change (Gly²⁶⁹Ser) in the S5 segment of the voltage-gated cardiac ion channel, KvLQT1, leads to long QT syndrome, which is an arrhythmic disorder (2).

- Cell adhesion and motility in living cells is altered by a single aa change in E-cadherin (3).

- In humans, one variant of the catestatin peptide, ³⁵²SSMKLSFRARGYS³⁶⁴FRGPGPQL³⁷² with Gly³⁶⁴Ser, profoundly alters autonomic activity in both the parasympathetic and sympathetic branches of the nervous system and protects against the future development of hypertension (4); and

- a single aa change (Lys¹⁴¹Asn) can allow a chimeric Theiler's virus to persist and demyelinate (5).

Most importantly, the degree of freedom for aa substitutions is the lowest in immunology. In general, a single aa substitution may have profound effects on immune responses and recognition. For example:

- A synthetic peptide containing a single aa change (Leu >His) was more than l000-fold more stimulatory to T cells than the wild-type peptide (6).

- A Ile-to-Phe substitution in the SCILLYIVI nonapeptide defines the antigenic polymorphism and T-cell specificity of the murine H47 locus (7).

- Myelin oligodendrocyte glycoprotein (MOG) is a myelin target antigen, and MOG peptides have been shown to have encephalitogenic activity. Strikingly, an Ala substitution at position 44 in the MOG_39-48WYRSPFSRVV peptide almost totally abrogates (96% reduction) the capacity to stimulate encephalitogenic line T cells (8); and

- a Lys¹⁴⁹Arg mutation in the antigenic site of hemagglutinin can alter the recognition of H9N2 influenza virus by a monoclonal antibody (MAb) (9).

However, when aa changes are analyzed, the specific link between aa substitutions and altered immuno-biological function(s) remains elusive. For example:

- Presenilin-1 is a subunit of the gamma-secretase complex, and, when altered, is involved in Alzheimer disease type 3. It has been reported that an aa substitution of Glu for Asp at position 385 abolishes gamma-secretase activity. Likewise, a change in the hydrophobic, bulky, aromatic acid (Tyr) to another hydrophobic, bulky, aromatic acid (Phe) at position 256 alters gamma-secretase cleavage specificity (10, 11). In contrast, changing the negatively charged Asp to the positively charged Asn at position 373 has no effect on caspase cleavage (11). In summary, it seems that maintaining the acid (or hydrophobic) features does not protect the functional characteristics, whereas changing from an acidic to a basic residue does not affect the function.

- Even changes directed towards the identical aa may affect function. Golgi coiled-coil protein GCC185 (also called renal carcinoma antigen NY-REN-53) was characterized following mutagenesis of Leu/Tyr to Ala with two opposite outcomes. Specifically, an aa substitution of Ala for Leu at position 1494 (Leu¹⁴⁹⁴Ala) decreases the RAB6A binding affinity two-fold, strongly decreases the RAB9 binding affinity, and abolishes Golgi localization. However, an aa substitution of Ala for Tyr at position 1517 (Tyr¹⁵¹⁷Ala) has no effect on RAB6A or RAB9 binding (12).

- Tuberin aa Arg⁶¹¹Gln, Arg⁶¹¹Trp, Ala⁶¹⁴Asp, Phe⁶¹⁵Ser, Cys⁶⁹⁶Tyr, and Val⁷⁶⁹Glu substitutions inhibit the phosphorylation of tuberin by the Ser/Thr protein kinase B (PKB, also known as Akt) (13). Specifically, disparate substitutions - i.e., introducing basic, hydrophobic, acid, or polar residues - produce the same final outcome in inhibiting tuberin phosphorylation, a pathogenic reaction that disrupts tuberin-hamartin interactions.

- Further paradigmatic examples occur in the polymorphic epithelial mucin (also called episialin or breast carcinoma-associated antigen DF3), which is an antigen that is overexpressed in tumors and shows cell-adhesive properties and participates in cell signalling. Mutagenesis experiments involving the same aa change in five different mucin positions produced a range of functional outcomes. More specifically, changing Tyr to Phe i) has no effect on EGFR-mediated phosphorylation at aa positions 1191, 1203, and 1218 (14-16); ii) causes some reduction in EGFR-mediated phosphorylation at aa position 1209 (14); and iii) greatly reduces EGFR- and Src-mediated phosphorylation at aa position 1229 (14, 17, 18).

Likewise, physico-chemical parameters are not a stringent enough criterion to predict/evaluate the ability of aa changes to affect the immunogenic potential of a peptide sequence:

- The H13a and H13b alleles encode for the minor histocompatibility antigen (mHAgs), which are presented in the context of the H2D^b MHC class I molecule. These alleles are pathologically important because they evoke reciprocal T-cell responses in H13a and H13b congenic strains. The molecular basis of the reciprocal T-cell responses between the two mHAg alleles resides in a conservative single aa substitution (Val/Ile) (19-22).

- A single Leu residue at position 1858 in the helicase domain of the pestivirus NS3 protein is critical for the genesis of an epitope recognized by the MAb P1D8. Changing the aliphatic Leu to aliphatic Val (Leu¹⁸⁵⁸Val) or polar Ser (Leu¹⁸⁵⁸Ser) destroys the NS3 epitope (23). That is, the maintenance of aliphatic characteristics or the acquisition of a polar feature results in, but does not explain, the same event: epitope destruction.

The data cited above raise an important question; how can a single aa change cause a gain/loss in the immunological content of a peptide sequence? During the last decade, Kanduc (24-27) reported data suggesting that the level of similarity to the host proteome modulates the immunogenicity of a peptide sequence. Kanduc hypothesized that the immunological information contained in an amino acid sequence is directly related to its rarity (28). As an extension of these studies, the present investigation explores whether there may be a link between single aa substitutions, sequence-similarity changes, and altered antigenicity/immunogenicity of a peptide.

3. METHODS

Published studies on single aa substitutions in peptide sequences were obtained from PubMed (http://www.ncbi.nlm.nih.gov). Peptide sequences were analyzed for similarity changes using the Protein Information Resource perfect match program (www.pir.georgetown.edu) (29). The rabbit proteome was downloaded from www.ensembl.org. Peptides were dissected into pentapeptides shifted by one residue and then each pentapeptide was analyzed for exact matches in the host proteome. The pentapeptide was used as a basic unit because a grouping of five residues represents a sufficient minimal antigenic determinant of immune responses and recognition (24, 30-35). Pentapeptide similarity is defined by the number of exact pentameric matches in the host proteome (24, 36), and can range from zero to hundreds (37). Based on previous data (38-42), a pentapeptide fragment that has about five perfect matches to the host proteome can be considered a low-similarity pentapeptide, i.e., a rare fragment.

4. RESULTS

4.1. Single amino acid changes, proteomic similarity, and immune recognition

In studying the particular epitope amino acids conferring specificity for the reaction with an antibody, Getzoff et al. (43, 44) demonstrated that substituting all 20 amino acids at the Ile⁴ and Tyr⁸ positions within the antigenic myohemerythrin_4-9IPEPYV sequence exerts a differential effect on peptide antigenicity (Figure 1). Specifically, the authors found that Ile⁴ was replaceable (Figure 1, panel A), whereas Tyr⁸ was essential for immune reactions with rabbit antisera (Figure 1, panel B). Indeed, data from Panel A appear to indicate that the epitope actually corresponds to the pentapeptide PEPYV. We explored the sequence similarity of the homologous epitope sequences versus the rabbit proteome and found that aa changes that do not alter the low similarity level of the epitope (Fig 1. panel C) do not change its antigenicity potential (Figure 1, panel A). Conversely, with few exceptions, aa changes leading to an altered epitope similarity level (Figure 1, panel D) are associated with altered antigenicity potential (Figure 1, panel B).

The relationship between a single aa change, an antigenicity shift, and peptide rarity appears evident also when different residues in an epitope are substituted with an identical residue, as shown in Ala scanning experiments. Recently, Hjelm et al. (45) defined a sequence of 11 amino acids (RIERATGQRPH) as the core epitope of a MAb against human tryptophanyl-tRNA synthetase, and found that homologues derived from Ala substitutions in the epitopic sequence (RIEaATGQR, RIERAaGQR, and RIERATaQR, with Ala substitutions in lowercase) did not bind to the antibody. Our similarity analyses of the binding epitope RIERATGQR and its non-epitopic homologues (Figure 2) show that Ala substitution transforms the low-similarity central pentapeptides into high-similarity homologous sequences. That is, loss in sequence antigenicity appears to be accompanied by a gain in proteomic similarity.

Analogous data are reported in Figure 3, which illustrates the similarity profiles of the Denque virus ¹⁰¹WGNGCGLFG¹⁰⁹ epitope and its homologue ¹⁰¹WGNGgGLFG¹⁰⁹ sequence containing a Cys¹⁰⁵Gly change (given in lowercase), versus the murine proteome. It can be observed that a substitution of a C with a G transforms the low-similarity sequence (Figure 3, panel A) into a high-similarity sequence (Figure 3, panel B). Again, it is worth noting that Dengue reactive mAbs react more strongly with the flavivirus-conserved low-similarity ¹⁰¹WGNGCGLFG¹⁰⁹ fusion sequence than with the high-similarity homologue (46, 47).

A similar picture emerges from the analysis of a single aa substitution in the five core residues (⁷¹AGNVN⁷⁵) of the immunodominant mycobacterial epitope p61-80/PT19 (⁶¹VTGSVVCTTAAGNVNIAIGG⁸⁰) (45). An Asn⁷³Ala change abolishes T immune recognition of the target epitope and, concomitantly, changes the proteomic similarity level of the five core residues from one match (⁷¹AGNVN⁷⁵) to eight matches (⁷¹AGaVN⁷⁵, with the Asn⁷³Ala substitution given in lowercase) (32, 48).

Alexander et al. also reported data linking changes in similarity level to changes in immunological information (49). These authors found that peptides differing in only 1 of 19 amino acids (Q versus R) are able to induce allospecific antisera. Specifically, Thy1.2 peptide 88 (KDEGDYFCELQVSGANPMS) elicits rabbit antisera recognizing only the Thy1.2 allotype, whereas Thy1.1 peptide 90 (KDEGDYFCELrVSGANPMS, with the Q⁸⁹R change in lowercase) elicits rabbit antisera recognizing Thy1.2 and Thy1.1 allotypes (46, and further details therein). Of note, the conservative aa substitution (Q versus R) changes the pentapeptide CELQV (characterized by two matches in the rabbit proteome) into the pentapeptide CELrV (occurring six times in rabbit proteins). In other words, the loss in immunological allospecificity appears to be associated with a gain in proteomic similarity.

4.2. Single amino acid changes, proteomic similarity, and immunogenicity/ pathogenicity

In Lewis rats, the peptide ²⁷³TWEGSGVLPCV²⁸³ of the rat interphotoreceptor retinoid-binding protein (IRBP) causes experimental autoimmune uveoretinitis (EAU) at 200 nmol/rat (50). A homologous peptide, in which residues 277 and 282 (i.e., terminal sequence residues in which MHC binding potential is allocated), were substituted with V and D, respectively, drastically increased (i) MHC affinity; and (iii) pathogenicity, by causing disease even at 0.02 nmol/rat. In addition, Figure 4 shows that the pentapeptide similarity profiles of IRBP epitope ²⁷³TWEGSGVLPCV²⁸³ (Figure 4, panel A) and its homologue ²⁷³TWEGvGVLPdV²⁸³ (Figure 4, panel B, with substitutions given in lowercase) are different. Specifically, the Ser²⁷⁷Val substitution decreases the proteomic similarity level of the epitopic central pentapeptides (see arrows in Figure 4), in which the epitope immunogenicity lies (32, 51). Consistent with the data patterns discussed above, Figure 4 appears to indicate a link between the decreased similarity of the core pentapeptides and the increased peptide immunogenicity.

4.3. Single amino acid changes, proteomic similarity, and therapeutic peptides

No less importantly, single aa substitutions can generate immunotherapeutic peptide analogues. Caspi et al. (52) described two peptide analogues derived from a major uveitogenic epitope, ¹⁶¹SGIPYVISYLHPGNTVMHVD¹⁸⁰, from the murine retinal antigen IRBP, which were able to protect against antiretinal autoimmunity. The two inhibitory homologous peptides, namely 169A and 171A, differed from the pathogenic parent by single Ala substitutions at positions 169 and 171, respectively. In applying our similarity rationale, we expected an increase in the sequence-similarity score of the two protective peptides as compared to the pathogenic precursor. Figure 5 confirms this hypothesis and shows that Ala substitution at position 169 or 171 causes an increase in the proteomic similarity of the uveitogenic epitope sequence (Figure 5, panels B and C, respectively) as compared to the pathogenic peptide (Figure 5, panel A).

Likewise, a single amino substitution marking the transformation of the encephalitogenic MOG_39-48WYRSPFSRVV peptide into the non-encephalitogenic MOG_39-48WYRSPaSRVV peptide (8, 53) is accompanied by a change in the proteomic similarity level of the five core residues (aa 42-46) from 5 matches (SPFSR) to 32 matches (SPASR).

5. DISCUSSION

A fundamental aspect in the study of proteins is the understanding of the effect(s) that mutations have on protein function and/or structure. In immunology, this aspect is crucial. Indeed, the present data demonstrate that the degree of "immunological freedom" is restricted for a peptide sequence. As discussed in detail above, a single aa change can result in a wide spectrum of outcomes in an immunological context, such as reversing inhibition of host innate immune response pathways (54), destroying or improving protein immunogenicity (55, 56), and impairing T-cell activation (57).

Therefore, the essential question remains: how can a single aa residue dramatically alter the immunological properties of a peptide? In this regard, research has annotated many possible explanations. It has been proposed that the changed sequence might contain a determinant that serves to amplify a separate population of lymphocytes (58, 59). Accordingly, Th2 shifts in the immune response and induction of regulatory T cells by altered peptides have been suggested (52). Changes involving anchor residues have been postulated to cause an impact on binding affinity (60, 61). Although of interest, this suggestion does not reconcile the heavy degeneracy of MHC recognition (62-64). With respect to antigenicity, substituted peptides have been suggested to cause perturbation/enhancement of the stereochemical fit between the antigenic peptide and antibody molecules, hence indicating loss/gain of important chemical contributions to antibody interactions (43). However, this mechanism does not explain, for example, why a conservative substitution equates a non-conservative change in destroying a B cell epitope (23).

Here, we analyzed and illustrated data to determine whether a single aa change can modify the immunological information of a peptide module when is accompanied by a loss/gain of sequence similarity. As a matter of fact, a basic concept emerging from our analyses is that a change in the proteomic similarity of a mutated peptide may explain the change in its immunological information. Amino acid changes that transform rare peptide sequences into sequences that repeatedly occur in human proteins "dilute" the immune reaction, which becomes scattered and dispersed among numerous homologous epitopes instead of being concentrated towards one specific target. It appears that a single aa residue substitution can switch on or off the antigenicity/immunogenicity/pathogenicity of proteins and small peptide modules, provided that the aa substitution modifies the sequence similarity level. In essence, as previously advocated by Kanduc (24-28), an increase in the rarity level of a peptide appears to be associated with an increase in its immunological information content. In contrast, becoming a common motif hosted by numerous proteins causes a loss of immunological specificity. The relationship between an aa substitution and a similarity change offers a mathematical mechanism, based on the law of mass action, to explain the shift in immune reactivity. The specificity and strength of peptide-peptide interactions during immune responses do not depend only on the nature of the immune reactants (e.g., antigens, antibodies, T-cell receptors, MHC molecules), but also on the amount of each player in the immune reaction. Logically, a quantitatively/qualitatively high immune response evoked by a rare sequence loses efficacy when fragmented toward numerous homologous sequences (e.g., antibodies reacting with multiple epitopic homologues).

From a clinical point of view, the fine-tuning of peptide sequence similarity might represent a powerful tool for designing peptides with single aa changes for use in the treatment of specific autoimmune pathologies (e.g., down-regulating the extent of autoimmune attack) and cancer diseases (e.g., up-regulating the specificity of the attack against tumour cells) (24, 32, 34, 35, 65). Likewise, it might be useful in designing peptide-based approaches against infectious microbial organisms, which appear to escape immune surveillance because of their widespread similarity to the human proteins (31-34, 66-74).

6. CONTRIBUTIONS

GL, AAS and DK have equally contributed to data analyses and interpretation. DK conceived the study and wrote the manuscript. All authors discussed and approved the final manuscript.

7. REFERENCES

1. T Sato, A Nakashima, L Guo, K Coffman, F Tamanoi: Single amino-acid changes that confer constitutive activation of mTOR are discovered in human cancer. Oncogene 29, 2746-2752 (2010)
doi:10.1038/onc.2010.28

2. R Verma, JK Ghosh: Structural and functional changes in a synthetic S5 segment of KvLQT1 channel as a result of a conserved amino acid substitution that occurs in LQT1 syndrome of human. Biochim Biophys Acta 1798, 461-470 (2010)
doi:10.1016/j.bbamem.2009.12.014

3. M Fuchs, P Hutzler, G Handschuh, C Hermannstädter, I Brunner, H Höfler, B Luber: Dynamics of cell adhesion and motility in living cells is altered by a single amino acid change in E-cadherin fused to enhanced green fluorescent protein. Cell Motil Cytoskeleton 59, 50-61 (2004)
doi:10.1002/cm.20019

4. F Rao, G Wen, JR Gayen, M Das, SM Vaingankar, BK Rana, M Mahata, BP Kennedy, RM Salem, M Stridsberg, K Abel, DW Smith, E Eskin, NJ Schork, BA Hamilton, MG Ziegler, SK Mahata, DT O'Connor: Catecholamine release-inhibitory peptide catestatin (chromogranin A(352-372)): in human autonomic activity and alters risk for hypertension. Circulation 115, 2271-2281 (2007)
doi:10.1161/CIRCULATIONAHA.106.628859

5. N Jarousse, RA Grant, JM Hogle, L Zhang, A SenKowski, RP Roos, T Michiels, M Brahic, A McAllister: A single amino acid change determines persistence of a chimeric Theiler's virus. J Virol 68, 3364-3368 (1994)

6. PA Monach, SC Meredith, CT Siegel, H Schreiber: A unique tumor antigen produced by a single amino acid substitution. Immunity 2, 45-59 (1995)
doi:10.1016/1074-7613(95)90078-0

7. LM Mendoza, G Villaflor, P Eden, D Roopenian, N Shastri: Distinguishing self from nonself: immunogenicity of the murine H47 locus is determined by a single amino acid substitution in an unusual peptide. J Immunol 166, 4438-4445 (2001)

8. A Ben-Nun, NK de Rosbo, N Kaushansky, M Eisenstein, L Cohen, JF Kaye, I Mendel: Anatomy of T cell autoimmunity to myelin oligodendrocyte glycoprotein (MOG): prime role of MOG44F in selection and control of MOG-reactive T cells in H-2b mice. Eur J Immunol 36, 478-493 (2006)
doi:10.1002/eji.200535363

9. J Ping, C Li, G Deng, Y Jiang, G Tian, S Zhang, Z Bu, H Chen: Single-amino-acid mutation in the HA alters the recognition of H9N2 influenza virus by a monoclonal antibody. Biochem Biophys Res Commun 371, 168-171 (2008)
doi:10.1016/j.bbrc.2008.04.045

10. JD Wrigley, EJ Nunn, O Nyabi, EE Clarke, P Hunt, A Nadin, B De Strooper, MS Shearman, D Beher: Conserved residues within the putative active site of gamma-secretase differentially influence enzyme activity and inhibitor binding. J Neurochem 90, 1312-1320 (2004)
doi:10.1111/j.1471-4159.2004.02596.x

11. J Grunberg, J Walter, H Loetscher, U Deuschle, H Jacobsen, C Haass: Alzheimer's disease associated presenilin-1 holoprotein and its 18-20 kDa C-terminal fragment are death substrates for proteases of the caspase family. Biochemistry 37, 2263-2270 (1998)
doi:10.1021/bi972106l

12. AS Burguete, TD Fenn, AT Brunger, SR Pfeffer: Rab and Arl GTPase family members cooperate in the localization of the golgin GCC185. Cell 132, 286-298 (2008)
doi:10.1016/j.cell.2007.11.048

13. M Nellist, O Sancak, MA Goedbloed, C Rohe, D van Netten, K Mayer, A Tucker-Williams, AM van den Ouweland, DJ Halley: Distinct effects of single amino-acid changes to tuberin on the function of the tuberin-hamartin complex. Eur J Hum Genet 13, 59-68 (2005)
doi:10.1038/sj.ejhg.5201276

14. Y Li, J Ren, W Yu, Q Li, H Kuwahara, L Yin, KL 3rd Carraway, D Kufe: The epidermal growth factor receptor regulates interaction of the human DF3/MUC1 carcinoma antigen with c-Src and beta-catenin. J Biol Chem 276, 35239-35242 (2001)
doi:10.1074/jbc.C100359200

15. CL Kinlough, PA Poland, JB Bruns, KL Harkleroad, RP Hughey: MUC1 membrane trafficking is modulated by multiple interactions. J Biol Chem 279, 53071-53077 (2004)
doi:10.1074/jbc.M40936020016. PK Singh, Y Wen, BJ Swanson, K Shanmugam, A Kazlauskas, RL Cerny, SJ Gendler, MA Hollingsworth: Platelet-derived growth factor receptor beta-mediated phosphorylation of MUC1 enhances invasiveness in pancreatic adenocarcinoma cells. Cancer Res 67, 5201-5210 (2007)
doi:10.1158/0008-5472.CAN-06-4647

17. L Huang, J Ren, D Chen, Y Li, S Kharbanda, D Kufe: MUC1 cytoplasmic domain coactivates Wnt target gene transcription and confers transformation. Cancer Biol Ther 2, 702-706 (2003)
doi:10.4161/cbt.2.6.610

18. Y Li, H Kuwahara, J Ren, G Wen, D Kufe. The c-Src tyrosine kinase regulates signaling of the human DF3/MUC1 carcinoma-associated antigen with GSK3 beta and beta-catenin. J Biol Chem 276, 6061-6064 (2001)
doi:10.1074/jbc.C000754200

19. LM Mendoza, P Paz, A Zuberi, G Christianson, D Roopenian, N Shastri: Minors held by majors: the H13 minor histocompatibility locus defined as a peptide/MHC class I complex. Immunity 7, 461-472 (1997)
doi:10.1016/S1074-7613(00)80368-4

20. T Higuchi, T Maruyama, A Jaramillo, T Mohanakumar: Induction of obliterative airway disease in murine tracheal allografts by CD8+ CTLs recognizing a single minor histocompatibility antigen. J Immunol 174, 1871-1878 (2005)

21. J Yang, A Jaramillo, W Liu, B Olack, Y Yoshimura, S Joyce, Z Kaleem, T Mohanakumar: Chronic rejection of murine cardiac allografts discordant at the H13 minor histocompatibility antigen correlates with the generation of the H13-specific CD8+ cytotoxic T cells. Transplantation 76, 84-91 (2003)
doi:10.1097/01.TP.0000072013.21336.64

22. DA Ostrov, MM Roden, W Shi, E Palmieri, GJ Christianson, L Mendoza, G Villaflor, D Tilley, N Shastri, H Grey, SC Almo, D Roopenian, SG Nathenson: How H13 histocompatibility peptides differing by a single methyl group and lacking conventional MHC binding anchor motifs determine self-nonself discrimination. J Immunol 168, 283-289 (2002)

23. LM Brown, RA Papa, MJ Frost, SG Mackintosh, X Gu, RJ Dixon, AD Shannon: A single amino acid is critical for the expression of B-cell epitopes on the helicase domain of the pestivirus NS3 protein. Virus Res 84, 111-124 (2002)
doi:10.1016/S0168-1702(01)00444-0

24. D Kanduc: Immunogenicity in peptide-immunotherapy: from self/nonself to similar/dissimilar sequences. Adv Exp Med Biol 640, 198-207 (2008)
doi:10.1007/978-0-387-09789-3_15

25. D Kanduc: Correlating low-similarity peptide sequences and allergenic epitopes. Curr Pharm Des 14, 289-295 (2008)
doi:10.2174/138161208783413257

26. D Kanduc: "Self-nonself" peptides in the design of vaccines. Curr Pharm Des 15, 3283-3289 (2009)
doi:10.2174/138161209789105135

27. D Kanduc: The self/nonself issue: a confrontation between proteomes. Self Nonself 1, 255-258 (2010)

28. D Kanduc: Protein information content resides in rare peptide segments. Peptides 31, 983-988 (2010)
doi:10.1016/j.peptides.2010.02.003

29. CH Wu, LS Yeh, H Huang, L Arminski, J Castro-Alvear, Y Chen, Z Hu, P Kourtesis, RS Ledley, BE Suzek, CR Vinayaka, J Zhang, WC Barker: The Protein Information Resource. Nucleic Acids Res 31, 345-347 (2003)
doi:10.1093/nar/gkg040

30. G Lucchese, A Stufano, B Trost, A Kusalik, D Kanduc: Peptidology: short amino acid modules in cell biology and immunology. Amino Acids 33, 703-707 (2007)
doi:10.1007/s00726-006-0458-z

31. G Lucchese, A Stufano, D Kanduc: Proteome-guided search for influenza A B-cell epitopes. FEMS Immunol Med Microbiol 57, 88-92 (2009)
doi:10.1111/j.1574-695X.2009.00582.x

32. G Lucchese, A Stufano, D Kanduc: Proposing low-similarity peptide vaccines against Mycobacterium tuberculosis. J Biomed Biotechnol 832341 (2010)

33. A Kusalik, M Bickis, C Lewis, Y Li, G Lucchese, FM Marincola, D Kanduc: Widespread and ample peptide overlapping between HCV and Homo sapiens proteomes. Peptides 28, 1260-1267 (2007)
doi:10.1016/j.peptides.2007.04.001

34. G Lucchese, A Stufano, M Calabro', D Kanduc: Charting the peptide crossreactome between HIV-1 and the human proteome. Front Biosci 3, 1385-1400 (2011)

35. G Lucchese, A Stufano, D Kanduc: Searching for an effective, safe and universal anti-HIV vaccine: Finding the answer in just one short peptide. Self Nonself 2, 49-54 (2011)

36. AC May: Percent sequence identity; the need to be explicit. Structure 12, 737-738 (2004)
doi:10.1016/j.str.2004.04.001

37. D Kanduc, G Capone, VP Delfino, G Losa: The fractal dimension of protein information. Adv Stud Biol 2, 53-62 (2010)

38. A Mittelman, R Tiwari, G Lucchese, J Willers, R Dummer, D Kanduc: Identification of monoclonal anti-HMW-MAA antibody linear peptide epitope by proteomic database mining. J Invest Dermatol 123, 670-675 (2004)
doi:10.1111/j.0022-202X.2004.23417.x

39. R Dummer, A Mittelman, FP Fanizzi, G Lucchese, J Willers, D Kanduc: Non-self-discrimination as a driving concept in the identification of an immunodominant HMW-MAA epitopic peptide sequence by autoantibodies from melanoma cancer patients. Int J Cancer 111, 720-726 (2004)
doi:10.1002/ijc.20310

40. D Kanduc, FP Fanizzi, G Lucchese, S Stevanovic, AA Sinha, A Mittelman: NMR probing of in silico identification of anti-HPV16 E7 mAb linear peptide epitope. Peptides 25, 243-250 (2004)
doi:10.1016/j.peptides.2003.12.004
41. D Kanduc, L Tessitore, G Lucchese, A Kusalik, E Farber, FM Marincola: Sequence uniqueness and sequence variability as modulating factors of human anti-HCV humoral immune response. Cancer Immunol Immunother 57, 1215-1223 (2008)
doi:10.1007/s00262-008-0456-y

42. L Polimeno, A Mittelman, L Gennero, A Ponzetto, G Lucchese, A Stufano, A Kusalik, D Kanduc: Sub-epitopic dissection of HCV E1315-328HRMAWDMMMNWSPT sequence by similarity analysis. Amino Acids 34, 479-484 (2008)
doi:10.1007/s00726-007-0539-7

43. ED Getzoff, HM Geysen, SJ Rodda, H Alexander, JA Tainer JA, RA Lerner: Mechanisms of antibody binding to a protein Science 235, 1191-1196 (1987)
doi:10.1126/science.3823879

44. HM Geysen, JA Tainer, SJ Rodda, TJ Mason, H Alexander, ED Getzoff, RA Lerner: Chemistry of antibody binding to a protein. Science 235, 1184-1190 (1987)
doi:10.1126/science.3823878

45. B Hjelm, CD Fernández, J Löfblom, S Ståhl, H Johannesson, J Rockberg, M Uhlén: Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase. N Biotechnol 27, 129-137 (2010)
doi:10.1016/j.nbt.2009.11.001

46. AK Falconar: Monoclonal antibodies that bind to common epitopes on the dengue virus type 2 nonstructural-1 and envelope glycoproteins display weak neutralizing activity and differentiated responses to virulent strains: implications for pathogenesis and vaccines. Clin Vaccine Immunol 15, 549-561 (2008)
doi:10.1128/CVI.00351-07

47. AK Falconar: Use of synthetic peptides to represent surface-exposed epitopes defined by neutralizing dengue complex- and flavivirus group-reactive monoclonal antibodies on the native dengue type-2 virus envelope glycoprotein. J Gen Virol 89, 1616-1621 (2008)
doi:10.1099/vir.0.83648-0

48. P Harris, M Hill, HM Vordermeier, M Jones, G Hewinson, H Thangaraj, J Ivanyi: Mutagenesis of an immunodominant T cell epitope can affect recognition of different T and B determinants within the same antigen. Mol Immunol 34, 315-322 (1997)
doi:10.1016/S0161-5890(97)00041-2

49. H Alexander, DA Johnson, J Rosen, L Jerabek, N Green, IL Weissman, RA Lerner: Mimicking the alloantigenicity of proteins with chemically synthesized peptides differing in single amino acids. Nature 306, 697-699 (1983)
doi:10.1038/306697a0

50. AT Kozhich, RR Caspi, JA Berzofsky, I Gery: Immunogenicity and immunopathogenicity of an autoimmune epitope are potentiated by increasing MHC binding through residue substitution. J Immunol 158, 4145-4151 (1997)

51. R Tiwari, J Geliebter, A Lucchese, A Mittelman, D Kanduc: Computational peptide dissection of Melan-a/MART-1 oncoprotein antigenicity. Peptides 25, 1865-1871 (2004)
doi:10.1016/j.peptides.2004.07.004

52. LM Cortes, D Avichezer, PB Silver, D Luger, MJ Mattapallil, CC Chan, RR Caspi: Inhibitory peptide analogs derived from a major uveitogenic epitope protect from antiretinal autoimmunity by inducing type 2 and regulatory T cells. J Leukoc Biol 84, 577-585 (2008)
doi:10.1189/jlb.0308189

53. I Mendel, NK de Rosbo, A Ben-Nun: Delineation of the minimal encephalitogenic epitope within the immunodominant region of myelin oligodendrocyte glycoprotein: diverse V beta gene usage by T cells recognizing the core epitope encephalitogenic for T cell receptor V beta b and T cell receptor V beta a H-2b mice. Eur J Immunol 26, 2470-2479 (1996)
doi:10.1002/eji.1830261030

54. AL Hartman, L Ling, ST Nichol, ML Hibberd: Whole-genome expression profiling reveals that inhibition of host innate immune response pathways by Ebola virus can be reversed by a single amino acid change in the VP35 protein. J Virol 82, 5348-5358 (2008)
doi:10.1128/JVI.00215-08

55. V Kim, WR Green: A single amino acid variation within an immunodominant AKR/Gross MuLV cytotoxic T-lymphocyte epitope leads to a loss in immunogenicity. Viral Immunol 11, 197-213 (1998)
doi:10.1089/vim.1998.11.197

56. MW Schreurs, EW Kueter, KB Scholten, FA Lemonnier, CJ Meijer, E Hooijberg: A single amino acid substitution improves the in vivo immunogenicity of the HPV16 oncoprotein E7(11-20) cytotoxic T lymphocyte epitope. Vaccine 23, 4005-4010 (2005)
doi:10.1016/j.vaccine.2005.03.014

57. JL Fornek, L Gillim-Ross, C Santos, V Carter, JM Ward, LI Cheng, S Proll, MG Katze, K Subbarao: A single-amino-acid substitution in a polymerase protein of an H5N1 influenza virus is associated with systemic infection and impaired T-cell activation in mice. J Virol 83, 11102-11115 (2009)
doi:10.1128/JVI.00994-09

58. DE McFarlin, SE Blank, RF Kibler, S McKneally, R Shapira: Experimental allergic encephalomyelitis in the rat: response to encephalitogenic proteins and peptides. Science 179, 478-480 (1973)
doi:10.1126/science.179.4072.478

59. RF Kibler, RB Fritz, F Chou, CH Jen Chou, NY Peacocke, NM Brown, DE McFarlin: Immune response of Lewis rats to peptide C1 (residues 68-88) of guinea pig and rat myelin basic proteins. J Exp Med 146, 1323-1331 (1977)
doi:10.1084/jem.146.5.1323

60. M Dayan, Z Sthoeger, A Neiman, J Abarbanel, M Sela, E Mozes: Immunomodulation by a dual altered peptide ligand of autoreactive responses to the acetylcholine receptor of peripheral blood lymphocytes of patients with myasthenia gravis. Hum Immunol 65, 571-577 (2004)
doi:10.1016/j.humimm.2004.02.030

61. Z Illes, H Waldner, J Reddy, E Bettelli, LB Nicholson, VK Kuchroo: T cell tolerance induced by cross-reactive TCR ligands can be broken by superagonist resulting in anti-inflammatory T cell cytokine production. J Immunol 175, 1491-1497 (2005)

62. ML Ford, BD Evavold: Degenerate recognition of T cell epitopes: impact of T cell receptor reserve and stability of peptide:MHC complexes. Mol Immunol 40, 1019-1025 (2004)
doi:10.1016/j.molimm.2003.11.004

63. A Mittelman, G Lucchese, A Stufano, D Kanduc: Degenerate binding of tyrosinase peptides to MHC II Ad/Ed molecules. J Exp Ther Oncol 6, 231-239 (2007)

64. E Martinez-Hackert, N Anikeeva, SA Kalams, BD Walker, WA Hendrickson, Y Sykulev: Structural basis for degenerate recognition of natural HIV peptide variants by cytotoxic lymphocytes. J Biol Chem 281, 20205-20212 (2006)
doi:10.1074/jbc.M601934200

65. D Kanduc: Peptimmunology: immunogenic peptides and sequence redundancy. Curr Drug Discov Technol 2, 239-244 (2005)
doi:10.2174/157016305775202946

66. B Trost, A Kusalik, G Lucchese, G Kanduc: Bacterial peptides are intensively present throughout the human proteome. Self Nonself 1, 71-74 (2010)

67. B Trost, G Lucchese, A Stufano, M Bickis, A Kusalik, D Kanduc D: No human protein is exempt from bacterial motifs, not even one. Self Nonself 1, 328-334 (2010)

68. D Kanduc, A Stufano, G Lucchese, A Kusalik: Massive peptide sharing between viral and human proteomes. Peptides 29, 1755-1766 (2008)
doi:10.1016/j.peptides.2008.05.022

69. D Kanduc: Quantifying the possible cross-reactivity risk of an HPV16 vaccine. J Exp Ther Oncol 8, 65-76 (2009)

70. D Kanduc: Penta- and hexapeptide sharing between HPV16 and Homo sapiens proteomes. Int J Med Med Sci 1, 387 (2009)

71. R Ricco, D Kanduc: Hepatitis B virus and Homo sapiens proteome-wide analysis: A profusion of viral peptide overlaps in neuron-specific human proteins. Biologics 4, 75-81 (2010)

72. D Kanduc: Describing the hexapeptide identity platform between the influenza A H5N1 and Homo sapiens proteomes. Biologics 4, 245-261 (2010)

73. SL Bavaro, D Kanduc: Pentapeptide commonality between Corynebacterium diphtheriae toxin and the Homo sapiens proteome. Immunotherapy 3, 49-58 (2011)
doi:10.2217/imt.10.83

74. SL Bavaro, M Calabro', D Kanduc: Pentapeptide sharing between Corynebacterium diphtheria toxin and the human neural protein network. Immunopharmacol Immunotoxicol 33, 360-372 (2011)
doi:10.3109/08923973.2010.518618

Key Words: Epitopic peptides, Immunological information, Single amino acid change, Sequence similarity change, Immune response, Immune recognition, Rare peptides