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Proteomic definition of a desmoglein linear determinant common to Pemphigus vulgaris and Pemphigus foliaceous.

Lucchese A, Mittelman A, Tessitore L, Serpico R, Sinha AA, Kanduc D - J Transl Med (2006)

Bottom Line: The relationship between sequence redundancy and peptide immunoreactivity has been successfully validated in a number of experimental models.Computational analysis led to identifying a linear immunodominant desmoglein-3 epitope highly reactive with the sera from Pemphigus vulgaris as well as Pemphigus foliaceous.This study 1) validates sequence redundancy to autoproteome as a main factor in shaping desmoglein peptide immunogenicity; 2) offers a molecular mechanicistic basis in analyzing the commonality of autoimmune responses exhibited by the two forms of pemphigus; 3) indicates possible peptide-immunotherapeutical approaches for pemphigus diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept of Odontostomatology, University of Bari, Italy. alucchese@hotmail.com

ABSTRACT

Background: A number of autoimmune diseases have been clinically and pathologically characterized. In contrast, target antigens have been identified only in a few cases and, in these few cases, the knowledge of the exact epitopic antigenic sequence is still lacking. Thus the major objective of current work in the autoimmunity field is the identification of the epitopic sequences that are related to autoimmune reactions. Our labs propose that autoantigen peptide epitopes able to evoke humoral (auto)immune response are defined by the sequence similarity to the host proteome. The underlying scientific rationale is that antigen peptides acquire immunoreactivity in the context of their proteomic similarity level. Sequences uniquely owned by a protein will have high potential to evoke an immune reaction, whereas motifs with high proteomic redundancy should be immunogenically silenced by the tolerance phenomenon. The relationship between sequence redundancy and peptide immunoreactivity has been successfully validated in a number of experimental models. Here the hypothesis has been applied to pemphigus diseases and the corresponding desmoglein autoantigens.

Methods: Desmoglein 3 sequence similarity analysis to the human proteome followed by dot-blot/NMR immunoassays were carried out to identify and validate possible epitopic sequences.

Results: Computational analysis led to identifying a linear immunodominant desmoglein-3 epitope highly reactive with the sera from Pemphigus vulgaris as well as Pemphigus foliaceous. The epitopic peptide corresponded to the amino acid REWVKFAKPCRE sequence, was located in the extreme N-terminal region (residues 49 to 60), and had low redundancy to the human proteome. Sequence alignment showed that human desmoglein 1 and 3 share the REW-KFAK-RE sequence as a common motif with 75% residue identity.

Conclusion: This study 1) validates sequence redundancy to autoproteome as a main factor in shaping desmoglein peptide immunogenicity; 2) offers a molecular mechanicistic basis in analyzing the commonality of autoimmune responses exhibited by the two forms of pemphigus; 3) indicates possible peptide-immunotherapeutical approaches for pemphigus diseases.

No MeSH data available.


Related in: MedlinePlus

Cross-reactivity of human recombinant EC Dsg1 or EC Dsg3 proteins with PF and PV sera. Panels A and B: Western blot analysis of PF or PV serum immunoreactivity towards human recombinant EC Dsg1 or EC Dsg3 protein. Control: phosphorylase was used as a control protein. Panels C and D: Dot blot analysis of PF or PV serum immunoreactivity towards peptide: 1) Dsg3190–204LNSKIAFKIVSQEPA, 2) Dsg349–60 REWVKFAKPCRE, and 3) Dsg149–60REWIKFAAACRE.
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Figure 5: Cross-reactivity of human recombinant EC Dsg1 or EC Dsg3 proteins with PF and PV sera. Panels A and B: Western blot analysis of PF or PV serum immunoreactivity towards human recombinant EC Dsg1 or EC Dsg3 protein. Control: phosphorylase was used as a control protein. Panels C and D: Dot blot analysis of PF or PV serum immunoreactivity towards peptide: 1) Dsg3190–204LNSKIAFKIVSQEPA, 2) Dsg349–60 REWVKFAKPCRE, and 3) Dsg149–60REWIKFAAACRE.

Mentions: False positives as well as false negatives are a constant feature of immunoassays. Consequently, we controlled the dot-blot results reported in Fig. 3 by NMR spectroscopy to irrefutably demonstrate the immunoreactivity of Dsg349–60REWVKFAKPCRE peptide with both PV and PF AAbs. NMR spectroscopy measures the antigen-antibody binding reaction by monitoring nuclear chemical shifts, i.e. the motional freedom of nuclei. However, application of NMR technology to the analysis of the antigen-antibody interaction is not unproblematic, since the high number of amino acid residues complicates the assignment of specific chemical shift signals. Therefore, we enhanced the NMR detection limit by measuring one-bond proton-nitrogen shift correlations with two-dimensional (2-D) phase-sensitive pulsed field gradient HSQC experiments [26]. In addition, synthetic peptides containing 15N-labelled amino acids were used in order to obtain greater intensities and unequivocal resolution. Specifically, the non-redundant Dsg349–60REWVKFAKPCRE, Dsg3373–380QVINVREG, and Dsg3518–525NRYTGPYT peptides (with 15N-labelled amino acid residues given underlined) were synthesized and chemical shift changes following AAb addition were monitored in two-dimensional correlated spectroscopy spectra as reported in Fig. 4, 5, and 6, respectively. Each spot in the figures is an NMR signal representing the 1H-15N one-bond coupling of the labelled amino acid residues in the peptide.


Proteomic definition of a desmoglein linear determinant common to Pemphigus vulgaris and Pemphigus foliaceous.

Lucchese A, Mittelman A, Tessitore L, Serpico R, Sinha AA, Kanduc D - J Transl Med (2006)

Cross-reactivity of human recombinant EC Dsg1 or EC Dsg3 proteins with PF and PV sera. Panels A and B: Western blot analysis of PF or PV serum immunoreactivity towards human recombinant EC Dsg1 or EC Dsg3 protein. Control: phosphorylase was used as a control protein. Panels C and D: Dot blot analysis of PF or PV serum immunoreactivity towards peptide: 1) Dsg3190–204LNSKIAFKIVSQEPA, 2) Dsg349–60 REWVKFAKPCRE, and 3) Dsg149–60REWIKFAAACRE.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1590053&req=5

Figure 5: Cross-reactivity of human recombinant EC Dsg1 or EC Dsg3 proteins with PF and PV sera. Panels A and B: Western blot analysis of PF or PV serum immunoreactivity towards human recombinant EC Dsg1 or EC Dsg3 protein. Control: phosphorylase was used as a control protein. Panels C and D: Dot blot analysis of PF or PV serum immunoreactivity towards peptide: 1) Dsg3190–204LNSKIAFKIVSQEPA, 2) Dsg349–60 REWVKFAKPCRE, and 3) Dsg149–60REWIKFAAACRE.
Mentions: False positives as well as false negatives are a constant feature of immunoassays. Consequently, we controlled the dot-blot results reported in Fig. 3 by NMR spectroscopy to irrefutably demonstrate the immunoreactivity of Dsg349–60REWVKFAKPCRE peptide with both PV and PF AAbs. NMR spectroscopy measures the antigen-antibody binding reaction by monitoring nuclear chemical shifts, i.e. the motional freedom of nuclei. However, application of NMR technology to the analysis of the antigen-antibody interaction is not unproblematic, since the high number of amino acid residues complicates the assignment of specific chemical shift signals. Therefore, we enhanced the NMR detection limit by measuring one-bond proton-nitrogen shift correlations with two-dimensional (2-D) phase-sensitive pulsed field gradient HSQC experiments [26]. In addition, synthetic peptides containing 15N-labelled amino acids were used in order to obtain greater intensities and unequivocal resolution. Specifically, the non-redundant Dsg349–60REWVKFAKPCRE, Dsg3373–380QVINVREG, and Dsg3518–525NRYTGPYT peptides (with 15N-labelled amino acid residues given underlined) were synthesized and chemical shift changes following AAb addition were monitored in two-dimensional correlated spectroscopy spectra as reported in Fig. 4, 5, and 6, respectively. Each spot in the figures is an NMR signal representing the 1H-15N one-bond coupling of the labelled amino acid residues in the peptide.

Bottom Line: The relationship between sequence redundancy and peptide immunoreactivity has been successfully validated in a number of experimental models.Computational analysis led to identifying a linear immunodominant desmoglein-3 epitope highly reactive with the sera from Pemphigus vulgaris as well as Pemphigus foliaceous.This study 1) validates sequence redundancy to autoproteome as a main factor in shaping desmoglein peptide immunogenicity; 2) offers a molecular mechanicistic basis in analyzing the commonality of autoimmune responses exhibited by the two forms of pemphigus; 3) indicates possible peptide-immunotherapeutical approaches for pemphigus diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept of Odontostomatology, University of Bari, Italy. alucchese@hotmail.com

ABSTRACT

Background: A number of autoimmune diseases have been clinically and pathologically characterized. In contrast, target antigens have been identified only in a few cases and, in these few cases, the knowledge of the exact epitopic antigenic sequence is still lacking. Thus the major objective of current work in the autoimmunity field is the identification of the epitopic sequences that are related to autoimmune reactions. Our labs propose that autoantigen peptide epitopes able to evoke humoral (auto)immune response are defined by the sequence similarity to the host proteome. The underlying scientific rationale is that antigen peptides acquire immunoreactivity in the context of their proteomic similarity level. Sequences uniquely owned by a protein will have high potential to evoke an immune reaction, whereas motifs with high proteomic redundancy should be immunogenically silenced by the tolerance phenomenon. The relationship between sequence redundancy and peptide immunoreactivity has been successfully validated in a number of experimental models. Here the hypothesis has been applied to pemphigus diseases and the corresponding desmoglein autoantigens.

Methods: Desmoglein 3 sequence similarity analysis to the human proteome followed by dot-blot/NMR immunoassays were carried out to identify and validate possible epitopic sequences.

Results: Computational analysis led to identifying a linear immunodominant desmoglein-3 epitope highly reactive with the sera from Pemphigus vulgaris as well as Pemphigus foliaceous. The epitopic peptide corresponded to the amino acid REWVKFAKPCRE sequence, was located in the extreme N-terminal region (residues 49 to 60), and had low redundancy to the human proteome. Sequence alignment showed that human desmoglein 1 and 3 share the REW-KFAK-RE sequence as a common motif with 75% residue identity.

Conclusion: This study 1) validates sequence redundancy to autoproteome as a main factor in shaping desmoglein peptide immunogenicity; 2) offers a molecular mechanicistic basis in analyzing the commonality of autoimmune responses exhibited by the two forms of pemphigus; 3) indicates possible peptide-immunotherapeutical approaches for pemphigus diseases.

No MeSH data available.


Related in: MedlinePlus