Sequences of antigenic epitopes of streptokinase identified via random peptide libraries displayed on phage

Behnaz Parhami-Seren, Tracy Keel, Guy Reed

Research output: Contribution to journalArticle

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Abstract

Though streptokinase (SK) is widely used to treat humans with thrombotic disease, it is antigenic and anti-SK antibody causes allergic reactions and neutralizes SK's therapeutic effects. To pinpoint the fine structure of two immunodominant, continuous epitopes in SK, we used unconstrained 15 and 6-mer random peptide libraries displayed on phage (theoretical complexity of 3.2 x 1019 and 0.64 x 108 unique sequences). The first epitope, recognized by both human Ab and murine monoclonal (m)Abs, was previously localized to the amino terminus of SK. Repeated panning and selection experiments against a 15-mer peptide phage library, using a representative mAb (A2.5) to this epitope, identified a dominant structural motif (GP[R/L]WL) corresponding to amino acids 3 to 7 of native SK, which was consistent with previous epitope mapping. These findings were further confirmed by: (1) the fact that a synthetic peptide spanning the epitope of A2.5 (AGPEWLL) specifically inhibited the binding of A2.5 to SK and (2) the finding that mAb 9D10, which competes with mAb A2.5 for binding to SK, independently selected, from a different random hexamer library, an epitope sequence spanning residues 4 to 9 that overlaps the A2.5 epitope. Similar studies of the second epitope in SK, which is immunodominant for murine but not human antibodies, identified a consensus sequence KS(K/L)P(F/Y) corresponding to amino acids 59 to 63 of SK; this was confirmed by epitope peptide binding experiments. This epitope is cleaved and destroyed when SK reacts with human but not murine plasminogen. Thus, pinpointing the sequences of antigenic epitopes of SK: (1) provides a potential explanation for species differences in SK's antigenicity, (2) demonstrates the overlapping, fine structure of epitopes recognized by competitive mAbs, (3) confirms previous epitope mapping studies and (4) has the potential to identify antigenic sequences that lead to allergic reactions in patients treated with SK.

Original languageEnglish (US)
Pages (from-to)333-341
Number of pages9
JournalJournal of Molecular Biology
Volume271
Issue number3
DOIs
StatePublished - Aug 22 1997

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Peptide Library
Streptokinase
Bacteriophages
Epitopes
Epitope Mapping
Hypersensitivity
Amino Acids
Immunodominant Epitopes
Peptides
Plasminogen
Consensus Sequence
Therapeutic Uses
Libraries
Anti-Idiotypic Antibodies

All Science Journal Classification (ASJC) codes

  • Molecular Biology

Cite this

Sequences of antigenic epitopes of streptokinase identified via random peptide libraries displayed on phage. / Parhami-Seren, Behnaz; Keel, Tracy; Reed, Guy.

In: Journal of Molecular Biology, Vol. 271, No. 3, 22.08.1997, p. 333-341.

Research output: Contribution to journalArticle

Parhami-Seren, Behnaz ; Keel, Tracy ; Reed, Guy. / Sequences of antigenic epitopes of streptokinase identified via random peptide libraries displayed on phage. In: Journal of Molecular Biology. 1997 ; Vol. 271, No. 3. pp. 333-341.
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abstract = "Though streptokinase (SK) is widely used to treat humans with thrombotic disease, it is antigenic and anti-SK antibody causes allergic reactions and neutralizes SK's therapeutic effects. To pinpoint the fine structure of two immunodominant, continuous epitopes in SK, we used unconstrained 15 and 6-mer random peptide libraries displayed on phage (theoretical complexity of 3.2 x 1019 and 0.64 x 108 unique sequences). The first epitope, recognized by both human Ab and murine monoclonal (m)Abs, was previously localized to the amino terminus of SK. Repeated panning and selection experiments against a 15-mer peptide phage library, using a representative mAb (A2.5) to this epitope, identified a dominant structural motif (GP[R/L]WL) corresponding to amino acids 3 to 7 of native SK, which was consistent with previous epitope mapping. These findings were further confirmed by: (1) the fact that a synthetic peptide spanning the epitope of A2.5 (AGPEWLL) specifically inhibited the binding of A2.5 to SK and (2) the finding that mAb 9D10, which competes with mAb A2.5 for binding to SK, independently selected, from a different random hexamer library, an epitope sequence spanning residues 4 to 9 that overlaps the A2.5 epitope. Similar studies of the second epitope in SK, which is immunodominant for murine but not human antibodies, identified a consensus sequence KS(K/L)P(F/Y) corresponding to amino acids 59 to 63 of SK; this was confirmed by epitope peptide binding experiments. This epitope is cleaved and destroyed when SK reacts with human but not murine plasminogen. Thus, pinpointing the sequences of antigenic epitopes of SK: (1) provides a potential explanation for species differences in SK's antigenicity, (2) demonstrates the overlapping, fine structure of epitopes recognized by competitive mAbs, (3) confirms previous epitope mapping studies and (4) has the potential to identify antigenic sequences that lead to allergic reactions in patients treated with SK.",
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