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Updated: Jul 20, 2026
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
Epitope topology and removal of mouse acrosomal plasma membrane by P12-targeted immunoaggregation
Han-Jia Lin1, Ching-Wei Luo, Chia-Yih Wang
1Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
Abstract:
P12 is a Kazal-type trypsin inhibitor that has been purified from mouse seminal vesicle secretion. We observed a slight impact of P12 on sperm capacitation, and demonstrated the removal of plasma membrane overlaying the acrosome region by immunoaggregation of P12 on mouse sperm. Further, we compared the immunoreactivity of P12 antibody to ten P12 variants, including six single-site mutated mutants (R19L, Y21V, D22G, R43G, K44S, and R45T), two multisite mutated mutants (R43G/K44S/R45T and L50H/R52G/K53A), and two deletion mutants (Nd10 and Cd8) in which 10 and 8 residues were deleted from the N- and C-terminals, respectively. We found that the N-terminal region, 43RKR45, and the C-terminal region, but not R19, Y21, and D22, are involved in the three epitopes that reside on one side and are three-dimensionally distant from R19, Y21, and D22 on the P12 molecule. Based on the epitope topology, we elucidated the structural basis by which P12 antibody immunoaggregated P12 on sperm head.
Insights
Mouse seminal vesicle secretion protein P12 impacts sperm capacitation and acrosome membrane removal. Its N- and C-terminal regions, specifically 43RKR45, are crucial for antibody binding and sperm head immunoaggregation.
Area of Science:
- Reproductive Biology
- Biochemistry
- Immunology
Background:
- P12 is a Kazal-type trypsin inhibitor found in mouse seminal vesicle secretion.
- Sperm capacitation and the acrosome reaction are critical for fertilization.
Purpose of the Study:
- To investigate the role of P12 in mouse sperm capacitation and acrosome membrane removal.
- To identify the specific regions and epitopes on P12 involved in antibody binding and sperm interaction.
Main Methods:
- Purification of P12 from mouse seminal vesicle secretion.
- Immunoaggregation assays using P12 antibodies on mouse sperm.
- Analysis of P12 variants (single-site, multi-site, and deletion mutants) to determine epitope mapping.
Main Results:
- P12 exhibited a slight effect on sperm capacitation and facilitated plasma membrane removal from the acrosome.
- Antibody binding and subsequent immunoaggregation were primarily mediated by the N-terminal region and the 43RKR45 motif, along with the C-terminal region.
- Specific residues R19, Y21, and D22 were found not to be involved in the main epitopes.
Conclusions:
- The study elucidates the structural basis of P12-antibody interaction on the sperm head.
- The N-terminal and C-terminal regions, particularly 43RKR45, are critical for P12's function in sperm capacitation and acrosome membrane removal via antibody-mediated aggregation.
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