A keratin antibody recognizing a heterotypic complex: epitope mapping to complementary locations on both components
A Waseem1, E B Lane, D Harrison
1Department of Craniofacial Development, Guy's Hospital, London, United Kingdom.
Experimental Cell Research
|March 15, 1996
Summary
The monoclonal antibody LE61 recognizes a complex keratin epitope, not individual keratin proteins. This antibody
Area of Science:
- Cell Biology
- Protein Biochemistry
- Immunology
Background:
- Keratin filaments are crucial cytoskeletal components in epithelial cells.
- Keratin 8 (K8) and Keratin 18 (K18) form heteropolymers in simple epithelia.
- The monoclonal antibody LE61 is widely used for keratin expression studies.
Purpose of the Study:
- To characterize the epitope recognized by the monoclonal antibody (MAb) LE61.
- To determine if MAb LE61 binds to individual keratin polypeptides or keratin complexes.
- To precisely map the epitope location on K8 and K18.
Main Methods:
- Recombinant expression of keratin 8 (K8) and keratin 18 (K18) polypeptides and fragments.
- Nitrocellulose binding assays and Enzyme-Linked Immunosorbent Assay (ELISA).
- Glutathione S-transferase (GST) fusion protein expression to analyze keratin fragments.
Main Results:
- MAb LE61 does not react with individual K8 or K18 proteins.
- MAb LE61 binds to K8/K18 heterotypic complexes, indicating an epitope formed by the complex.
- The epitope was mapped to specific residues on both K8 (353-367) and K18 (357-385).
- The epitope is conserved across different keratin types, explaining cross-reactivity.
Conclusions:
- MAb LE61 recognizes a discontinuous epitope formed by the keratin 8/keratin 18 complex.
- The precise mapping of the epitope provides insights into keratin structure and antibody recognition.
- The conserved nature of the epitope contributes to MAb LE61's utility in studying various keratin complexes.
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