Mutational Analysis of Two Stefin A Epitopes
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N. Kopitar-Jerala
Abstract
Stefin A, an intracellular inhibitor of cysteine proteinases, is expressed most abundantly in epithelial cells and in cells of lymphatic origin. In order to study its role in normal and pathological conditions we have prepared and characterized monoclonal antibodies against recombinant stefin A. Two high affinity monoclonal antibodies (mAbs) (A22 and C52) were tested for binding to free and papain-complexed stefin A and to a chimeric inhibitor, consisting of 61 amino acid residues of stefin A and 37 carboxy-terminal residues of stefin B. mAb A22 recognized not only free stefin A but also stefin A in complex with papain. The mAbs were further tested for their cross-reactivity against stefin A and B isolated from different mammalian species. On the basis of sequence similarity and tertiary structure of human stefin A we have prepared three mutants–Glu33Lys, Asp61Gly and Asn62Tyr–and their reactivity with the mAbs was tested. The binding affinities of mAb A22 for the Asp61Gly and Asn62Tyr mutants were significantly lower, indicating that the two amino acids are part of the stefin A epitope recognized by A22. The binding of both mAbs to the mutants Gly4Arg and Gly4Glu was comparable to wild-type stefin A.
Copyright © 1999 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- To Our Authors, Readers and Subscribers
- Michael Strauss. January 12, 1950 April 29, 1999
- Monoallelic Expression of Pax5: A Paradigm for the Haploinsufficiency of Mammalian Pax Genes?
- Adeno-Associated Viral Vectors for Gene Transfer and Gene Therapy
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- Dequalinium TM Vesicles Form Stable Complexes with Plasmid DNA which Are Protected from DNase Attack
- On the Role of Thymopoietins in Cell Proliferation. Immunochemical Evidence for New Members of the Human Thymopoietin Family
- Functional Characterization of Atherosclerosis-Associated Ser128Arg and Leu554Phe E-Selectin Mutations
- Cycloheximide, a New Tool to Dissect Specific Steps in ER-Associated Degradation of Different Substrates
- Localization of Rat Cathepsin K in Osteoclasts and Resorption Pits: Inhibition of Bone Resorption and Cathepsin K-Activity by Peptidyl Vinyl Sulfones
- Oxidation of 4-tert-Butylcatechol and Dopamine by Hydrogen Peroxide Catalysed by Horseradisch Peroxidase
- A Non-Radioactive Method for Inexpensive Quantitative RT-PCR
- Activation of the Transcription Factor ISGF3 by Interferon-gamma
- Acquisition of Myogenic Specificity through Replacement of One Amino Acid of MASH-1 and Introduction of an Additional alpha -Helical Turn
- The Heavy Metal-Responsive Transcription Factor-1 (MTF-1) Is Not Required for Neural Differentiation
- Human Fc gamma Receptor IIb Expressed in <I>Escherichia coli</I> Reveals IgG Binding Capability
- Mutational Analysis of Two Stefin A Epitopes
