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Recombinant antibodies recognize conformation-dependent epitopes of the leucine zipper of misfolding-prone myocilin.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2021 Sep; Vol. 297 (3), pp. 101067. Date of Electronic Publication: 2021 Aug 09. - Publication Year :
- 2021
-
Abstract
- Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of myocilin and that are cross-reactive for human and mouse myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.<br />Competing Interests: Conflict of interest A. C. P.-O., A. N. Q., L. R. A., S. E. H., J. A. M., and R. L. L. are coinventors of US Provisional Patent 62/776,799.<br /> (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Subjects :
- Animals
Antibodies immunology
Cytoskeletal Proteins metabolism
Epitopes metabolism
Eye Proteins metabolism
Female
Glaucoma metabolism
Glycoproteins metabolism
Humans
Male
Mice
Mice, Inbred BALB C
Molecular Conformation
Protein Conformation
Protein Domains immunology
Recombinant Proteins immunology
Reproducibility of Results
Trabecular Meshwork metabolism
Cytoskeletal Proteins immunology
Epitopes immunology
Eye Proteins immunology
Glycoproteins immunology
Leucine Zippers immunology
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 297
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 34384785
- Full Text :
- https://doi.org/10.1016/j.jbc.2021.101067