An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein.
A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states.
*Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BN, U.K.
James A
JA
Irving
†Department of Biology and Biotechnologies 'Charles Darwin' and Pasteur Institute-Cenci Bolognetti Foundation, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.
Elena
E
Miranda
*Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BN, U.K.
Imran
I
Haq
‡Department of Cell Biology, Genetics and Animal Physiology, University of Malaga, Campus de Teatinos, 29071 Malaga, Spain.
Juan
J
Perez
§Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers University, 675 Hoes Lane Piscataway, NJ 08854, U.S.A.
Vadim R
VR
Kotov
*Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BN, U.K.
Sarah V
SV
Faull
*Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BN, U.K.
Neda
N
Motamedi-Shad
*Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BN, U.K.
David A
DA
Lomas
D019943:Amino Acid Substitution; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D055503:Protein Multimerization / Q000276:immunology; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D013816:Thermodynamics; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*
D058846:Antibodies, Monoclonal, Murine-Derived; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; C043392:SERPINA1 protein, human; D000515:alpha 1-Antitrypsin