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.

An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein / J. A., Irving; MIRANDA BANOS, MARIA ELENA; I., Haq; J., Perez; V. R., Kotov; S. V., Faull; N., Motamedi Shad; D. A., Lomas. - In: BIOCHEMICAL JOURNAL. - ISSN 0264-6021. - STAMPA. - 468:1(2015), pp. 99-108. [10.1042/BJ20141569]

An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein

MIRANDA BANOS, MARIA ELENA;
2015

Abstract

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.
aggregation; antibody; alpha(1)-antitrypsin; polymerization; potein stability
01 Pubblicazione su rivista::01a Articolo in rivista
An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein / J. A., Irving; MIRANDA BANOS, MARIA ELENA; I., Haq; J., Perez; V. R., Kotov; S. V., Faull; N., Motamedi Shad; D. A., Lomas. - In: BIOCHEMICAL JOURNAL. - ISSN 0264-6021. - STAMPA. - 468:1(2015), pp. 99-108. [10.1042/BJ20141569]
File allegati a questo prodotto
File Dimensione Formato  
Irving_Antibody_2015

accesso aperto

Note: Articolo principale
Tipologia: Versione editoriale (versione pubblicata con il layout dell'editore)
Licenza: Creative commons
Dimensione 1.26 MB
Formato Adobe PDF
1.26 MB Adobe PDF Visualizza/Apri PDF

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/780798
Citazioni
  • ???jsp.display-item.citation.pmc??? 10
  • Scopus 18
  • ???jsp.display-item.citation.isi??? 18
social impact