Exceptional Amyloid β Peptide Hydrolyzing Activity of Nonphysiological Immunoglobulin Variable Domain Scaffolds

Hiroaki Taguchi,Stephanie Planque,Gopal Sapparapu,Stephane Boivin,Mariko Hara,Yasuhiro Nishiyama,Sudhir Paul

doi:10.1074/jbc.m806766200

Hiroaki Taguchi, Stephanie Planque + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m806766200

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Abstract

Nucleophilic sites in the paired variable domains of the light and heavy chains (VL and VH domains) of Ig can catalyze peptide bond hydrolysis. Amyloid beta (Abeta)-binding Igs are under consideration for immunotherapy of Alzheimer disease. We searched for Abeta-hydrolyzing human IgV domains (IgVs) in a library containing a majority of single chain Fv clones mimicking physiological VL-VH-combining sites and minority IgV populations with nonphysiological structures generated by cloning errors. Random screening and covalent selection of phage-displayed IgVs with an electrophilic Abeta analog identified rare IgVs that hydrolyzed Abeta mainly at His14-Gln15. Inhibition of IgV catalysis and irreversible binding by an electrophilic hapten suggested a nucleophilic catalytic mechanism. Structural analysis indicated that the catalytic IgVs are nonphysiological structures, a two domain heterodimeric VL (IgVL2-t) and single domain VL clones with aberrant polypeptide tags (IgVL-t'). The IgVs hydrolyzed Abeta at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain VL with VH or VL domains resulted in reduced Abeta hydrolysis, suggesting catalysis by the unpaired VL domain.Angstrom level amino acid displacements evident in molecular models of the two domain and unpaired VL domain clones explain alterations of catalytic activity. In view of their superior catalytic activity, the VL domain IgVs may help attain clearance of medically important antigens more efficiently than natural Igs.

Highlights

The antigen-combining sites of immunoglobulins found in higher organisms are composed of the variable domains of light and heavy chain subunits (VL and VH domains)
We report here the search for efficient A␤-hydrolyzing Ig fragments in a human IgV domains (IgVs) domain (IgV) library in which the majority of clones are single chain Fv constructs
amyloid ␤ peptide (A␤)40 Hydrolyzing IgVs—We reported previously the hydrolysis of A␤-(1– 40) (A␤40) by polyclonal IgM purified from humans without dementia [18]

Summary

Introduction

The antigen-combining sites of immunoglobulins found in higher organisms are composed of the variable domains of light and heavy chain subunits (VL and VH domains). Ig adaptive selection is geared toward noncovalent immune complexation (the ground state stabilization step), and the ability of Igs to recognize the high energy transition state complex that must be stabilized to accelerate chemical reactions is limited This is supported by observations that IgMs, the first and least diversified Ig class produced during B cell differentiation, express superior catalytic rate constants than IgGs produced by the cells at later stages of their adaptive differentiation [12]. We reported naturally occurring IgMs and isolated Ig light chain subunits (IgLs) that hydrolyze A␤ impede A␤ aggregation and inhibit A␤-induced neurotoxicity [18, 19] These Igs hydrolyze A␤ slowly, and development of more efficient catalysts will help advance the use of catalytic Igs for A␤ clearance. The observations suggest that novel Ig structures freed of constraints imposed by the physiological organization of V domains can be the source of efficient catalysts to medically important antigens

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