A Human Germ Line Antibody Light Chain with Hydrolytic Properties Associated with Multimerization Status

Vikram Sharma,William Heriot,Kirk Trisler,Vaughn Smider

doi:10.1074/jbc.m109.036087

Abstract

Antibodies with nucleophilic or catalytic properties often have these characteristics encoded in their germ line genes. Because hydrolytic activity has been reported to be associated with light chain V regions, we have begun an analysis of germ line light chain proteins that could be the basis for affinity maturation into hydrolytic or other reactive antibodies. We produced the germ line A18b light chain and characterized its hydrolytic, nucleophilic, and tertiary structural activities. This light chain was purified to >99% purity and found to hydrolyze aminomethylcoumarin-peptide and larger protein substrates and bind a fluorophosphonate probe. Mutation of putative catalytic residues only resulted in loss of activity of a tetrameric but not dimeric form of the light chain. These biochemical properties provide a framework for understanding the structure-function relationships of germ line antibodies.

Highlights

Ple conformations and bind different antigens (4 – 6)
Endogenous hydrolytic activity has been mapped to the light chain of the c23.5 mouse anti-VIP antibody [24, 25], and other homologous light chains with hydrolytic activity have been reported [26, 27]
Any nucleophilic or hydrolytic activity attributable to A18b could be compared with the negative control purifications to rule out contaminating activity that may be below the electrophoretic detection threshold

Summary

Introduction

Ple conformations and bind different antigens (4 – 6). The structural basis for catalytic activity is more obscure, structural studies of induced catalytic antibodies show multiple structures in their germ line precursors [4, 7, 8]. Bence-Jones light-chain proteins can contribute to pathogenesis of the disease and in some cases have been reported to contain catalytic activity [16, 17]. When A18b and the serine 27a to alanine mutant were purified, they were seen to migrate as ϳ66% dimer and 33% monomer by silver stain analysis on non-reducing SDS-PAGE (Fig. 1A, top gel, lanes 1 and 2).

Results

Conclusion