Recovering Antibody Secretion Using a Hapten Ligand as a Chemical Chaperone

Abstract

Engineered antibodies have come to the forefront as research reagents and clinical therapeutics. However, reduced stability or expression levels pose a major problem with many engineered antibodies. As a model for understanding functional consequences of variable region mutation, we have studied the assembly and trafficking of anti-phenylphosphocholine antibodies. Previously, we identified severe secretion defects because of mutations in the heavy chain second complementarity determining region, which is involved in antigen binding. Here we demonstrate that immunoglobulin secretion is increased up to 27-fold by incubating stably transfected PCG1-1 cells with cognate hapten p-nitrophenylphosphocholine. Secretion was unaffected by nonbinding analogs. Radiotracer and metabolic labeling experiments demonstrated specific cellular uptake of p-nitrophenylphosphocholine and increased intracellular heavy and light chain assembly. Brefeldin A inhibited hapten-mediated immunoglobulin secretion but not assembly, indicating that assembly occurs early within the biosynthetic pathway. Recovery of secretion correlated with antigen binding capacity, suggesting that the rescue mechanism involves stabilization of heavy and light chain variable domains. This model system provides the first demonstration that cognate ligands can increase intracellular assembly of functional anti-hapten antibody within mammalian cells and suggests that small molecules of appropriate specificity and affinity acting as chemical chaperones may find application for increasing or regulating immunoglobulin expression.

Chemical Chaperones: A Pharmacological Strategy for Disorders of Protein Folding and Trafficking

“…The paradigm has also been used to enhance assembly and secretion of immunoglobulin using a hapten ligand as chemical chaperone (37). Treatment of a stably transfected cell line with phenylphosphocholine, but not with a nonbinding analog, resulted in a 27-fold increase in secretion of anti-phenylphosphocholine antibody.…”

Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli

“…In one mechanism, the chemical chaperones function as osmolytes, promoting solvation of unfolded states or of the native conformation (Qu et al 1998). In the other mechanism, chemical chaperones function as ligands that are specific for the native protein conformation, as, for example, in hapten‐induced folding of antibodies (Wiens et al 2001). ThDP is likely to interact with knob through the second mechanism for the following reasons: The total concentration of ThDP in the E. coli cytoplasm was estimated at ∼0.15 mM (Makarchikov et al 2003), but most of these molecules are present in complexes with ThDP‐dependent enzymes.…”

“…The interfacial surfaces that give rise to strong subunit interactions in the knob trimer would be exposed on monomer and dimer assembly intermediates, raising the question whether these highly interactive surfaces require stabilization or shielding from nonspecific interaction with cellular components. While molecular chaperones potentially could provide this function, various small solutes, collectively termed chemical chaperones (Perlmutter 2002), also have been shown to facilitate the folding of proteins in vitro and in vivo, either by promoting solvation of unfolded or native protein conformations (Qu et al 1998) or by trapping native protein conformations through specific ligand interactions (Wiens et al 2001). Here we present evidence that thiamine diphosphate (ThDP), the intracellular coenzyme derivative of vitamin B1, functions as a chemical chaperone in the assembly of recombinant knob trimers in E. coli , possibly by stabilizing assembly intermediates through interaction with exposed surfaces on folded subunits.…”

Repertoire Shift in the Humoral Response to Phosphocholine-Keyhole Limpet Hemocyanin: VH Somatic Mutation in Germinal Center B Cells Impairs T15 Ig Function

“…Thus, it is formally possible that compensatory mutations in the L chain may have existed. Studies are currently underway to examine directly the proliferative potential and in vivo fate of B cells harboring deleterious Ig mutations and to determine whether compensatory mechanisms may rescue Ab function during an immune response as we have demonstrated in our in vitro studies (65)(66)(67).…”