Recovering antibody secretion using a hapten ligand as a chemical chaperone.
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.
Wiens
G D
GD
Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon 97201-3098, USA. wiensg@ohsu.edu
O'Hare
T
T
Rittenberg
M B
MB
D001120:Arginine / Q000737:chemistry; D002460:Cell Line; D006241:Haptens / Q000378:metabolism*; D007136:Immunoglobulins / Q000737:chemistry / Q000378:metabolism*; D007532:Isoleucine / Q000737:chemistry; D008024:Ligands; D009578:Nitrobenzenes / Q000378:metabolism*; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000378:metabolism*; D014162:Transfection
C440032:4-nitrophenylphosphocholine; D006241:Haptens; D007136:Immunoglobulins; D008024:Ligands; D009578:Nitrobenzenes; D007532:Isoleucine; D010767:Phosphorylcholine; D001120:Arginine