Efficient vasoactive intestinal polypeptide hydrolyzing autoantibody light chains selected by phage display.
An immunoglobulin light chain (L chain) library derived from the peripheral blood lymphocytes of a patient with asthma was cloned into a phagemid vector. Phage particles displaying L chains capable of binding vasoactive intestinal polypeptide (VIP) were isolated by affinity chromatography. Two VIP binding L chains were expressed in Escherichia coli in soluble form and purified to electrophoretic homogeneity by metal chelating and protein L affinity chromatography. Both L chains catalyzed the hydrolysis of [tyr10-125I]VIP substrate. The catalytic activity eluted at the molecular mass of the monomer form of the L chain (28 kDa) from a gel filtration column. The activity was bound by immobilized anti-kappa-chain antibody. A control recombinant L chain displayed no catalytic activity. Hydrolysis of VIP by the catalytic L chains was saturable and consistent with Michaelis-Menten kinetics. The turnover of the L chains was moderate (0.22 and 2.21/min) and their Km values indicated comparatively high affinity recognition of VIP[111 and 202 nM), producing catalytic efficiencies comparable to or greater than trypsin. Unlike trypsin, the L chains did not display detectable cleavage of casein, suggesting a catalytic activity specialized for VIP. Comparisons of the nucleotide sequences of the L chain cDNA with their putative germ-line counterparts suggested the presence of several replacement mutations in the complementarity determining regions (CDRs). These observations suggest: (a) Retention or acquisition of catalytic activity by the L chains is compatible with affinity maturation of antibodies; and (b) The autoimmune L chain repertoire can serve as a source of substrate-specific and efficient catalysts.
Tyutyulkova
S
S
Department of Anesthesiology, University of Nebraska Medical Center, Omaha, USA.
Gao
Q S
QS
Thompson
A
A
Rennard
S
S
Paul
S
S
D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000235:genetics / Q000378:metabolism*; D001249:Asthma / Q000276:immunology; D001323:Autoantibodies / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D005803:Genes, Immunoglobulin; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*
D017151:Antibodies, Catalytic; D001323:Autoantibodies; D017931:DNA Primers; D007145:Immunoglobulin kappa-Chains; D011994:Recombinant Proteins; D014660:Vasoactive Intestinal Peptide