EFFICIENT VASOACTIVE INTESTINAL POLYPEPTIDE HYDROLYZING AUTOANTIBODY LIGHT-CHAINS SELECTED BY PHAGE DISPLAY

An immunoglobulin light chain (L chain) library derived from the perip heral blood lymphocytes of a patient with asthma was cloned into a pha gemid vector. Phage particles displaying L chains capable of binding v asoactive intestinal polypeptide (VIP) were isolated by affinity chrom atography. 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 catalyz ed the hydrolysis of [tyr(10-125)I]VIP substrate. The catalytic activi ty 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 immobili zed anti-lc-chain antibody. A control recombinant L chain displayed no catalytic activity. Hydrolysis of VIP by the catalytic L chains was s aturable and consistent with Michaelis-Menten kinetics. The turnover o f the L chains was moderate (0.22 and 2.21/min) and their K-m values i ndicated comparatively high affinity recognition of VIP [111 and 202 n M), producing catalytic efficiencies comparable to or greater than try psin. Unlike trypsin, the L chains did not display detectable cleavage of casein, suggesting a catalytic activity specialized for VIP, Compa risons of the nucleotide sequences of the L chain cDNA with their puta tive germ-line counterparts suggested the presence of several replacem ent mutations in the complementarity determining regions (CDRs). These observations suggest: (a) Retention or acquisition of catalytic activ ity by the L chains is compatible with affinity maturation of antibodi es; and (b) The autoimmune L chain repertoire can serve as a source of substrate-specific and efficient catalysts.