Document Type
Article
Journal Title
Biochimica et Biophysica Acta
Publication Date
8-23-1996
Volume
1316
Abstract
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.
MeSH Headings
Amino Acid Sequence, Antibodies, Catalytic, Asthma, Autoantibodies, Base Sequence, DNA Primers, Genes, Immunoglobulin, Humans, Immunoglobulin kappa-Chains, Kinetics, Molecular Sequence Data, Recombinant Proteins, Vasoactive Intestinal Peptide
DOI Link
ISSN
0006-3002
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Recommended Citation
Tyutyulkova, Sonia; Gao, Qing-Sheng; Thompson, Austin; Rennard, Stephen I.; and Paul, Sudhir, "Efficient Vasoactive Intestinal Polypeptide Hydrolyzing Autoantibody Light Chains Selected by Phage Display" (1996). Journal Articles: Pulmonary & Critical Care Med. 19.
https://digitalcommons.unmc.edu/com_pulm_articles/19
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