Efficient vasoactive intestinal polypeptide hydrolyzing autoantibody light chains selected by phage display

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE

卷:1316

Article

Tyutyulkova, S ; Gao, QS ; Thompson, A ; Rennard, S ; Paul, S

关键词: catalytic antibody;    autoantibody;    light chain;    vasoactive intestinal polypeptide;    asthma;    phage display;   

DOI  :  10.1016/0925-4439(96)00028-2

来源: Elsevier

【 摘 要 】

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 [tyr(10-125)I]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-lc-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 K-m 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.

【 授权许可】

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