CLEAVAGE SPECIFICITY OF A PROTEOLYTIC ANTIBODY LIGHT-CHAIN AND EFFECTS OF THE HEAVY-CHAIN VARIABLE DOMAIN

The recombinant light chain (L chain) of an antibody raised by immuniz ation with vasoactive intestinal polypeptide (VIP) cleaved this peptid e on the C-terminal side of basic residues. The major sites of cleavag e in VIP were two adjacent peptide bonds, Lys20-Lys21 and Lys21-Tyr22. Lower levels of cleavage were evident at Arg14-Lys15 and Lys15-Gln16. Hydrolysis of radiolabeled VIP by the L chain was inhibited by two se rine protease inhibitors, diisopropylfluorophosphate and aprotinin, bu t not by soybean or lima bean trypsin inhibitors or inhibitors of othe r classes of proteases. To probe the role of the V-H domain, single ch ain F-v constructs composed of the V-L domain of the anti-VIP L chain linked via a 14-residue peptide to its natural V-H domain partner or a n irrelevant anti-lysozyme V-H domain (hybrid F-v) were prepared. The anti-VIP F-v hydrolyzed VIP with K-s 21.4-fold lower than the L chain and 250-fold lower than the hybrid F-v, suggesting increased affinity for the substrate ground state due to the anti-VIP V-H domain. The kin etic efficiency (k(cat)/K-s) of the anti-VIP F-v was 6.6-fold greater compared to the L chain and 29.4-fold greater compared to the hybrid F -v. Peptide-MCA substrates unrelated in sequence to VIP were hydrolyze d by the anti-VIP F-v and L chain at equivalent rates. These observati ons lead to a model of catalysis by the anti-VIP F-v in which the esse ntial catalytic residues are located in the V-L domain and additional residues from the V-H domain are involved in high affinity binding of the substrate. (C) 1997 Academic Press Limited.