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 immunization with vasoactive intestinal polypeptide (VIP) cleaved this peptide on the C-terminal side of basic residues. The major sites of cleavage 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 serine protease inhibitors, diisopropylfluorophosphate and aprotinin, but not by soybean or lima bean trypsin inhibitors or inhibitors of other classes of proteases. To probe the role of the VH domain, single chain Fv constructs composed of the VL domain of the anti-VIP L chain linked via a 14-residue peptide to its natural VH domain partner or an irrelevant anti-lysozyme VH domain (hybrid Fv) were prepared. The anti-VIP Fv hydrolyzed VIP with Ks 21.4-fold lower than the L chain and 250-fold lower than the hybrid Fv, suggesting increased affinity for the substrate ground state due to the anti-VIP VH domain. The kinetic efficiency (kcat/Ks) of the anti-VIP Fv was 6.6-fold greater compared to the L chain and 29.4-fold greater compared to the hybrid Fv. Peptide-MCA substrates unrelated in sequence to VIP were hydrolyzed by the anti-VIP Fv and L chain at equivalent rates. These observations lead to a model of catalysis by the anti-VIP Fv in which the essential catalytic residues are located in the VL domain and additional residues from the VH domain are involved in high affinity binding of the substrate.
Sun
M
M
Department of Anesthesiology and Eppley Cancer Research Institute, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6830, USA.
Gao
Q S
QS
Kirnarskiy
L
L
Rees
A
A
Paul
S
S
D000595:Amino Acid Sequence; D017151:Antibodies, Catalytic / Q000276:immunology / Q000378:metabolism*; D007611:Aprotinin / Q000494:pharmacology; D001483:Base Sequence; D002384:Catalysis; D004247:DNA; D010450:Endopeptidases / Q000276:immunology / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000276:immunology / Q000378:metabolism*; D007147:Immunoglobulin Light Chains / Q000276:immunology / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000378:metabolism*; D007531:Isoflurophate / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014660:Vasoactive Intestinal Peptide / Q000276:immunology*
D017151:Antibodies, Catalytic; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D010455:Peptides; D015842:Serine Proteinase Inhibitors; C055966:immunoglobulin Fv; D007531:Isoflurophate; D014660:Vasoactive Intestinal Peptide; D004247:DNA; D007611:Aprotinin; D010450:Endopeptidases