Amino acid sequences and hapten binding of catalytic and noncatalytic antibodies against N(alpha)-(5'-phosphopyridoxyl)-L-lysine.
Upon immunization with a transition-state analog, only a minority of the hapten-binding antibodies will possess catalytic activity, which will vary in efficacy and substrate specificity. Here, the amino acid sequences of the variable domains of two pyridoxal-5'-phosphate-dependent catalytic and five noncatalytic hapten-binding antibodies raised by immunization with protein-conjugated N(alpha)-(5'-phosphopyridoxyl)-L-lysine (Gramatikova, S., Christen, P., 1997. J. Biol. Chem. 272, 9779-9784) were determined by sequencing their cDNAs. The analysis revealed that the light chains of this set of antibodies were closely related (pairwise identity 65-80%), whereas the heavy chains could be traced back to two different but related groups (intergroup identity 50-54%). The majority of the antibodies proved not to be clonally related, a finding which correlates with their differences in enantiomeric selectivity in ligand binding and reaction specificity. Only one noncatalytic antibody was found to be clonally related with a catalytic antibody, the sequence identity being >95% in both the V(H) and V(L) domains. The complementarity-determining regions were invariably abundant in tyrosine residues. Nitration of three to four tyrosine residues with tetranitromethane abolished hapten binding and catalytic activity. Partial protection by pyridoxal-5'-phosphate against inactivation suggested the presence of functionally important tyrosine residues in the binding sites of the antibodies.
Mouratou
B
B
Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Gramatikova
S
S
Kunz
B
B
Christen
P
P
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D002384:Catalysis; D006241:Haptens / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology; D008239:Lysine / Q000276:immunology*; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000276:immunology*
D000911:Antibodies, Monoclonal; D006241:Haptens; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011732:Pyridoxal Phosphate; D008239:Lysine