Broadly Distributed Chemical Reactivity of Natural Antibodies Expressed in Coordination with Specific Antigen Binding Activity

Abstract

Antibody (Ab) nucleophilic reactivity was studied using hapten and polypeptide antigens containing biotinylated phosphonate diester groups (covalently reactive antigen analogs, CRAs). Polyclonal IgG from healthy donors formed covalent adducts with a positively charged hapten CRA at levels superior to trypsin. Each of the 16 single chain Fv clones studied expressed a similar reactivity, indicating the V domain location of the nucleophiles and their broad distribution in diverse Abs. The formation of hapten CRA-Fv adducts was correlated with Fv proteolytic activity determined by cleavage of a model peptide substrate. Despite excellent nucleophilicity, proteolysis by IgG proceeded at lower rates than trypsin, suggesting that events occurring after nucleophilic attack on the substrate limit the rate of Ab proteolysis. The extracellular domain of the epidermal growth factor receptor with phosphonate diester groups at Lys side chains and a synthetic peptide corresponding to residues 421-431 of human immunodeficiency virus glycoprotein (gp) 120 with the phosphonate diester at the C terminus formed covalent adducts with specific polyclonal and monoclonal Abs raised by immunization with epidermal growth factor receptor and synthetic gp120-(421-436) devoid of phosphonate diester groups, respectively. Adduct formation was inhibited by extracellular domain of the epidermal growth factor receptor (exEGFB) and synthetic gp120-(421-436) devoid of phosphonate groups, suggesting that the nucleophiles are located within the antigen binding sites. These results suggest the innate character of the Ab nucleophilic reactivity, its functional coordination with non-covalent adaptive binding interactions developing over the course of B cell maturation, and novel routes toward permanent inhibition of Abs.Many enzymes exploit covalent interactions with substrates to catalyze chemical transformations. On the other hand, most studies on Ab 1 catalysis have focused on non-covalent binding forces as the mechanism by which the energy barrier between reactants and products is lowered, e.g. the electrostatic forces that stabilize the negatively charged oxyanionic transition state of ester hydrolysis (reviewed in Refs. 1 and 2). The underlying assumption has been that Abs interact with their ligands exclusively by non-covalent means. Initial indications that natural Abs express chemical reactivity indistinguishable from enzymes came from reports of proteolytic and nuclease activity of autoantibodies (3, 4). Similar activities were later found in Ab light chains from multiple myeloma patients (5), alloantibodies from patients with transfusion-induced hemophilia (6), Abs raised by routine immunization with polypeptides (7, 8), and anti-idiotypic Abs to anti-enzyme Abs (9). From mutagenesis and inhibitor studies, it appears that the proteolytic activity of natural Abs originates from nucleophilic mechanisms similar to those utilized by conventional serine proteases (10, 11). The catalytic activity of natural Abs could be construed to violate the pri...

Ontogeny of Proteolytic Immunity

“…the activation of Ser/Thr side chain hydroxyl groups by hydrogen bonding to His residues, and can be detected by covalent binding to electrophilic phosphonate diesters (9,10). Using these compounds as covalently reactive analogs of antigens (CRAs), we observed that IgG Abs express nucleophilic reactivities comparable to trypsin (11). Despite their nucleophilic competence, IgG Abs display low efficiency proteolysis, presumably due to deficiencies in steps occurring after formation of the acyl-Ab intermediate, viz., water attack on the intermediate and product release.…”

“…Splenocyte-CRA Binding-Synthesis of compounds I-IV and confirmation of their chemical identity have been published (11,18). Compounds I, III, and IV are diphenyl phosphonate esters reactive with nucleophilic sites (9,10,18).…”

“…Formation of Ab-CRA adducts was determined by SDS-electrophoresis as in the preceding section. Band intensities are expressed in arbitrary area units (AAU) determined by densitometry (11). Initial velocities were computed as the slopes of progress curves plotted as a function of time (initial 60 min).…”

“…The cell extract (1.4 ml; diluted to 1 mM CHAPS; from 3 ϫ 10 6 cells) was passed through the column, the column washed with PBS-CHAPS (9 volumes) and bound proteins were eluted with 100 mM glycine-HCl, pH 2.7 (8 column volumes) and subjected to reducing SDS-polyacrylamide gel electrophoresis (4 -20%, Bio-Rad). Protein-CRA adducts were visualized by staining nitrocellulose electroblots of the gels with streptavidin-peroxidase as in (11). For immunoblotting, the blots were stained with goat polyclonal Abs (IgG) to mouse , ␥, ␦, , and chains followed by peroxidase-conjugated rabbit anti-goat IgG (Fc specific, 1:1000; Pierce) as in Ref.…”

“…To evaluate the nucleophilic reactivity expressed on the surface of B cells in the preimmune repertoire (viz., the repertoire developed spontaneously without purposeful immunological challenge), viable splenocytes from BALB/c mice were treated with hapten CRA I. The control compound II is identical in structure to hapten CRA I, except that the phosphonate group is not esterified, which results in loss of covalent reactivity with nucleophilic residues (11,18). Treatment with hapten CRA I resulted in staining of most of cells at levels greater than compound II, with a minority of the cells displaying intense staining (11 Ϯ 2, n ϭ 3 experiments; determined by counting 400 lymphocytes using a UV microscope).…”