Phosphonate Ester Probes for Proteolytic Antibodies

Abstract

The reactivity of phosphonate ester probes with several available proteolytic antibody (Ab) fragments was characterized. Irreversible, active site-directed inhibition of the peptidase activity was evident. Stable phosphonate diester-Ab adducts were resolved by column chromatography and denaturing electrophoresis. Biotinylated phosphonate esters were applied for chemical capture of phage particles displaying Fv and light chain repertoires. Selected Ab fragments displayed enriched catalytic activity inhibitable by the selection reagent. Somewhat unexpectedly, a phosphonate monoester also formed stable adducts with the Abs. Improved catalytic activity of phage Abs selected by monoester binding was evident. Turnover values (k cat ) for a selected Fv construct and a light chain against their preferred model peptide substrates were 0.5 and 0.2 min ؊1, respectively, and the corresponding Michaelis-Menten constants (K m ) were 10 and 8 M. The covalent reactivity of Abs with phosphonate esters suggests their ability to recapitulate the catalytic mechanism utilized by classical serine proteases. Abs1 and Ab L chains are reported to catalyze the cleavage of VIP (1, 2), the HIV coat proteins gp41 (3) and gp120 (4), Arg-vasopressin (5), thyroglobulin (6), factor VIII (7), prothrombin (8), and various model peptidase substrates (5, 9, 10). Recent studies suggest that the peptidase activity is a heritable function encoded by a germ line variable region gene(s) (11,12). In principle, the immune system may be capable of recruiting the catalyst-encoding germ line V gene(s) to elaborate specific proteolytic Abs directed to diverse polypeptide antigens, much as noncatalytic Abs capable of high affinity binding to different antigens can be developed by somatic sequence diversification of the same germ line V genes. Introduction of single replacement mutations in Ab combining sites can result in gain of proteolytic (13) and esterase (14) activities, underscoring the potential contributions of variable region diversification in maturation of Ab catalytic activities.The presence of a serine protease-like catalytic triad in a model proteolytic Ab L chain has previously been deduced from site-directed mutagenesis studies (15). Formation of a covalent complex between the nucleophilic serine residue and the substrate (the acyl-enzyme intermediate) is an essential step en route to peptide bond cleavage by non-Ab serine proteases (16). Phosphonate diesters, like the classical inhibitor DFP, can bind the active site of non-Ab serine proteases and serine esterases covalently (17)(18)(19). In comparison, negatively charged phosphonate monoesters have traditionally been assumed to bind esterolytic Abs (20, 21) and non-Ab serine esterases (22) via noncovalent electrostatic interactions. The aim of the present study was to characterize the reactivity of recombinant proteolytic Abs with phosphonate diesters and monoesters. Irreversible, active site-directed inhibition of catalytic activity by the phosphonate diesters was evident; stable Ab-phosphon...

Ontogeny of Proteolytic Immunity

“…The extent of irreversible CRA binding activity correlates approximately with the catalytic activity (11,29). In the present study, hapten CRA I adducts were located in close proximity to CD19 on the surface of B cells.…”

Specific HIV gp120-cleaving Antibodies Induced by Covalently Reactive Analog of gp120

“…Electrophoresis of Ab-CRA Complexes-Irreversible binding of biotinylated CRAs by purified IgG was determined by denaturing electrophoresis (6). Briefly, the reaction mixtures were incubated at 37°C in 50 mM Tris-HCl, 0.1 M glycine, pH 8.0.…”

“…N-terminal sequencing of protein bands from electrophoresis gels was done as described previously (17). Hydrolysis of peptide-MCA substrates (Peptide International, Louisville, KY or Bachem Biosciences, King of Prussia, PA) was determined in 96-well plates by fluorimetric detection of aminomethylcoumarin (Varian Cary Eclipse; ex 360 nm, em 470 nm) with authentic aminomethylcoumarin as standard (6). Cleavage of [Tyr 10 -125 I]VIP by mAb c23.5 was measured as the radioactivity rendered soluble in trichloroacetic acid (17).…”

“…1) and their covalent reactivity with naturally occurring proteolytic Abs has been described previously (6,7). The electrophilic phosphonate mimics the peptide bond carbonyl group susceptible to nucleophilic attack, the positively charged amidino group adjacent to the phosphonate diester serves as a mimic of Lys/Arg P1 residues at which cleavage by germ line-encoded proteolytic Abs is observed (6), and the biotin group in I permits sensitive detection of Ab-phosphonate adducts. gp120-CRA III contains phosphonate diester groups in spatial proximity with antigenic epitopes presented by the protein.…”

“…Originally developed as irreversible inhibitors of conventional serine proteases, haptenic phosphonate esters are reported to bind the nucleophilic sites of natural proteolytic Abs covalently (6,7). The haptenic phosphonates could potentially serve as covalently reactive analogs (CRAs) for inducing the synthesis of Abs with improved nucleophilicity.…”