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

2003; Elsevier BV; Volume: 278; Issue: 22 Linguagem: Inglês

10.1074/jbc.m300870200

1083-351X

Sudhir Paul, Stephanie Planque, Yong-Xin Zhou, Hiroaki Taguchi, Gita Bhatia, Sangeeta Karle, Carl V. Hanson, Yasuhiro Nishiyama,

HIV/AIDS drug development and treatment

We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314–28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and Km for this reaction was ∼200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization. We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314–28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and Km for this reaction was ∼200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization. Promiscuous cleavage of small peptide substrates is a heritable function of Abs 1The abbreviations used are: Ab, antibody; mAb, monoclonal Ab; Bt, biotin; CRA, covalently reactive antigen analog; MCA, methylcoumarinamide; VIP, vasoactive intestinal peptide; HIV, human immunodeficiency virus; exEGFR, extracellular domain of enhanced green fluorescent protein; ELISA, enzyme-linked immunosorbent assay.1The abbreviations used are: Ab, antibody; mAb, monoclonal Ab; Bt, biotin; CRA, covalently reactive antigen analog; MCA, methylcoumarinamide; VIP, vasoactive intestinal peptide; HIV, human immunodeficiency virus; exEGFR, extracellular domain of enhanced green fluorescent protein; ELISA, enzyme-linked immunosorbent assay. encoded by germ line gene variable domains (for review, see Ref. 1Tramontano A. Golobov G. Paul S. Paul S. Chemical Immunology: Catalytic Antibodies. 77. S. Karger GmbH, Basel, Switzerland2000: 1-17Google Scholar). Peptide bond cleaving Abs with specificity for individual polypeptides have been identified in patients with autoimmune (1Tramontano A. Golobov G. Paul S. Paul S. Chemical Immunology: Catalytic Antibodies. 77. S. Karger GmbH, Basel, Switzerland2000: 1-17Google Scholar) and alloimmune disease (2Lacroix-Desmazes S. Moreau A. Sooryanarayana-Bonnemain C. Stieltjes N. Pashov A. Sultan Y. Hoebeke J. Kazatchkine M.D. Kaveri S.V. Nat. Med. 1999; 5: 1044-1047Crossref PubMed Scopus (179) Google Scholar). Specific monoclonal Abs and Ab light chain subunits displaying proteolytic activities can be raised by routine immunization with polypeptides (3Paul S. Sun M. Mody R. Tewary H.K. Stemmer P. Massey R.J. Gianferrara T. Mehrotra S. Dreyer T. Meldal M. Tramontano A. J. Biol. Chem. 1992; 267: 13142-13145Abstract Full Text PDF PubMed Google Scholar, 4Hifumi E. Okamoto Y. Uda T. J. Biosci. Bioengin. 1999; 88: 323-327Crossref PubMed Scopus (30) Google Scholar). Under ordinary circumstances, however, adaptive maturation of the catalytic activity may not be a favored event. B cell clonal selection occurs by sequence diversification of genes encoding the Ab variable domains followed by selective binding of the antigen to cell surface Abs with the greatest affinity, which drives proliferation of the B cells (5Nossal G.J. Immunol. Rev. 2002; 185: 15-23Crossref PubMed Scopus (9) Google Scholar). Catalysis entails chemical transformation of the antigen and release of products from the Ab, which may cause cessation of B cell proliferation when the catalytic rate exceeds the rate of transmembrane signaling necessary to stimulate cell proliferation. Originally developed as irreversible inhibitors of conventional serine proteases, haptenic phosphonate esters are reported to bind the nucleophilic sites of natural proteolytic Abs covalently (6Paul S. Tramontano A. Gololobov G. Zhou Y.X. Taguchi H. Karle S. Nishiyama Y. Planque S. George S. J. Biol. Chem. 2001; 276: 28314-28320Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 7Taguchi H. Burr G. Karle S. Planque S. Zhou Y.X. Paul S. Nishiyama Y. Bioorg. Med. Chem. Lett. 2002; 12: 3167-3170Crossref PubMed Scopus (13) Google Scholar). The haptenic phosphonates could potentially serve as covalently reactive analogs (CRAs) for inducing the synthesis of Abs with improved nucleophilicity. To the extent that Ab nucleophilicity is rate-limiting in proteolysis, its enhancement may permit more rapid peptide bond cleavage, i.e. if the subsequent steps in the catalytic reaction cycle (hydrolysis of the acyl-Ab complex and product release) do not pose significant energetic hurdles (see Fig. 1). The innate character of Ab nucleophilic reactivity is the central element of this approach, and there is no requirement for de novo formation of chemically reactive sites over the course of variable domain sequence diversification. Most previous attempts to program the structure of catalytic sites in Abs in comparison have relied on noncovalent stabilization of the oxyanionic transition state (i.e. by immunization with transition state analogs; Refs. 8Tramontano A. Janda K.D. Lerner R.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6736-6740Crossref PubMed Scopus (98) Google Scholar and 9Schultz P.G. Lerner R.A. Science. 1995; 269: 1835-1842Crossref PubMed Scopus (419) Google Scholar). An Ab with esterase activity (10Wagner J. Lerner R.A. Barbas III, C.F. Science. 1995; 270: 1797-1800Crossref PubMed Scopus (418) Google Scholar) and another with aldolase activity (11Zhou G.W. Guo J. Huang W. Fletterick R.J. Scanlan T.S. Science. 1994; 265: 1059-1064Crossref PubMed Scopus (190) Google Scholar) utilize covalent catalytic mechanisms, but the relationship of these activities to innate Ab nucleophilicity is unclear. An ideal antigen-specific proteolytic Ab may be conceived to combine traditional noncovalent binding interactions in the ground state of the Ab-antigen complex with nucleophilic attack on the peptide backbone. The ground state interactions are desirable to obtain specificity for individual polypeptide antigens. No impediments for catalysis are presented by the stable ground state complexes, provided the noncovalent interactions are carried over into the transition state complex and are properly coordinated with nucleophilic attack at the reaction center. In theory, synthesis of antigen-specific proteolytic Abs could be induced by an analog that presents a mimetic of the chemical reaction center in the context of classical antigenic epitopes available for noncovalent binding interactions. If the reaction proceeds by a lock-and-key stereochemical mechanism, the mimetic must be located precisely at the position of the intended scissile bond in the backbone of the polypeptide antigen. In the instance of large proteins, locating the mimetic within the protein backbone is outside the range of present-day synthetic technologies. A potential solution is to place the mimetic group at amino acid side chains using chemical linker techniques. An Ab nucleophile that recognizes the side chain mimetic could facilitate proteolysis if it enjoys sufficient conformational freedom to approach the polypeptide backbone of the substrate and form the acyl-Ab complex (see Fig. 1). We describe here the characteristics of Abs induced by a CRA of the HIV-1 coat protein gp120 (gp120-CRA) consisting of phosphonate diester groups located in Lys side chains of the protein. Enhanced serine protease-like nucleophilic reactivity of the Abs was observed. One monoclonal Ab cleaved gp120 slowly and specifically, it displayed preference for cleavage on the C-terminal side of Lys/Arg residues, and the catalytic reaction was susceptible to CRA inhibition. These findings are the first indications that Abs with proteolytic activity specific for individual proteins can be raised on demand. Hapten, gp120-CRAs, and Biotinylated Proteins—Synthesis of hapten CRAs I and II (see Fig. 1) and their characterization by electrospray ionization-mass spectroscopy and elemental analyses have been described previously (12Nishiyama Y. Taguchi H. Luo J.Q. Zhou Y.X. Burr G. Karle S. Paul S. Arch. Biochem. Biophys. 2002; 402: 281-288Crossref PubMed Scopus (28) Google Scholar). For preparation of gp120-CRA III, the precursor diphenyl-N-[O-(3-sulfosuccinimidyl)suberoyl]amino(4amidinophenyl)methanephosphonate (IV) was synthesized by mixing a solution of diphenylamino(4-amidinophenyl)methanephosphonate (79 mg, 0.13 mmol) in N,N-dimethylformamide (2 ml) containing N,N-diisopropylethylamine (0.11 ml, 0.63 mmol) and bis(sulfosuccinimidyl)suberate disodium salt (150 mg, 0.26 mmol; Pierce) for 2 h. IV was obtained by reversed-phase high performance liquid chromatography (12Nishiyama Y. Taguchi H. Luo J.Q. Zhou Y.X. Burr G. Karle S. Paul S. Arch. Biochem. Biophys. 2002; 402: 281-288Crossref PubMed Scopus (28) Google Scholar) and lyophilized to give a colorless powder (yield 54%, 50 mg; m/z 715 (MH+) by electrospray ionization mass spectroscopy). IV (1.1 mg) was reacted with electrophoretically pure gp120 (0.5 mg; Immunodiagnostic Inc., MN strain, purified from baculovirus expression system) in 5 ml of 10 mm HEPES, 25 mm NaCl, 0.1 mm CHAPS, pH 7.5 buffer (2 h, 25 °C). Excess IV was removed by gel filtration (Micro Bio-Spin 6 disposable column, Bio-Rad), and the concentration of free amines in the initial protein and CRA-derivitized protein was measured using fluorescamine (13Udenfriend S. Stein S. Bohlen P. Dairman W. Leimgruber W. Weigele M. Science. 1972; 178: 871-872Crossref PubMed Scopus (2193) Google Scholar). The density of labeling was varied as needed from 4.0 to 32.6 mol of CRA/mol of gp120 by varying the concentration of IV. Preparation of gp120 labeled at Lys residues with biotin (Bt-gp120) was by similar means using 6-biotinamidohexanoic acid N-hydroxysuccinimide ester (Sigma). The reaction time and reactant concentrations were controlled to yield biotin/gp120 molar ratios 0.8–1.9. Unreacted biotinylation reagent was removed using a disposable gel filtration column in 50 mm Tris-HCl, 100 mm glycine, 0.1 mm CHAPS, pH 7.8. The biotin content was determined using 2-(4′-hydroxyazobenzene)benzoic acid (14Green N.M. Biochem. J. 1965; 94: 23-24Crossref PubMed Google Scholar). Total protein measurements were done using the BCA method (Pierce kit). Biotinylated III was prepared from Bt-gp120 as described for III. With increasing incorporation of the hapten groups, biotinylated III tended to form dimers and trimers evident in SDS electrophoresis gels as bands at ∼ 240 and 380 kDa (nominal mass of monomer gp120, 120 kDa). Biotinylated III at hapten density similar to the non-biotinylated III employed as immunogen (23 mol/mol of gp120) contained the monomer, dimer, and trimer species at proportions of 50, 21, and 29%, respectively. Protein-CRAs were lyophilized and stored at -20 °C until used. Bt-gp120 was stored at -70 °C in 50 mm Tris-HCl, pH 8.0, 0.1 m glycine, 0.1 mm CHAPS. Storage of I and II was at -70 °C as 10 mm solutions in N,N-dimethylformamide. The extracellular domain of EGFR (exEGFR) obtained from Dr. Maureen O'Connor (15Brown P.M. Debanne M.T. Grothe S. Bergsma D. Caron M. Kay C. O'Connor-McCourt M.D. Eur. J. Biochem. 1994; 225: 223-233Crossref PubMed Scopus (56) Google Scholar) was biotinylated as described for gp120 (0.9 mol of biotin/mol of exEGFR). Antibodies—mAbs were prepared from female MRL/MpJ-Faslpr mice (The Jackson Laboratory, Bar Harbor, ME; 4–5 weeks) immunized with gp120-CRA III (23 mol of phosphonate diester/mol of gp120). The mice were injected intraperitoneally on days 0, 14, and 28 days with gp120-CRA III (11 μg) in Ribi adjuvant (monophosphoryl lipid A + trehalose dicorynomycolate emulsion; Sigma) followed by a fourth intravenous booster without adjuvant on day 55. Blood was obtained from the retroorbital plexus over the course of the immunization schedule. Three days after the final injection, hybridomas were prepared by fusion of splenocytes with myeloma cell line (NS-1; Ref. 3Paul S. Sun M. Mody R. Tewary H.K. Stemmer P. Massey R.J. Gianferrara T. Mehrotra S. Dreyer T. Meldal M. Tramontano A. J. Biol. Chem. 1992; 267: 13142-13145Abstract Full Text PDF PubMed Google Scholar). After identification of wells secreting the desired Abs by ELISA, monoclonal cell lines were prepared by two rounds of cloning by limiting dilution. Monoclonal IgG was prepared from tissue culture supernatants containing mAbs (200 ml) by affinity chromatography on immobilized protein G (3Paul S. Sun M. Mody R. Tewary H.K. Stemmer P. Massey R.J. Gianferrara T. Mehrotra S. Dreyer T. Meldal M. Tramontano A. J. Biol. Chem. 1992; 267: 13142-13145Abstract Full Text PDF PubMed Google Scholar). Control mAbs (anti-VIP clone c23.5 and anti-yellow fever virus antigen clone CRL 1689; ATCC) and serum IgG were purified similarly. The IgG preparations were electrophoretically homogeneous, determined by silver staining of overloaded IgG and immunoblotting with specific Abs to mouse IgG (3Paul S. Sun M. Mody R. Tewary H.K. Stemmer P. Massey R.J. Gianferrara T. Mehrotra S. Dreyer T. Meldal M. Tramontano A. J. Biol. Chem. 1992; 267: 13142-13145Abstract Full Text PDF PubMed Google Scholar). Additional immunizations of female BALB/c mice (Jackson; 4–5 weeks) with gp120 or gp120-CRA were carried out similarly. mAb heavy and light chain isotypes were determined by ELISA as described (3Paul S. Sun M. Mody R. Tewary H.K. Stemmer P. Massey R.J. Gianferrara T. Mehrotra S. Dreyer T. Meldal M. Tramontano A. J. Biol. Chem. 1992; 267: 13142-13145Abstract Full Text PDF PubMed Google Scholar). ELISA—Maxisorp 96-well microtiter plates (Nunc) were coated with gp120 or gp120-CRA (40–100 ng/well) in 100 mm bicarbonate buffer, pH 8.6. Routine ELISAs were carried out as described (16Karle S. Nishiyama Y. Zhou Y.X. Luo J. Planque S. Hanson C. Paul S. Vaccine. 2003; 21: 1213-1218Crossref PubMed Scopus (21) Google Scholar). For assay of irreversible binding, the Abs were allowed to bind the plates, and the wells were treated for 30 min with 2% SDS in 10 mm sodium phosphate, 137 mm NaCl, 2.7 mm KCl, 0.05% Tween 20, pH 7.4 (PBS-Tween) or PBS-Tween without SDS (control wells for measurement of total binding). The wells were then washed three times with PBS-Tween, and bound IgG was determined as usual using a peroxidase conjugate of goat anti-mouse IgG (Fc-specific; Sigma). Observed values of binding were corrected for nonspecific binding in wells containing nonimmune IgG or nonimmune mouse serum (A490 < 0.03). Percent residual binding in SDS-treated wells was computed as (A490, SDS-treated wells) × 100/(A490, PBS-Tween-treated wells). Electrophoresis of Ab-CRA Complexes—Irreversible binding of biotinylated CRAs by purified IgG was determined by denaturing electrophoresis (6Paul S. Tramontano A. Gololobov G. Zhou Y.X. Taguchi H. Karle S. Nishiyama Y. Planque S. George S. J. Biol. Chem. 2001; 276: 28314-28320Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Briefly, the reaction mixtures were incubated at 37 °C in 50 mm Tris-HCl, 0.1 m glycine, pH 8.0. SDS was added to 2%, and the mixtures were boiled (5 min) and then subjected to SDS-PAGE (4–20%, Bio-Rad, or 8–25% Phast gels, Amersham Biosciences). After electro-blotting onto nitrocellulose membranes (0.22 μm, Bio-Rad), the membranes were blocked with 5% skim milk in PBS-Tween and processed for detection of IgG or biotin using peroxidase-conjugated goat anti-mouse IgG (Sigma) or peroxidase-conjugated streptavidin, respectively. Imaging and quantification were using x-ray film (Eastman Kodak Co.) with Unscan-it software (Silk scientific, Orem, UT) or a Fluoro-STM MultiImager (Bio-Rad). Biotinylated bovine serum albumin (11 mol of biotin/mol of bovine serum albumin; Sigma) was employed to construct a standard curve (0.06–1.5 pmol of biotin/lane). Hydrolysis Assays—Biotinylated proteins were incubated with IgG in 50 mm Tris-HCl, 0.1 m glycine, 0.1 mm CHAPS, pH 8, at 37 °C, the reaction was terminated by addition of SDS to 2%, and the samples were boiled (5 min) and then analyzed by reducing SDS-gel electrophoresis (4–20%, Bio-Rad). Biotin containing protein bands in blots of the gel were identified and quantified as in the preceding section. In some blots, reaction products were identified by immunoblotting using peroxidase-conjugated goat anti-gp120 Abs (Fitzgerald, Concord, MA; catalog #60-H14) (16Karle S. Nishiyama Y. Zhou Y.X. Luo J. Planque S. Hanson C. Paul S. Vaccine. 2003; 21: 1213-1218Crossref PubMed Scopus (21) Google Scholar). N-terminal sequencing of protein bands from electrophoresis gels was done as described previously (17Sun M. Gao Q.S. Kirnarskiy L. Rees A. Paul S. J. Mol. Biol. 1997; 271: 374-385Crossref PubMed Scopus (62) Google Scholar). 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; λex 360 nm, λem 470 nm) with authentic aminomethylcoumarin as standard (6Paul S. Tramontano A. Gololobov G. Zhou Y.X. Taguchi H. Karle S. Nishiyama Y. Planque S. George S. J. Biol. Chem. 2001; 276: 28314-28320Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Cleavage of [Tyr10-125I]VIP by mAb c23.5 was measured as the radioactivity rendered soluble in trichloroacetic acid (17Sun M. Gao Q.S. Kirnarskiy L. Rees A. Paul S. J. Mol. Biol. 1997; 271: 374-385Crossref PubMed Scopus (62) Google Scholar). Kinetic parameters for cleavage of increasing concentrations of peptide-MCA substrates were determined from the Michaelis-Menten equation, v = (Vmax[S])/(Km + [S]). Because of the expense of studying gp120 cleavage at large concentrations of the protein, Kd (∼Km) and kcat for this reaction were obtained from the general quadratic equation (17Sun M. Gao Q.S. Kirnarskiy L. Rees A. Paul S. J. Mol. Biol. 1997; 271: 374-385Crossref PubMed Scopus (62) Google Scholar) [CS]2 - [CS] ([Ct] + [St] + Kd) + [Ct] [SCt] = 0, where [Ct] and [St] are the total concentrations of catalyst and substrate, and [CS] is the catalyst-substrate concentration. The method consists of calculation of [CS] at a series of assumed Kd values. The assumed Kd value yielding the best fit (by linear regression) between the observed reaction velocity and [CS] represents the experimentally determined Kd. kcat is computed as the slope of the observed velocity versus [CS] plot. gp120-CRA Design and Validation—Synthesis of hapten CRAs I and II (Fig. 1) and their covalent reactivity with naturally occurring proteolytic Abs has been described previously (6Paul S. Tramontano A. Gololobov G. Zhou Y.X. Taguchi H. Karle S. Nishiyama Y. Planque S. George S. J. Biol. Chem. 2001; 276: 28314-28320Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 7Taguchi H. Burr G. Karle S. Planque S. Zhou Y.X. Paul S. Nishiyama Y. Bioorg. Med. Chem. Lett. 2002; 12: 3167-3170Crossref PubMed Scopus (13) Google Scholar). 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 (6Paul S. Tramontano A. Gololobov G. Zhou Y.X. Taguchi H. Karle S. Nishiyama Y. Planque S. George S. J. Biol. Chem. 2001; 276: 28314-28320Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), 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. Multiple phosphonate diester groups were available per molecule of gp120, allowing presentation of the electrophilic hapten in conjunction with diverse antigenic epitopes. Robust polyclonal Ab responses in MRL/lpr and BALB/c mice immunized with III were observed by routine ELISA. Abs raised to III were bound at somewhat greater levels by immobilized III than control gp120 devoid of phosphonate diester groups (Fig. 2). Conversely, Abs raised to control gp120 recognized immobilized III, but the binding was 3–4-fold lower than by immobilized gp120 (e.g. at serum dilution of 1:1000, A490 0.44 ± 0.03 for immobilized III and 1.40 ± 0.03 for immobilized gp120). III binding by nonimmune Abs was negligible, indicating that indiscriminate covalent binding at the hapten groups was not a problem. The observed differences in the antigenic reactivity of gp120 and III were held to be sufficiently small to proceed with further Ab studies. To facilitate high throughput screening, the feasibility of measuring irreversible III binding by Abs was studied by ELISA. After binding of polyclonal Abs anti-III Abs to the immobilized antigens, ELISA plates were treated with the denaturant SDS to remove reversibly bound Abs. SDS treatment allowed essentially complete removal of anti-III Abs bound by control gp120 devoid of hapten phosphonate groups. In comparison, 13–40% of the overall anti-III Ab binding activity consistently remained bound to immobilized III after SDS treatment in three repeat experiments. SDS-electrophoresis and immunoblotting with Abs to mouse IgG confirmed formation of irreversible Ab-III complexes in boiled reaction mixtures (Fig. 2B, inset, lane 3, estimated mass from extrapolated standard curve of molecular mass standards, ∼400 kDa; large complexes can be formed by binding of multiple Abs to hapten groups in III). Catalytic Activity—mAbs were prepared from MRL/lpr mice immunized with gp120-CRA III. This mouse strain develops lupus-like autoimmune disease attributable to the dysfunctional Fas-receptor gene. Spontaneous development of proteolytic Abs (18Bangale Y. Karle S. Zhou Y.X. Lan L. Kalaga R. Paul S. FASEB J. 2003; 17: 628-635Crossref PubMed Scopus (36) Google Scholar) and increased synthesis of esterase Abs in response to immunization with phosphonate monoester haptens (19Tawfik D.S. Chap R. Green B.S. Sela M. Eshhar Z. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2145-2149Crossref PubMed Scopus (92) Google Scholar, 20Sun J. Takahashi N. Kakinuma H. Nishi Y. J. Immunol. 2001; 167: 5775-5785Crossref PubMed Scopus (8) Google Scholar) have been reported in this mouse strain. Supernatants from 712 hybridoma wells (two splenocyte-myeloma cell fusions) were screened for SDS-resistant binding to III. IgG from seven wells was positive for this activity. After cloning of the cells by limiting dilution, monoclonal IgG from the supernatants of the seven cell lines was purified, and the binding assays were repeated (Fig. 3; clones YZ18, IgG2a,κ; YZ19, IgG2b,κ; YZ20, IgG2a,κ; YZ21, IgG2a,κ; YZ22, IgG2a,κ; YZ23, IgG2a,κ, and YZ24, IgG1,κ). Of total binding observed without SDS treatment of the ELISA plates, residual binding after the detergent treatment was 43–83% in 4 repeat assays. All seven mAbs were also bound by gp120 devoid of hapten CRA groups determined by routine ELISA without SDS treatment, indicating that they are not directed to neoepitopes generated by chemical modification procedures used for III preparation. An irrelevant mAb (clone CRL 1689) displayed no detectable binding of III or gp120. Of seven mAbs with irreversible III binding activity, slow cleavage of Bt-gp120 by three mAbs was detected (YZ18, YZ20, YZ24), determined by the appearance of biotin-containing fragments of the protein in SDS-electrophoresis gels. The electrophoretic pattern of Bt-gp120 cleaved by mAbs YZ18 and YZ24 were similar to that shown for mAb YZ20 in Fig. 4. mAb YZ20 was further studied as it cleaved Bt-gp120 ∼5-fold more rapidly than the other two mAbs. The consumption of gp120 was time-dependent (Fig. 4A). Major biotin-containing cleavage products with apparent mass 55 and 50 kDa were observed along with less intensely stained bands at 27 and 15 kDa. A band at 35 kDa was visible in overexposed gels, but this does not represent a product of mAb cleavage, as it was present at similar density in control incubations of Bt-gp120 in diluent. A control-irrelevant mAb (clone CRL 1689) did not cleave Bt-gp120. Immunoblotting using polyclonal anti-gp120 Abs confirmed that non-biotinylated gp120 is also susceptible to cleavage by the mAb (55-kDa cleavage product, Fig. 4B). Both detection methods allow quantification of gp120 cleavage by measuring depletion of intact gp120. Neither method provides guidance about the complete product profile or product concentration, because Bt-gp120 contains minimal amounts of biotin (∼1 mol/molgp120), and the polyclonal Abs used for immunoblotting do not react equivalently with the cleavage products. mAb YZ20 did not cleave biotinylated bovine serum albumin or the extracellular domain of the epidermal growth factor (exEGFR), indicating selectivity for gp120 (Fig. 5A). Attempts to identify the bonds cleaved by mAb YZ20 were unsuccessful. N-terminal sequencing of the 55- and 50-kDa bands yielded identical sequences (TEKLWVTVYY), corresponding to the N-terminal residues of gp120. Sequencing of the 15-kDa band from the YZ20 reaction mixture did not yield detectable phenylthiohydantoin derivatives of amino acids, possibly because of a blocked N terminus. Identification of the 27-kDa gp120 fragment is complicated because of its comigration with the Ab light chain in reducing gels. Because identification of the precise bonds in gp120 cleaved by the mAb was not central to the present study, we turned to the use of model peptide substrates for determination of scissile bond preferences. A fluorimetric assay was employed to determine mAb-catalyzed cleavage of the amide bond linking aminomethylcoumarin to the C-terminal amino acid in a panel of peptide-MCA substrates (Fig. 5B). The peptide-MCA substrates were used at excess concentration (200 μm), permitting detection of even weakly cross-reactive catalytic Abs. Selective cleavage at Arg-MCA and Lys-MCA was observed, with no evident cleavage on the C-terminal side of neutral or acidic residues. To confirm that the rate differences are because of recognition of the basic residue at the cleavage site (as opposed to remote residues), we studied two tripeptide substrates identical in sequence except for the N-terminal residue at the scissile bond, Gly-Gly-Arg-MCA and Gly-Gly-Leu-MCA. The former substrate was cleaved at detectable levels by Ab YZ20 (0.31 ± 0.01 (S.D.) μm aminomethylcoumarin/19 h/μm IgG), whereas the fluorescence intensity in reaction mixtures of the latter substrate and the Ab was statistically indistinguishable from background values observed in assay diluent (0.02 ± 0.04 μm MCA/19 h/μm IgG; p > 0.05; student's t test, unpaired; Fig. 5C). The basic residue preference is consistent with the presence of positively charged amidino groups neighboring the phosphonate groups in the immunogen (III) and selective cleavage on the C-terminal side of Arg/Lys residues by germ line-encoded proteolytic Abs observed previously (21Kalaga R. Li L. O'Dell J.R. Paul S. J. Immunol. 1995; 155: 2695-2702PubMed Google Scholar, 22Gololobov G. Sun M. Paul S. Mol. Immunol. 1999; 36: 1215-1222Crossref PubMed Scopus (51) Google Scholar). Attainment of the desired catalytic properties, i.e. the ability to combine high affinity for individual antigens with rapid turnover, can be judged from the Km and kcat parameters (mol of antigen cleaved/mol of Ab/unit time). The Km of mAb YZ20 for Bt-gp120 was about 200-fold smaller than its preferred peptide-MCA substrate (EAR-MCA; Table I; single letter