Broadly Distributed Chemical Reactivity of Natural Antibodies Expressed in Coordination with Specific Antigen Binding Activity
2003; Elsevier BV; Volume: 278; Issue: 22 Linguagem: Inglês
10.1074/jbc.m301468200
ISSN1083-351X
AutoresStephanie Planque, Hiroaki Taguchi, Gary S. Burr, Gita Bhatia, Sangeeta Karle, Yong-Xin Zhou, Yasuhiro Nishiyama, Sudhir Paul,
Tópico(s)Immunodeficiency and Autoimmune Disorders
ResumoAntibody (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. 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 1The abbreviations used are: Ab, antibody; BSA, bovine serum albumin; HIV, human immunodeficiency virus; CRA, covalently reactive antigen analog; gp, glycoprotein; DFP, diisopropyl fluorophosphate; EGFR, epidermal growth factor receptor; exEGFR, extracellular domain of human EGFR; MCA, methylcoumarinamide; V domain, variable domain; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AAU, arbitrary area units; ELISA, enzyme-linked immunosorbent assay; Fv, fragment variable; scFv, single chain fragment variable.1The abbreviations used are: Ab, antibody; BSA, bovine serum albumin; HIV, human immunodeficiency virus; CRA, covalently reactive antigen analog; gp, glycoprotein; DFP, diisopropyl fluorophosphate; EGFR, epidermal growth factor receptor; exEGFR, extracellular domain of human EGFR; MCA, methylcoumarinamide; V domain, variable domain; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AAU, arbitrary area units; ELISA, enzyme-linked immunosorbent assay; Fv, fragment variable; scFv, single chain fragment variable. 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. 1Schultz P.G. Lerner R.A. Science. 1995; 269: 1835-1842Crossref PubMed Scopus (420) Google Scholar and 2Stewart J.D. Benkovic S.J. Nature. 1995; 375: 388-391Crossref PubMed Scopus (114) Google Scholar). 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 (3Paul S. Volle D.J. Beach C.M. Johnson D.R. Powell M.J. Massey R.J. Science. 1989; 244: 1158-1162Crossref PubMed Scopus (384) Google Scholar, 4Shuster A.M. Gololobov G.V. Kvashuk O.A. Bogomolova A.E. Smirnov I.V. Gabibov A.G. Science. 1992; 256: 665-667Crossref PubMed Scopus (358) Google Scholar). Similar activities were later found in Ab light chains from multiple myeloma patients (5Matsuura K. Sinohara H. Biol. Chem. 1996; 377: 587-589PubMed Google Scholar), alloantibodies from patients with transfusion-induced hemophilia (6Lacroix-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), Abs raised by routine immunization with polypeptides (7Paul 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, 8Hifumi E. Okamoto Y. Uda T. J. Biosci. Bioengin. 1999; 88: 323-327Crossref PubMed Scopus (30) Google Scholar), and anti-idiotypic Abs to anti-enzyme Abs (9Izadyar L. Friboulet A. Remy M.H. Roseto A. Thomas D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8876-8880Crossref PubMed Scopus (146) Google Scholar). 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 (10Gao Q.-S. Sun M. Rees A. Paul S. J. Mol. Biol. 1995; 253: 658-664Crossref PubMed Scopus (86) Google Scholar, 11Paul 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). The catalytic activity of natural Abs could be construed to violate the principles of B cell clonal selection theory. Antigen-specific Abs develop over the course of the immune response by sequence diversification of germ line genes encoding the V domains followed by selective antigen binding by B cell receptors with the greatest affinity, which stimulates clonal proliferation. Catalysis entails chemical transformation of the antigen and release of products (surface immunoglobulins associated with Igα and Igβ subunits), which is predicted to result in the cessation of B cell selection. Therefore, adaptive selection of Ab catalytic activity may be a disfavored event. For this reason, catalysis by naturally occurring Abs is often assumed to be a molecular accident arising from sequence variability of the V domains as opposed to a general phenomenon with functional implications. The foregoing restrictions do not apply to the initial step in the catalytic cycle of serine proteases. In analogy with conventional enzymes, a nucleophile belonging to a proteolytic Ab (Fig 1A, Nu) is conceived to initiate nucleophilic attack on the antigen following the formation of the non-covalent ground state complex. Adaptive development of Ab nucleophilicity is fully compatible with B cell clonal selection if the outcome is the formation of a covalent acyl-Ab complex as occupancy of the B cell receptor will be maintained. Whether the catalytic cycle is completed depends on the efficiency of hydrolysis of the acyl-Ab complex and release of the product. Recently, hapten phosphonate esters have been developed as probes for covalent binding to the active site nucleophiles in Abs displaying serine protease and serine esterase activity (11Paul 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, 12Kolesnikov A.V. Kozyr A.V. Alexandrova E.S. Koralewski F. Demin A.V. Titov M.I. Avalle B. Tramontano A. Paul S. Thomas D. Gabibov A.G. Friboulet A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13526-13531Crossref PubMed Scopus (106) Google Scholar) (designated CRAs). These compounds can be applied for direct study of Ab chemical activity independent of additional activities needed for accomplishment of catalysis. In addition, the phosphonates can be placed within peptides and proteins (Fig. 1, B and C) for studying the interplay between Ab nucleophilic reactivity and non-covalent forces permitting specific recognition of individual polypeptides. We describe here observations suggesting the broad distribution of nucleophilic reactivity in IgG and recombinant Fv preparations at levels exceeding that of the conventional serine protease trypsin. Originally prepared as probes for catalytic Abs, CRA analogs of EGFR and an HIV gp120-synthetic peptide were observed to form covalent adducts with ordinary Abs raised by immunization with antigens devoid of the phosphonate groups, suggesting that adaptive maturation processes favor the expression of nucleophilic reactivity. These observations argue for Ab nucleophilicity as a force responsible for shaping the expressed Ab repertoire and suggest novel routes toward permanent inactivation of Abs. Abs—Human polyclonal IgG was prepared by affinity chromatography on protein G-Sepharose (Amersham Biosciences) from the sera of six healthy human subjects (laboratory codes 1086, 1087, 1088, 1091, 1092, and 1518). IgG from pooled serum from eight BALB/c mice (4–5 weeks) was obtained similarly. Preparation of polyclonal Abs by hyperimmunization with synthetic Cys-gp120-(421– 436) (KQIINMWQEVGKAMYA, residues 421– 436 of gp120 HIV SF2 strain) conjugated to keyhole limpet hemocyanin is described by Karle et al. (13Karle S. Nishiyama Y. Zhou Y.-X. Luo J. Planque S. Hanson C. Paul S. Vaccine. 2003; 21: 1213-1218Crossref PubMed Scopus (21) Google Scholar). Polyclonal Abs to exEGFR were raised by immunizing female BALB/c mice (5– 6 weeks) intraperitoneally with exEGFR (10 μg/injection) on days 0, 27, and 41 in RIBI™ adjuvant and with A431 tumor cells (107 cells in saline) on day 14. Monoclonal Abs to exEGFR (clones C225, H11, and C111.6) were purchased from Labvision (Fremont, CA). A control monoclonal anti-BSA IgG (clone BGN/H8) was from Biogenesis (Kingston, NH). Single chain Fv constructs (n = 15) were picked randomly from a human Fv library derived from lupus patients as described by Paul et al. (11Paul 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) (MM series clones: 12, 14, 18, 20, 24, F1, F2, F4, F5, F6, F7, F11, F12, F14, F17, and F18). The scFv proteins were purified to electrophoretic homogeneity (27-kDa band) by metal affinity chromatography on nickel-nitrilotriacetic acid columns (11Paul 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). Expression levels were 0.3–5.7 mg/liter bacterial culture. The library contains diverse scFv clones determined by nucleotide sequencing (11Paul 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), assuring a broad sampling of Ab V domains. One of the scFv clones examined in this study, MM-F4, was sequenced (GenBank™ accession number AF522073), the V domains of the light and heavy chains were determined to belong to families X and I, respectively, and the germ line gene counterparts were V1–13 and VH1–2, respectively. Confirmation of scFv band identities in SDS-electrophoresis gels was by immunoblotting using a monoclonal Ab to c-Myc (10Gao Q.-S. Sun M. Rees A. Paul S. J. Mol. Biol. 1995; 253: 658-664Crossref PubMed Scopus (86) Google Scholar). Probes for Nucleophiles—Synthesis of hapten CRA I (Fig. 1) and its covalent reactivity with naturally occurring proteolytic Abs has been described previously (11Paul 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, 14Nishiyama 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). The electrophilic phosphonate diester mimics the peptide bond, the positively charged amidino group mimics the Lys/Arg P1 preference of naturally occurring proteolytic Abs (11Paul 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 permits sensitive detection of Ab phosphonate adducts. CRA II was prepared by condensation of diphenylamino(phenyl)methanephosphonate (compound a) and 6-biotinamidohexanoic acid-N-hydroxysuccinimide ester (Sigma) as described for CRA I. For the preparation of CRA III, compound a (160 mg, 0.34 mmol) was treated with 30% hydrogen bromide/CH3COOH (5 ml). The resulting diphenylamino(phenyl)methanephosphonate hydrobromide (100 mg, 0.24 mmol) was dissolved in 0.5 m sodium methoxide in methanol (9.5 ml), and the solution was stirred under N2 (room temperature, 2 h). After removing solvent under reduced pressure, the residue was extracted with CH2Cl2 (50 ml) and the extract was washed with water (5 ml × 3), dried over Na2SO4, and evaporated to dryness. The yellowish oily residue was dissolved in diethyl ether (30 ml). HCl (1 m) in diethyl ether (0.25 ml) was added, yielding a precipitate that was collected by filtration and washed with diethyl ether (yield 35 mg, 68%; tR 11.8 min; >97% purity; C18 column, 5– 80% acetonitrile in 0.1% trifluoroacetic acid, 50 min, 1.0 ml/min; m/z by electrospray ionization mass spectroscopy 216 (MH+)). Biotinylation of this compound was done as usual (14Nishiyama 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). To prepare CRA IV, diphenyl-N-[O-(3-sulfosuccinimidyl)suberoyl]-amino(4amidinophenyl)methane phosphonate (compound b) was first synthesized by mixing diphenylamino(4-amidinophenyl)methanephosphonate (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. Compound b was purified by reversed-phase high pressure liquid chromatography and lyophilized to give a colorless powder (yield 54%, 50 mg; m/z 715 (MH+)). Electrophoretically pure exEGFR (0.5 mg, kindly provided by Dr. O'Connor-McCourt) (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 reacted with 6-biotinamidohexanoic acid N-hydroxysuccinimide ester (59 nmol, Sigma) in 0.53 ml of 10 mm HEPES, 150 mm NaCl, 0.1 mm CHAPS, pH 7.5, buffer (50 min, 25 °C). Unreacted biotinylation reagent was removed by gel filtration (Micro Bio-Spin 6 column, Bio-Rad). Biotinylated exEGFR (0.33 mg) was then reacted with compound b (136 nmol) in 3.3-ml buffer for 2 h. Following the removal of excess compound b by gel filtration in 50 mm Tris-HCl, 100 mm glycine, 0.1 mm CHAPS, pH 7.8, the concentration of free amines in the initial and CRA-derivitized proteins was measured using fluorescamine (16Udenfriend S. Stein S. Bohlen P. Dairman W. Leimgruber W. Wegele M. Science. 1972; 178: 871-872Crossref PubMed Scopus (2194) Google Scholar). Biotin content determined using 2-(4′-hydroxyazobenzene)benzoic acid (17Green N.M. Biochem. J. 1965; 94: 23C-24CCrossref PubMed Google Scholar) was 1.1 mol/mol exEGFR. The density of phosphonate diester labeling was 19 mol/mol exEGFR. Total protein was measured using BCA (Pierce). Some experiments were done using exEGFR CRA IVa. This compound is identical to CRA IV but for the presence of a disulfide bond in linker. To prepare CRA IVa, the precursor diphenyl-N-((3-sulfosuccinimidyl)-3,3′-dithiobispropionyl)amino-(4-amidinophenyl)methanephosphonate (compound c) was obtained as described for compound b using 3,3′-dithiobis(sulfosuccinimidyl-propionate) (Pierce) (yield 6.0 mg, 21.4%; tR 24.49 min, >98 purity; 20–50% acetonitrile in 0.1% trifluoroacetic acid, 60 min); m/z 751 (MH+)). Labeling with biotin and compound c was as described for CRA IV (biotin and phosphonate diester content of CRA IVa, respectively, 2.3 mol and 18.3 mol/mol exEGFR). Synthesis of peptidyl-CRAs V and Va and their chemical characterization are described by Taguchi et al. (18Taguchi 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). CRA V was conjugated with BSA using γ-maleimidobutyric acid N-hydroxysuccinimide ester as described previously (13Karle S. Nishiyama Y. Zhou Y.-X. Luo J. Planque S. Hanson C. Paul S. Vaccine. 2003; 21: 1213-1218Crossref PubMed Scopus (21) Google Scholar). BSA was pre-treated with diphenyl-N-(benzyloxycarbonyl)amino(4-amidinophenyl)-methanephosphonate (BSA, 21.3 μm; phosphonate, 0.5 mm; solvent, 10 mm PBS containing 5% Me2SO; 15.5 h) to block potential CRA V binding sites. CRA V/BSA molar ratio was 3.9 determined from consumption of -SH groups using Ellman's reagent. The storage of CRAs I-III was at -70 °C as 10 mm solutions in N,N-dimethylformamide. CRAs IV and IVa were stored at -70 °C in 50 mm Tris-HCl, pH 8.0, 0.1 m glycine, 0.1 mm CHAPS. CRAs V and Va were stored at -70 °C as 10 mm solutions in N,N-dimethylformamide. ELISA—Maxisorp 96-well microtiter plates (Nunc) were coated with gp120-(421– 436) conjugated to BSA (20-ng peptide equivalent/well (for details regarding peptide conjugation method see Ref. 13Karle S. Nishiyama Y. Zhou Y.-X. Luo J. Planque S. Hanson C. Paul S. Vaccine. 2003; 21: 1213-1218Crossref PubMed Scopus (21) Google Scholar). CRA V was conjugated to BSA (20 ng of peptide-CRA equivalent/well), exEGFR (200 ng/well), or exEGFR-CRA V (200 ng of protein equivalent/well) in 100 mm sodium bicarbonate buffer, pH 8.6, for 2 h. ELISA procedures were essentially as described previously (13Karle S. Nishiyama Y. Zhou Y.-X. Luo J. Planque S. Hanson C. Paul S. Vaccine. 2003; 21: 1213-1218Crossref PubMed Scopus (21) Google Scholar). Bound murine IgG was detected with goat anti-mouse IgG-horseradish peroxidase conjugate (1:1000, specific for the constant domain of the heavy chain, Sigma). Irreversible CRA Binding—Following incubation of biotinylated CRAs with Abs or trypsin (porcine, type IX, Sigma) in 50 mm Tris, HCl, 100 mm glycine, 0.1 mm CHAPS, pH 7.7, at 37 °C, the reaction mixtures were boiled (5 min) in 2% SDS and subjected to SDS-PAGE (4–20%, Bio-Rad, or 8–25% Phast gels, Amersham Biosciences). Electroblotting and biotin detection procedures using streptavidin-horseradish peroxidase and a chemiluminescent substrate (Supersignal, Pierce) have been described previously (11Paul 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). Imaging and quantification was on x-ray film (Eastman Kodak Co.) using Unscan-it software (Silk Scientific, Orem, UT) or Fluoro-STM MultiImager (Bio-Rad). Band intensities are expressed in arbitrary area units (AAU). Valid comparisons of band intensities from different experiments is not possible because exposure and development times were not held constant. Diisopropyl fluorophosphate (DFP) (Sigma) was kept at 4 °C until used. In some experiments, biotinylated BSA (8 mol of biotin/mol protein, Pierce) was electrophoresed at several concentrations in parallel with the samples and the biotin content of the CRA adducts was determined. Pseudo-first order rate constants (kobs) were computed from reaction progress curves by fitting to the equation Bt = Bmax(1 - exp(-kobst)), where Bt represents adduct concentration at various times and Bmax represents the initial Ab concentration. Immunoblotting with goat anti-mouse IgG Abs was as described previously (7Paul 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). Proteolysis Assay—Catalytic activity was measured by fluorimetric determination (λex 360 nm, λem 470 nm; Varian Cary Eclipse) of the cleavage of amide bond linking aminomethylcoumarin to the C-terminal amino acid in short peptide-MCA substrates (Pro-Phe-Arg-MCA, Boc-Glu-Ala-Arg-MCA, and Boc-Ile-Glu-Ala-Arg-MCA (200 μm), Peptide International) (10Gao Q.-S. Sun M. Rees A. Paul S. J. Mol. Biol. 1995; 253: 658-664Crossref PubMed Scopus (86) Google Scholar). Catalysts were incubated with peptide-MCA substrates in 50 mm Tris HCl, 0.1 m glycine, 0.025% Tween 20, pH 8.0, at 37 °C in 96-well plates. In some assays, a comparison of IgG and trypsin proteolytic activity was done in 10 mm sodium phosphate, pH 7.4, 0.137 m NaCl, 2.7 mm KCl, 0.1 mm CHAPS. Authentic aminomethylcoumarin (Peptide International) was used to construct a standard curve from which product release was computed in molar values. Ab Nucleophilicity Identified with Hapten CRAs—Phosphonate hapten CRAs I-III (Fig. 1) are analogs of known active site-directed inhibitors of serine proteases (19Oleksyszyn J. Powers J.C. Methods Enzymol. 1994; 244: 423-441Crossref PubMed Scopus (93) Google Scholar). Similar to the serine protease trypsin, IgG from a healthy human subject formed adducts with CRA I that were resistant to boiling and the denaturant SDS (Fig. 2, IgG, 150-kDa adducts; trypsin, 21-kDa adducts). Pooled IgG from immunologically unmanipulated BALB/c mice formed similar CRA I adducts. The positively charged amidino group in CRA I was originally incorporated in this compound to allow selective recognition of trypsin, which displays preference for basic residues at the P1 site (the residue immediately adjacent to the cleavage site in peptide substrates) (for review see Ref. 20Oleksyszyn J. Boduszek B. Kam C.M. Powers J.C. J. Med. Chem. 1994; 37: 226-231Crossref PubMed Scopus (102) Google Scholar). CRA II lacks the positively charged amidino group adjacent to the covalently reactive phosphorus atom. IgG was 240-fold less reactive with CRA II than with CRA I, suggesting the trypsin-like P1 specificity of Abs. CRA III, which contains a weaker leaving group than CRA II did not form detectable adducts with IgG (the presence of methoxy-leaving groups reduces the electrophilicity of the phosphorus atom, and methoxy-containing phosphonate diesters are reported to bind weakly with certain serine proteases) (for review see Ref. 21Zhao Q. Kovach I.M. Bencsura A. Papathanassiu A. Biochemistry. 1994; 33: 8128-8138Crossref PubMed Scopus (33) Google Scholar). Increasing formation of covalent CRA I adducts with IgG and trypsin was evident as a function of reaction time (Fig. 2B). The velocity of the reaction for IgG was 14.5-fold greater than for trypsin measured under identical conditions (172.7 ± 14.2 and 11.9 ± 0.6 AAU/min, respectively, from linear regression of Fig. 2B data). Assuming that hydrolysis of the phosphonylated-protein complex is equivalent (see reaction schemes in Fig. 1), it may be concluded that the nucleophilic efficiency of IgG is superior to that of trypsin. IgG preparations from healthy humans and immunologically unmanipulated mice have been documented to cleave small model peptide substrates on the C-terminal side of basic residues. The cleavage activity was observed in each of several IgG preparations examined. The activity co-migrated with intact 150-kDa IgG in denaturing gel filtration studies, and it was expressed by Fab (fragment antigen binding) preparations made by papain digestion (22Kalaga R. Li L. O'Dell J.R. Paul S. J. Immunol. 1995; 155: 2695-2702PubMed Google Scholar). In this study, we compared the proteolytic activity of trypsin and IgG from a healthy human subject (the same preparation as in nucleophilicity studies illustrated in Fig. 2). With Glu-Ala-Arg-MCA and Pro-Phe-Arg-MCA substrates, initial rates of proteolysis by IgG were, respectively, 1.8 × 105- and 6.8 × 105-fold smaller than by trypsin (Fig. 3, A and B, determined from the slopes of the progress curves). Glu-Ala-Arg-MCA is the preferred substrate for trypsin. Glu-Ala-Arg-MCA and Pro-Phe-Arg-MCA are the preferred substrates for human IgG determined from previous screening of a panel of peptide-MCA substrates (22Kalaga R. Li L. O'Dell J.R. Paul S. J. Immunol. 1995; 155: 2695-2702PubMed Google Scholar). The magnitude of proteolysis by this IgG preparation falls within the range reported previously for other human IgG preparations. Despite its superior nucleophilic reactivity, the IgG is evidently a poor catalyst compared with trypsin. CRA I and DFP (another active site-directed inhibitor of serine proteases) inhibited the catalytic activity of IgG-catalyzed peptide-MCA cleavage (Fig. 3C), and DFP inhibited the irreversible binding of CRA I by the IgG (by 95%). These results provide assurance that CRA I binds the catalytic sites of IgG. As DFP binds the active site of serine proteases, its inhibitory effect confirms the serine protease character of the CRA I binding sites of IgG. Electrophoresis of CRA I-IgG adducts under reducing conditions revealed labeling of both subunits by the hapten CRA, evident as biotin-containing bands at 50-kDa heavy chain bands and 25-kDa light chain bands (Fig. 3D). Irreversible CRA I binding activity of IgG was lost by preheating the protein at 60 °C for 10 min, indicating the dependence of the nucleophilic reactivity on the native protein conformation. Each of the five polyclonal IgG preparations from healthy humans displayed irreversible binding to CRA I (Table I). Each of 16 randomly picked scFv clones from a human library formed CRA I-adducts (see example in Fig. 4A), indicating the V domain location of the binding site and suggesting that the nucleophilic reactivity is a shared property of diverse Abs. 91% of the total protein available in Fv MM-F4 shown in Fig 4A (GenBank™ accession number AF522073) displayed nucleophilic reactivity (computed as mol biotin/mol Fv protein in the 27-kDa CRA I adduct band; Fv valency 1; reaction conditions as shown in Fig. 4). Analyzed by electrophoresis under non-reducing conditions, some scFv reaction mixtures contained CRA I adducts at 55–90 kDa in addition to the monomer scFv adducts at 27 kDa. All of the CRA-adduct bands were also stainable with Ab to c-Myc, confirming the presence of scFv in the adducts (the recombinant proteins contain a 10-residue c-Myc peptide) (10Gao Q.-S. Sun M. Rees A. Paul S. J. Mol. Biol. 1995; 253: 658-664Crossref PubMed Scopus (86) Google Scholar). The tendency of scFv to form aggregates has been reported previously (23Whitlow M. Bell B.A. Feng S.L. Filpula D. Hardman K.D. Hubert S.L. Rollence M.L. Wood J.F. Schott M.E. Milenic D.E. Yokota T. Schlom J. Protein Eng. 1993; 6: 989-995Crossref PubMed Scopus (241) Google Scholar). Diminished levels of CRA I-adducts were detected when an scFv clone was treated with DFP prior to CRA I treatment (by 72%). The rate of covalent adduct formation by different Fv clones was variable over a 34-fold range (Table I), indicating distinct levels of nucleophilic reactivity of different Abs. The reactivity of the five polyclonal IgG samples, which represent mixtures of different Abs, was less variable (by 5.4). A comparison of the peptide-MCA cleaving activity (Glu-Ala-Arg-MCA substrate) and irreversible CRA I binding by the scFv clones indicated a strong correlation (p < 0.005, r2 = 0.77) (Fig. 4B), confirming the functional importance of superior nucleophilic reactivity.Table IBroad distribution of hapten I-irreversible binding by antibodiesAntibodyHapten I-irreversible binding, AAU × 103/μm proteinMean ± S.D.MedianRangeNHuman serumIgG33.7 ± 20.430.112.4-67.15scFv928 ± 688105055-190016 Open table in a new tab Specific Covalent Binding of Peptidyl and Protein CRA— Protein CRA IV and peptide CRA Va were analyzed to assess
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