Antigen-specific IgG Abs 1 in autoimmune and alloimmune disease are described to catalyze chemical reactions (1-3). Examples of catalytic Abs raised by routine experimental immunization with ordinary antigens have also been published (4 -7). However, no consensus has developed whether naturally occurring catalytic Abs represent rare accidents arising from adaptive sequence diversification processes or genuine enzymes with important functional roles. The major reason is that the turnover (k cat ) of antigen-specific IgG Abs is low. Some catalytic Abs express catalytic efficiencies (k cat /K m ) comparable to conventional enzymes, but this is due to high affinity recognition of the antigen ground state (reviewed in Ref. 8).Certain enzymes cleave peptide bonds by a mechanism involving the formation of a transient covalent intermediate of the substrate and a nucleophilic residue present in the active site. The nucleophiles are generated by intramolecular activation mechanisms, e.g. 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. Occupancy of the B cell receptor (BCR, surface Ig complexed to ␣ and ␤ subunits along with other signal transducing proteins) by the antigen drives B cell clonal selection. Proteolysis by the BCR is compatible with clonal selection, therefore, only to the extent that the release of antigen fragments is slower than the rate of antigeninduced transmembrane signaling necessary for induction of cell division. Immunization with haptens mimicking the charge characteristics of the transition state (12) has been suggested as a way to surmount the barrier to adaptive improvement of catalytic rate constants. Catalysis by designer IgG Abs obtained by these means, however, also proceeds only slowly.In mice and humans, the initial Ab repertoire consists of ϳ100 heritable VL and VH genes. Adaptive maturational processes expand the repertoire by several orders of magnitude. The initial BCR complex on the pre-B cell surface contains V-(D)-J rearranged Ig chains as a complex with surrogate L chains (reviewed in Ref. 13). Precise assignment of the B cell differentiation stage at which cell division becomes antigen-dependent is somewhat ambiguous, but it is generally believed that non-covalent antigen binding to the pre-BCR is not required for initial cell growth. / chains replace the surrogate L chain at the later stages of antigen-driven B cell differentiation, which is accompanied by diversification via somatic hypermutation processes and continued gene rearrangements (14,15). V-(D)-J gene ...

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...

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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 K m for this reaction was ϳ200-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.

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...

Antibody hydrolysis of the superantigenic gp120 site and HIV-1 neutralization was studied as a potential anti-HIV mechanism in uninfected humans. gp120 hydrolysis by purified serum and salivary antibodies was determined by electrophoresis and peptide sequencing, the proteolytic mechanism was analyzed using electrophilic peptide analogs, and viral neutralization was studied using peripheral blood mononuclear cells as hosts. Polyclonal and monoclonal IgA but not IgG preparations selectively catalyzed the cleavage of HIV gp120 at rates sufficient to predict biologically relevant protection against the virus. The IgA hydrolytic reaction proceeded by noncovalent recognition of gp120 residues 421-433, a component of the superantigenic site of gp120, coordinated with peptide bond cleavage via a serine protease-like mechanism. The Lys-432-Ala-433 bond was one of the cleavage sites. Infection of peripheral blood mononuclear cells by a primary isolate of HIV was neutralized by the IgA but not IgG fractions. The neutralizing activity was specifically inhibited by an electrophilic inhibitor of the catalytic activity. The existence of catalytic IgAs to gp120 in uninfected humans suggests their role in resistance to HIV.

Objective-To identify an HIV epitope suitable for vaccine development.Design-Diverse HIV-1 strains express few structurally constant regions on their surface vulnerable to neutralizing antibodies. The mostly-conserved CD4 binding site (CD4BS) of gp120 is essential for host cell binding and infection by the virus. Antibodies that recognize the CD4BS are rare, and one component of the CD4BS, the 421-433 peptide region, expresses B cell superantigenic character, a property predicted to impair the anti-CD4BS adaptive immune response.Methods-IgA samples purified from the plasma of subjects with HIV infection were analyzed for the ability to bind synthetic mimetics containing the 416-433 gp120 region and full-length gp120. Infection of peripheral blood mononuclear cells by clinical HIV isolates was measured by p24 ELISA.Results-IgA preparations from 3 subjects with subtype B infection for 19-21 years neutralized heterologous, coreceptor CCR5-dependent subtype A, B, C, D and AE strains with exceptional potency. The IgAs displayed specific binding of a synthetic 416-433 peptide mimetics dependent on recognition of the CD4 binding residues located in this region. Immunoadsorption, affinity chromatography and mutation procedures indicated that HIV neutralization occurred by IgA recognition of the CD4BS. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Following initial noncovalent binding of antigen, some Igs proceed to catalyze its chemical transformation. Examples of Ig-catalyzed reactions include hydrolysis of polypeptide antigens (2, 3), hydrolysis of nucleic acids (4, 5), and various acyl transfer reactions of other antigen classes (6). Proteolytic Igs that utilize serine protease-like covalent hydrolytic pathways have been described (7,8). Serine protease-like catalytic triads have been identified in the V domains of Igs by site-directed mutagenesis and crystallography (9, 10). The catalytic mechanism involves nucleophilic attack on the electrophilic carbonyl of peptide bonds. Electrophilic phosphonate diesters originally developed as covalent probes for the nucleophilic site of serine proteases bind catalytic Igs irreversibly and inhibit their catalytic activity (7,11,12). The strength of Igantigen noncovalent binding often exceeds that of enzyme-substrate binding. An important limitation holding back the application of catalytic Igs for clearance of undesirable antigens is that their catalytic rate constants (turnover number; k cat ) are small compared with enzymes. Evidently, Ig adaptive selection is geared toward noncovalent immune complexation (the ground state stabilization step), and the ability of Igs to recognize the high energy transition state complex that must be stabilized to accelerate chemical reactions is limited. This is supported by observations that IgMs, the first and least diversified Ig class produced during B cell differentiation, express superior catalytic rate constants than IgGs produced by the cells at later stages of their adaptive differentiation (12).Accumulation of amyloid ␤ peptide (A␤) 2 aggregates in the brain is thought to be a central contributor to neurodegenera-* This work was supported, in whole or in part, by National Institutes of Health Grant R01AG025304. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) FJ231714, FJ231715, FJ231716, FJ231717, FJ231718, FJ231719, FJ231720, FJ231721, FJ231722, and FJ231723