Loss of p53 activates thyroid hormone via type 2 deiodinase and enhances DNA damage.

The Thyroid Hormone (TH) activating enzyme, type 2 Deiodinase (D2), is functionally required to elevate the TH concentration during cancer progression to advanced stages. However, the mechanisms regulating D2 expression in cancer still remain poorly understood. Here, we show that the cell stress sensor and tumor suppressor p53 silences D2 expression, thereby lowering the intracellular THs availability. Conversely, even partial loss of p53 elevates D2/TH resulting in stimulation and increased fitness of tumor cells by boosting a significant transcriptional program leading to modulation of genes involved in DNA damage and repair and redox signaling. In vivo genetic deletion of D2 significantly reduces cancer progression and suggests that targeting THs may represent a general tool reducing invasiveness in p53-mutated neoplasms.

Epigenome Chaos: Stochastic and Deterministic DNA Methylation Events Drive Cancer Evolution.

Cancer evolution is associated with genomic instability and epigenetic alterations, which contribute to the inter and intra tumor heterogeneity, making genetic markers not accurate to monitor tumor evolution. Epigenetic changes, aberrant DNA methylation and modifications of chromatin proteins, determine the "epigenome chaos", which means that the changes of epigenetic traits are randomly generated, but strongly selected by deterministic events. Disordered changes of DNA methylation profiles are the hallmarks of all cancer types, but it is not clear if aberrant methylation is the cause or the consequence of cancer evolution. Critical points to address are the profound epigenetic intra- and inter-tumor heterogeneity and the nature of the heterogeneity of the methylation patterns in each single cell in the tumor population. To analyze the methylation heterogeneity of tumors, new technological and informatic tools have been developed. This review discusses the state of the art of DNA methylation analysis and new approaches to reduce or solve the complexity of methylated alleles in DNA or cell populations.

LSD1: more than demethylation of histone lysine residues.

Lysine-specific histone demethylase 1 (LSD1) represents the first example of an identified nuclear protein with histone demethylase activity. In particular, it plays a special role in the epigenetic regulation of gene expression, as it removes methyl groups from mono- and dimethylated lysine 4 and/or lysine 9 on histone H3 (H3K4me1/2 and H3K9me1/2), behaving as a repressor or activator of gene expression, respectively. Moreover, it has been recently found to demethylate monomethylated and dimethylated lysine 20 in histone H4 and to contribute to the balance of several other methylated lysine residues in histone H3 (i.e., H3K27, H3K36, and H3K79). Furthermore, in recent years, a plethora of nonhistone proteins have been detected as targets of LSD1 activity, suggesting that this demethylase is a fundamental player in the regulation of multiple pathways triggered in several cellular processes, including cancer progression. In this review, we analyze the molecular mechanism by which LSD1 displays its dual effect on gene expression (related to the specific lysine target), placing final emphasis on the use of pharmacological inhibitors of its activity in future clinical studies to fight cancer.

Targeted DNA oxidation by LSD1-SMAD2/3 primes TGF-ß1/ EMT genes for activation or repression.

The epithelial-to-mesenchymal transition (EMT) is a complex transcriptional program induced by transforming growth factor ß1 (TGF-ß1). Histone lysine-specific demethylase 1 (LSD1) has been recognized as a key mediator of EMT in cancer cells, but the precise mechanism that underlies the activation and repression of EMT genes still remains elusive. Here, we characterized the early events induced by TGF-ß1 during EMT initiation and establishment. TGF-ß1 triggered, 30-90 min post-treatment, a nuclear oxidative wave throughout the genome, documented by confocal microscopy and mass spectrometry, mediated by LSD1. LSD1 was recruited with phosphorylated SMAD2/3 to the promoters of prototypic genes activated and repressed by TGF-ß1. After 90 min, phospho-SMAD2/3 downregulation reduced the complex and LSD1 was then recruited with the newly synthesized SNAI1 and repressors, NCoR1 and HDAC3, to the promoters of TGF-ß1-repressed genes such as the Wnt soluble inhibitor factor 1 gene (WIF1), a change that induced a late oxidative burst. However, TGF-ß1 early (90 min) repression of transcription also required synchronous signaling by reactive oxygen species and the stress-activated kinase c-Jun N-terminal kinase. These data elucidate the early events elicited by TGF-ß1 and the priming role of DNA oxidation that marks TGF-ß1-induced and -repressed genes involved in the EMT.

Methylation of the Suppressor Gene p16INK4a: Mechanism and Consequences.

Tumor suppressor genes in the CDKN2A/B locus (p15INK4b, p16INK4a, and p14ARF) function as biological barriers to transformation and are the most frequently silenced or deleted genes in human cancers. This gene silencing frequently occurs due to DNA methylation of the promoter regions, although the underlying mechanism is currently unknown. We present evidence that methylation of p16INK4a promoter is associated with DNA damage caused by interference between transcription and replication processes. Inhibition of replication or transcription significantly reduces the DNA damage and CpGs methylation of the p16INK4a promoter. We conclude that de novo methylation of the promoter regions is dependent on local DNA damage. DNA methylation reduces the expression of p16INK4a and ultimately removes this barrier to oncogene-induced senescence.

Tracing and tracking epiallele families in complex DNA populations.

DNA methylation is a stable epigenetic modification, extremely polymorphic and driven by stochastic and deterministic events. Most of the current techniques used to analyse methylated sequences identify methylated cytosines (mCpGs) at a single-nucleotide level and compute the average methylation of CpGs in the population of molecules. Stable epialleles, i.e. CpG strings with the same DNA sequence containing a discrete linear succession of phased methylated/non-methylated CpGs in the same DNA molecule, cannot be identified due to the heterogeneity of the 5'-3' ends of the molecules. Moreover, these are diluted by random unstable methylated CpGs and escape detection. We present here MethCoresProfiler, an R-based tool that provides a simple method to extract and identify combinations of methylated phased CpGs shared by all components of epiallele families in complex DNA populations. The methylated cores are stable over time, evolve by acquiring or losing new methyl sites and, ultimately, display high information content and low stochasticity. We have validated this method by identifying and tracing rare epialleles and their families in synthetic or in vivo complex cell populations derived from mouse brain areas and cells during postnatal differentiation. MethCoresProfiler is written in R language. The software is freely available at https://github.com/84AP/MethCoresProfiler/.

RNA Stabilizes Transcription-Dependent Chromatin Loops Induced By Nuclear Hormones.

We show that transcription induced by nuclear receptors for estrogen (E2) or retinoic acid (RA) is associated with formation of chromatin loops that juxtapose the 5' end (containing the promoter) with the enhancer and the 3' polyA addition site of the target gene. We find three loop configurations which change as a function of time after induction: 1. RA or E2-induced loops which connect the 5' end, the enhancer and the 3' end of the gene, and are stabilized by RNA early after induction; 2. E2-independent loops whose stability does not require RNA; 3. Loops detected only by treatment of chromatin with RNAse H1 prior to hormonal induction. RNAse H1 digests RNA that occludes the relevant restriction sites, thus preventing detection of these loops. R-loops at the 5' and 3' ends of the RA or E2-target genes were demonstrated by immunoprecipitation with anti-DNA-RNA hybrid antibodies as well as by sensitivity to RNAse H1. The cohesin RAD21 subunit is preferentially recruited to the target sites upon RA or E2 induction of transcription. RAD21 binding to chromatin is eliminated by RNAse H1. We identified E2-induced and RNase H1-sensitive antisense RNAs located at the 5' and 3' ends of the E2-induced transcription unit which stabilize the loops and RAD21 binding to chromatin. This is the first report of chromatin loops that form after gene induction that are maintained by RNA:DNA hybrids.

EGFR activation triggers cellular hypertrophy and lysosomal disease in NAGLU-depleted cardiomyoblasts, mimicking the hallmarks of mucopolysaccharidosis IIIB.

Mucopolysaccharidosis (MPS) IIIB is an inherited lysosomal storage disease caused by the deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The defective lysosomal clearance of undigested HS results in dysfunction of multiple tissues and organs. We recently demonstrated that the murine model of MPS IIIB develops cardiac disease, valvular abnormalities, and ultimately heart failure. To address the molecular mechanisms governing cardiac dysfunctions in MPS IIIB, we generated a model of the disease by silencing NAGLU gene expression in H9C2 rat cardiomyoblasts. NAGLU-depleted H9C2 exhibited accumulation of abnormal lysosomes and a hypertrophic phenotype. Furthermore, we found the specific activation of the epidermal growth factor receptor (EGFR), and increased phosphorylation levels of extracellular signal-regulated kinases (ERKs) in NAGLU-depleted H9C2. The inhibition of either EGFR or ERKs, using the selective inhibitors AG1478 and PD98059, resulted in the reduction of both lysosomal aberration and hypertrophy in NAGLU-depleted H9C2. We also found increased phosphorylation of c-Src and a reduction of the hypertrophic response in NAGLU-depleted H9C2 transfected with a dominant-negative c-Src. However, c-Src phosphorylation remained unaffected by AG1478 treatment, posing c-Src upstream EGFR activation. Finally, heparin-binding EGF-like growth factor (HB-EGF) protein was found overexpressed in our MPS IIIB cellular model, and its silencing reduced the hypertrophic response. These results indicate that both c-Src and HB-EGF contribute to the hypertrophic phenotype of NAGLU-depleted cardiomyoblasts by synergistically activating EGFR and subsequent signaling, thus suggesting that EGFR pathway inhibition could represent an effective therapeutic approach for MPS IIIB cardiac disease.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair.

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.

Aldehyde modification and alum coadjuvancy enhance anti-TNF-α autovaccination and mitigate arthritis in rat.

Experimental vaccination to induce antibodies (Abs) capable of cytokine antagonism shows promise as a novel immunotherapy for chronic inflammatory disease. We prepared a hybrid antigen consisting of residues 141-235 of rat TNF-α fused to the C-terminus of glutathione-S-transferase (GST), chemically modified to incorporate aldehyde residues, for development of an auto-vaccine eliciting anti-rTNF-α Abs. In rat immunization the soluble aldehyde-modified fusion protein did not generate observable Ab responses. By contrast, vaccination with the aldehyde-modified fusion protein adsorbed on alum induced anti-TNF-α autoAbs with high titer and neutralizing activity. Induction of adjuvant arthritis in rats pre-immunized with unmodified fusion protein or a control protein in alum resulted in severe inflammation and joint damage, whereas the disease induced in rats immunized with the aldehyde-bearing fusion protein in alum was markedly attenuated. Similar results were obtained in a collagen-induced rat arthritis model. Anti-collagen II IgG Ab titers did not deviate significantly in groups pre-immunized with modified fusion protein and control protein, suggesting that anti-TNF vaccination did not skew the immune response related to disease induction. This study demonstrates synergy between particulate alum and protein bound carbonyl residues for enhancement of protein immunogenicity. The antigen-specific co-adjuvant system could prove advantageous for breaking tolerance in emerging auto-vaccination therapies targeting inflammatory cytokines as well as for enhancing a broader category of subunit vaccines. Aldehyde adduction introduces a minimal modification which, together with the established use of alum as a safe adjuvant for human use, could be favorable for further vaccine development.

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics.

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (V(L)) and variable heavy (V(H)) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.

Idiopathic membranous nephropathy: an autoimmune disease.

For more than 50 years researchers have debated the evidence for an autoimmune basis of human idiopathic membranous nephritis (MN). Work published in the past 2 years has substantially strengthened the belief that MN is indeed an autoimmune disease of the kidney. Autoantibodies of the IgG4 subclass to at least three podocyte membrane proteins including phospholipase A(2)-receptor, aldose reductase, and manganese superoxide dismutase have been detected by immunoblotting in sera as well as in acid eluates prepared from renal biopsy tissue of patients with this disease, using either whole tissue or microdissected glomeruli from frozen sections. In each case the podocyte antigen has been shown to co-localize with the subepithelial glomerular immune deposits in renal tissue of the same patients. It is not certain if any of these podocyte proteins is an inciting/primary autoantigen or whether they are secondary antigens recruited by intermolecular epitope-spreading, initiating from a yet-to-be-discovered autoantigen. Although it is clear that autoantibodies to podocyte membrane proteins are elicited in idiopathic MN and contribute to the formation of the subepithelial deposits, many questions remain concerning the triggers for their development and their contribution toward proteinuria and progression of the disease.

Combinatorial antibody library from multiple sclerosis patients reveals antibodies that cross-react with myelin basic protein and EBV antigen.

Multiple sclerosis (MS) is a widespread neurodegenerative autoimmune disease with unknown etiology. It is increasingly evident that, together with pathogenic T cells, autoreactive B cells are among the major players in MS development. The analysis of myelin neuroantigen-specific antibody repertoires and their possible cross-reactivity against environmental antigens, including viral proteins, could shed light on the mechanism of MS induction and progression. A phage display library of single-chain variable fragments (scFvs) was constructed from blood lymphocytes of patients with MS as a potential source of representative MS autoantibodies. Structural alignment of 13 clones selected toward myelin basic protein (MBP), one of the major myelin antigens, showed high homology within variable regions with cerebrospinal fluid MS-associated antibodies as well as with antibodies toward Epstein-Barr latent membrane protein 1 (LMP1). Three scFv clones showed pronounced specificity to MBP fragments 65-92 and 130-156, similar to the serum MS antibodies. One of these clones, designated E2, in both scFv and full-size human antibody constructs, was shown to react with both MBP and LMP1 proteins in vitro, suggesting natural cross-reactivity. Thus, antibodies induced against LMP1 during Epstein-Barr virus infection might act as inflammatory trigger by reacting with MBP, suggesting molecular mimicry in the mechanism of MS pathogenesis.

Reactive immunization suppresses advanced glycation and mitigates diabetic nephropathy.

Agents that inhibit glycation end products by reducing the carbonyl load from glycation and glycoxidation are an emerging pharmacologic approach to treat complications of diabetes. We previously demonstrated that antibodies generated to the glycoprotein keyhole limpet hemocyanin (KLH) can cross-link with reactive carbonyl residues on protein conjugates. Here, we immunized streptozotocin-induced diabetic rats with KLH to assess the capacity of the elicited antibodies to intercept carbonyl residues on glycated proteins and to mitigate glycation-related pathology. Compared with diabetic rats immunized with adjuvant alone, KLH-immunized diabetic rats had decreased levels of glycated peptides in sera and demonstrated a reduction in albuminuria, proteinuria, deposition of glycation end products in the kidney, and histologic damage. In vitro, low molecular weight glycated peptides from rat serum reacted with anti-KLH antibodies at a faster rate than normal IgG and selectively modified the lambda chains. The reaction products contained peptide sequences from type I collagen alpha chain, albumin, and LDL receptor-related protein. These adduction reactions were inhibited by free KLH and by reduction of glycated peptides with borohydride. In summary, these results suggest that inherent reactivity of Ig light chains provides a natural mechanism for the removal of cytotoxic glycation products. This reactivity can be augmented by glycoprotein-specific reactive immunization, a potential biopharmaceutical approach to glycation-related pathology.

Intramolecular epitope spreading in Heymann nephritis.

Immunization with megalin induces active Heymann nephritis, which reproduces features of human idiopathic membranous glomerulonephritis. Megalin is a complex immunological target with four discrete ligand-binding domains (LBDs) that may contain epitopes to which pathogenic autoantibodies are directed. Recently, a 236-residue N-terminal fragment, termed "L6," that spans the first LBD was shown to induce autoantibodies and severe disease. We used this model to examine epitope-specific contributions to pathogenesis. Sera obtained from rats 4 weeks after immunization with L6 demonstrated reactivity only with the L6 fragment on Western blot, whereas sera obtained after 8 weeks demonstrated reactivity with all four recombinant fragments of interest (L6 and LBDs II, III, and IV). We demonstrated that the L6 immunogen does not contain the epitopes responsible for the reactivity to the LBD fragments. Therefore, the appearance of antibodies directed at LBD fragments several weeks after the primary immune response suggests intramolecular epitope spreading. In vivo, we observed a temporal association between increased proteinuria and the appearance of antibodies to LBD fragments. These data implicate B cell epitope spreading in antibody-mediated pathogenesis of active Heymann nephritis, a model that should prove valuable for further study of autoimmune dysregulation.

Routes to covalent catalysis by reactive selection for nascent protein nucleophiles.

Reactivity-based selection strategies have been used to enrich combinatorial libraries for encoded biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites. The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied to a phage display library of antibody variable fragments (scFv) to sample the scope and mechanism of chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent and noncovalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2. Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus for covalent catalysis.

Albumin-stimulated TGFbeta-1 in renal tubular cells is associated with activation of MAP kinase.

BACKGROUND: Proteinuric renal diseases are often associated with progressive tubulointerstitial fibrosis that usually defines the degree and rate of progression of renal failure. Glomerular filtration of excess albumin, the dominant protein in proteinuria, into proximal tubule could provide the stimulus to induce certain fibrogenic cytokines from proximal tubular cells (PTC), which may account for fibrosis in the interstitium. To explore this hypothesis we tested the effect of bovine albumin in PTC in culture on the expression and secretion of transforming growth factor (TGF) beta-1, a prominent fibrogenic cytokine. METHODS: TGFbeta-1 expressed by cultured PTC was measured by enzyme-linked immunosorbent assay (ELISA) and indirectly by reverse-transcription polymerase chain reaction (RT-PCR). Relative messenger ribonucleic acid (mRNA) levels were measured in ethidium bromides stained gels, by comparison to transcripts for 18s ribosomal RNA. Activated mitogen-activated protein (MAP) kinase was estimated by Western blot with phosphotyrosine-specific antibody. RESULTS: Following incubation of PTC with albumin determination of TGF beta-1 mRNA in PTC and TGF beta-protein in culture medium both indicated a time- and dose-dependent increase. MAP kinase (p44/42) was activated within 5 min of exposure to albumin. Inhibition of the MAP kinase cascade by PD98059 attenuated the effect of albumin on TGF beta-1 expression. CONCLUSIONS: These observations suggest that overexpression of TGF beta-1 by PTC in response to albumin is regulated through a MAP kinase signaling pathway. This mechanism may play a role in the development of interstitial fibrosis in proteinuric states.

Aldehyde modification of peptide immunogen enhances protein-reactive antibody response to toxic shock syndrome toxin-1.

Introduction of aldehyde groups into protein conjugates enhanced the immune response to a coupled peptide without the use of strong adjuvants. Synthetic peptides representing the N-terminal (residues 1-16) and internal (residues 53-65) epitopes of toxic shock syndrome toxin-1 (TSST-1) were coupled to carrier protein, and carbonyl tags were introduced by Amadori reaction with glycolaldehyde. Modified and unmodified antigens in alum were used to immunize rabbits and the reactivities of antisera were compared. Aldehyde modification augmented the response detected by ELISA, which included enhanced binding to peptides and to native TSST-1. In western blot, TSST-1 was detected by antiserum elicited to the N-terminal peptide, but not that generated to the peptide representing the internal sequence. The same antiserum also neutralized TSST-1 activity in a lymphocyte proliferation assay. The circular dichroism spectrum of the N-terminal peptide indicated a propensity for helical conformation, similar to the structure at the corresponding sequence of the native protein. These data suggest that aldehyde modification can boost immunogenicity of peptide-based vaccines, generating epitope-specific immune responses against the cognate protein antigens without using potent adjuvants.

Nested N-terminal megalin fragments induce high-titer autoantibody and attenuated Heymann nephritis.

It was shown previously that an N-terminal fragment (nM60) that encompasses amino acid residues 1 to 563 of megalin could induce active Heymann nephritis (AHN) as efficiently as the native protein. For delineation of a minimal structure within this fragment that is sufficient to induce AHN, smaller protein fragments that encompass residues 1 to 236 (L6), 1 to 195 (L5), 1 to 156 (L4), and 1 to 120 (L3), representing successive C-terminal truncations within ligand-binding repeats of nM60, were cloned and produced in a baculovirus insect cell expression system. Protein fragments L4, L5, and L6 clearly were glycosylated. All four fragments stimulated proliferation of megalin-sensitized lymph node cells and induced high-titer anti-megalin autoantibodies in Lewis rats. A full-blown disease, as assessed by severity of proteinuria, was observed in rats that were immunized with L6 and L5, whereas animals that were immunized with L4 and L3 developed only mild disease. The proteinuria levels correlated with staining for complement (C3, C5b-9) and IgG1 isotype in glomerular immune deposits. The results suggest that one or more molecular determinants on the region that comprises amino acid residues 157 to 236 contribute to the induction of a full-blown form of AHN. Study of the structure, conformation, and posttranslational modifications of these determinants could provide greater insight into the molecular correlates of immunopathogenesis in this disease model.

Induction of covalent binding antibodies.

Covalent interactions between antibody combining site residues and substrates have been implicated in the catalytic activity of abzymes elicited by design or occurring naturally in autoimmune disease. In this study, the potential for covalent binding by antibodies (Abs) was investigated by the induction of immune responses against molecules presenting chemically reactive haptenic groups. Immunogenic conjugates containing a phosphonate diester or a pyruvate carbonyl group were used to elicit antibodies that could specifically react with the electrophilic moieties. Products formed by covalent binding were detected by a western blot technique or by differential ELISA on reduced or unreduced carbonyl haptens. Antisera to the diphenylphosphonate contained antibodies with covalent reactivity, which increased with immunization. The reactivity was specific to the anti-phosphonate response and not to control immune sera induced against the unmodified carrier protein. Reactivity was focused on the antibody light (L) chain. Antisera to the phenylpyruvate hapten appeared to bind strongly to proteins modified by the carbonyl group hapten. However, anti-carrier antisera and non-immune sera had similar reactivity, indicating that the pyruvate moiety reacts nonspecifically with immunoglobulins. This suggested that affinity maturation of antibodies for reversible binding through hemiacetal or Schiff base adducts with antigens requires a less reactive carbonyl in the antigen. On the other hand, the induction of antibodies with enhanced nucleophilic reactivity toward phosphonate esters implies that irreversible binding to the B cell receptor can drive clonal expansion and antibody selection. These results support a designer strategy for generating nucleophilic abzymes and could also account for the occurrence of chemically reactive or catalytic antibodies in natural immunity or autoimmunity.