Logo of jbcAbout JBCASBMBSubmissionsSubscriptionsContactJBCThis Article
J Biol Chem. 2012 Oct 19; 287(43): 36096–36104.
Published online 2012 Sep 4. doi:  10.1074/jbc.M112.401075
PMCID: PMC3476277

Constant Domain-regulated Antibody Catalysis*An external file that holds a picture, illustration, etc.
Object name is sbox.jpg

Abstract

Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies.

Keywords: Antibodies, Antibody Engineering, Enzyme Catalysis, Lymphocyte, Scaffold Proteins, Catalytic Antibodies, Constant Domains, First-line Defense, HIV gp120, Variable Domains

Introduction

Antibodies (immunoglobulins) are generated by linking the constant (C)4-domain genes encoding the heavy chain (μ, δ, γ, α, ϵ) or light chain (κ, λ) to the variable (V)-domain genes, that in turn are generated by rearrangement of about 500 V, (D) and J germ line genes. The C-domains define the antibody class and subclass. The paired heavy and light chain V-domains (VH, VL domains) contain the antigen combining site. The C-domains contain the “effector” sites mediating complement fixation and Fc receptor activation. Despite their spatial separation, the V- and C-domain sites display inter-dependent functional relationships. Antigen binding at the V-domains stimulates complement and Fc receptor binding by the C-domain sites (1). Noncovalent antigen binding to the V domains generally occurs without dependence on the C-domains, but subtle effects of the IgG C-domains are documented (for review, see Ref. 2). Placing the same VH-VL domain pair into the differing C-domain scaffold of various IgG subclasses can induce alterations of antigen binding affinity and fine specificity (3). The V-domains are subject to conformational transitions upon linkage to different constant domains. Identical VH-VL domains cloned into different IgG subclasses are bound nonequivalently by an anti-idiotypic antibody directed to V-domain epitopes (3). Similarly, the circular dichroism spectra of the differing IgG isotypes containing the same V-domains in the presence of antigen are nonidentical (4).

Antibodies are initially expressed on the B cell surface as IgM or IgD B cell receptors (BCRs) complexed to signal transducing proteins. The V-(D)-J gene rearrangement and combinatorial VL-VH pairing events produce a diverse innate repertoire composed of >1012 secreted IgMs that is shaped by various epigenetic factors and contact with self-antigens (5, 6). The V- and C-domains go through programmed structural changes during B cell differentiation. Accumulation of V-domain somatic mutations in the immunogen-driven differentiation phase improves the immunogen binding affinity. C-domain class switching required for production of IgGs and IgAs can occur in the absence of immunogen, but this process often takes place contemporaneously with V-domain hypermutation over the course of cellular development. IgMs are thought to fulfill a first line defense function by virtue of multivalent, high avidity recognition of microbial antigens containing repeat antigen epitopes. IgGs are the predominant blood-borne antibodies and represent the primary antibody class responsible for adaptive humoral immunity to microbes.

The V domains contain conformation-dependent nucleophilic sites that are fully competent in completing the first step in catalytic hydrolysis of amide and peptide bonds, nucleophilic attack on electrophilic carbonyl groups (see reaction scheme in supplemental Fig. S1A). The nucleophilic sites were identified by their covalent bonding to strongly electrophilic, nonhydrolyzable phosphonate probes (supplemental Fig. S1B) (7), loss of hydrolytic activity upon site-directed mutagenesis at the catalytic residues (8, 9), and structural localization of amino acid triads that are appropriately positioned to express an activated nucleophile (10, 11). Antibodies with V-domains containing no or sparse somatic mutations express the nucleophilic catalytic sites (12, 13), showing that catalysis is an innate antibody function. The same conclusion may be reached from the substrate classes sensitive to catalytic antibodies that are produced constitutively with no requirement for exposure to an external immunogen. Very small peptide “microantigens”' are hydrolyzed by antibody V-domains without the involvement of traditional high affinity noncovalent binding interactions that are developed adaptively by immunogen-driven B cell clonal selection (14, 15). The same hydrolytic reaction is catalyzed frequently by Bence Jones proteins, light chains produced by multiple myeloma patients (16, 17). Similarly, constitutive antibodies produced without exposure to microbial proteins designated B cell superantigens bind and hydrolyze these proteins specifically (1820).

The polyclonal IgM mixture in blood is composed of antibodies with diverse V-domains. The V-domain diversity in IgG class antibodies is even greater by virtue of the somatic mutation mechanism. IgGs generally express the adaptively developed noncovalent antigen binding function better than IgMs. We reported robust catalytic hydrolysis of small model peptides by polyclonal IgM preparations (19). Despite the frequent presence of nucleophilic sites in their V-domains, IgGs from humans and mice express only low level catalytic activity (7, 19, 21). Nucleophilic antibodies can complete the catalytic reaction only if the active site also supports the subsequent steps of water attack on the acyl-antibody complex and product release (supplemental Fig. S1A). We show here the superior catalytic activity of a recombinant IgM compared with its IgG and single chain Fv counterparts containing the same VH-VL domain pair. In addition, all 13 monoclonal IgMs tested expressed catalytic activity. The data suggest a contribution from the C-domain scaffold in enhancing V-domain catalysis by IgM antibodies and loss of catalytic activity induced by class switching to IgG.

MATERIALS AND METHODS

Polypeptides and Haptens

The fluorogenic peptide Glu-Ala-Arg-7-amino-4-methylcoumarin (AMC) was from Peptides International (Louisville, KY). Recombinant gp120 (MN strain) from Immunodiagnostics (Woburn, MA) was linked to biotin (1.8 mol of biotin/mol) at Lys residues (22). Synthetic nonelectrophilic peptide 421–436 conjugated to BSA (6 mol of peptide/mol) and electrophilic E-421–433 with biotin at the N terminus contained the consensus subtype B gp120 421–433 residues (7, 23). Control biotin-E-VIP was prepared as in Ref. 24. The chemical identity of all peptides was verified by electrospray ionization mass spectrometry. Electrophilic haptens (E-Hapten-1, E-Hapten-3) and the nonelectrophilic hapten (Hapten-2) were synthesized as in Refs. 19, 25. E-Hapten-1 and E-Hapten-3 are diphenylphosphonate esters reactive with nucleophilic sites (7, 26). E-Hapten-1 and Hapten-2 contain biotin to permit detection of adducts by electrophoresis. Hapten-2 is the unesterified phosphonic acid analog of E-Hapten-1 devoid of electrophilic reactivity.

Antibodies

The panel of monoclonal IgMs from 13 patients with Waldenström macroglobulinemia (WM; lymphoplasmacytic lymphoma) is described in Ref. 19 (identification codes 1718, 1801, 1805, 1808–1810, 1812–1814, 1816–1819, serum IgM 9.2–55.7 mg/ml). Polyclonal IgM and IgG pools were purified from the sera of 34 humans without evidence of disease by affinity chromatography on immobilized anti-human IgM and protein G columns, respectively (17 females, 17 males; age 17–65; identification codes 679, 681–689, and 2058–2081, Gulf Coast Blood Bank) (19, 27). Protein concentrations were determined using bicinchoninic acid. Purity was evaluated by SDS-gel electrophoresis under nonreducing and reducing conditions followed by staining with Coomassie Blue or specific peroxidase-conjugated antibodies to the λ, γ, and μ subunits (19). All protein bands present in the IgM and IgG preparations were stainable with these antibodies. scFv JL427 was isolated from a phage library from humans without HIV infection by binding to immobilized gp120, expressed in bacteria, and purified by metal-affinity chromatography to electrophoretic homogeneity (28). A molecular model of the scFv was constructed using a homology-based method (WAM). Potential nucleophilic sites were located using the previously described algorithm for identifying enzyme-like amino acid triads and diad (21). To construct full-length antibodies, the VL and VH domain cDNA (corresponding to amino acid residues 1–128 and 1–127, respectively, IMGT numbering) were amplified by the polymerase chain reaction and expressed linked to the human IgG1 constant domain scaffold as in Refs. 29, 30 except that the VL domain was cloned on the 5′ side of the λ constant domain gene (corresponding to IGLC domain amino acids 1–106 in the IMGT system) in the pLC-huCλ vector using the BglII/NotI site. Similar procedures were used to construct the JL427 μ chain. The leader-VH cDNA excised from the JL427 pHC-huCγ1 vector using NheI/HindIII was cloned on the 5′ side of the μ constant domain genes (corresponding to IGHM CH1 residues 1–104, CH2 residues 1–100, CH3 residues 1–106, CH4 residues 1–131) in pHC-huCμ vector via the NheI/HindIII sites. To accommodate insert cloning and vector shuttling procedures the following antibody residues were mutated: the two N-terminal VH residues from QV to EF, the μ CH1 residue 3 from A to L, the γ1 CH1 residue 3 from T to F, and the Cλ residues 2–4 from QPK to RTA. PCR primers are listed in supplemental Table S1. Dideoxy nucleotide sequencing of the IgG and IgM V-domains in both directions yielded identical sequences that matched the parental VL- and VH-domain sequences. Full-length IgG and IgM were obtained in the supernatants of stable NS0 cell lines coexpressing the light and heavy chain vectors grown at a density of 1.6 × 106 cells/ml in CELLine flasks (Wilson Wolf Corp., New Brighton, MN) (30). IgG-depleted FBS was used for cell culture. The concentration of bovine IgM in FBS is insignificant. Secreted IgG and IgM were measured by capture ELISA on wells coated with anti-λ antibody (200 ng/well; Sigma-Aldrich) and peroxidase-conjugated goat anti-IgG1 or anti-IgM antibodies (1:1000; Sigma-Aldrich) with IgG1 and IgM as standards. Expression levels were ∼3.3 mg of IgM/liter of IgM and 4.0 mg/liter IgG. The antibodies were purified from 10-fold concentrated tissue culture supernatants (Centriprep YM10; Millipore) by chromatography on immobilized anti-IgM antibody and protein G columns as before. Further IgM size exclusion chromatography was on a Superose-6 FPLC column in 50 mm Tris-HCl, pH 7.8, 0.1 m glycine, 0.15 m NaCl, 0.1 mm CHAPS (0.4 ml/min) or the same buffer containing the denaturant 6 m guanidine-HCl adjusted to pH 6.5 with HCl (0.13 ml/min) (19). The retention volumes of the pentamer and monomer IgM fractions recovered from the nondenaturing column were 9.0 ml and 15.8 ml, respectively, and from the denaturing column, 5.7 ml and 8.8 ml, respectively. The shorter retention times in the denaturing column are consistent with lesser permeation of the gel pores by nonglobular unfolded proteins. Column calibration was with thyroglobulin (660 kDa), IgG (150 kDa), and albumin (67 kDa) (Sigma-Aldrich).

Binding and Catalysis Assays

scFv JL427 binding to the immobilized BSA-conjugated gp120 peptide 421–436 (230 ng/well) was measured using an antibody to the c-myc tag located at the scFv C terminus (28). scFv binding to electrophilic probes was determined by SDS-electrophoresis using boiled reaction mixtures followed by staining of blots with peroxidase-conjugated streptavidin (7). Hydrolysis of the amide bond linking AMC to the C-terminal Arg in Glu-Ala-Arg-AMC (Peptides International or Bachem, King of Prussia, PA) was measured by fluorometry (λex 360 nm, λem 470 nm; Varian Cary Eclipse) (19). Authentic AMC was used to construct a standard curve. Kinetic parameters were obtained by fits of rate data to the Michaelis-Menten-Henri equation. Hydrolysis of biotinylated gp120 was measured by reducing SDS-electrophoresis, staining of blots with streptavidin and densitometry (19). In tests of inhibition by electrophilic probes, control reaction mixtures contained equivalent concentrations of the solvents in which the probe stock solutions had been prepared (dimethyl sulfoxide). Purified antibodies were dialyzed against the appropriate assay buffer prior to measurement of binding and catalytic activity.

RESULTS

Monoclonal IgM Catalytic Activity

Hydrolysis of the amide bond linking the fluorophore AMC group to small model peptide substrates is a convenient surrogate for peptide bond hydrolysis by antibodies. The reaction occurs preferentially on the C-terminal side of Arg/Lys residues and does not require high affinity binding to an antigenic epitope (19). All 13 monoclonal human IgMs from patients with WM hydrolyzed Glu-Ala-Arg-AMC detectably (Fig. 1A). The IgMs contain an identical C-domain scaffold. Yet, the hydrolysis rate of individual IgMs varied >91-fold, indicating that the catalytic activity is a V-domain function. This is consistent with the finding of catalysis by the Fab fragment of an IgM antibody (19). In addition, previous studies have identified nucleophilic sites in the C-domain-free V-domains with varying levels of catalytic activity, including recombinant scFvs (VL-linker-VH constructs) (7) and isolated VL domains (31). Fig. 1B reports the comparative Glu-Ala-Arg-AMC hydrolytic rates of the monoclonal IgMs with the highest and lowest activities along with pooled polyclonal IgM and IgG from the same sera. The hydrolytic rates for serum IgM and IgG from individual human donors have been reported previously (14, 19, 27). The polyclonal IgG pool displayed detectable but low catalytic activity (0.33 μm substrate/μm IgG at 21 h, the final observation point). Even the least catalytic monoclonal IgM (1801) hydrolyzed Glu-Ala-Arg-AMC more rapidly than polyclonal IgG (by 18-fold). The hydrolytic rate of polyclonal IgM was 939-fold superior to polyclonal IgG.

FIGURE 1.
Proteolytic activities of human IgMs. A, scatter plot of Glu-Ala-Arg-AMC hydrolysis by monoclonal IgMs from WM patients. Each symbol is a monoclonal IgM. Reaction rates were determined as the slope of the linear progress curve over 21 h. Reaction conditions: ...

Source V Domain Properties

The VH-VL domain pair from scFv JL427 was used to prepare IgM and IgG as described in the next section. The scFv was isolated by fractionating a human scFv library displayed on phages using immobilized HIV gp120 as the selection reagent. The JL427 V domains contain a large number of somatic mutations (supplemental Table S2; GenBank accession number AF329462). Fig. 2A shows the noncovalent binding of scFv JL427 to synthetic peptide 421–436, similar to the specificity of other gp120-binding scFv clones isolated from this library (28). The scFv formed 32-kDa covalent adducts with the electrophilic analog of peptide 421–433 (E-421–433) but not an irrelevant electrophilic peptide (E-VIP) (Fig. 2, B and C). The adducts were stable to denaturing conditions that dissociate noncovalent binding (boiling, SDS treatment), indicating a covalent nucleophile-electrophile reaction. No scFv adducts were formed at an equivalent electrophilic hapten phosphonate diester 1 concentration (E-Hapten-1). The adducts were visible at a 10-fold higher concentration of E-Hapten-1 but not the control hapten phosphonic acid 2 devoid of electrophilic reactivity (Fig. 2D, lanes 1 and 2, respectively). The data suggest a nucleophilic site that reacts with the electrophile guided by noncovalent epitope binding. The nucleophilic sites are formed by conformation-dependent hydrogen bonding between amino acid triads and diads (32, 33). Molecular modeling suggested that the scFv conformation was permissive for forming candidate nucleophilic sites (supplemental Table S3).

FIGURE 2.
scFv JL427 binding specificity and nucleophilic reactivity. A, binding of immobilized gp120 peptide 421–436 conjugated to BSA (230 ng/well) by the scFv purified from a bacterial periplasmic extract and an equivalently purified control extract ...

Catalytic Activity of IgM and IgG JL427

To eliminate V-domain differences as a contributory factor in catalysis, we cloned the same V domains from scFv JL427 into the full-length IgM and IgG scaffolds. The antibodies were obtained from culture supernatants of NS0 lymphoid cells coexpressing vectors containing the V-domain cDNA cloned adjacent to the μ, λ or γ1 C-domain genes. The cells express the J chain needed for assembling pentameric IgM (900 kDa) constitutively. The IgM and IgG preparations purified using immobilized anti-IgM antibody and protein G, respectively, contained heavy and light chain subunit bands with the anticipated mass in reducing SDS-electrophoresis gels (Fig. 3A). A major, correctly assembled 150-kDa band along with two incompletely assembled oligomer bands were evident in the IgG preparation in a nonreducing SDS-gel (Fig. 3A, lanes 3 and 4). Gel filtration of the IgM in a denaturing solvent (6 m guanidine hydrochloride) indicated a majority species with nominal mass of pentamer IgM (52% of recovered protein; mass measured by comparison with marker proteins, 1070 kDa), along with a minority monomer IgM species (29%; nominal mass, 195 kDa) and free subunits (Fig. 3B).

FIGURE 3.
IgM and IgG electrophoresis and gel filtration. A, SDS-electrophoresis gels of anti-IgM-purified IgM JL427 and protein G-purified IgG JL427. Lanes 1 and 2, reducing SDS-gels of the IgG stained with Coomassie Blue or anti-γ/λ antibodies, ...

scFv JL427 and its IgG counterpart hydrolyzed Glu-Ala-Arg-AMC at detectable but low levels (Fig. 4A). The hydrolytic activity of IgM JL427 was superior to the scFv and IgG (Fig. 4A, by 740-fold and 202-fold, respectively). Ten repeat assays using two independent IgM preparations and four assays using three IgG preparations confirmed consistently more rapid catalysis by the IgM (expressed per mole of antibody, by 328 ± 218-fold, p < 0.0001, unpaired t test). To preclude noncovalently associated trace contaminants, the IgM purified by anti-IgM affinity chromatography was subjected to denaturing gel filtration. Following renaturation, the majority pentamer IgM species from the column displayed robust hydrolytic activity that was only 1.4-fold lower compared with the pentamer-monomer mixture loaded on the column (Fig. 4A). Fractionation of a second IgM preparation by gel filtration in a nondenaturing solvent yielded the individual pentamer and monomer species (Fig. 4B; to compensate for the difference in molecular valence, the data are expressed per unit combining site). The monomer IgM was substantially more hydrolytic than monomer IgG. The difference in hydrolytic rates of monomer IgM and pentamer IgM (1.8-fold) is within the range of error encountered while handling of the proteins at low concentrations. Inclusion of E-Hapten-1 in the reaction mixture completely inhibited the hydrolysis of Glu-Ala-Arg-AMC (Fig. 4C), consistent with the nucleophilic catalytic mechanism deduced for previously reported IgMs (19). Over 24 h, 737 substrate molecules were hydrolyzed per IgM molecule over the reaction duration in Fig. 4A, indicating regeneration of active IgM that was reused for multiple catalytic cycles. Saturation kinetics consistent with the Michaelis-Menten equation were observed (Fig. 4D). The equilibrium dissociation constant for the noncovalent binding step is approximated by the Km value. The IgM and IgG Km values were comparable (respectively, 105 and 113 μm). The IgM turnover number (catalytic rate constant kcat) was 135-fold greater than IgG on a molar basis (respectively, 0.42 and 0.0031/min). If all 10 IgM and 2 IgG valences are filled, the turnover number/valence for IgM is 27-fold greater than IgG. As the V-domains of the IgM and IgG are identical, the superior catalytic activity of IgM is attributable to a favorable C-domain effect on V-domain catalysis.

FIGURE 4.
Model peptide hydrolytic properties of IgM, IgG, and scFv JL427. A, time-dependent hydrolysis of Glu-Ala-Arg-AMC. IgM, native is the IgM purified by affinity chromatography on immobilized anti-IgM antibody. IgM, renatured pentamer is the yellow fraction ...

We also measured the hydrolysis of biotinylated gp120 to verify hydrolysis of true peptide bonds. Because the gp120 is available only in limited quantities, the assays were conducted at a nonsaturating gp120 concentration (100 nm). No hydrolytic activity of the IgM is detectable using Glu-Ala-Arg-AMC at this substrate concentration. scFv JL427 V-domains employed for full-length antibody construction bind gp120 noncovalently. Previously described catalytic antibodies with noncovalent gp120 recognition capability hydrolyzed gp120 more rapidly compared with the Glu-Ala-Arg-AMC substrate (19). Depletion of the parent gp120 band and appearance of product fragments was evident upon treatment with IgM JL427 (Fig. 5). The scFv and IgG JL427 counterparts did not hydrolyze gp120 detectably, confirming their poor catalytic activity observed using the Glu-Ala-Arg-AMC substrate. The mass of the observed product bands was similar to the gp120 fragments generated by previously described catalytic antibody preparations (34), as determined by comparison with the overexposed gp120 digest lane in Fig. 5 (lane 7; 80, 55, 39, 25, and 17 kDa bands).

FIGURE 5.
gp120 hydrolysis by IgM, IgG, and scFv JL427. Biotinylated gp120 was treated with diluent or increasing antibody concentrations followed by measurement of hydrolysis by reducing SDS-electrophoresis. Percentage of gp120 hydrolysis was computed as: 100×([gp120] ...

DISCUSSION

Individual antibody species within a given antibody class can express varying catalytic activities because of their differing V-domain structures, illustrated by the finding of widely divergent activities of monoclonal IgMs with identical C-domain structures. Polyclonal antibody studies indicated that the average catalytic activity of IgMs far exceeds that of IgGs (15, 19, 35). IgGs develop from IgMs by a switch of C-domain gene usage, usually accompanied by immunogen-driven adaptive accumulation of V-domain mutations. The same VL-VH domain pair from scFv JL427 expressed in the IgM scaffold displayed superior catalytic activity compared with the IgG scaffold. This indicates a favorable effect of the remote IgM C-domains in expression of the catalytic function, independent of the adaptive V-domain sequence diversification process. Structural differences in the scFv, IgG, and IgM scaffolds are shown in Fig. 6A.

FIGURE 6.
Differing scFv, IgG, and IgM structures and proposed functional contributions of catalytic IgMs. A, schematic scFv, IgG, and monomer IgM structures. A scaled-down pentamer IgM model is included. Catalysis occurs at the V-domain nucleophilic site. Remote ...

The catalytic rate constant (turnover number) per combining site was superior for IgM JL427 compared with IgG JL427. This indicates that V-domain linkage to the IgM C-domains accelerates a reaction step after completion of noncovalent substrate binding. Moreover, noncovalent substrate binding by the IgM and IgG judged from the Km values was comparable, indicating that more avid noncovalent binding due to differing antibody valence is not a factor (note: multivalent binding of substrates devoid of repeat epitopes, e.g. Glu-Ala-Arg-AMC, is precluded in solution-state assays). Loss of substrate binding affinity (increased Km) is described to improve the catalytic rate constant due to a decrease of the reaction activation energy (36). The IgM C-domains exert a favorable effect on the catalytic rate constant without an alteration of the Km value, indicating improved catalysis independent of the initial noncovalent binding step. In addition to the model peptide substrate, IgM JL427 hydrolyzed gp120 more efficiently than the IgG containing the same V-domains. The V-domains employed for IgM construction bind the 421–433 gp120 epitope specifically. Catalytic antibodies that hydrolyze gp120 with specificity derived from noncovalent binding to the 421–433 epitope were described previously (19, 34).

Catalysis is a germ line BCR-encoded function that is expressed with no requirement for B cell encounter with an immunogen (12, 13). According to the B cell clonal selection theory, immunogen-BCR binding drives synthesis of antibodies with somatically mutated V-domains. BCR-catalyzed immunogen hydrolysis will cause release of product fragments, depriving B cells of the stimulatory binding signal. Although adaptive selection of sequence-diversified V-domains may well reduce the germ line-encoded catalytic activity, this factor alone does not explain satisfactorily the observation of superior IgM catalysis. IgM JL427 contains V-domains with extensive deviations from their germ line gene sequences due to the V-(D)-J gene rearrangement and somatic mutation processes. The level of somatic sequence deviations is comparable with adaptively generated IgGs with strong antigen binding activity (supplemental Table S2). Nonetheless, IgM JL427 was more hydrolytic than its IgG counterpart with the same V-domains, suggesting that the constant domain scaffold regulates catalysis regardless of the V-domain sequence diversification status. As the two V-domains originated from a combinatorial VH/VL library, they may represent a nonphysiological pair. However, there is no reason to believe that nonphysiological VH-VL pairing contributes to differential expression of catalytic activity by the two antibody classes. Moreover, polyclonal IgM displayed better catalytic activity compared with polyclonal IgG, indicating that the observation of superior IgM catalysis extends broadly to diverse physiological antibodies. The WM monoclonal IgM studies also indirectly support the hypothesis of C-domain-facilitated catalysis by IgMs with diverse V-domains. Like other antibodies originating from different B cell clones, WM IgMs contain distinct VH/VL CDR3 sequences due to V-(D)-J junctional diversification (37). Moreover, the V-domains of WM IgMs continue to accumulate large number of V-domain somatic mutations due to defective class switching, and their V-domain mutation level (3740) is comparable with adaptively generated IgGs (e.g. an average 7.1% VH mutations for 20 WM IgMs in Ref. 40, compared with 6.6% VH mutations for 12 adaptively generated IgG in Ref. 41). The average catalytic activity of the WM IgMs was 278-fold greater than pooled polyclonal IgG from healthy humans (respectively, 8.9 ± 11.6 and 0.032 ± 0.003 μm Glu-Ala-Arg-AMC cleaved/μm antibody per h), and all 13 WM IgMs were more hydrolytic than polyclonal IgG. If increasing V-domain mutation is the sole reason for deteriorated IgG catalysis, the WM IgMs and polyclonal IgG should be comparably hydrolytic.

Mutagenesis and crystallography studies have identified V-domain catalytic sites in which a Ser or Tyr side chain nucleophile is activated by H-bonding to a general base (e.g. His, Arg) (811). Consistent with the nucleophilic catalytic mechanism, the electrophilic E-Hapten-1 inhibited the hydrolytic activity of IgM JL427. The same probe was previously shown to inhibit other catalytic antibodies irreversibly (19), including antibody V-domains devoid of C-domains (31, 42). We did not identify the IgM catalytic site or determine the mechanistic basis of the C-domain effect on catalysis, but a conceptual framework for further analysis is available. The poorly catalytic scFv JL427 contained a V-domain nucleophilic site based on formation of irreversible complexes with electrophilic probes. A V-domain structural change caused by linkage to the spatially distant IgM C-domains that accelerates a rate-limiting step needed to complete the catalytic cycle will explain the observed improvement of catalysis, e.g. water attack on the acyl-antibody intermediate and product release (supplemental Fig. S1A). Sub-Ångstrom alterations of the catalytic site topography can be induced by remote structural changes in enzymes (43) and antibodies (31). Catalytic sites accomplish nucleophilic attack on peptide bonds, acyl-enzyme hydrolysis, and product release by virtue of precisely positioned functional groups and small conformational rearrangements occurring during the catalytic reaction. For instance, the trypsin Ser nucleophile is deprotonated by the H-bonded His, the proton is donated to the C-terminal substrate leaving fragment, and the same His in an altered orientation deprotonates the water molecule responsible for hydrolysis of the acyl-enzyme intermediate (33). Interfacial bound water has been identified in crystal structures of antigens complexed to high affinity antibody fragments (44), but no structural information is available about limitations in antibody catalysis at the water attack step.

IgMs are found at blood concentrations of ∼2 μm compared with picomolar-nanomolar concentrations of classical proteases (45, 46). Peptide bond hydrolysis often results in inactivation of polypeptides. The gain in biological efficacy due to catalysis can be illustrated from the observed hydrolytic rates of polyclonal IgM from healthy humans at saturating concentrations of gp120 and small peptide substrates (2.1–2.8/min) (19). Large amounts of these antigens will be hydrolyzed over a single half-life of blood-borne IgM (>9000 mol of antigen/mol of IgM over 3 days). In comparison, a maximum of 2 mol of antigen is bound per mol of stoichiometric IgGs with noncovalent binding activity. We described antibodies and antibody fragments to HIV gp120 (34), the Staphylococcus aureus virulence factor Efb (20) and the autoantigen amyloid β (47) with neutralizing activity attributable to the catalytic function. The group of Uda and Hifumi has described catalytic antibody fragments to bacterial and viral target antigens that reduce infection in experimental animal models (48, 49). Autoimmune disease is associated with increased catalytic antibodies to autoantigens, and numerous examples of pathogenic catalytic autoantibodies have been reported (5054; for review see Ref. 55). Conversely, examples of beneficial (physiological) catalytic autoantibodies to toxic amyloid β aggregates (47) and the coagulation enzyme Factor IX (56) are available. Maintenance of homeostatic levels of antibodies that hydrolyze small model peptides is associated with reduced death in septic shock (57), transplant rejection (58), and autoimmune disease incidence (14, 59). The phenomenon of naturally occurring antibody catalysis was discovered from studies on IgG class autoantibodies (50). Contrary to the assumption that catalysis improves with adaptive maturation of antibodies, our findings suggest that catalysis is a first line defense function of immature, minimally mutated IgM antibodies (Fig. 6). Somatic maturation without class switching is also compatible with expression of catalysis, supporting consideration of the catalytic IgMs with mutated V-domains as functionally important adaptive mediators.

In summary, the findings show that the C-domain scaffold is an important factor influencing expression of V-domain catalytic activity. Future functional studies hold the potential of generating more precise insight to the beneficial and pathogenic functions of catalytic IgMs. In addition, monoclonal and polyclonal catalytic IgMs are of interest as potential therapeutic reagents.

Acknowledgments

We thank Dr. Beverly Handy for providing monoclonal IgMs from Waldenström macroglobulinemia; Drs. Carl Hanson and Richard Massey for discussions; and Yogesh Bangale, Mukulesh Baruah, Stephane Boivin, Robert Dannenbring, Sangeeta Karle, and Dipanjan Gosh for collaborative assistance.

*This work was supported, in whole or in part, by National Institutes of Health Grants AI071951, AI058865, AI067020, AI087527, and AI093261. This work was also supported by The Richard J. Massey Foundation for Arts and Sciences and Covalent Bioscience Inc. Stephanie Planque, Yasuhiro Nishiyama, and Sudhir Paul have a financial interest in patents covering the catalytic antibody area. These individuals are also advisors for Covalent Bioscience Inc. and have a financial interest in the company.

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThis article contains supplemental Fig. S1, Tables S1–S3, and additional references.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF329462 and JX390613.

4The abbreviations used are:

C-domain
constant domain
AMC
7-amino-4-methylcoumarin
BCR
B cell receptor
gp120
glycoprotein 120
scFv
single chain Fv
V-domain
variable domain
VH
heavy chain variable domain
VL
light chain variable domain
VIP
vasoactive intestinal peptide
WM
Waldenström macroglobulinemia.

REFERENCES

1. Raghavan M., Bjorkman P. J. (1996) Fc receptors and their interactions with immunoglobulins. Annu. Rev. Cell Dev. Biol. 12, 181–220 [PubMed]
2. Torres M., Casadevall A. (2008) The immunoglobulin constant region contributes to affinity and specificity. Trends Immunol. 29, 91–97 [PubMed]
3. Torres M., May R., Scharff M. D., Casadevall A. (2005) Variable-region-identical antibodies differing in isotype demonstrate differences in fine specificity and idiotype. J. Immunol. 174, 2132–2142 [PubMed]
4. Janda A., Casadevall A. (2010) Circular dichroism reveals evidence of coupling between immunoglobulin constant and variable region secondary structure. Mol. Immunol. 47, 1421–1425 [PMC free article] [PubMed]
5. Haury M., Sundblad A., Grandien A., Barreau C., Coutinho A., Nobrega A. (1997) The repertoire of serum IgM in normal mice is largely independent of external antigenic contact. Eur. J. Immunol. 27, 1557–1563 [PubMed]
6. Murre C. (2007) Epigenetics of antigen-receptor gene assembly. Curr. Opin. Genet. Dev. 17, 415–421 [PMC free article] [PubMed]
7. Planque S., Taguchi H., Burr G., Bhatia G., Karle S., Zhou Y. X., Nishiyama Y., Paul S. (2003) Broadly distributed chemical reactivity of natural antibodies expressed in coordination with specific antigen binding activity. J. Biol. Chem. 278, 20436–20443 [PubMed]
8. Gao Q. S., Sun M., Rees A. R., Paul S. (1995) Site-directed mutagenesis of proteolytic antibody light chain. J. Mol. Biol. 253, 658–664 [PubMed]
9. Sharma V., Heriot W., Trisler K., Smider V. (2009) A human germ line antibody light chain with hydrolytic properties associated with multimerization status. J. Biol. Chem. 284, 33079–33087 [PMC free article] [PubMed]
10. Ramsland P. A., Terzyan S. S., Cloud G., Bourne C. R., Farrugia W., Tribbick G., Geysen H. M., Moomaw C. R., Slaughter C. A., Edmundson A. B. (2006) Crystal structure of a glycosylated Fab from an IgM cryoglobulin with properties of a natural proteolytic antibody. Biochem. J. 395, 473–481 [PMC free article] [PubMed]
11. Smirnov I., Carletti E., Kurkova I., Nachon F., Nicolet Y., Mitkevich V. A., Débat H., Avalle B., Belogurov A. A., Jr., Kuznetsov N., Reshetnyak A., Masson P., Tonevitsky A. G., Ponomarenko N., Makarov A. A., Friboulet A., Tramontano A., Gabibov A. (2011) Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics. Proc. Natl. Acad. Sci. U.S.A. 108, 15954–15959 [PMC free article] [PubMed]
12. Gololobov G., Sun M., Paul S. (1999) Innate antibody catalysis. Mol. Immunol. 36, 1215–1222 [PubMed]
13. Le Minoux D., Mahendra A., Kaveri S., Limnios N., Friboulet A., Avalle B., Boquet D., Lacroix-Desmazes S., Padiolleau-Lefèvre S. (2012) A novel molecular analysis of genes encoding catalytic antibodies. Mol. Immunol. 50, 160–168 [PubMed]
14. Kalaga R., Li L., O'Dell J. R., Paul S. (1995) Unexpected presence of polyreactive catalytic antibodies in IgG from unimmunized donors and decreased levels in rheumatoid arthritis. J. Immunol. 155, 2695–2702 [PubMed]
15. Kamalanathan A. S., Goulvestre C., Weill B., Vijayalakshmi M. A. (2010) Proteolysis activity of IgM antibodies from rheumatoid arthritis patients' sera: evidence of atypical catalytic site. J. Mol. Recognit. 23, 577–582 [PubMed]
16. Matsuura K., Yamamoto K., Sinohara H. (1994) Amidase activity of human Bence Jones proteins. Biochem. Biophys. Res. Commun. 204, 57–62 [PubMed]
17. Paul S., Li L., Kalaga R., Wilkins-Stevens P., Stevens F. J., Solomon A. (1995) Natural catalytic antibodies: peptide-hydrolyzing activities of Bence Jones proteins and VL fragment. J. Biol. Chem. 270, 15257–15261 [PubMed]
18. Berberian L., Goodglick L., Kipps T. J., Braun J. (1993) Immunoglobulin VH3 gene products: natural ligands for HIV gp120. Science 261, 1588–1591 [PubMed]
19. Planque S., Bangale Y., Song X. T., Karle S., Taguchi H., Poindexter B., Bick R., Edmundson A., Nishiyama Y., Paul S. (2004) Ontogeny of proteolytic immunity: IgM serine proteases. J. Biol. Chem. 279, 14024–14032 [PubMed]
20. Brown E. L., Nishiyama Y., Dunkle J. W., Aggarwal S., Planque S., Watanabe K., Csencsits-Smith K., Bowden M. G., Kaplan S. L., Paul S. (2012) Constitutive production of catalytic antibodies to a Staphylococcus aureus virulence factor and effect of infection. J. Biol. Chem. 287, 9940–9951 [PMC free article] [PubMed]
21. Nishiyama Y., Mitsuda Y., Taguchi H., Planque S., Hara M., Karle S., Hanson C. V., Uda T., Paul S. (2005) Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activity. J. Mol. Recognit. 18, 295–306 [PubMed]
22. Paul S., Planque S., Zhou Y. X., Taguchi H., Bhatia G., Karle S., Hanson C., Nishiyama Y. (2003) Specific HIV gp120-cleaving antibodies induced by covalently reactive analog of gp120. J. Biol. Chem. 278, 20429–20435 [PubMed]
23. Karle S., Nishiyama Y., Taguchi H., Zhou Y. X., Luo J., Planque S., Hanson C., Paul S. (2003) Carrier-dependent specificity of antibodies to a conserved peptide determinant of gp120. Vaccine 21, 1213–1218 [PubMed]
24. Nishiyama Y., Bhatia G., Bangale Y., Planque S., Mitsuda Y., Taguchi H., Karle S., Paul S. (2004) Toward selective covalent inactivation of pathogenic antibodies: a phosphate diester analog of vasoactive intestinal peptide that inactivates catalytic autoantibodies. J. Biol. Chem. 279, 7877–7883 [PubMed]
25. Nishiyama Y., Taguchi H., Luo J. Q., Zhou Y. X., Burr G., Karle S., Paul S. (2002) Covalent reactivity of phosphonate monophenyl esters with serine proteinases: an overlooked feature of presumed transition state analogs. Arch. Biochem. Biophys. 402, 281–288 [PubMed]
26. Oleksyszyn J., Powers J. C. (1994) Amino acid and peptide phosphonate derivatives as specific inhibitors of serine peptidases. Methods Enzymol. 244, 423–441 [PubMed]
27. Mitsuda Y., Planque S., Hara M., Kyle R., Taguchi H., Nishiyama Y., Paul S. (2007) Naturally occurring catalytic antibodies: evidence for preferred development of the catalytic function in IgA class antibodies. Mol. Biotechnol. 36, 113–122 [PubMed]
28. Karle S., Planque S., Nishiyama Y., Taguchi H., Zhou Y. X., Salas M., Lake D., Thiagarajan P., Arnett F., Hanson C. V., Paul S. (2004) Cross-clade HIV-1 neutralization by an antibody fragment from a lupus phage display library. AIDS 18, 329–331 [PubMed]
29. McLean G. R., Nakouzi A., Casadevall A., Green N. S. (2000) Human and murine immunoglobulin expression vector cassettes. Mol. Immunol. 37, 837–845 [PubMed]
30. Sapparapu G., Planque S. A., Nishiyama Y., Foung S. K., Paul S. (2009) Antigen-specific proteolysis by hybrid antibodies containing promiscuous proteolytic light chains paired with an antigen-binding heavy chain. J. Biol. Chem. 284, 24622–24633 [PMC free article] [PubMed]
31. Taguchi H., Planque S., Sapparapu G., Boivin S., Hara M., Nishiyama Y., Paul S. (2008) Exceptional amyloid β peptide hydrolyzing activity of nonphysiological immunoglobulin variable domain scaffolds. J. Biol. Chem. 283, 36724–36733 [PMC free article] [PubMed]
32. Auclair S. M., Bhanu M. K., Kendall D. A. (2012) Signal peptidase I: cleaving the way to mature proteins. Protein Sci. 21, 13–25 [PMC free article] [PubMed]
33. Radisky E. S., Lee J. M., Lu C. J., Koshland D. E., Jr. (2006) Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates. Proc. Natl. Acad. Sci. U.S.A. 103, 6835–6840 [PMC free article] [PubMed]
34. Planque S., Mitsuda Y., Taguchi H., Salas M., Morris M. K., Nishiyama Y., Kyle R., Okhuysen P., Escobar M., Hunter R., Sheppard H. W., Hanson C., Paul S. (2007) Characterization of gp120 hydrolysis by IgA antibodies from humans without HIV infection. AIDS Res. Hum. Retroviruses 23, 1541–1554 [PubMed]
35. Odintsova E. S., Baranova S. V., Dmitrenok P. S., Calmels C., Parissi V., Andreola M. L., Buneva V. N., Nevinsky G. A. (2012) Anti-integrase abzymes from the sera of HIV-infected patients specifically hydrolyze integrase but nonspecifically cleave short oligopeptides. J. Mol. Recognit. 25, 193–207 [PubMed]
36. Paul S. (1996) Natural catalytic antibodies. Mol. Biotechnol. 5, 197–207 [PubMed]
37. Kriangkum J., Taylor B. J., Treon S. P., Mant M. J., Reiman T., Belch A. R., Pilarski L. M. (2007) Molecular characterization of Waldenstrom's macroglobulinemia reveals frequent occurrence of two B-cell clones having distinct IgH VDJ sequences. Clin. Cancer Res. 13, 2005–2013 [PubMed]
38. Sahota S. S., Forconi F., Ottensmeier C. H., Provan D., Oscier D. G., Hamblin T. J., Stevenson F. K. (2002) Typical Waldenstrom macroglobulinemia is derived from a B-cell arrested after cessation of somatic mutation but prior to isotype switch events. Blood 100, 1505–1507 [PubMed]
39. Walsh S. H., Laurell A., Sundström G., Roos G., Sundström C., Rosenquist R. (2005) Lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia derives from an extensively hypermutated B cell that lacks ongoing somatic hypermutation. Leuk. Res. 29, 729–734 [PubMed]
40. Rollett R. A., Wilkinson E. J., Gonzalez D., Fenton J. A., Short M. A., Evans P. A., Rawstron A. C., Owen R. G. (2006) Immunoglobulin heavy chain sequence analysis in Waldenstrom's macroglobulinemia and immunoglobulin M monoclonal gammopathy of undetermined significance. Clin. Lymphoma Myeloma 7, 70–72 [PubMed]
41. Bose B., Sinha S. (2005) Problems in using statistical analysis of replacement and silent mutations in antibody genes for determining antigen-driven affinity selection. Immunology 116, 172–183 [PMC free article] [PubMed]
42. Paul S., Tramontano A., Gololobov G., Zhou Y. X., Taguchi H., Karle S., Nishiyama Y., Planque S., George S. (2001) Phosphonate ester probes for proteolytic antibodies. J. Biol. Chem. 276, 28314–28320 [PubMed]
43. Bock P. E., Panizzi P., Verhamme I. M. (2007) Exosites in the substrate specificity of blood coagulation reactions. J. Thromb. Haemost. 5, 81–94 [PMC free article] [PubMed]
44. Lo Conte L., Chothia C., Janin J. (1999) The atomic structure of protein-protein recognition sites. J. Mol. Biol. 285, 2177–2198 [PubMed]
45. Chen T. Y., Huang C. C., Tsao C. J. (1993) Hemostatic molecular markers in nephrotic syndrome. Am. J. Hematol. 44, 276–279 [PubMed]
46. Alvarez-Twose I., Vañó-Galván S., Sánchez-Muñoz L., Morgado J. M., Matito A., Torrelo A., Jaén P., Schwartz L. B., Orfao A., Escribano L. (2012) Increased serum baseline tryptase levels and extensive skin involvement are predictors for the severity of mast cell activation episodes in children with mastocytosis. Allergy 67, 813–821 [PMC free article] [PubMed]
47. Taguchi H., Planque S., Nishiyama Y., Symersky J., Boivin S., Szabo P., Friedland R. P., Ramsland P. A., Edmundson A. B., Weksler M. E., Paul S. (2008) Autoantibody-catalyzed hydrolysis of amyloid β peptide. J. Biol. Chem. 283, 4714–4722 [PubMed]
48. Hifumi E., Morihara F., Hatiuchi K., Okuda T., Nishizono A., Uda T. (2008) Catalytic features and eradication ability of antibody light-chain UA15-L against Helicobacter pylori. J. Biol. Chem. 283, 899–907 [PubMed]
49. Hifumi E., Honjo E., Fujimoto N., Arakawa M., Nishizono A., Uda T. (2012) Highly efficient method of preparing human catalytic antibody light chains and their biological characteristics. FASEB J. 26, 1607–1615 [PubMed]
50. Paul S., Volle D. J., Beach C. M., Johnson D. R., Powell M. J., Massey R. J. (1989) Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody. Science 244, 1158–1162 [PubMed]
51. Shuster A. M., Gololobov G. V., Kvashuk O. A., Bogomolova A. E., Smirnov I. V., Gabibov A. G. (1992) DNA hydrolyzing autoantibodies. Science 256, 665–667 [PubMed]
52. Polosukhina D. I., Kanyshkova T. G., Doronin B. M., Tyshkevich O. B., Buneva V. N., Boiko A. N., Gusev E. I., Favorova O. O., Nevinsky G. A. (2004) Hydrolysis of myelin basic protein by polyclonal catalytic IgGs from the sera of patients with multiple sclerosis. J. Cell Mol. Med. 8, 359–368 [PubMed]
53. Ponomarenko N. A., Durova O. M., Vorobiev I. I., Belogurov A. A., Jr., Kurkova I. N., Petrenko A. G., Telegin G. B., Suchkov S. V., Kiselev S. L., Lagarkova M. A., Govorun V. M., Serebryakova M. V., Avalle B., Tornatore P., Karavanov A., Morse H. C., 3rd, Thomas D., Friboulet A., Gabibov A. G. (2006) Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen. Proc. Natl. Acad. Sci. U.S.A. 103, 281–286 [PMC free article] [PubMed]
54. Yang Y. H., Chang C. J., Chuang Y. H., Hsu H. Y., Chen P. P., Chiang B. L. (2010) Identification of anti-prothrombin antibodies in the anti-phospholipid syndrome that display the prothrombinase activity. Rheumatology 49, 34–42 [PubMed]
55. Wootla B., Lacroix-Desmazes S., Warrington A. E., Bieber A. J., Kaveri S. V., Rodriguez M. (2011) Autoantibodies with enzymatic properties in human autoimmune diseases. J. Autoimmun. 37, 144–150 [PMC free article] [PubMed]
56. Wootla B., Christophe O. D., Mahendra A., Dimitrov J. D., Repessé Y., Ollivier V., Friboulet A., Borel-Derlon A., Levesque H., Borg J. Y., Andre S., Bayry J., Calvez T., Kaveri S. V., Lacroix-Desmazes S. (2011) Proteolytic antibodies activate factor IX in patients with acquired hemophilia. Blood 117, 2257–2264 [PubMed]
57. Lacroix-Desmazes S., Bayry J., Kaveri S. V., Hayon-Sonsino D., Thorenoor N., Charpentier J., Luyt C. E., Mira J. P., Nagaraja V., Kazatchkine M. D., Dhainaut J. F., Mallet V. O. (2005) High levels of catalytic antibodies correlate with favorable outcome in sepsis. Proc. Natl. Acad. Sci. U.S.A. 102, 4109–4113 [PMC free article] [PubMed]
58. Wootla B., Nicoletti A., Patey N., Dimitrov J. D., Legendre C., Christophe O. D., Friboulet A., Kaveri S. V., Lacroix-Desmazes S., Thaunat O. (2008) Hydrolysis of coagulation factors by circulating IgG is associated with a reduced risk for chronic allograft nephropathy in renal transplanted patients. J. Immunol. 180, 8455–8460 [PubMed]
59. Paul S., Li L., Kalaga R., O'Dell J., Dannenbring R. E., Jr., Swindells S., Hinrichs S., Caturegli P., Rose N. R. (1997) Characterization of thyroglobulin-directed and polyreactive catalytic antibodies in autoimmune disease. J. Immunol. 159, 1530–1536 [PubMed]
60. Paul S., Karle S., Planque S., Taguchi H., Salas M., Nishiyama Y., Handy B., Hunter R., Edmundson A., Hanson C. (2004) Naturally occurring proteolytic antibodies: selective immunoglobulin M-catalyzed hydrolysis of HIV gp120. J. Biol. Chem. 279, 39611–39619 [PubMed]
61. Roux K. H., Strelets L., Brekke O. H., Sandlie I., Michaelsen T. E. (1998) Comparisons of the ability of human IgG3 hinge mutants, IgM, IgE, and IgA2, to form small immune complexes: a role for flexibility and geometry. J. Immunol. 161, 4083–4090 [PubMed]
62. Ramsland P. A., Brock C. R., Moses J., Robinson B. G., Edmundson A. B., Raison R. L. (1999) Structural aspects of human IgM antibodies expressed in chronic B lymphocytic leukemia. Immunotechnology 4, 217–229 [PubMed]

Articles from The Journal of Biological Chemistry are provided here courtesy of American Society for Biochemistry and Molecular Biology

Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Nucleotide
    Nucleotide
    Primary database (GenBank) nucleotide records reported in the current articles as well as Reference Sequences (RefSeqs) that include the articles as references.
  • Protein
    Protein
    Protein translation features of primary database (GenBank) nucleotide records reported in the current articles as well as Reference Sequences (RefSeqs) that include the articles as references.
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem chemical substance records that cite the current articles. These references are taken from those provided on submitted PubChem chemical substance records.

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...