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Clinical Trial
. 2014 May 9;289(19):13243-58.
doi: 10.1074/jbc.M114.557231. Epub 2014 Mar 19.

Physiological IgM Class Catalytic Antibodies Selective for Transthyretin Amyloid

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Free PMC article
Clinical Trial

Physiological IgM Class Catalytic Antibodies Selective for Transthyretin Amyloid

Stephanie A Planque et al. J Biol Chem. .
Free PMC article

Abstract

Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded β-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetrameric TTR (phyTTR). IgM class B cell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid β peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function.

Keywords: Aging; Amyloid; Amyloidosis; Antibodies; Catalytic Antibody; Innate Immunity; Protein Evolution; Superantigen; Transthyretin.

Figures

FIGURE 1.
FIGURE 1.
Preaggregated and non-aggregated TTR properties. A, ThT binding. ThT fluorescence values for non-radiolabeled TTR (no label) and 125I-TTR (125I) were small compared with TTR aggregated over 5 days (TTR, 7.1 μm; ThT, 15 μm). B, turbidity. The turbidity of non-radiolabeled TTR (no label) and 125I-TTR (125I) was marginal compared with the TTR aggregated over 5 days. C, SDS-gel electrophoresis. Left box, non-aggregated 125I-TTR without (lane 2) or with boiling (lane 1). Right box, preaggregated 125I-TTR without (lane 4) or with boiling (lane 3). Bands labeled 4mer, 2mer, and 1mer are the tetramer (54 kDa), dimer (27 kDa), and monomer (14 kDa), respectively. Minor 20 and 26 kDa bands are impurities from the source TTR (<0.5%). The 4-mer phyTTR species is dominant in non-boiled, non-aggregated TTR, whereas the 1-mer derived from soluble and insoluble misTTR is present in large amounts in the non-boiled, preaggregated TTR. misTTR content of the preaggregated and non-aggregated 125I-TTR preparation, respectively, is 70 and <0.5%. D, TTR distribution in particulate and soluble fractions. Nearly equivalent radioactivity amounts from preaggregated 125I-TTR were recovered in the pellet (particulate fraction) and supernatant (soluble fraction) obtained by centrifugation. E, misTTR distribution in particulate and soluble TTR fractions. The non-boiled particulate and soluble fractions prepared from preaggregated 125I-TTR contained the radioactive 1-mer band derived from misTTR as the majority and minority species, respectively. Plotted values computed as 100 × (1-mer band intensity/sum of 1-mer and 4-mer band intensities). Inset, lane 1 shows preaggregated 125I-TTR without separation into particulate and soluble fractions (radioactivity in 1-mer and 4-mer bands, respectively, 43 and 57%). The 1-mer band is dominant in particulate 125I-TTR (95% of total radioactivity; lane 2) and is also present in smaller amounts in soluble 125I-TTR (27% of total radioactivity; lane 3). Error bars, S.D.
FIGURE 2.
FIGURE 2.
Selective misTTR hydrolysis by polyclonal IgM. A, IgM class-restricted hydrolytic activity. Boiled reaction mixtures of preaggregated or non-aggregated 125I-TTR (red and blue bars, respectively; 100 nm; misTTR content, 40%) treated for 18 h with diluent or IgM, IgG, and IgA purified from human serum (130 μg/ml, pooled from sera of 12 healthy humans) were electrophoresed. Hydrolysis was determined from depletion of the 1-mer band intensity after antibody treatment relative to the band intensity after diluent treatment. Inset, electrophoresis lanes showing depletion of the 1-mer band in preaggregated 125I-TTR treated with IgM (lane 2) compared with diluent (lane 1). Products are visible as 13, 10, and 7 kDa bands. No 1-mer band depletion or products were evident in the pIgM-treated, non-aggregated 125I-TTR (lane 4) compared with the diluent-treated substrate (lane 3). B, hydrolysis of non-radiolabeled, misTTR by polyclonal IgM. Silver-stained electrophoresis gel of preaggregated TTR (4.5 μm) treated for 52 h with diluent (lane 1) or pooled pIgM (lane 2; 570 μg/ml). From densitometry, 85% of the 1-mer band derived from misTTR was depleted by pIgM treatment. The 10- and 7-kDa product fragments are observed. The 13-kDa product is not visible because of poor silver stainability. C, hydrolysis of soluble and particulate misTTR species by pIgM. The soluble and particulate fractions of preaggregated 125I-TTR (misTTR content, 37 and >95%, respectively) were treated for 18 h with diluent or polyclonal IgM (pIgM) and IgG (pIgG) purified from the same human serum pool (130 μg/ml). Non-boiled reaction mixtures were analyzed. D, misTTR selectivity. The 1-mer band (14 kDa) derived from misTTR but not the 4-mer phyTTR band (54 kDa) of preaggregated 125I-TTR (100 nm; misTTR content, 40%) was depleted as a function of time by treatment with pooled pIgM (130 μg/ml). Inset, 4-mer, 1-mer, and 10 kDa band cut-outs from electrophoresis gels; non-boiled reaction mixtures. Error bars, S.D.
FIGURE 3.
FIGURE 3.
Characterization of polyclonal IgM hydrolytic activity. A, comparison of hydrolytic activity of individual pIgM preparations (90 μg/ml) from non-aged (<35 years, n = 12) and aged humans (>70 years, n = 20) determined by electrophoresis. Shown are the hydrolysis levels estimated using the preaggregated 125I-TTR substrate (160 nm; misTTR content, 60%; 18-h incubation) and non-boiled reaction mixtures. B, rates of preaggregated 125I-TTR hydrolysis by MMP9 and pIgM (left axis). MMP9 and pIgM concentrations were 133 nm. Other conditions were as in A. T50 (right axis) is the time required to hydrolyze 50% of 1 nm misTTR at the MMP9 or pIgM concentrations in blood (3.2 nm and 2.2 μm, respectively, computed from the equation, S = S0(1 − e−[Eo]kt), where S0 is substrate concentration at time 0, E0 is the enzyme concentration at time 0, k is the second order rate constant derived from the equilibrium binding and catalytic constants, and t is reaction time. Inset, SDS gel showing reduced 1-mer misTTR band following MMP9 treatment (lane 2) compared with diluent treatment (lane 1). C, pIgM immunoadsorption. misTTR hydrolysis by unfractionated human serum and Ab-free serum was measured as in A. The protein content of unfractionated serum (diluted 1:100) and the antibody-free serum was adjusted to be identical (1.14 mg/ml). IgM concentrations in hydrolysis assays using unfractionated serum and antibody-free serum were 20 and <0.01 μg/ml, respectively. Data are expressed as percentage depletion of the 1-mer misTTR band compared with the control substrate reaction mixture treated with diluent (100% value = 22.5 ± 5.6 nm). Inset, electrophoresis showed identical product fragments (13, 10, and 7 kDa bands) generated by digesting preaggregated 125I-TTR with unfractionated serum (lane 3, 1:100 dilution) and purified IgM (lane 4, 120 μg/ml). Lane 1, substrate treated with diluent. Lane 2, substrate treated with antibody-free serum. D, distribution of hydrolytic activity of unfractionated serum. Only the IgM-containing 900-kDa fraction (retention volume 8–11 ml) obtained by FPLC gel filtration of human serum (blue trace) displayed detectable misTTR hydrolytic activity assayed as in A (red bars). IgM concentration in the assay, 20 μg/ml (equivalent to IgM concentration in 1% unfractionated serum). Elution of reference purified IgM (black trace) is coincident with elution of the serum hydrolytic activity. Non-IgM serum fractions were tested as two pools at a concentration corresponding to 1% unfractionated serum equivalents (retention volume 0–8.0 and 11.0–22.0 ml; concentrated using a 3-kDa ultrafilter). Inset, reducing SDS gels of 900-kDa serum IgM fraction showing anti-μ antibody-stained 70-kDa heavy chain band (lane 1) and anti-λ/κ antibody-stained 25-kDa light chain band (lane 2). Lanes 3 and 4, respectively, are similarly stained heavy and light chain bands of the reference IgM, respectively. H, heavy chain; L, light chain. E, protease inhibitor effects. misTTR hydrolysis was measured using IgM pretreated (1 h) with diluent or the indicated inhibitors of metalloproteases, cysteine proteases, acid proteases, and serine proteases. The structure of phosphonate 1 is shown. SAP is an amyloid-binding protein. Residual hydrolytic activity of inhibitor-treated IgMs was computed as a percentage of IgM activity after treatment with diluent (100% value = 24 ± 4 nm TTR). Hydrolysis was measured using boiled reaction mixtures of 100 nm preaggregated 125I-TTR incubated with 60 μg IgM/ml for 18 h. F, unimpeded hydrolysis of preaggregated 125I-TTR by pIgM in the presence of Ab-free serum. Plotted are 1-mer misTTR hydrolytic activities expressed as a percentage of the activity of purified (130 μg/ml) alone (90 ± 3 nm). Reaction conditions were as in A. Error bars, S.D.
FIGURE 4.
FIGURE 4.
Selective misTTR recognition by B cells and monoclonal IgMs. A, selective E-misTTR binding by human IgM+ B cells. Profile of PECy7-SA-stained IgM+ B cells treated with E-misTTR in diluent or excess non-biotinylated misTTR. The dotted area represents IgM+ B cells with saturable E-misTTR binding activity (45% of total IgM+ B cells). E-misTTR binding was not affected in the presence of excess ovalbumin (Ova). B, nearly equivalent binding of the cells to E-phyTTR was evident in diluent and in the presence of excess phyTTR, suggesting the absence of saturable E-phyTTR reactivity. C, E-misTTR binding to IgM+ B cells. Deconvoluted images show an IgM+ B cell stained with FITC-conjugated antibody to IgM (image 1, green), the cell stained with PECy7-SA to visualize E-misTTR binding (image 2, red), the merged rendition (image 3), and the reconstituted three-dimensional model suggesting E-misTTR binding to cell surface BCRs (image 4). Blue counterstain, 4′,6-diamidino-2-phenylindole; magnification, ×60. D, misTTR hydrolysis by monoclonal IgM 1802. The reaction mixtures of preaggregated or non-aggregated 125I-TTR (100 nm) treated with diluent or hydrolytic mIgM 1802 (18 h, 60 μg/ml) were boiled, thereby dissociating both phyTTR and misTTR into 1-mers (14 kDa). IgM 1819 is one of four IgMs devoid of misTTR hydrolytic activity. Inset, electrophoresis gel showing preaggregated 125I-TTR treated with IgM 1802 (lane 1) or mIgM 1819 (lane 2) and non-aggregated 125I-TTR treated with mIgM 1802 (lane 3) or mIgM 1819 (lane 4). Product profiles were similar to the polyclonal IgM digests. E, time-dependent misTTR hydrolysis. mIgM 1802 treatment (120 μg/ml) depleted the 1-mer band derived from misTTR but not the 4-mer phyTTR band visualized by electrophoresis (assay conditions as in A). Insets, electrophoresis gel cut-outs of the 4-mer phyTTR band and 1-mer misTTR band at the four time points tested (left to right: 0, 18, 54, and 154 h). Error bars, S.D.
FIGURE 5.
FIGURE 5.
Relationship between EAR-AMC and misTTR hydrolysis by monoclonal IgMs. A, split site catabody model. Catabody selectivity derives from initial epitope recognition by the noncovalently binding subsite followed by peptide bond hydrolysis at a spatially proximate catalytic subsite. Small peptide substrates are hydrolyzed at the catalytic subsite without dependence on the noncovalent binding subsite. B, misTTR and EAR-AMC hydrolysis at a shared catalytic subsite. Hydrolysis of preaggregated 125I-TTR (100 nm; misTTR content, 40%) by mIgM Yvo (300 μg/ml) was measured in the presence of the alternate substrates EAR-AMC or AAA-AMC (1 mm; incubation for 18 h). IgM hydrolytic activity is expressed as a percentage of activity in diluent instead of alternate substrate (45 ± 3 nm). Inset, data from Ref. showing stoichiometric inhibition of mIgM Yvo-catalyzed EAR-AMC hydrolysis at varying phosphonate 1/IgM molar ratios. The x intercept is the number of catalytic sites/mIgM molecule. C, correlated EAR-AMC and misTTR hydrolysis by mIgMs. EAR-AMC hydrolysis by the panel of 16 mIgMs was measured fluorimetrically (1). misTTR hydrolysis was measured as in Fig. 4 using boiled reaction mixtures. Solid and dotted lines show the least-square regression fit (Pearson r2 = 0.44, p < 0.005) and 95% confidence band, respectively. Error bars, S.D.
FIGURE 6.
FIGURE 6.
Monoreactive and oligoreactive misTTR hydrolyzing monoclonal IgMs. A, no hydrolysis of non-amyloid and non-superantigen proteins. Shown are SDS electrophoresis gels containing reaction mixtures of the following biotinylated proteins treated for 22 h with pIgM (pooled from 12 humans) or mIgM 1802 (130 μg/ml): extracellular domain of epidermal growth factor receptor (exEGFR), bovine serum albumin (BSA), ovalbumin (OVA), transferrin (Trans), and two S. aureus virulence factors, LukS and collagen adhesion protein CNA. mIgM-P1, control reaction mixture containing IgM 1802 inhibited by electrophilic phosphonate 1. B–D, no fibrillar Aβ hydrolysis by mIgM 1802. FPLC gel filtration profiles of formic acid-solubilized reaction mixtures containing fibrillar 125I-Aβ42 (30,000 cpm) treated for 16 h with diluent (B), monoclonal mIgM 1802 (120 μg/ml) (C), or the Aβ-hydrolyzing immunoglobulin V domain fragment (IgV 2E6, 10 μg/ml) (D). No depletion of intact 125I-Aβ42 (computed mass 4,630 Da) or product appearance was evident by mIgM treatment, whereas IgV 2E6 generated a radioactive fragment with a mass of 1654 Da corresponding to hydrolysis at the His14–Gln15 bond (29). E, discordant misTTR and Aβ hydrolytic activities of mIgMs (n = 9). Aβ40 hydrolysis data are from Ref. . Connecting lines identify individual mIgMs. In parentheses are the number of mIgMs without detectable activity. misTTR-monoreactive mIgM 1814 and oligoreactive mIgM Yvo are identified. F, discordant misTTR and Protein A hydrolytic activities of mIgMs (n = 16). Hydrolysis of biotinylated Protein A (72 nm) following IgM treatment (45 μg/ml, 72 h) was determined electrophoretically. Solid and dotted lines, least-square regression fit and 95% confidence bands, respectively (p > 0.05, r2 < 0.001; two-tailed Pearson analysis). misTTR-monoreactive mIgMs 1802 and 1814 and oligoreactive mIgM Yvo are identified. Inset, SDS-gel electrophoresis lanes of Protein A treated with diluent (lane 1), non-hydrolytic mIgM 1801 (lane 2), and hydrolytic mIgM Yvo showing depletion of intact Protein A and the appearance of smaller mass products (lane 3). G, hydrolysis of biotinylated gp120 (100 nm) following IgM treatment (45 μg/ml, 18 h) was determined electrophoretically. Solid and dotted lines are the least-square regression fit and 95% confidence bands, respectively (p > 0.05, Pearson r2 = 0.007). misTTR-monoreactive mIgMs 1802 and 1814 and oligoreactive mIgM Yvo are identified. Inset, SDS-gel electrophoresis of gp120 treated with diluent (lane 1), non-hydrolytic mIgM 1801 (lane 2), and hydrolytic mIgM Yvo showing depletion of intact gp120 and appearance of smaller mass product bands (lane 3). H, inhibition of mIgM Yvo catalyzed misTTR hydrolysis by alternate substrates. Only Aβ inhibited the misTTR hydrolytic activity. Like the irrelevant protein ovalbumin (OVA), the superantigens Protein A and gp120 were non-inhibitory. Concentration of alternate substrates was 1 μm, and that of mIgM Yvo was 130 μg/ml. Other reaction conditions were as in Fig. 4A. Error bars, S.D.
FIGURE 7.
FIGURE 7.
Dissolution of preaggregated TTR by mIgM 1802. A, reduced turbidity. Preaggregated TTR (7.1 μm) was incubated in the presence of hydrolytic mIgM 1802 (120 μg/ml, 0.67 μm), an equivalent non-hydrolytic IgM 1819 concentration, or diluent. mIgM 1802 treatment reduced the turbidity to the basal value observed for control non-aggregated TTR treated with mIgM 1802. Inset, photograph showing turbid misTTR suspension (white precipitates against black background) following incubation in diluent and misTTR dissolution after treatment with the hydrolytic IgM. B, reduced ThT fluorescence. Shown are ThT fluorescence values after treating preaggregated TTR with hydrolytic mIgM 1802, non-hydrolytic mIgM, or diluent for 120 h as in A. mIgM 1802 treatment reduced the ThT fluorescence value to the basal value observed for non-aggregated TTR treated with diluent. Data are corrected for ThT fluorescence of IgMs alone without TTR substrate (84 ± 5 fluorescence units (FU)). Error bars, S.D.
FIGURE 8.
FIGURE 8.
Catabody defense system (CADSys). A, regions of TTR with the best sequence similarity to Aβ, gp120, and Protein A. 1, TTR and Aβ42 (e = 9.4). 2 and 3, TTR and gp120 (e = 2.6 and 6.4, respectively). 4, TTR and Protein A (e = 23). Dots, identities. Red, conservative substitutions. Alignments were obtained with BLAST using the BLOSUM62 matrix and expect (e) threshold of 1,000. The sequence similarities are weak (large e values, p > 0.05 for all alignments). The indicated Protein A region, but not the Aβ42 and gp120 regions, has a β-turn-forming propensity. The TTR region aligned with Protein A also tends to form a β-turn. Substrate regions with the greatest β-turn forming propensity are as follows: TTR, 95–101 and 109–115; Aβ, 5–11 and 23–29; gp120, 146–147e and 362–368 (HXB2 numbering); Protein A, 259–265 and 299–305. Accession numbers are as follows: human TTR, AAA73473; human Aβ42, NP000475; HIV gp120MN, AF075721; S. aureus Protein A, 88193823. B, the catabody defense system concept is based on discovery of innate antibodies that clear pathogenic amyloids and microbial superantigens. Because amyloid removal is predicted to confer a survival advantage to the organism, the emergence of germ line anti-amyloid catabodies in immune evolution is proposed. Superantigen epitopes leave microbes susceptible to innate catabodies and reversibly binding antibodies. However, superantigens help microbes evade acquired immunity, and microbial superantigen-host immune system interactions probably reflect competing factors favoring survival of microbes and the host. C, amyloid protein conservation in humans and jawed fish of the Batoidea superorder (Leucoraja erinacea and Narke japonica). Top, TTR; bottom, Aβ42. Dots, amino acid identities. Red, conservative substitutions. The full-length human TTR and Aβ42 sequences are shown. Accession numbers are as follows: L. erinacea TTR, CV221819; N. japonica amyloid precursor protein 699 residues 601–642, BAA24230.1. Jawed fish are the first extant organisms with human antibody-like molecules.

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