Metal-dependent amyloid β-degrading catalytic antibody construct

Abstract

Catalytic antibodies (catabodies) that degrade target antigens rapidly are rare. We describe the metal-dependence of catabody construct 2E6, an engineered heterodimer of immunoglobulin light chain variable domains that hydrolyzes amyloid β peptides (Aβ) specifically. In addition to the electrophilic phosphonate inhibitor of serine proteases, the metal chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline completely inhibited the hydrolysis of Aβ by catabody 2E6. Formation of catabody-electrophilic phosphonate inhibitor adducts was unaffected by EDTA, suggesting that the metal exerts a favorable effect on a catalytic step after the initial catabody nucleophilic attack on Aβ. The EDTA inactivated catabody failed to disaggregate fibrillar Aβ, indicating the functional importance of the Aβ hydrolytic activity. Treating the EDTA-inactivated catabody with Zn(2+) or Co(2+) restored the Aβ hydrolytic activity, and Zn(2+)-induced catabody conformational transitions were evident by fluorescence emission spectroscopy. The studies reveal the absolute catabody dependence on a metal cofactor.

Keywords: Alzheimer disease; Amyloid β clearance; Catalytic antibody light chain; Metal activation.

Fig. 1. EDTA inactivation of IgV 2E6 hydrolytic activity

(a) Enzyme inhibitor effects. ¹²⁵I-Aβ40 was treated (18 h) with IMAC-purified IgV 2E6 (10 μg/mL) that had been preincubated (1 h) with phosphonate 1a (1 mM), Pepstatin A (0.01 mM), 1,10-phenanthroline (0.5 mM), EDTA (1 mM) or iodoacetamide (1 mM). Data show % inhibition of hydrolytic activity compared to ¹²⁵I-Aβ40 hydrolysis by the IgV in diluent (7,165±193 CPM). Inset, heterodimeric IgV composed of V_L1 and V_L2 domains. Filled and unfilled boxes correspond to framework regions (FRs) and complementarity determining regions (CDRs), respectively. (b) Phosphonate inhibitors and EDTA. 1a, 1b contain the electrophilic phosphonate diester. The biotin group of 1b allows detection of irreversible protein adducts. EDTA chelates divalent metals. (c) Irreversible IgV inhibition by EDTA. IMAC-purified IgV 2E6 was pretreated with EDTA (10 mM, 1 h). ¹²⁵I-Aβ40 hydrolysis was measured without or with removal of EDTA (- removal and + removal, respectively). Also shown are hydrolytic activities of EDTA-pretreated paIgV 2E6 (10 mM, 1 h) with and without EDTA removal (1:2,000 dilution of paIgV prepared by anion exchange chromatography). (d) ¹²⁵I-Aβ40 hydrolysis by IgV 2E6-containing bacterial culture supernatants (diluted 1:30). Control culture supernatants displayed negligible hydrolysis (bacteria without vector or empty vector, bacteria expressing IgV MMF6 or IgV 2E6 with mutated FR1-FR4 segments). The activity of reference IMAC purified IgVs is also shown (10 μg/mL).¹²⁵I-Aβ40 treated for 18 h with culture supernatants (mean±SD of 3 replicates). Inset, Lane 1, anti-myc antibody stained SDS-electrophoresis gel of IgV 2E6-containing bacterial culture supernatant. Lanes 2 and 3, respectively, anti-c-myc antibody-stained and silver-stained SDS-gels of IMAC-purified IgV 2E6. The 30 kDa and 18 kDa bands correspond to the hydrolytically active heterodimeric IgV and its inactive C-terminal V_L fragment produced by IgV autolysis respectively (Taguchi et al. 2008a). (e) Peptide bond hydrolysis pathway. R_N-CONH-R_C, substrate peptide; HNu-CAb, catabody containing a nucleophile (HNu-); NH₂-R_C, C-terminal substrate fragment; Wact-CAb, catabody containing a water activating group; R_N-CO₂H, N-terminal substrate fragment. The initial catabody reaction consists of nucleophilic attack on a peptide bond. Hydrolysis of the acyl-catabody intermediate requires a water activating group e.g., a metal ion that facilitates water nucleophilic attack or stabilizes the oxyanionic ester bond transition state (not shown). (f) 1b-IgV 2E6 adducts. The 1b adduct band (30 kDa) formed by IgV 2E6 (22 μg/mL) that had been pretreated with diluent or EDTA (1 mM, 1 h) was comparable. 1b, 0.1 mM, 18 h incubation. Inset, streptavidin-peroxidase stained SDS-gels showing adduct bands formed by EDTA-treated IgV (lane 1) and diluent-treated IgV (lane 2). (g) Reduced fibrillar Aβ disaggregation by EDTA-pretreated paIgV 2E6. Following EDTA pretreatment as in panel C, the EDTA was removed by dialysis, and fibrillar Aβ42 (20 μM) was incubated for 24 h with the EDTA-pretreated paIgV 2E6 (+ bar), control paIgV pretreated with diluent (− bar) or non-catalytic paIgV MMF6 (4 μg/mL). Residual fibrillar Aβ42 was measured by the ThT fluorescence method. Data are expressed as % of ThT fluorescence observed using control fibrillar Aβ42 incubated with diluent (113 ±6 FU). Mean ± SD of two replicates.

Fig. 2. Reactivation of EDTA-treated IgV 2E6 by Co²⁺ and Zn²⁺

(a) Screening of IgV reactivation by divalent metals. IgV 2E6 was inactivated by EDTA (10 mM, 1 h) and the EDTA was removed by dialysis. The EDTA-inactivated IgV 2E6 treated with Zn²⁺ and Co²⁺ but not the remaining metal ions hydrolyzed ¹²⁵I-Aβ40 detectably. Data are % of ¹²⁵I-Aβ40 hydrolysis by the IgV incubated in diluent (no EDTA/no metal, 8,850±486 CPM). IgV 2E6 10 μg/mL, metal dichlorides 0.075 mM, incubation for 18h. (b) Restoration by Co²⁺. IgV 2E6 was inactivated with EDTA as in panel A. ¹²⁵I-Aβ40 hydrolysis by the EDTA-inactivated treated IgV 2E6 (20 μg/mL, open circles) and the control IgV (closed circles) without EDTA treatment was measured in the presence of CoCl₂ (0.75, 0.225, 0.075, 0.0225 mM). (c) Restoration by Zn²⁺. Experimental details as in panel B.

Fig. 3. Zn²⁺-induced IgV conformational transitions

(a) Fluorescence emission spectra of EDTA-inactivated IgV 2E6 (46 μg/ml, 1 h) treated with varying Zn²⁺ concentrations. (b) Fluorescence emission intensity at 336 nm plotted versus Zn²⁺ concentration. Data are from panel A.