Modulation of lysyl oxidase-like 2 enzymatic activity by an allosteric antibody inhibitor

Abstract

In this report, we assessed the steady-state enzymatic activity of lysyl oxidase-like 2 (LOXL2) against the substrates 1,5-diaminopentane (DAP), spermine, and fibrillar type I collagen. We find that both DAP and spermine are capable of activating LOXL2 to the same extent and have similar Michaelis constants (K(m) approximately 1 mm) and catalytic rates (k(cat) approximately 0.02 s(-1)). We also show that LOXL2 is capable of being inhibited by a known suicide inhibitor of lysyl oxidase (LOX), beta-aminopropionitrile, which we find is a potent inhibitor of LOXL2 activity. The modality of inhibition of beta-aminopropionitrile was also examined and found to be competitive with respect to the substrates DAP and spermine. In addition, we identified an antibody inhibitor (AB0023) of LOXL2 enzymatic function and have found that the inhibition occurs in a non-competitive manner with respect to both spermine and DAP. The binding epitope of AB0023 was mapped to the scavenger receptor cysteine-rich domain four of human LOXL2. AB0023 binds to a region remote from the catalytic domain making AB0023 an allosteric inhibitor of LOXL2. This affords AB0023 several advantages, because it is specific for LOXL2 and inhibits the enzymatic function of LOXL2 in a non-competitive manner thereby allowing inhibition of LOXL2 regardless of substrate concentration. These results suggest that antibody allosteric modulators of enzymatic function represent a novel drug development strategy and, in the context of LOXL2, suggest that inhibitors such as these might be useful therapeutics in oncology, fibrosis, and inflammation.

FIGURE 1.

Purity of proteins used. (A), SDS-PAGE 4–12% showing the purity of the proteins used in this study. The marker is denoted by M, and molecular weights are labeled next to corresponding band. Lane 1 is hLOX, lane 2 is hLOXL1, lane 3 is hLOXL2, lane 4 is hLOXL3, and lane 5 is hLOXL4. (B), Western blot of proteins shown in A (0.5 μg/well) probed using and anti-LOXL2 antibody (1 μg/ml) produced at Arresto. The marker is denoted by M, and molecular weights are labeled next to the corresponding band. Lane 1 is hLOX, lane 2 is hLOXL1, lane 3 is hLOXL2, lane 4 is hLOXL3, and lane 5 is hLOXL4. (C), Western blot of the proteins shown in A probed with anti-histidine antibody. The marker is denoted by M, and molecular weights are labeled next to corresponding band. Lane 1 is hLOX, lane 2 is hLOXL1, lane 3 is hLOXL2, lane 4 is hLOXL3, and lane 5 is hLOXL4.

FIGURE 2.

The steady-state enzymatic rate was measured over a concentration range of substrate for 1,5-diaminopentane (○) and spermine (●). The steady-state values k_cat and K_m were determined for DAP and spermine by fitting the data (line) to the Michaelis-Menten equation (see “Experimental Procedures”). K_m = 1.01 ± 0.18 mm and k_cat = 0.015 ± 0.001 s⁻¹ for DAP and K_m = 1.05 ± 0.32 mm and k_cat = 0.014 ± 0.001 s⁻¹ for spermine.

FIGURE 3.

Inhibition of LOLX2 by BAPN where the substrate is DAP (IC₅₀ = 5. 0 ± 1.4 μm) and spermine (IC₅₀ = 3.8 ± 0.2 μm). The final reaction contained 25 nm LOXL2, 15 mm DAP, or spermine. Data were normalized to the “no inhibitor control.”

FIGURE 4.

Inactivation kinetics of LOXL2. A, a representative example of the dependence of the rate of inactivation against the concentration of BAPN when DAP is used as substrate at different concentrations. The concentrations of DAP are given. B, the second order rate constant (k_inact/K_I) as a function of DAP concentration. The k_inact/K_I values are the slope of the data in A. The plotted data are the average of the replicates, and the error bars represent the standard deviations. C, plot representative of the rate of inactivation as a function of BAPN over varying spermine concentrations. D, the dependence of k_inact/K_I on spermine concentration. Shown are the average of the replicates, and the error bars are the standard deviations.

FIGURE 5.

Binding specificity of AB0023 against the five members of the lysyl oxidase family (A), LOXL2 using surface Plasmon resonance (B). The ordinate describes the signal, and the abscissa is time in seconds. The figure shows kinetic traces of AB0023 binding to LOXL2 with AB0023 at concentrations of 25 nm, 12.5 nm, 6.25 nm, and 3.125 nm at 25 °C and the different SRCR domain fragments of LOXL2 (C). Binding against the lysyl oxidase family and SRCR fragments was detected using an ELISA-based assay and data fit as described under “Experimental Procedures.” Surface plasmon resonance experiments were conducted on a ProteOn XPR system, and data were analyzed using the ProteOn Manager software. (D), binding assessment of AB0023 against the intact SRCR domains of hLOXL2, hLOXL3 and (E) hLOXL4 using an ELISA-based method.

FIGURE 6.

Inhibition of LOXL2 by AB0023 in a reaction where DAP (IC₅₀′ = 62 ± 5.8 nm) and spermine (IC₅₀′ = 55 ± 11 nm) (A) or collagen I (IC₅₀′ = 60.9 ± 3.9 nm) (B) are used as substrate. LOXL2 concentration was at 25 nm when DAP and spermine are used. For collagen I reactions, LOXL2 was at 100 nm. C, the effect of AB0023 on the enzymatic activity of active hLOXL3 as a function of AB0023 concentration. The final reaction mixture contained 20 nm hLOXL3, 15 mm DAP, and the indicated concentration of AB0023. D, inhibition of hLOXL3 by BAPN where the substrate is DAP. Final conditions are as described in C. The observed IC₅₀ for BAPN against hLOXL3 was 3.4 μm ± 1.9 μm. Data for all figures were normalized to the enzymatic rate in the absence of AB0023 or inhibitor.

FIGURE 7.

AB0023 inhibition modality against DAP (α = 1. 02 ± 0.05, β = 0.53 ± 0.04) (A) and spermine (α = 1.13 ± 0.26, β = 0.51 ± 0.06) (B) in the presence of varying concentrations of AB0023 (as shown). The response with both substrates is consistent with that of a non-competitive inhibitor (α = 1, see text).