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.
Highlights
20964 JOURNAL OF BIOLOGICAL CHEMISTRY and elastin [4]
We identified a novel antibody that binds to lysyl oxidase-like 2 (LOXL2) and inhibits enzymatic function through a non-competitive inhibitory mechanism, which may serve as an important therapeutic in a variety of cancers and fibrosis-related diseases
In the case of AB0023-mediated inhibition of LOXL2, the inhibitory effect can be exerted on the substrate-bound or free
Summary
Chemicals and Reagents—1,5-Diaminopentane dihydrochloride, spermine, horseradish peroxidase type XII (5000 units), antifoam 204, -aminopropionitrile fumarate salt (BAPN), and 3,3Ј,5,5Ј-tetramethylbenzidine were purchased from Sigma. Washed membrane was probed with anti-LOXL2 antibody generated by Arresto at a concentration of 1 g/ml antibody in the 5% milk solution described above for 1 h at ambient temperature. Resolubilization was centrifuged at 14,000 rpm for 30 min, and the soluble sample was loaded onto Ni-Sepharose resin equilibrated with 16 mM sodium phosphate and 6 M urea, pH 7.8, and batch-bound for 1 h at 4 °C. The bound protein was eluted from resin with 16 mM sodium phosphate, 6 M urea, 0.4 M imidazole, pH 7.8. The experimental setup involves a substrate titration at differing concentrations of inhibitor These data are globally fit using Equation 4 to give the values relating to the kinetics scheme (Scheme 1). Km is the Michaelis constant, Ki is the dissociation constant of the inhibitor, S is substrate concentration, I is the inhibitor concentration, Vmax is the maximal velocity, and  is usually set to zero for linear inhibitors, but in the case of the inhibitory antibody  was allowed to float as the inhibitory effect is only partial and nonlinear
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