Functional Significance of Calcium Binding to Tissue-Nonspecific Alkaline Phosphatase

<div><p>The conserved active site of alkaline phosphatases (AP) contains catalytically important Zn²⁺ (M1 and M2) and Mg²⁺-sites (M3) and a fourth peripheral Ca²⁺ site (M4) of unknown significance. We have studied Ca²⁺ binding to M1-4 of tissue-nonspecific AP (TNAP), an enzyme crucial for skeletal mineralization, using recombinant TNAP and a series of M4 mutants. Ca²⁺ could substitute for Mg²⁺ at M3, with maximal activity for Ca²⁺/Zn²⁺-TNAP around 40% that of Mg²⁺/Zn²⁺-TNAP at pH 9.8 and 7.4. At pH 7.4, allosteric TNAP-activation at M3 by Ca²⁺ occurred faster than by Mg²⁺. Several TNAP M4 mutations eradicated TNAP activity, while others mildly influenced the affinity of Ca²⁺ and Mg²⁺ for M3 similarly, excluding a catalytic role for Ca²⁺ in the TNAP M4 site. At pH 9.8, Ca²⁺ competed with soluble Zn²⁺ for binding to M1 and M2 up to 1 mM and at higher concentrations, it even displaced M1- and M2-bound Zn²⁺, forming Ca²⁺/Ca²⁺-TNAP with a catalytic activity only 4–6% that of Mg²⁺/Zn²⁺-TNAP. At pH 7.4, competition with Zn²⁺ and its displacement from M1 and M2 required >10-fold higher Ca²⁺ concentrations, to generate weakly active Ca²⁺/Ca²⁺-TNAP. Thus, in a Ca²⁺-rich environment, such as during skeletal mineralization at pH 7.4, Ca²⁺ adequately activates Zn²⁺-TNAP at M3, but very high Ca²⁺ concentrations compete with available Zn²⁺ for binding to M1 and M2 and ultimately displace Zn²⁺ from the active site, virtually inactivating TNAP. Those <i>ALPL</i> mutations that substitute critical TNAP amino acids involved in coordinating Ca²⁺ to M4 cause hypophosphatasia because of their 3D-structural impact, but M4-bound Ca²⁺ is catalytically inactive. In conclusion, during skeletal mineralization, the building Ca²⁺ gradient first activates TNAP, but gradually inactivates it at high Ca²⁺ concentrations, toward completion of mineralization.</p></div

Role of Ca²⁺ binding to M4 in TNAP activity.

<p>Dose-response of stimulation of AP activity (mean mA405nm/min) in steady-state (i.e. the slope measured between 60–90 min) at pH 9.8 (10 mM pNPP) for increasing [CaCl₂], added to AbM2-bound Zn²⁺-TNAP and the indicated mutants, after pre-treatment with EDTA (2 h) and loading with 20 μM Zn²⁺; correlation between calculated apparent K_ds for the functionally relevant Mg²⁺ and Ca²⁺ binding to TNAP and the three indicated TNAP mutants. Lines constructed in the absence and presence of 20 μM ZnCl₂ are as indicated; Apparent K_ds are represented with their respective SD.</p

Kinetic Parameters of PLAP, TNAP and the M4-site mutants, measured in 1M DEA buffer, pH 9.8, with pNPP as a substrate.

<p>* based on historical values [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119874#pone.0119874.ref013" target="_blank">13</a>]</p><p>Kinetic Parameters of PLAP, TNAP and the M4-site mutants, measured in 1M DEA buffer, pH 9.8, with pNPP as a substrate.</p

Representation of M4 ligands in the modeled structure of TNAP.

<p>Rendering of the 3D structure of the entire TNAP dimer in front view (a) and coordinating residues, detailed in a lateral zoom of the shoulder region harboring M4, in larger detail (b) and in ribbon representation (c).</p

Allosteric activation of Zn²⁺-TNAP by MgCl₂.

<p>a. Progressive AbM2-bound Zn²⁺-TNAP activation, visualized as increasing slopes in plots of A405 nm <i>vs</i>. time, for the indicated [Mg²⁺], added to Chelex-pretreated pNPP (10 mM) at pH 9.8 (left panel); repeat of the experiment in (a) at 15-fold lower [TNAP] over a time-interval 0–90 min, for the indicated [Mg²⁺] (right panel); b. Slopes (first derivatives) to the lines in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119874#pone.0119874.g001" target="_blank">Fig. 1A</a>, right panel <i>vs</i>. time, for the indicated [Mg²⁺], describing formation of Mg²⁺/Zn²⁺-TNAP as a function of time (left panel); plots of k_app (calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119874#pone.0119874.g001" target="_blank">Fig. 1B</a> left panel) <i>vs</i>. [Mg²⁺] and determination of k_a and k_d for binding of Mg²⁺ to Zn²⁺-TNAP (right panel);. Experiments representative of at least three replicates with variable enzyme and MgCl₂ concentrations.</p

Allosteric activation of Zn²⁺-TNAP by CaCl₂.

<p>a. Progressive AbM2-bound Zn²⁺-TNAP activation, measured as A405 nm <i>vs</i>. time, for the indicated [Ca²⁺], added to Chelex-pretreated pNPP (10 mM) at pH 9.8, showing dose-dependent activation (left panel) and inhibition at high concentrations (right panel); b. Dose-response of generated AP activity (mean mA405nm/min) in steady-state (i.e. the slope measured between 60–90 min in Figs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119874#pone.0119874.g001" target="_blank">1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119874#pone.0119874.g002" target="_blank">2A</a>) for increasing [MgCl₂] and [CaCl₂] at identical AbM2-bound [Zn²⁺-TNAP], reflecting the plateau and pseudo-plateau at high [MgCl₂] and [CaCl₂] respectively, followed by a steep drop of the TNAP activity in the case of CaCl₂ (right panel). Activities were measured in Chelex-treated pNPP (10 mM) at pH 9.8, supplemented with MgCl₂ and CaCl₂, as indicated. Experiments representative of at least three replicates with variable enzyme and MgCl₂ concentrations.</p

Consequences of Ca²⁺ binding to TNAP at pH 7.4.

<p><b>a</b>. Kinetics of TNAP activation by low [Mg²⁺]) and acceleration of activation during formation of Mg²⁺/Zn²⁺-TNAP by increasing [CaCl₂] as indicated, measured as A405 nm (left panel); additive effects for various combinations of Mg²⁺ and Ca²⁺ during TNAP activation (measured in steady-state, right panel); <b>b</b>. Dose-response of activation and partial inhibition of TNAP by CaCl₂, at the indicated concentrations (left panel); TNAP activity recovery in chelex-treated pNPP, containing the indicated metal ion composition (0: no metal ion; [Zn²⁺] = 20 μM; [Mg²⁺] = 1 mM; Zn²⁺/Mg²⁺ = 20 μM Zn²⁺ + 1 mM Mg²⁺) after 3 h of TNAP binding to AbM2 in the presence of co-incubated CaCl₂ (0–20 mM, as indicated (right panel); black bars: corresponding activity for maximally active TNAP, (pre-incubation for 3 h in TBS, containing 20 μM ZnCl₂ + 1 mM CaCl₂). Results represent mean ± SD for 3 identical experiments. (*p<0.05 and **p<0.01 <i>vs</i>. plateau, § p<0.005 vs. [CaCl₂] = 0).</p

Structural mapping of TNAP M4 mutants.

<p>Degree of binding of TNAP and the indicated TNAP mutants to four epitope-mapped monoclonal anti-TNAP antibodies (of 19 studied). Binding was analyzed in the absence (black bars) and presence (white bars) of 1 mM CaCl₂. TNAP was bound to microtiter plates coated with AbM2 and bound TNAP (mutant) was detected with AbM2, recognizing the FLAG tag.</p

TNAP inactivation by high [CaCl₂] at pH 7.4.

<p><b>a</b>. Reconstitution of TNAP activity by 20 μM Zn²⁺ + 1 mM Mg²⁺, but not by the individual metal ions, added to fully demetalated holo-TNAP, starting after 60 min (left panel); comparison of specific TNAP activity of (holo)-TNAP, after reconstitution with the indicated metal ion composition, after overnight treatment with TBS, with or without added EDTA (250 μM); b. Dose-dependency of holo-TNAP reconstitution by CaCl₂ (0–10 mM), in the absence or presence of the indicated [ZnCl₂] (0–20 μM, left panel); competition between the indicated concentrations of Zn²⁺ (2 μM and 20 μM) and increasing concentrations of CaCl₂; minor displacement of TNAP-bound Zn²⁺ (overnight Zn²⁺ preloading indicated as “Pre”) by increasing concentrations of CaCl₂, independently of the presence of free Zn²⁺.(0–20 μM). Results represent mean ± SD for 3 identical experiments (*p<0.003, **p<0.0001,).</p

Activation <i>vs</i>. inhibition of Zn²⁺-TNAP by MgCl₂ and CaCl₂.

<p><b>a</b>. Dose-response of generated AP activity (mean mA405nm/min) in steady-state (i.e. the slope measured between 60–90 min) for increasing [MgCl₂] and [CaCl₂] at identical AbM2-bound [Zn²⁺-TNAP], measured in Chelex-treated pNPP (1 mM) at pH 9.8; <b>b</b>. TNAP inhibition at high [MgCl₂] and [CaCl₂], measured in Chelex-treated pNPP (1 or 10 mM as indicated) at pH 9.8, in the presence of the indicated concentrations of MgCl₂. Results represent mean ± SD for 3 identical experiments or are representative examples of experiments, performed in 3-fold (b, middle and right panel).</p