Synergistic Ca2+ and Cu2+ requirements of the FGF1–S100A13 interaction measured by quartz crystal microbalance: An initial step in amlexanox-reversible non-classical release of FGF1

Abstract

It is known that fibroblast growth factor-1 (FGF1) lacking a conventional signal peptide sequence shows non-classical release independent of the endoplasmic reticulum-Golgi system. Recent studies reveal that FGF1 is co-released with S100A13, a Ca 2+ -binding protein that acts as an extracellular cargo molecule. Although both FGF1 and S100A13 are Cu 2+ -binding proteins, the role of Cu 2+ , as well as that of Ca 2+ , in non-classical release, remains to be clarified. In the present study we examined the requirements of both metal ions for the interaction between these two proteins. The addition of Ca 2+ significantly increased the k a value, while decreasing the K D value, for the interaction between Strep-tagII-S100A13 and GST-FGF1; both values were obtained by use of a quartz crystal microbalance, a real-time mass measuring device.The EC 50 of Ca 2+ to enhance the interaction was 10.11 µM. Although the addition of Cu 2+ alone had no effect, it caused a marked potentiation of the Ca 2+ -enhanced interaction. The EC 50 of Cu 2+ for the potentiation was 50.45 nM. On the other hand, the EC 50 of Ca 2+ and the K D value were decreased from 11.69 to 2.07 µM and 0.75 to 0.38 x 10 -7 M, respectively, by the addition of 200 nM Cu 2+ . The Cu 2+ -induced potentiation of this interaction was abolished by amlexanox, which inhibits non-classical release of FGF1. All of these findings suggest that synergistic effects of Ca 2+ and Cu 2+ play a key role in the interaction between FGF1 and S100A13, which is the initial step in non-classical release of FGF1.

Stress-induced non-vesicular release of prothymosin-α initiated by an interaction with S100A13, and its blockade by caspase-3 cleavage

“…[15][16][17] It has been reported that S100A13 is involved in the non-classical extracellular release of target molecules containing fibroblast growth factor-1 (FGF-1) and interleukin-1a. [9][10][11]19,34,35 In this study, we successfully showed that ProTa is another example of stress-induced non-vesicular extracellular release, using S100A13, a cargo molecule. Furthermore, we revealed that the C-terminal regions of ProTa and S100A13 are essential for their interaction, which precedes extracellular release of both proteins, and that caspase-3 cleaves off C-terminal regions of ProTa.…”

“…The addition of Strep-tagII-S100A13 to GST-ProTa immobilized on the sensor tip of a quartz crystal microbalance (QCM) decreased the quartz oscillation, as quantified by the oscillation unit (OU: DF in Hz), which represents the degree of interaction between the two proteins, as reported earlier. 19 The interaction between proteins was enhanced in the presence of Ca 2 þ , but not in the presence of Mg 2 þ or Cu 2 þ , and this enhancement was Ca 2 þ dependent in the range 0.1-200 mM (Table 1; Figure 4b). As the Ca 2 þ -dependent interaction was further enhanced by the addition of Cu 2 þ (Table 1), which has substantial binding affinity for S100A13, we used the addition of both Ca 2 þ and Cu 2 þ (100 mM and 100 nM, respectively) to determine the best ionic conditions for the interaction between GST-ProTa and Strep-tagII-S100A13.…”

“…As the Ca 2 þ -dependent interaction was further enhanced by the addition of Cu 2 þ (Table 1), which has substantial binding affinity for S100A13, we used the addition of both Ca 2 þ and Cu 2 þ (100 mM and 100 nM, respectively) to determine the best ionic conditions for the interaction between GST-ProTa and Strep-tagII-S100A13. From the kinetic analysis, 19 the saturated OU max for the interaction of Strep-tagII-S100A13 with immobilized GST-ProTa (430 fmol) was 201.44±3.53 OU, which corresponds to 435.63 ± 7.63 fmol. Therefore, it is evident that ProTa and S100A13 interact at a ratio of 1 : 1.…”

“…Detailed procedures have been described earlier. 19 AT-cut quartz crystals coated with a thin gold surface layer with a fundamental frequency of 27 MHz were used. Immediately before use, the gold surface of the quartz resonator was cleaned with piranha solution (H 2 SO 4 : 30% H 2 O 2 ¼ 3 : 1) for 5 min, and thoroughly washed with double-distilled water.…”

A survey of the 2006–2009 quartz crystal microbalance biosensor literature

“…Fibroblast growth factor‐1 (FGF1) was co‐released with S100A13, a Ca 2+ ‐binding protein that acts as an extracellular cargo molecule. QCM was used to determine affinities and kinetics in the presence of varying concentrations of Ca 2+ or Cu 2+ (Matsunaga and Ueda, 2008).…”