Abstract
The antiangiogenic activity of the H/P domain of histidine–proline-rich glycoprotein is mediated by its binding with tropomyosin, a protein exposed on endothelial cell-surface during the angiogenic switch, in presence of zinc ions. Although it is known that copper ion serum concentration is significantly increased in cancer patients, its role in the interaction of H/P domain with tropomyosin, has not yet been studied. In this paper, by using ELISA assay, we determined the modulating effect of TetraHPRG peptide, a sequence of 20 aa belonging to H/P domain, on the binding of Kininogen (HKa) with tropomyosin, both in absence and presence of copper and zinc ions. A potentiometric study was carried out to characterize the binding mode adopted by metal ions with TetraHPRG, showing the formation of complex species involving imidazole amide nitrogen atoms in metal binding. Moreover, circular dichroism showed a conformational modification of ternary systems formed by TetraHPRG, HKa and copper or zinc. Interestingly, slight pH variation influenced the HKa-TetraHPRG-tropomyosin binding. All these results indicate that both metal ions are crucial in the interaction between TetraHPRG, tropomyosin and HKa.
Highlights
The histidine–proline-rich glycoprotein (HPRG/HRG) is a single chain (75 kDa) protein, produced by the liver and present at high concentrations in plasma of vertebrates
The antiangiogenic activity of the His/Pro rich domain (H/P) domain of histidine–proline-rich glycoprotein is mediated by its binding with tropomyosin, a protein exposed on endothelial cell-surface during the angiogenic switch, in presence of zinc ions
The plate was coated by human cardiac tropomyosin, while HKa* and the TetraHPRG were added in buffer as competitive agents
Summary
The histidine–proline-rich glycoprotein (HPRG/HRG) is a single chain (75 kDa) protein, produced by the liver and present at high concentrations in plasma of vertebrates. The previous mentioned HKa, requires a direct binding to tropomyosin to produce antiangiogenic activity, and it is characterized by a domain (D5) that shows a high similarity with H/P of HPRG suggesting analogous functions Both HKa-D5 and H/P domain of HPRG display a primary sequence rich of positive charges and lack of disulfide bonds that favor the binding to heparin and zinc ions [14]. The activated platelets release free Zn2+ from α-granules [15], and the local concentration of metal ions reaches the levels (approximately 10–50 μmol/L) needed to support the binding of HPRG to tropomyosin on endothelial cells This interaction is believed essential to mediate the HPRG antiangiogenic effects. The complex formation of Zn2+ TetraHPRG was compared to that of Cu2+ by means of potentiometric study [27], to assess the relevance of pH and metal complex species in HKa/Tropomyosin/TetraHPRG binding
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