Trace metal-protein interactions in plasma of relevance to health and immune functioning

Trace metal ions play many critical roles in biological functions in humans. Plasma proteins such as human serum albumin (HSA) are primarily responsible for the circulatory transport of metal ions and as such are relevant to health and immune functioning. To further understand the biological processes and some diseases associated with defective homeostasis of metal ions (including Co²⁺, Zn²⁺, Cd²⁺ and Cu²⁺), a deeper understanding of the properties of protein-metal ion interactions is required. On HSA, three metal-binding sites involving in binding to these metal ions are known, including sites A (His67 site), B (His9 site) and the N-terminal site (His3 site), which play different roles in binding different metal ions. Additionally, metal ion states are of clinical importance in immune system. Among these metal ions, defective homeostasis of Cu²⁺ has been proposed to be related to the development of celiac disease (CD), by regulating tissue transglutaminase (TG2) activity in extracellular environment. Thus, the aims of this project are to elucidate properties of metal ion-HSA interactions and investigate potential role for Cu²⁺ in TG2 activity regulation. Employing site-directed mutagenesis, isothermal titration calorimetry (ITC) and circular dichroism (CD), the results presented here indicate that Co²⁺ preferentially binds to HSA at site B, followed by site A and subsequentially binds to multiple weak-affinity sites. Additionally, the presence of 5 molar equivalents of palmitate can trigger structural changes and diminish Co²⁺ binding to sites A and B of HSA. An investigation of Zn²⁺ binding using mutagenesis and ITC revealed that this ion preferentially binds to site A, followed by site B, which is in firm agreement with previous data. In addition, Zn²⁺ binding to site A was dramatically reduced by addition of 5 molar equivalents of palmitate. ITC results in combination with ¹¹¹Cd-NMR spectroscopy suggest sites B and A are the two major binding sites participating in Cd²⁺ binding to HSA. Additionally, the results confirm that His9 is the indispensable histidine residue contributing to site B. Intriguingly, inspection of these data reveals that binding modes of different metal ions in different metal-binding sites (especially in site A) vary substantially. Both His67 and His247 participate in the "intact" site A in Zn²⁺ binding; however, His247 may play a less important role than His67 in Co²⁺ and Cd²⁺ binding to site A. Regarding to Cu²⁺ binding to HSA, it primarily binds to the N-terminal site and secondly binds to site A, supported by the results from ITC and EPR spectroscopy. Compellingly, the data presented here also demonstrate that Cu²⁺ is a potent inhibitor of TG2, providing an insight into deeper understanding of the development of CD. Collectively, these data provide a more comprehensive understanding of the properties of Co²⁺, Zn²⁺, Cd²⁺ and Cu²⁺ interactions with HSA and their interplay with fatty acids, contributing to more accurate insights into metal ion distribution and speciation mediated by HSA and their implications in health.