The Dynamics of Circulatory Zinc Handling and Transport

There are 25 chemical elements that are required for mammalian life, 15 of these elements are metals. Zinc, in its ionised form Zn2+, is an essential metal ion in mammals and performs a wide range of important physiological functions by allowing many vital chemical reactions to occur. Zinc is known to play roles in fertility and development, the immune system, ageing, and major diseases such as Alzheimer's, diabetes, and cancer. Unfortunately however, the molecular mechanisms that enable zinc to get to where it is needed after it enters the bloodstream, following sequestration from the diet, are not fully understood.

It is known that a particular protein called serum albumin (that is highly abundant in the blood) plays an important role in transporting zinc (and other metals including calcium and magnesium) in the blood. Serum albumin not only carries zinc through the circulatory system but also mediates its uptake into cells. The molecular mechanisms that control the interaction between zinc and serum albumin and its uptake into cells are largely unknown. It is important that these interactions are understood. Albumin also transports other types of molecules (e.g. fatty acids, hormones) and binding of one particular molecule can affect binding of another at a separate site. For example, we previously identified a zinc site that is perturbed by fatty acid-binding elsewhere on the molecule. Physiological events that alter the small molecule composition of blood can therefore alter metal transport processes. Such events may be short-term (e.g. fasting, eating, infection, stroke) or long-term (obesity, disease). Long-term alterations in blood chemistry are particularly likely to have serious consequences due to the knock-on effects caused by altered metal binding/delivery.

We have identified three regions on the molecule that are likely to form novel metal binding sites. We will examine whether these regions that are involved in or impact upon zinc transport by synthesising mutant serum albumin proteins with chemical alterations at each site. These alterations will remove important chemical groups that are likely to participate in metal binding. The ability of each "altered protein" to bind zinc and other relevant metals will be examined and compared to the native (or "normal") protein using complementary approaches. We will also examine whether binding of molecules (including metals) at other sites alters the binding properties of each site.

The importance of known and newly identified zinc-binding sites in mediating zinc uptake into cultured vascular endothelial cells (a particular type of cell that lines the blood vessels) will be determined by incubating these cells with albumins that contain alterations at each zinc site and physiological levels of zinc. The amount of zinc taken up by the cells will then be measured.

The outcome of these experiments will allow a better understanding of how metal ions are transported in the circulatory system. Particularly with regard to the events that can alter this process and the importance of albumin in mediating cellular zinc uptake. This provides important physiological information that will help us to understand a variety of physiological and disease processes that involve metals and will aid in their study.

Discerning the mechanisms Zn homeostasis, speciation, and distribution is essential in understanding the diverse roles of this metal in biological systems. However, the molecular mechanisms that mediate Zn speciation and distribution in the circulation are not well understood.

The majority of exchangeable Zn2+ in plasma is bound to serum albumin. The location and structure of the major zinc site on albumin has only recently been reported, and has enabled the applicants to show that this protein links the levels and distribution of metabolites as diverse as fatty acids and Zn2+, with potentially far-reaching physiological consequences.

Several metal ion transport sites on albumin are yet to be located and characterised. Analysis of known X-ray structures has enabled us to identify 3 new putative metal ion-binding sites that are likely to be relevant for circulatory metal ion transport in mammals. To identify which sites impact upon zinc binding and transport, we will generate albumins that possess mutations in key residues and determine their metal ion binding properties. We will examine co-operative effects between sites and whether binding of other types of molecule, including fatty acids, alter binding properties at each site. This will be achieved using complementary biophysical approaches including NMR, ITC and EXAFS. To test whether Zn distribution in plasma is modulated by levels of fatty acids, metalloproteomic studies will be conducted by analysing commercially available plasma in the presence/absence of added fatty acid.

Albumin is also proposed to be important for cellular Zn2+ uptake in endothelial cells via an endocytotic mechanism. We will therefore examine the role of albumin in this process. This will be achieved using primary and transformed vascular endothelial cells incubated with wild-type albumin or mutants that exhibit defective Zn2+-binding characteristics. The impact of fatty acid levels on cellular uptake will also be studied.

Zinc is an essential nutrient which impacts upon on almost all major physiological and disease processes in mammals. The proposed work will provide detailed and reliable qualitative and quantitative data relating to Zn2+ handling in the circulation. This includes the binding properties of serum albumin toward Zn2+ (and other metal ions) in various physiologically relevant states and the significance of this interaction in mediating the cellular uptake of Zn2+. As well as the considerable academic benefits provided by this research at the systems level, this work will deliver positive economic and societal impacts on animal health and agriculture, as well as human health (including reproduction and development). The relevance of zinc for all aspects of biology remains greatly under-appreciated; hence, generating a raised awareness of the importance of zinc biology for these areas is expected to have long-term impacts on the livestock farming industry and veterinary practitioners. We hope that a more thorough understanding of the molecular and cellular biology of zinc will stimulate further investigations, including dietary studies. This may help to avoid subclinical hypozincemia or zinc toxicity. The former is estimated to affect 2 billion people worldwide and to be widespread amongst domestic animals and may result in impaired reproduction, reduced weight gain, and an impaired immune system. Hence, an adequate provision of zinc may ultimately not only impact on animal welfare, but also lead to better economic returns from healthier animals.

The outcomes of this work may also be exploited directly within the commercial private sector, primarily by biotechnology, biomanufacturing and biopharmaceutical companies. The development of albumin mutants with altered metal-binding properties may be used to develop new in vitro cell systems, for the development of serum replacement media for clinical use, and/or for the formulation of vaccines and other drugs. It is also possible that albumin could be engineered to provide a first generation of selective biocompatible protein-based chelation agents. Recombinant serum albumin is currently used clinically and its manufacture is a vibrant global industry with key players in Europe, USA and Asia. Such beneficiaries (Novozymes Biopharma) have been previously engaged in discrete projects with the applicants. It is envisaged that there will be key junctures (either during or after the project) whereby the involvement of a commercial partner could add great value in taking the research forward. At these points potential partners will be directly contacted by the applicants with a view to entering partnership agreements.