Gelsolins Metal Binding Studies

Patrick Wilson, MS in Chemistry candidate Advisor: Kestutis Bendinskas

Abstract:
Gelsolin is a Ca2+ regulated actin binding protein[1], which has been shown to be capable of degrading the potentially harmful A protein implicated in Alzheimers disease[2]. Alzheimers disease is known to be exacerbated by elevated levels of mercury in the body[3]. Interestingly, two proteomic studies have shown gelsolin so be down regulated in the presence of mercury[4][5]. It is hypothesized by this research group that mercury may regulate or bind to gelsolin in order to prevent its proper functioning to degrade A proteins, and thus promote Alzheimers disease. In order to investigate this claim, an experiment was designed utilizing the quenching of the intrinsic fluorescence of tryptophan residues of gelsolin by mercury (II) chloride. Similar protocols have been successfully performed when using the model protein bovine serum albumin. The binding of gelsolin to mercury remains to be confirmed.

Introduction:
Gelsolin is the foremost protein involved in the assembly and severing of actin filaments. Actin is a major component of the cellular cytoskeleton and is considered one of the most abundant proteins in eukaryotes. Three common isoforms of gelsolin exist in humans, plasma gelsolin (pGSN), cytoplasmic gelsolin (cGSN), and the less studied isoform 3 which is present in the brain, testes, and lungs. Inside the cell, its most studied role is in the construction and disassembly of actin filaments. By transference, this means cGSN is involved in cell structure, motility, division and apoptosis. The extracellular form differs from the cytoplasmic by the addition of a 25-amino acid export sequence and the presence of a disulfide bond between

cysteines 188 and 201[6]. Plasma gelsolin is known to assist the vitamin D-binding protein, Gcglobulin, as part of a two-protein actin scavenging system capable of degrading filaments which have escaped from ruptured or lysed cells[6].The importance of this system is easily discerned as actin comprises 10% of total human body protein, and its polymerization is thermodynamically favored outside the cell[6]. Among other recently discovered roles, gelsolin has also been shown to bind and degrade fibrillar amyloid beta-protein (A) implicated in the development of Alzheimers disease (AD)[2]. Alzheimers disease (AD) is the most common neurodegenerative disease, affecting roughly 25 million people around the world. It is characterized by development of amyloid fibers in senile plaques in the walls of blood vessels and the accumulation of neurofibrillary tangles in neurons, which ultimately results in neuronal cell loss[2]. A protein is a major component of these senile plaques in patients with AD. Both plasma gelsolin and cytoplasmic gelsolin have been shown to complex with A[2]. Additionally they have been show to inhibit fibrillization by up to 90% as well as defibrillize the preformed fibrils of A in vitro[2]. Independently, increased levels of plasma and cytoplasmic gelsolin resulted in a significant reduction of A load in the brains of APP/PS-transgenic mice[2]. Collectively, this evidence suggests a protective role of gelsolin against the development of AD. Exposure to heavy metals has also been linked to the progression of Alzheimers disease[3]. In a proteomic assessment of the effects of mercury on the blood serum proteome of 34 children, gelsolin was found to be down-regulated by 12.07% (2.21 %RSD)[4]. In an expanded study of 100 children, gelsolin was found to be a mediator of the effect of mercury on the decreased salivary cortisol[5]. In light of the fact that gelsolin contains multiple calcium binding sites and its function is partially dependent of Ca2+ concentration, we hypothesize here that gelsolin may also

bind to heavy metal pollutants, which may reduce its overall function, including its ability to degrade the potentially harmful amyloid plaques; alternatively, these pollutants might affect gelsolin production or elimination from our bodies. Fluorescence quenching is a technique that has been utilized to determine binding relationships of quencher-protein complexes[7][8]. This technique utilizes uses the intrinsic fluorescence of the aromatic amino acids in proteins. The most commonly used amino acid is tryptophan, which has a maximum wavelength of absorbance of 280 nm, and has an emission maximum between 330 and 370 nm. There are two main types of fluorescence quenching, dynamic and static. Dynamic quenching is the result of a collision between a quencher and a fluorophore so that upon relaxation to the ground state, the fluorophore does not fluoresce. Static quenching is the result of the quencher binding the fluorophore so that it is unable to emit fluorescence. The investigation of static quenching can be used in order to determine the binding relationship between potentially harmful substances, such as heavy metals, and proteins. Specifically, it is possible to determine the binding constant of such a metal-protein complex, as well as the number of metal binding sites per protein. It is the overarching goal of this project to study the relationship between Alzheimers, mercury, and gelsolin further. Our first specific goal is to see if mercury can bind gelsolin.

Methods:
Given that gelsolin costs roughly 800$ for 1 mg, we decided to perfect our methods on the model protein bovine serum albumin (BSA). The experimental procedure was adapted from Meng Le et alia[7] and the results were analyzed in a method similar to Belatik et alia[8]. Fluorescence experiments were performed using a constant BSA concentration of 125 M in 0.1 M Tris-HCl with 0.1 M NaCl (pH 7) at 35 C. HgCl2 concentrations were varied from 50 M to 2.25 mM.

The emission and excitation slits were both 2 nm. The experiment was run on a Horiba Fluoromax-4 spectrofluorometer at SUNY-Upstate Medical University. The excitation wavelength was 280 nm and the spectra were obtained from 290 nm to 450 nm in accordance with tryptohans wavelengths of maximum absorbance and emission. Samples were prepared the day of the experiment, and mercury was added roughly 10 minutes prior to each experiment to allow equilibration. Additionally, an experiment utilizing 2.5 M gelsolin (Sigma-Aldrich) in 0.14 M Tris-HCl with 0.14 M KCl and 0.0026 M MgCl2 was prepared. In this experiment, HgCl2 concentrations were varied from 0.25 M to 25 M. The presence of K+ and Mg2+ is thought to be necessary for gelsolin binding to Ca2+ with high affinity (1.09 106 M-1)[9]. A similar experiment, in which all

conditions were replicated, except the inclusion of BSA instead of gelsolin, was run using a Perkin-Elmer LS fluorescence spectrometer at SUNY-Oswego. Unfortunately this experiment was unable to obtain quantitative results. The gelsolin experiment is currently being stored at -80 C while our research group attempts to obtain reproducible, quantitative data from our instrumentation.

Results and Discussion:

The fluorescence intensity related count number, ranged from about 9 million to about 500 thousand, (Figure 1). An inverse relationship between fluorescence intensity of BSA and concentration of Hg2+ is evident.

A
10000000 9000000 8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 290 310 330 350 370 390 410 water 0.00 4.92E-05 1.00E-04 2.00E-04 3.00E-04 4.00E-04 5.00E-04 1.00E-03 1.25E-03 2.25E-03

B
1.00E+07 9.00E+06 8.00E+06 7.00E+06 Fluorescence 6.00E+06 5.00E+06 4.00E+06 3.00E+06 2.00E+06 1.00E+06 0.00E+00 0 0.0005 0.001 [Hg2+] (M)
2+

0.0015

0.002

0.0025

Figure 1: Relationship between fluorescence intensity of BSA and concentration of Hg . (A): The legend represents the 2+ varying concentrations of Hg for each sample with a constant BSA concentration of 125 M. (B): Maximum fluorescence 2+ of each sample from 330 nm to 370 nm is plotted against that samples concentration of Hg .

Results are analyzed via the Stern-Volmer method according to the equation:

[ ]

[ ]

Where KSV is the Stern-Volmer quenching constant and is representative of the dynamic quenching constant, and Ka is representative of the static binding constant and is taken as the association constant of the complex. A plot of vs [Q] results in a straight line with a slope

equal to KD, where KD is a combination of the static and dynamic quenching constants and is equal to kqo (Figure 2). In this representation, kq is known as the bimolecular quenching constant and o is the lifetime of fluorescence in the absence of quencher. The value of o for tryptophan in BSA is 5.9 ns[8]. Therefore the value of kq is calculated to be 4.7 1011 M-1 s-1.

Since this value is more than 10 fold greater than the maximum dynamic quenching constant of 1.0 1010, we can assume that the contribution of dynamic quenching is negligible:

2.5

1.5 Fo/F

y = 2783.3x + 0.8108 R = 0.979

0.5

0 0 0.0001 0.0002 0.0003 [Hg] (M) 0.0004 0.0005 0.0006

Figure 2: Plot of a modified Stern-Volmer equation in order to determine major contributor (static vs dynamic) to fluorescence quenching.

Utilizing an additional modified Stern-Volmer equation, it is possible to determine the association constant of the complexed quencher and fluorescent molecule:

A plot of

[]

and a slope of . Using this, the

gives a straight line with an intercept of

association constant for the BSA-Hg2+ complex is calculated to be 1380 M-1 (Figure 3).

3 2.5 2 Fo/(Fo-F) 1.5 1 0.5 0 0 500 1000 1500 1/[Hg] 2000 (M-1) 2500 3000 3500

y = 5.72E-04x + 7.23E-01 R = 9.90E-01

Figure 3: Modified Stern-Volmer equation for determination of the binding constant, Ka.

It is possible to determine the number binding sites for the quencher-fluorescent molecule complex by plotting a third modified Stern-Volmer equation:

log

log[ ]

log

Where n is the number of quencher molecules bound to the fluorescent molecule. For the BSAHg2+ complex this number is shown to be 1.80, which is taken as 2 Hg2+ binding sites for each BSA molecule (Figure 4).

0.5

0 -4.5 -4.3 -4.1 -3.9 -3.7 -3.5 -3.3 -3.1

log[(Fo-F)/F]

-0.5

-1 y = 1.80x + 6.07 R = 0.99 -1.5

log([Hg]) (M)

-2

Figure 4: Modified Stern-Volmer plot for the determination of the number Hg binding sites for BSA. The slope is the number 2+ 2+ of Hg bound per BSA molecule. The slope is rounded to the closest whole number, to show 2 Hg binding sites for BSA.

2+

Given the success of the BSA experiments, I am confident in performing the gelsolin experiment on the same Fluoromax-4 instrument. The fluorescence spectra showed good separation between samples of varying Hg2+ concentration, with no overlap or deviations from the inverse relationship. However, given that the protein concentration of the Hg2+-BSA experiment is 50 fold larger than the prepared concentrations for the Hg2+-gelsolin experiment (125 M vs. 2.5 M respectively), it would be wise to optimize a similar BSA experiment beforehand. It is important to note, that since the assay is based on the intrinsic fluorescence of tryptophan residues, the number of these residues per protein will likely affect the response of the instrument. BSA contains only two tryptophan residues, while bovine and human gelsolin each contain 15[10][11]. This means it may be possible to run gelsolin experiments at much lower concentrations than BSA, however we will not know for sure until the first gelsolin experiment is completed. The 2.5 M BSA experiment performed on Perkin-Elmer instrument at SUNY-

Oswego was not successful and needs to be repeated. However, given that the Horiba Fluoromax-4 is roughly 9,000 times more responsive than the Perkin-Elmer LS fluorometer (9 million counts compared to 1 thousand count maximum respectively), I am not confident it will be possible to obtain quantitative results on the Perkin-Elmer instrument.

Conclusions:
The relationship between Alzheimers disease, gelsolin, and mercury remains to be elucidated. Determining the binding constant of the gelsolin-Hg2+ complex, as well as the number of Hg2+ atoms bound per gelsolin will be a good first step in this process. In the future, it would be important to determine if this binding inhibits gelsolins ability to degrade actin filaments and even degrade the A plaques present in Alzheimers disease, which may be possible using in vitro techniques in our lab. Finally, the effects of mercury on the expression of gelsolin need to be determined. If any of these relationships are confirmed, they will have significant implications on the modern day research of Alzheimers disease.

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