Cindy Lee
1041791
Biochemistry 499
Affinity Chromatography: Homemade Microcystin-Sepharose
Cindy Lee, Marcia Craig
May 1, 2006
- 5 -
Cindy Lee
1041791
Summary
Reversible phosphorylation of proteins is known to be a method of regulation of many cellular pathways. The enzymes involved in the reactions in reversible phosphorylation are protein kinases, which catalyze phosphorylation, and protein phosphatases, which catalyze dephosphorylation. Protein phosphatases must be tightly regulated because they are relatively few in number in the cell and must counterbalance the actions of many different protein kinases. It has been recognized that abnormal phosphorylation can cause diseases, such as cancer. To assist in the study of the serine/threonine protein phosphatase class of protein phosphatases, an affinity column made from microcystin-LR, a toxin and inhibitor of protein phosphatase-1 and -2A, linked to Sepharose can be used for their purification and determination of their regulatory subunits. In this project, a microcystin-Sepharose affinity column was created and shown to bind protein phosphatase-1 and -2A and demonstrated how it could be used for the purification of these enzymes.
Introduction
Reversible phosphorylation of proteins is a very important form of regulation in almost every aspect of cell life [1]. The enzymes that catalyze reactions involved in reversible phosphorylation are protein kinases and protein phosphatases. Protein kinases catalyze the phosphorylation of proteins and phosphatases catalyze the dephosphorylation of proteins. One family of phosphatases are the serine/threonine phosphatases, which includes protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A).
Abnormal phosphorylation has been known as a cause or consequence of many human diseases, such as cancer. This stresses the importance of regulation of the enzymes involved in reversible phosphorylation, especially the protein phosphatases. There are relatively few Ser/Thr phosphatases in the cell and one catalytic subunit must counterbalance the activity of many different protein kinases. Thus protein phosphatases are subjected to spatial and temporal regulation.
The catalytic subunit of protein phosphatases do not exist as free molecules in vivo; they are usually complexed with other proteins. These proteins can target the phosphatases to particular subcellular locations or modify their activity [2]. PP1c (the catalytic subunit of PP1) is regulated by the association of regulatory proteins. Proteins that target PP1c commonly contain a four amino acid motif, RVXF. The catalytic subunit of PP2A is bound to two regulatory subunits: a constant regulatory subunit of 65kDa (PR65), and a variable regulatory subunit that can be one of many diverse proteins that respond to cell signaling [3].
There are a number of drugs and toxins that target protein phosphatases. One such toxin is microcystin. Microcystin is a cyclic heptapeptide produced by cyanobacteria, or blue-green algae. Microcystin is a potent inhibitor of PP1 and PP2A and a potent liver carcinogen [4]. This inhibition of protein phosphatases causing diseases implicates the importance of maintaining regulation of the enzymes involved in reversible phosphorylation.
Over 50 types of microcystin have been identified. The general structure of microcystin is cyclo(D-Ala-X-D-MeAsp-Y-Adda-D-Glu-Mdha-), where X and Y are variable amino acids (see Figure 1). Microcystins may vary in degrees of methylation, configuration of Adda, or amino acids in the X and Y positions [4]. One of the most common microcystins is microcystin-LR, where L-leucine is in the X position and L-arginine is in the Y position.
Microcystin-LR interacts with PP1 and PP2A in two stages. The initial stage is a fast (within minutes) binding of the Adda, glutamic acid, and methylaspartic acid residues to the acidic, C terminal and hydrophobic grooves of PP1. The active site of PP1 is located at the bifurcation point of these grooves [5]. The second stage is a slow (over hours) formation of a covalent bond via Michael Addition of the thiol group of cysteine 273 of PP1 (cysteine 266 of PP2A) to the a,b-unsaturated carbonyl of the Mdha group of microcystin. Figure 2a shows microcystin-LR in the active site of PP1 and the covalent bond with the cysteine 273 residue.
An affinity column has been developed to take advantage of the interaction between microcystin-LR and PP1 [2]. The a,b-unsaturated carbonyl of the Mdha group is used to link microcystin-LR to a Sepharose matrix, which also prevents a covalent bond formation with PP1, but does not interfere with toxin-enzyme interactions. This makes it possible for PP1 to bind to the microcystin-LR on the Sepharose matrix, and allows its elution. Another aspect of this microcystin-Sepharose column is that the RVXF motif binding site of PP1 is exposed because it is separate from the active site (see Figure 2b). As mentioned, many regulatory proteins of PP1 have a RVXF motif, thus the column allows for the association of these regulatory proteins.
A microcystin-Sepharose affinity column has the potential to purify PP1 and PP2A as well as show the association of regulatory proteins to these protein phosphatases. The purification of PP1 and identification of some of its regulatory subunits has been done before [2,6]. However, the purification of PP2A using this method was problematic in the literature [6].
The goal of this project was to create a microcystin-Sepharose column for purification of PP1 and PP2A and for determining their regulatory subunits. We can learn more about cell regulation from studying these proteins. In these experiments, we were successful creating a column for PP1 purification. Preliminary results showed that PP2A may be eluted with another toxin, okadaic acid, which inhibits PP2A more potently than microcystin-LR [4].
Experimental Procedures
Materials
Microcystin-LR was obtained by purification offrom blue-green algae from Little Beaver Lake in 1992 by Hue Anh Luu. The microcystin was purified by HPLC. Dried fractions from these purifications were dissolved in water and analyzed for microcystin by PNPP inhibition assays of PP1, and pooled [7]. PThe pooled fractions were run on the HPLC to determine the purity and quantitate the amount of microcystin LR. 2-aminoethanethiol, or cysteamine, was from Sigma. N-hydroxysuccinimide-activated CH-Sepharose 4B was from Amersham Biosciences/GE. PP1 and PP2A were purified by Hue Anh Luu and Andrea Fong.
Reverse Phase High-Performance Liquid Chromatography
All HPLC runs were on a Beckman with a microbore Vydac C18 column. The cHPLC columns were equilibrated with 0.1% TFA/water and eluted with linear AB gradient (0.3%B/minute), where solvent A is 0.1% TFA/water and solvent B is 0.1% TFA/acetonitrile, over 3 hours at a flow rate of 0.2 mL/minute. The wavelength for detection was 206nm.
Preparation of Aminoethanethiol-Microcystin
The reaction protocol of Moorehead was followed using 1mg of microcystin-LR in water. The product was purified with a Waters C18 Long Body Plus Sep-Pak cartiridges. The Sep-Pak was solvated with 10 mL of 0.1%TFA in acetonitrile, then equilibrated with 10 mL of 0.1% TFA in water. The sample was then loaded and washed with 10 mL of 0.1% TFA in 10% acetonitrile. AThe aminoethanethiol-MCLR was eluted off with 0.1%TFA in acetonitrile until no more eluted off as shown with PNPP inhibition assays. EThe eluted fractions containing aminoethanethiol-MCLR were run on the HPLC to determine the completion of the reaction. The eluted fractions were dried with a SpeedVac.
Preparation of Microcystin-thioethaneamino-Sepharose
AThe 1 mg of aminoethanethiol-MCLR (1 mg) was dissolved in 50mM sodium bicarbonate, pH 9.2 and reacted with 3.5 mL of N-hydroxysuccinimide-activated CH-Sepharose 4B resin end-over-end overnight at 4oC. The resin was then centrifuged and the supernatant was removed. An aliquot of the supernatant was run on the HPLC to determine the completeness of the reaction.
The resin was washed five times alternately with 50mM Tris-HCl pH 8, 0.5M NaCl and 50 mM sodium acetate pH 4, and 0.5M NaCl. The resin wais stored in 20% ethanol at 4oC.
Preparation of Tris-Sepharose
Activated CH Sepharose 4B was reacted with 50mM Tris-HCl, pH 7.5, end-over-end overnight at 4oC. The resin wais stored in 20% ethanol at 4oC.
Determination of the Binding Capacity of Microcystin-Sepharose
50mL of microcystin-Sepharose resin and of the Tris-Sepharose resin (control) were placed in Eppendorf tubes and washed with Buffer A (50mM Tris-HCl, 0,1mM EDTA, 0.5mM MnCl2, 0.2% beta-mercaptoethanol, pH 7.5). PP1 was diluted with Buffer A and incubated end-over-end with the resin for 1 hour. The resin wais then centrifuged and the supernatant is removed. The incubation step wais repeated with new PP1 until the resin wais saturated (no longer binds additional PP1). Aliquots of the supernatant from each step weare used in PNPP assays to determine the percentage of binding by comparing the microcystin-Sepharose resin to the Tris-Sepharose control resin.
Binding of PP1 to Microcystin-Sepharose
50mL of microcystin-Sepharose resin and of the Tris-Sepharose resin (control) were placed in Eppendorf tubes and washed with Buffer A. PP1 was diluted with Buffer A and incubated end-over-end with the resin for 1 hour. The resin wais washed four times with 0.3M NaCl in Buffer A. After the last wash, 5mL of resin slurry iwas removed for antesting with SDS PAGE gel. PP1 was eluted with 3M NaSCN in Buffer A (incubated end-over-end for 30 minutes at 4oC). An aliquot of the elution was used for an SDS PAGE gel. The resin wais then washed four times with 0.3M NaCl in Buffer A. After the last wash, 5mL of resin slurry wais removed for an SDS PAGE gel.
Binding of PP2A to Microcystin-Sepharose
The same procedure as the PP1 binding experiment was used with the exception of the elution step. Okadaic acid, in excess, was used to elute the PP2A fromoff the resin.
Results
Quantitation of Microcystin by HPLC
A standard of microcystin-LR was run on the HPLC to determine the retention time (Figure 3a). The retention time for microcystin-LR came off the column atwas 121.04 minutes. PThe pooled fractions of microcystin that were run on the HPLC showed a microcystin-LR peak at 121.27 minutes, but there were also impurities (Figure 3b). The total microcystin-LR in the pooled fractions was calculated to be ~10 mg based on comparison of peak heights in the chromatograms.
Preparation of Aminoethanethiol-Microcrystin
An aliquot of the reaction product ofbetween microcystin treated withand aminoethanethiol that was purified on the Sep-Pak cartridge was run on the HPLC (Figure 3c). There was a shift in the retention time of the microcystin-LR from 121.04 minutes before the reaction to 113.89/115.02 minutes after the reaction. There was no residual peak at 121.27 minutes, thus the reaction is assumed to be ~95100% complete.
Preparation of Microcystin-thioethaneamino-Sepharose
An aliquot of the supernatant after centrifuging the reacted aminoethanethiol-microcystin-LR and NHS-activated CH Sepharose 4B was run on the HPLC (Figure 3d). The missing peak at 113.89/115.02 minutes indicates the absence of aminoethanethiol-microcystin in solution. therefore it This was expected ifmust be aminoethanethiol-microcystin was linked to the Sepharose that was removed from solution by centrifugation. The absence of the peak at 113.89/115.02 minutes suggests the reaction went to ~95100% completion.
Binding Capacity of Microcystin-Sepharose
Activity assays of PNPP were carried out with aliquots of the supernatant after incubation of the resin with PP1. The percentage of activity compared to the control was used to calculate the quantity of PP1 that bound to the resin. After four additions of PP1, 59 mg of PP1 bound to 50 mL of resin, which wais calculated to contain approximately 14.3 mg of microcystin. However, the saturation point was not reached, thus 59mg is a lower limit of the binding capacity. (Unfortunately, there were time constraints.) 1 mg of microcystin wais calculated to bind 4.2mg of PP1.
PP1 Binding Experiments
Figure 4 shows the SDS PAGE gel of the experiment. Comparison between the control Tris-Sepharose resin and the microcystin-Sepharose resin shows that PP1 bound specifically to the microcystin-Sepharose resin. The markers are in lane 1. Lane 2 was the PP1 that was incubated with the resin. Lanes 3 and 4 were the resins after incubation with PP1 and then washed with 0.3M NaCl. Lanes 5 and 6 were the resins after elution with 3M NaSCN and lanes 7 and 8 were the elutions. The faint band in lane 6, the microcystin-Sepharose resin after elution, indicates there was residual PP1 that did not elute from the resinoff. The elution in lane 8 confirms that PP1 was eluted off with 3M NaSCN.
PP2A Binding Experiments
Figure 5 shows the SDS PAGE gel of the experiment. Comparison between the control Tris-Sepharose resin and the microcystin-Sepharose resin shows that PP2A bound specifically to the microcystin-Sepharose resin. The markers are in lane 1. Lane 2 was the PP2A that was incubated with the resin. Lanes 3 and 4 were the resins after incubation with PP2A and then washed with 0.3M NaCl. Lanes 5 and 6 were the resins after elution with excess okadaic acid and lanes 7 and 8 were the elutions. The band in lane 6, the microcystin-Sepharose resin after elution, indicates there was residual PP2A that was not eluted by okadaic aciddid not elute off. There is only a very faint band in lane 8, which represents the elution of PP2A by okadaic acid.
Discussion
This project was successful in creating a microcystin-Sepharose for purifying PP1 and PP2A. Based on the quantitation using HPLC, there are approximately 10mg of microcystin-LR in the pooled fractions that can be use for creating microcystin-Sepharose resin. From the binding capacity experiments, 10mg of microcystin-LR can bind 42 mg of PP1, which would make large-scale purification of PP1 possible.
The reactions involved in creating the resin, microcystin-LR with aminoethanethiol and the product of that reaction with NHS-activated Sepharose, were high yielding, ~95%. Though there were impurities in the pooled fractions (figure 3b) and appeared to react with aminoethanethiol (figure 3c shows that all peaks were shifted to the left), they did not react with the NHS-activated Sepharose (figure 3d). This showed that purification of microcystin-LR before any reactions was unnecessary because reactions with NHS-activated Sepharose are specific for microcystin.
The binding capacity experiments showed that at least 59 mg of PP1 bound to 50 mL of resin that contains ~14.3 mg of microcystin-LR. Unfortunately, the maximum capacity of the resin was not determined due to time constraints. Work can be continued to determine the saturation point. This is important to establish because it will allow more efficient purification of PP1 and more efficient binding of regulatory proteins to PP1.
The binding experiments with PP1 verified that it could be purified using microcystin-Sepharose resin, as shown in the literature [2]. Comparing lanes 3 and 4 of figure 4, PP1 bound to the microcystin-Sepharose resin but not the control (Tris-Sepharose) resin indicating that PP1 bound specifically and not non-specifically. Also, PP1 used in this experiment was not completely pure as there are visible faint bands at 15 and 75 kDa (lane 2, figure 4), but these proteins did not bind to the microcystin-Sepharose resin. This shows this resin can be used for purification.