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Summer Research Fellowship Programme of India's Science Academies

Effect of phosphorylation of αB Crystallin on its chaperone function

Disha Jayesh Shah

Department of Biotechnology, University of Calicut, Malappuram district, Thenhippalam, Kerala 673635

Guided by:

Dr. Ch Mohan Rao

Center for Cellular and Molecular Biology, Clinical Research Facility, Habsiguda, Hyderabad

Abstract

α Crystallin is a member of the small heat shock protein family. Members of this family have in common a C-terminal region, 80 to 100 amino acid residues long, called the α-crystallin domain. α crystallin is a major protein of the mammalian lens. It is isolated from the lens as a large, multimeric, polydisperse complex of two closely related polypeptides αA-crystallin and αB-crystallin (approximately 20kDa each). It has been shown to have both structural and functional roles in maintaining the eye-lens transparency. Previously thought to be found only in the lens, α crystallins are now known to be ubiquitous. The expression of αB-crystallin is induced under heat and other stresses and has a role in several neurodegenerative diseases. It acts as a molecular chaperone and has a role in apoptosis, cell differentiation, protein turnover and quality control. Phosphorylation is one of the most important post-translational modifications that could play an important role in the function of αB-crystallin. The role that phosphorylation plays in the chaperone activity is poorly understood.

Keywords: αB-crystallin, molecular chaperone, heat-shock protein, phosphorylation, role of phosphorylation, chaperone activity

INTRODUCTION

Background

Defective protein folding is being linked to a growing number of disease conditions. Protein aggregation has been implicated in pathological conditions like Cataract and in neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s and Huntington’s disease. Protein aggregation is one of the most common routes for protein instability, and is often a problem for therapeutic-protein production, as it renders the product unfit for use. Molecular chaperone is a term used for proteins that are known to assist protein folding and assembly in-vivo. An understanding of chaperone activity will be useful for developing therapeutic strategies for protein aggregation-related medical conditions.

A class of proteins that are up-regulated under stress conditions (though originally observed under heat shock conditions) are generally referred to as heat shock proteins (Hsps). Hsps have been classified into six major families according to their approximate molecular size: Hsp100, Hsp90, Hsp70, Hsp60, Hsp40, and sHsps.

α crystallin is a member of the small heat shock protein (sHsps) family. Members of this family have in common a C-terminal region, 80 to 100 amino acid residues long, called the α-crystallin domain. α-crystallin is a major protein of the mammalian lens. It is isolated from the lens as a large, multimeric, polydisperse complex of two closely related polypeptides αA-crystallin and αB-crystallin (approximately 20kDa each). It has been shown to have both structural and functional roles in maintaining eye-lens transparency. Previously thought to be found only in the lens, α-crystallins are now known to be ubiquitous. Considerable amounts of αB-crystallin are found in muscle, heart, lung, spinal cord, skin, brain, kidney and retina. The expression of αB-crystallin is induced under heat and other stresses and has a role in several neurodegenerative diseases. It acts as a molecular chaperone and has a role in apoptosis, cell differentiation, protein turnover and quality control.

Phosphorylation and chaperone-like activity of αB-crystallin

Phosphorylation is one of the most important post-translational modifications that could play an important role in the function of αB-crystallin. Phosphorylated αB-crystallin has an important role not only in the eye lens but also in other tissues. Phosphorylation is induced under a variety of stimuli and may have implications in the regulation of various cellular functions. αB-crystallin has three phosphorylation sites, S19, S45, and S59. S19 phosphorylation occurs in an age-dependent manner, S59 is phosphorylated under heat stress and S45 is known to be phosphorylated in the mitotic phase of the cell cycle. The role that phosphorylation plays in the chaperone activity is poorly understood. The effect of phosphorylation on chaperone activity and oligomeric size can be studied with phosphorylation-mimicking mutants. The triplet codon for serine can be replaced with another triplet-codon coding for another amino acid. This is achieved by Site-Directed Mutagenesis (SDMS) using the “Overlapping PCR strategy”.

Mutants such as S19D/S19E where the Serine-19 has been replaced by Aspartic acid (D) or glutamic acid (E) respectively have been previously studied.

If a serine residue is replaced by Glutamic acid (E) at 19th position and the serines at 45 and 59 are replaced by Alanine (A), then the resultant product is termed as EAA, similarly AEA and AAE can be generated by replacing 45th and 59th position of Serine by either Glutamic acid (E) or Alanine(A). If two positions are replaced by Glutamic acid, the resultant mutant is termed as EEA, AEE, and EAE. If all the serine residues are replaced by Glutamic acid (E), the resultant mutant will be termed as EEE. Similarly, if all the serine residues are replaced with Alanine, it makes AAA which is a fully non-phosphorylatable mutant.

OBJECTIVES OF THE RESEARCH

The aim of this project was to purify the native human αB-crystallin and its phosphomimicking-mutants and to perform chaperone assays to assess the effect that phosphorylation has on αB-Crystallin’s chaperone function. The clones of native αB-crystallin and its phosphorylation-mimic mutants made using site-directed mutagenesis (SDMS) and cloned in pET21a vector between the restriction sites NdeI and HindIII were already available in the lab.

LITERATURE REVIEW

Horwitz [1] showed that α-crystallin can prevent thermal aggregation of Beta and Gamma crystallins and a few other proteins like a molecular chaperone.

Reports regarding the role of phosphorylation in the chaperone function of α-crystallin are debatable. Studies performed in vitro have used either purified forms of the phosphorylated protein from lenses, or recombinant proteins that have been designed to mimic serine phosphorylation by replacing it with a negatively charged aspartic or glutamic acid at the same position.

Ito et al ​[2]​ have reported that the phosphorylation-mimicking mutant has decreased chaperone activity towards the heat-induced aggregation of lactate dehydrogenase and refolding of luciferase. Koteiche & Mchaourab ​[3]​ have reported that phosphorylation-mimicking mutations can lead to an increase in the chaperone activity of αB-crystallin in binding to the destabilized T4 lysozyme mutants. A study by Ecroyd et al ​[4]​ reported that mimicking phosphorylation increased the chaperone activity of αB-crystallin against one amyloid-forming target protein, but decreased it against another. On the other hand, Wang et al ​[5]​ have found that phosphorylated and unphosphorylated forms of αB-crystallin purified from rat lenses exhibited similar chaperone-like activity.

METHODOLOGY

Media and buffer compositions

1. Luria Bertani (LB) Medium

LB medium
Peptone10g
Yeast Extract5g
NaCl10g
100μl of 10N NaOH for adjusting pHpH should be 7.2
Make up the volume to 1 Litre with double distilled water

2. 2XYT Medium

LB medium
Peptone10g
Yeast Extract5g
NaCl10g
100μl of 10N NaOH for adjusting pHpH should be 7.2
Make up the volume to 1 Litre with double distilled water

3. 10X TE

LB medium
Peptone10g
Yeast Extract5g
NaCl10g
100μl of 10N NaOH for adjusting pHpH should be 7.2
Make up the volume to 1 Litre with double distilled water

4. 1X TNE buffer

LB medium
Peptone10g
Yeast Extract5g
NaCl10g
100μl of 10N NaOH for adjusting pHpH should be 7.2
Make up the volume to 1 Litre with double distilled water

5. 1X Phosphate Buffer Saline (PBS)

LB medium
Peptone10g
Yeast Extract5g
NaCl10g
100μl of 10N NaOH for adjusting pHpH should be 7.2
Make up the volume to 1 Litre with double distilled water

Preparation of BL21 competent cells

A primary culture of BL21 DE3 cells (2xYT medium) was set up and incubated at 37°C overnight. 100 ml of 2xYT medium was inoculated (1% inoculum) with the primary culture and kept for incubation at 37°C. The cells were taken off incubation once an O.D of 0.4 had been reached. The culture was then subjected to centrifugation at 5000 rpm for 5 minutes at 4°C. The pellet obtained was resuspended in half the culture volume of ice-cold 0.1M Calcium Chloride solution. The culture was then kept on ice for 30 minutes. The cells were harvested as before (5000 rpm, 5 minutes) and then gently resuspended in a 1/10th culture volume of 0.1M Calcium Chloride. The cells were aliquoted into micro-centrifuge tubes followed by the addition of glycerol (to a final concentration of 10%) and stored at -80 °C.

Transformation of DH5 α strain of E.coli

100 ng of plasmid was added to 100 microlitres of competent DH5α cells. The cells were subjected to heat-shock by placing them in a water bath set at 42°C for 90 seconds. This was immediately followed by incubation on ice for 5 minutes. 1 ml of LB broth was then added to the cells and was incubated at 37°C for 60 minutes. Cells were harvested by spinning at 5000 rpm for 3 minutes. Most of the supernatant was discarded, leaving some for resuspension. After resuspension of the pellets, the cells were spread plated onto an LB plate containing Ampicillin (100μg/ml). The plate was incubated at 37°C for 14 hours and then observed for transformed colonies. A patch plate of the transformed colonies was prepared.

Plasmid isolation

Plasmid isolation was carried out using the alkaline-lysis method (Nucleospin plasmid isolation kit (MN)). A 10ml primary culture of the transformed DH5 α cells was set up and incubated at 37°C for 14 hours. The cells were harvested by spinning at 15000 rpm for 5 minutes. The cells were resuspended in 500μL of the resuspension buffer (Buffer A1), mixed gently and then incubated at room temperature for 5 minutes. This was followed by addition of 600μL of the lysis buffer (Buffer A2). 600μL of Buffer A3 was then added. The solution was then spun at 12000 rpm for 5 minutes. The supernatant was added to the provided column (containing a silica membrane for binding DNA) placed in a collection tube and spun. The flow-through was discarded. The membrane was then washed by adding Buffer A4 (supplemented with ethanol) and spinning at 12000 rpm for 3 minutes. The membrane was then allowed to air dry to remove all residual ethanol. The plasmid was then eluted using autoclaved distilled water. The concentration of plasmid was estimated using Nanodrop (Thermofischer Scientific) and then stored at -80°C.

Transformation of BL21 cells

200 ng of plasmid was added to 100μL of competent BL21 DE3 cells. The cells were heat-shocked by placing them in a water bath set at 42°C for 90 seconds. This was immediately followed by incubation on ice for 5 minutes. 1 ml of LB broth was then added to the cells and they were incubated at 37°C for 60 minutes. Cells were harvested by spinning at 5000 rpm for 3 minutes. Most of the supernatant was discarded, leaving some for resuspension. After resuspension of the pellets, the cells were spread plated onto an LB plate containing Ampicillin (100μg/mL). The plate was incubated at 37°C for 14 hours and then observed for transformed colonies. A patch plate of the transformed colonies was prepared.

Protein purification

Primary culture

Five patches from the patch plate were used to inoculate 40ml of LB broth and incubated at 37°C overnight.

Secondary culture

After 12-15 hours of incubation, 1% inoculum secondary cultures were set up in 4 flasks each holding 1L LB broth containing Ampicillin (100μg/mL) and incubated at 37°C, 200rpm.

IPTG induction

The OD (at 600nm) of the secondary cultures was periodically checked using a Spectrophotometer. Once an OD of 0.6 had been reached, IPTG was added to a final concentration of 1mM. Incubation at 37°C, 200rpm was continued for 5 hours after IPTG addition.

Harvesting of cells

The secondary cultures were spun at 10,000 rpm for 10 minutes in Nalgene bottles using the Beckman Avanti centrifuge to harvest the cells. The cell pellets hence obtained were stored at -20°C until further use.

Cell lysis

The cell pellets from the secondary cultures were resuspended in approximately 40ml of 1X TNE buffer. For lysis, PMSF and lysozyme were added to final concentrations of 15μM and 200μg/ml respectively, followed by a 30-minute incubation on the ice. The cell lysate thus obtained was a viscous solution, owing to release of high molecular weight DNA from the cells.

Sonication

The cell lysate was then subjected to ultrasonication to fragment the DNA. This was done for 10 minutes (3 cycles of 3.33 minutes, 21% amplitude). To prevent excess heating, the pulse was set to 5 seconds on and 10 seconds off and the solution

was kept on ice all through the process. The sonicated cell lysate was notably less viscous. To remove cell debris, the sonicated cell lysate was spun at 15,000 rpm for 45 minutes. The pellet was discarded and the soluble fraction was saved for further processing.

Selective salting out using Ammonium Sulphate

The supernatant was subjected to two rounds of Ammonium Sulphate precipitation. In the first round, starting concentration of zero leading up to a final saturation of 30%, the pellet was discarded and the supernatant saved. In the second round, going from 30% to 60% ammonium sulfate saturation, the pellet was saved and the supernatant discarded. The pellets were stored at -20°C.

Gel-Exclusion Chromatography

The second cut ammonium sulfate pellets were resuspended in 5ml of 1x TNE buffer. The solution was filtered using a 0.45-micrometer filter and loaded onto a gel exclusion column (S-300) for purification fitted to an FLPC system. Fractions over which the 280nm absorbance peak extended were pooled and stored at 4°C.

Ion Exchange Chromatography

The pooled fractions were spun at 15,000rpm for 30 minutes. The supernatant was loaded onto an ion-exchange column. The column used was an Anion-exchanger, packed with a Q-sepharose matrix, with a bed volume of roughly 20ml. The column was first equilibrated with 100ml of 1x TNE buffer. The sample was then passed through the column, followed by 20ml of 1x TNE buffer to elute any residual protein. Finally, the column was washed with 100ml of 5M NaCl solution.

Assessing purity using SDS PAGE

An SDS-PAGE gel was cast (12% resolving gel and 5% stacking gel). Samples collected at each of the previous stages were loaded onto the gel and observed for the relevant band corresponding to the 20 kDa band from the protein marker.

Protein estimation

The concentration of protein after passing through the ion exchange column was estimated using a UV-Visible Spectrophotometer. The 0.1% Absorbance was used to calculate the concentration of the protein from the absorbance value measured at 280nm. αB-crystallin’s 0.1% absorbance is known to be 0.693.

Buffer exchange

Using a gel exclusion column (Desalting column) in an FPLC (Fast Performance Liquid Chromatography) set up, the protein, originally in 1X TNE buffer was subjected to buffer exchange and collected in 20mM PBS.

Chaperone assay

The chaperone activity of the purified, buffer-exchanged proteins was assessed using a reduction dependent Insulin aggregation system. DTT-induced aggregation of Insulin in the presence of αB-crystallin and the phosphorylation-mimicking mutants was studied by measuring the light scatter in a fluorescence spectrophotometer at 37°C in 20mM phosphate buffer containing 100mM NaCl.

The working concentration of Insulin was 0.2 mg/ml. The αB-crystallin and its phospho-mimicking mutant proteins were added in a 1:0.25 Insulin to protein ratio.

RESULTS AND DISCUSSION

Purification of wild-type αB-crystallin and its phosphorylation-mimicking mutants

The proteins were over-expressed in LB cultures, the cells were pelleted, and proteins were isolated from them as mentioned previously. The various stages of protein purification have been shown in Figure 2.1. The last two bands from left correspond to proteins eluted from anion exchange 1st pass and 2nd pass and the presence of a single band signifies the purity of the protein.

PurificationSDSGel.png
    A 15% SDS-PAGE image of samples taken at the various steps of protein purification. The numbers indicate the steps of purification, as listed out below.
    Well contents (from left to right)
    1Protein marker (Thermo Scientific PageRuler)
    2Post sonication supernatant
    3Post-sonication pellet
    4Ammonium Sulphate first cut supernatant
    5Ammonium Sulphate first cut pellet
    6Ammonium Sulphate second cut supernatant
    7Ammonium Sulphate second cut pellet
    8Gel filtration fractions
    9Anion exchange first pass
    10Anion exchange second pass

    Gel filtration chromatography

    In order to detect the fractions containing αB-crystallin or its mutants, O.D of each fraction at 280 nm was measured and the fractions showing significant absorbance values were loaded onto SDS-PAGE as shown. Both, in the case of wild-type recombinant αB-crystallin and its phosphorylation-mimicking mutants, no considerable difference was observed in the time of elution.

    The gel filtration chromatography fractions which showed the presence of the protein of interest in SDS-PAGE were pooled and subjected to further purification by passing through ion exchange chromatography (Q-Sepharose column; bed volume = ~20 ml).

    Gel Filtration PAGE.jpg
      A 15% SDS-PAGE image of gel filtration elusions collected in different fractions of αB-S59E protein. The numbers indicate the fractions of elution.

      Chaperone assays

      Chaperone assays were performed using the reduction dependent insulin aggregation system at 465 nm emission and excitation wavelengths, 2.5 nm slit width, a PMT voltage of 600V at 37°C for 1500 seconds.

      Chaperone assays_1.jpg
        The graph shows reduction dependent insulin aggregation chaperone assay curves of wild type αB Crystallin, S59E (AAE), S19E(EAA) and EEE. 

        Comparison of the curves obtained by the chaperone assays of wild-type αB-Crystallin and the phosphomimicking-mutants revealed that the phosphomimetic mutants EEE and EAA showed improvement in activity compared to wild-type αB, while AAE had reduced activity.

        CONCLUSION

        The effect of phosphorylation on the chaperone activity of αB-Crystallin was studied. Phosphorylation at all the three sites (EEE) enhances the chaperone activity thereby preventing protein aggregation to a significant level. Phosphorylation at S-19 (EAA) also showed better protection than wild-type αB-Crystallin. Phosphorylation at S-59 (AAE) showed lesser protection than wild-type αB-Crystallin.

        REFERENCES

        ACKNOWLEDGMENTS

        Let me begin by thanking the Indian Academy of Sciences for organizing this Summer Research Fellowship Programme and giving me the opportunity to be a part of it and also for introducing me to the online portal Authorcafe, which I expect will make the process of any future academic writing a whole lot more convenient.

        I would like to thank my guide Dr. Ch Mohan Rao for being easilyapproachable and for all the knowledge and inspiration.

        My mentor, Mr. Kranthi Kiran, has been a pleasure to work with. I would like to thank him for being so patient with me and for all that he taught me.

        Much gratitude is due to my lab mate, Miss. Madhusmita Rawooth, for being, in equal parts, easy and fun to work with.

        I am grateful to all the people at CCMB for their hospitability and for all the effort that went into making this summer training a fruitful experience for us.

        Let me wrap up by extending a final thanks to all the fellow summer trainees and other students who have made this a memorable summer.

        References

        • Horwitz, J. 1992, Proc. Natl. Acad. Sci. U. S. A., 89, 10449.

        • Ito, H., Kamei, K., Iwamoto, I., Inaguma, Y., Nohara D. & Kato K. (2001). Phosphorylation-induced change of the oligomerization state of αB-crystallin. J. Biol.Chem. 276, 5346–5352.

        • Koteiche, H. A. & McHaourab, H. S. (2003). Mechanism of chaperone function in small heat-shock proteins. Phosphorylation-induced activation of two-mode binding in αB-crystallin. J. Biol. Chem. 278, 10361–10367.

        • Ecroyd, H., Meehan, S., Horwitz, J., Aquilina, J. A., Benesch, J. L., Robinson, C. V. et al. (2007). Mimicking phosphorylation of αB-crystallin affects its chaperone activity. Biochem. J. 401, 129–141.

        • Wang, K., Ma, W. & Spector, A. (1995). Phosphorylation of α-crystallin in rat lenses is stimulated byH2O2 but phosphorylation has no effect on chaperone activity. Exp. Eye Res. 61, 115–124.

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