Investigating Chemical Chaperones that can improve the stability of Lysozymes under high thermal temperature. Sabastine Aliyu Zubairu1, Joseph Oyepata Simeon2, Isaac Ralph Elon1, Mahdi Mohammed1, Sunday Blessing

1. 1
Investigating Chemical Chaperones that can improve the stability of Lysozymes
under high thermal temperature.
Sabastine Aliyu Zubairu1
, Joseph Oyepata Simeon2
, Isaac Ralph Elon1
, Mahdi
Mohammed1
, Sunday Blessing3
.
1
Department of Pharmacology Gombe State University
2
Department of Pharmacology Bingham University
3
School of Medcine New Vision University Republic of Georgia.
Corresponding author: Sabastine A. Zubairu Department of Pharmacology, Faculty of
Pharmaceutical Sciences, college of Medicine Gombe State University, Gombe State
Nigeria. +234(0)8065201165, seb4christ@gmail.com sabastinezubairu@yahoo.com
Abstract
Keywords: Lysozymes, Chemical Chaperones, Type III galactosemia
Abstract
Conformational diseases such as, Type III galactosemia is a genetic disorder caused by mutations
of enzyme GALE, these mutations alter the stability of GALE by shifting the ligand UDP-
galactose and the cofactor NAD+
binding site in some variants, which are believed to improve the
stability of GALE, some disease-associated variants become more unstable than the wild type,
and some variants are more susceptible to proteolysis. There are no treatments for galactosemia,
and if left untreated complications could develop such as liver damage, early onset of cataract, it
can damage the ears, can cause mental retardation. In view of these challenges, we hypothesized
that Protein Stabilizing agents will increase the activity as well as the stability of Lysozymes and
diseaseassociated variants. We investigated the effect of glycerol and betaine on the rate of this

2. 2
reaction. by means of differential scanning fluorimeter (DSF) Thermal shift assay, we
investigated the melting temperature of Lysozymes (35µM) and have now studied the effect of
glycerol and betaine on the melting temperature of Lysozyme, comparative to control sample
without chaperone as assayed by differential scanning fluorimetry. The enzyme was stabilized by
glycerol at 50%v/v and betaine at 450mM. Significant effects were obtained by 50% glycerol
This innovative project gives insight into the possible treatment of Type III galactosemia and
other conformational diseases, as our results indicate that pharmacological chaperones aiming
the stability of Lysozymes could be used for the treatment of conformational disorders.
Introduction
There is increasing number of diseases that are hereditary in nature, most of these diseases
results from mutations that causes proteins to fold wrongly (1). Although rare, most of
these conformational diseases are detrimental and without cure (2, 3). Lately, groups of
minor molecules known as chemical/pharmacological chaperones are used to stabilize such
misfolded or mutant proteins and improve their transfer to their action site (1-3). Some
diseases such Alzheimer's, diabetes and non-neurogenic systemic diseases have turn out to
be serious public health challenges, they generally called neurodegenerative disorder.
These diseasesoccur because of deposition of abnormal proteins in tissues (4-7). They
canaccumulate both extracellularly, and intracellularly, consequently becoming
dysfunctional and are believed to have various pathogenic effects.
Certain types of Malignant polypeptides are often fashioned by folding errors, which are
enhanced by genetic mutations and numerous factors relating to the environment such as
oxidizing and toxic conditions, salt, heat, and molecular crowding (8-10). Misfolded
proteins are disposed to their hydrophobic regions that are suppressedwithin the interior
when the proteins are folded correctly. The visible hydrophobic surfaces facilitate

3. 3
theoligomerization and aggregation of proteins (11, 12)). There is normally acellular
response that copes with aberrant proteins to sustain cell homeostasis, thishas been
extensively investigated by a mechanism underlying a system known as the protein quality
control (14-17). A key aspect of the protein quality control system is located in the
endoplasmic reticulum (ER) (16). The ER obtains proteins that fail to undergo proper
folding, embraces them unto the step of ubiquitination and allocates them to further
processes like degradation by the system of the proteasome(18, 19). At times, stored
proteins in the cells can become a target of autophagy and eventually degradation (19).
Irregular proteins are sometimesisolated to a specific cellular compartment to produce an
intracellular inclusion body called the aggresome (23, 24). The most important factors in
the PQC system are the molecular chaperones such as heat shock proteins (Hsp) that
recognize abnormal proteins, prevent their aggregation, promote refolding and help to
reestablish the injured proteins [21]. It was stated that a reduction in the cell of molecular
chaperones and the loss of their function induced folding failure and an escalation in the
cell of aggregate proteins (22).
In a study, hereditary renal and non-neuropathic amyloidosis (23, 24), were recently
investigated using molecular and biochemical techniques, the pathogenesisof this type of
amyloidosis has been gradually elucidated.(24) To date, several types of amyloidosis,
including lysozyme have been revealed to cause this hereditary renal and non-neuropathic
amyloidosis (24). More recently, there are results indicating that renal amyloidosis (gene)
is associated with the newlydiscovered lysozyme mutation Phe57Ile (23, 24). During the
clinical courses of all patients in this family, the most noticeable clinical manifestation was
nephropathy caused byrenal amyloidosis (24). Our current work investigated the effect of
chaperones in restoring the stability of Lysozymes under very high thermal temperature

4. 4
(25-27). This provides a flat form to investigatepharmacological chaperones that can
improve the stability and folding of variant Lysozyme in any amyloidosis(27-28).
An example of chemical chaperone is Glycerol, it was used to correct the mutant TP53
conformation (29). The results suggest that glycerol is effective in reinstating several TP53
mutants to normal TP53 function, leading to normal CDKN1A expression after heat stress
(29).Betaine is very strong osmolyte (30), there was a study on betaine was carried out
with urea as a denaturising agent and it was discovered that betaine improved proteins
stability (30). This study is aim at Investigating pharmacological chaperones that have the
ability to improve the stability of Lysozymes
Materials/Methods
Differential Scanning Fluorimeter (DSF) Thermal Shift Assay of Lysozymes
This is a model set up to determine the melting temperature and the effect of chemical
chaperons (glycerol and Betaine) on lysozymes thermal stability (Noel et al., 2013).
Preliminarywork was carried out to determine the concentration of Lysozymes that will be
more sensitive to Sypro orange dye and to determine the melting temperature. The samples
include Lysozymes at a varying concentration of 2µM to 100µM, Spyro orange
fluorescence dye and PBS to a final volume of 20µl were loaded in a Rotor-Gene Q cycler
(7,8, 10).in a triplicate manner and a procedure was carefully chosen for the high-
resolution melting temperature (7,8). With increasing temperature from 25o
C to 95o
C, the
enzymes were subjected to a rise in temperature with 1o
C additions and a waiting period of
5seconds (9, 10). The fluorescence was subjected to an excitatory wavelength of 460 nm
and assessing emission at 510 nm (9, 10). The melting temperature (Tm) of each lysozymes
concentration was determined from the primary derivative of the melting curve to establish

5. 5
the right concentration of lysozyme with the best curve was used to investigate the effect
of chaperones.
Effect of Glycerol on the Differential Scanning Fluorimetry (DSF) of Lysozymes
Lysozyme enzyme (Sigma UK) at a concentration of 35µM, Sypro orange (Sigma UK) a
fluorescent dye protein was mixed in serial dilution as described in previous articles of
50X solution into a PBS 50mM, pH 7.5, each stock solution was thoroughly mixed before
use.The same protocol was followed as the above.
Effect of Betaine on the Differential Scanning Fluorimetry (DSF) of Lysozymes
Lysozyme enzyme (Sigma UK) at a concentration of 35µM, Sypro orange (Sigma UK) a
fluorescent dye protein was mixed in serial dilution as described in previous articles of
50X solution into a PBS 50mM, pH 7.5, each stock solution was thoroughly mixed before
each use. Sample of lysozyme (35µM) were prepared using 50mM PBS, betaine (at
concentrations of 50Mm, 100Mm, 150Mm, 200mM, 250mM, 300mM, 350mM, 400mM,
450mM and 500mM) were prepared using PBS. Same as the above.
Statistical analysis and data evaluation
One-way ANOVA used to determine the level of significance difference between glycerol
and control as well as the effect of various concentration of glycerol. Turkey`s post hoc test
was used to determine the significance of the of the difference in Tm due glycerol binding.
Results and Discussion
Differential Scanning Fluorimetery

6. 6
Previous studies described methods on ways to establish and carry out protein stability
measurement by means of a Rotor-Gene Q cycler (9, 10). There are couples of applications
data signifying the importance of discovering drugs using this technique. An initial model
was set up using Lysozymes, first a preliminary determination of the right concentration of
Lysozyme that correspond to the melting temperature from 5µM, 10µM, 15µM, 20µM,
25µM, 30,µM, 35µM 40µM, 45µM and 50µM. The effect of glycerol and betaine as
chaperones were tested as shown below
Effect of Glycerol on the Melting Temperature of
Lysozymes
Lysozymes 15µM
Lysozymes 20µM
Lysozymes 25µM
Lysozymes 30µM
Lysozymes 35µM
Temperature (o
C )
Fig. 1: Thermal shift assay, transition curves make available valuable information. Effect of temperature on
the stability lysozyme were measured by DSF at varying concentration of lysozyme and at a scan rate of
1o
C/min, the graph represent the lysozyme (15µM, 20µM, 25µM, 30µM and 35µM), each well contain 5µM
of spyro orange dye, 10µl of PBS 50mM (pH 7.5) to a total volume of 20µl.
Preliminary fluorescence of lysozymes at various concentrations; this model is to
determine the best concentration of lysozyme and melting temperature, each sample
contains the enzyme at various concentration in 50mM PBS (pH=7.5), Spiro orange dye at
50X to a final concentration of 5µM (2.5µL) to a final volume of 20μL. The test sample
and control were loaded into a 36 well plates. Thermal denaturation was then monitored,
by following the ANS fluorescence emission with excitation wavelength of 395nm, and
0 20 40 60 80 100
0
10
20
30
40
50
Flu
ore
sce
nce

7. emission wavelength of 500 nm. A: represents Lysozyme concentration from 2µM, 5µM,
10µM, 15µM and 25µM.
Fig. 2:Effect of glycerol and betaine on
concentration and at a scan rate of 1o
C/min. each well was set up in a triplicate manner containing lysozyme
only (without any ligand) as the control, then in the presence of glycerol at 5%
orange dye 5µM, PBS 50mM (pH 7.5). The melting temperature of each was compared to the control.
7
emission wavelength of 500 nm. A: represents Lysozyme concentration from 2µM, 5µM,
:Effect of glycerol and betaine on the stability of lysozyme measured by DSF 35µM lysozyme
C/min. each well was set up in a triplicate manner containing lysozyme
only (without any ligand) as the control, then in the presence of glycerol at 5%- 50% concentration. Spyro
orange dye 5µM, PBS 50mM (pH 7.5). The melting temperature of each was compared to the control.
emission wavelength of 500 nm. A: represents Lysozyme concentration from 2µM, 5µM,
the stability of lysozyme measured by DSF 35µM lysozyme
C/min. each well was set up in a triplicate manner containing lysozyme
ntration. Spyro
orange dye 5µM, PBS 50mM (pH 7.5). The melting temperature of each was compared to the control.

8. 8
Fig. 3: Effect of glycerol binding on the stability of lysozyme measured by DSF 35µM lysozyme
concentration and at a scan rate of 1o
C/min. each well was set up in a triplicate manner containing lysozyme
only (without any ligand) as the control, then in the presence of glycerol at 5%- 50% concentration. Spyro
orange dye 5µM, PBS 50mM (pH 7.5). The melting temperature of each was compared to the control.
This result indicates the melting temperature of Lysozyme in the presence of glycerol comparing
it with Lysozymes only as control and 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45% and 50%
Glycerol with corresponding melting temperature 62.5o
C, 62.5o
C, 61.5o
C, 63.5o
C, 62.5o
C,
62.5o
C, 64.5o
C, 65.5o
C and 68.5o
C respectively,
One-way ANOVA analysis of the melting temperature of lysozymes plus glycerol, it
revealed that the higher the concentration of glycerol the more it stabilizes the enzymes;
there is a significant different between the melting temperature of Lysozymes only and in
the presence of glycerol.

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Figure 4: This represents the effect of betaine on the melting temperature and stability of 35µM Lysozymes:
Each sample contains the enzyme at 35µM concentration in 50mM PBS, Spiro orange dye at 50X to a final
concentration of 5µM (2.5µL) to a final volume of 20μL. The test sample and control were loaded into a 36
well plates. Thermal denaturation was then monitored, by following the ANS fluorescence emission with
excitation wavelength of 395nm, and emission wavelength of 500nm shows the effect of betaine on the
melting temperature of Lysozymes and there appears to be no respond of betaine on the melting temperature
of lysozyme and statistical analysis revealed no significant difference between the control and betaine with p-
value <0.05 except for 400mM.
Preliminary to Determine Melting Temperature of Lysozyme
Lysozyme at low concentration of 2µM to 25µM does not appear to fluorescence. In the
preliminary, we investigated the response of lysozymes at different concentration, and we
did observe the melting temperature of lysozyme, at various concentrations of 5µM, 10
µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM and 50µM of lysozyme.
Interestingly, a melting curve appeared only from 35µM. pharmacological chaperones
effects were tested to see if they could improve the melting temperature of lysozyme. In

10. 10
fig. 4. Above, lysozyme melted at 65o
C average. The use of Lysozyme was to function as a
model to study the melting temperature and the stability of GALE.
Here, we demonstrated what concentration of Lysozyme can show a level of sensitivity to
temperature and because Lysozyme like most enzymes is supposed to denature at extreme
temperature and thus loss of activity. Therefore, if a temperature is significantly risen
above the physiology the protein tends to denature. Here, the melting temperature of
lysozyme was affected by 5%, 10%, 15%, 20% 25%, 30%, 35%, 40%, 45% of glycerol, as
the melting temperature remained even after these concentrations were added. However,
glycerol at 50% v/v tend to be improved the melting temperature of lysozyme as shown in
fig.7, this means that a very high concentration of glycerol can be used to improve the
stability of lysozyme.
On the other hand, betaine did not indicate any benefit in improving the stability and the
melting temperature of lysozyme. When compared with the control, betaine showed no
significant difference as a stabilizer of lysozyme, although there some effect is seen, but
the effect is not significant at these concentrations. This could be because the concentration
of betaine was too low, per haves a higher concentration should be use or that fact that
betaine has no stabilizing effect on the melting temperature of lysozyme. Surprisingly, at
400mM of betaine some effects were observed but this effect disappeared with higher
concentration of 500mM betaine, this is quite interesting and it suggest that there could
some error with 40mM of betaine or it could be that lysozyme does not response to betaine
at a higher concentration.
Conclusions

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• Pharmacological chaperones can improve the stability and activity of Lysozymes.
• Glycerol at high concentration can improve the melting temperature of enzymes.
• This innovative project discovered Pharmacological chaperones and other protein
stabilizing-agents that will possibly treat type III galactosemia by increasing enzyme
catalytic activity, increase protein stability, increase cofactor binding and decrease
proteolytic activity of the mutants. However, glycerol revealed some level of effect at a
very high concentration (50%v/v).
• Thermal shift assay is a potential technique for drug discovery.
References
1. Arakawa, T., Ejima, D., Kita, Y. and Tsumoto, K. (2006), "Small molecule
pharmacological chaperones: from thermodynamic stabilization to pharmaceutical
drugs", BBA - Proteins and Proteomics,1764 (11); 1677-1687.
2. Ellis, R.J. (2001), "Macromolecular crowding: an important but neglected aspect of the
intracellular environment", Current Opinion in Structural Biology. 11(4):500-500
3. Hayer-Hartl, M. and Hartl, F.U. (2009), "Converging concepts of protein folding in vitro
and in vivo", Nature Structural & Molecular Biology, vol. 16(6):574-581.
4. Blennow K, de Leon MJ, Zetterberg H. (2006). Alzheimer’s disease. Lancet. 368:
387–403.
5. Lynch MA. (2014). The impact of neuroimmune changes on development of amyloid
pathology; relevance to Alzheimer's disease. Immunology.45(141): 292–301.
6. El-Kaissi S, Sherbeeni S. (2011). Pharmacological management of type 2 diabetes
mellitus: an update. Curr Diabetes Rev. (7): 392–405.
7. Pinney JH, Hawkins PN. (2012) Amyloidosis. Ann ClinBiochem.(49): 229–41.
8. Valastyan JS, Lindquist S. (2014). Mechanisms of protein-folding diseases at a glance.
Dis Model Mech. (7): 9–14.
9. Gregersen N, Bolund L, Bross P. (2013). Protein misfolding, aggregation, and degradation
in disease. MolBiotechnol. (31): 141–50.
10.Chatani E, Imamura H, Yamamoto N, Kato M. M. Kato, (2014). Stepwise organization of
the β-structure identifies key regions essential for the propagation and cytotoxicity of
insulin amyloid fibrils. J Biol Chem. (289): 10399–410.
11.Gsponer J, Vendruscolo M. (2006). Theoretical approaches to protein aggregation.
Protein PeptLett. (13): 287–93.

12. 12
12.Lindgren M, Hammarström P. (2010). Amyloid oligomers: spectroscopic characterization
of amyloidogenic protein states. FEBS J. (277): 1380–88.
13.Kajava AV, Steven AC.(2006). Beta-rolls, beta-helices, and other beta-solenoid proteins.
Adv Protein Chem. (73): 55–96.
14.Chakrabarti A, Chen AW, Varner JD.(2011). A review of the mammalian unfolded protein
response.BiotechnolBioeng. (108): 2777–93.
15.Tyedmers A, Mogk B, Bukau B. (2010). Cellular strategies for controlling protein
aggregation. Nat Rev Mol Cell Biol. 20(11): 777–88.
16.Ruggiano A, Foresti O, Carvalho P. (2014). Quality control: ER-associated degradation:
protein quality control and beyond. J Cell Biol. 2(204): 869–79.
17.FU XL, Gao DS. (2014). Endoplasmic reticulum proteins quality control and the unfolded
protein response: the regulative mechanism of organisms against stress injuries.
Biofactors. J Cell Biol (40): 569–85.
18.Hoozemans JM, Scheper W. (2012). Endoplasmic reticulum: The unfolded protein
response is tangled in neurodegeneration. Int J Biochem Cell Biol.(44):1295–98.
19.Yang L, Zhao D, Ren J, Yang J. (2015). Endoplasmic reticulum stress and protein quality
control in diabetic cardiomyopathy. BiochimBiophysActa. 2(1852): 209–18.
20.Kopito R. (2000). Aggresomes, inclusion bodies and protein aggregation. Trends Cell
Biol.; (10): 524–30.
21.Wolff S, Weissman JS, Dillin A. (2014). Differential scales of protein quality control.
Cell,(157): 52–64.
22.Gregeren N, Bross P. (2010). Protein misfolding and cellular stress: an overview.
Methods Mol Biol.(648): 3–23.
23.Masahideyazaki, S. Farrell A., and Merrill DB, (2003). A novel lysozyme mutation
Phe57Ile associated with hereditary renal amyloidosis, Kidney International, (63)1652–
1657.
24.Muntau, A.C., Leandro, J., Staudigl, M., Mayer, F. and Gersting, S.W. (2014), "Innovative
strategies to treat protein misfolding in inborn errors of metabolism: pharmacological
chaperones and proteostasis regulators", Journal of Inherited Metabolic Disease, vol.
37(4):505-523
25.Calvo, A.C., Scherer, T., Pey, A.L., Ying, M., Winge, I., McKinney, J., Haavik, J.,
Thony, B. and Martinez, A. (2010), "Effect of pharmacological chaperones on brain
tyrosine hydroxylase and tryptophan hydroxylase 2", Journal of Neurochemistry,(3):
853.
26.Cantó, C., Menzies, K.J. and Auwerx, J. (2015), "NAD+
metabolism and the control
of energy homeostasis: a balancing act between mitochondria and the nucleus", Cell
Metabolism, 22 (1): 1-53.

13. 13
27.Convertino, M., Das, J. andDokholyan, N.V. (2016), "Pharmacological chaperones:
design and development of new therapeutic strategies for the treatment of
conformational diseases", ACS Chemical Biology. 11(6): 1471.
28.Ditlecadet, D., Short, C.E. and Driedzic, W.R. (2011), Glycerol loss to water
exceeds glycerol catabolism via glycerol kinase in freeze-resistant rainbow smelt
(Osmerusmordax), American Journal of Physiology - Regulatory, Integrative and
Comparative Physiology. 300(3): 674-684.
29.Gong, H., Croft, K., Driedzic, W.R. and Ewart, K.V. (2011), Chemical chaperoning
action of glycerol on the antifreeze protein of rainbow smelt", Journal of Thermal
Biology. 36 (1): 78-83.
30.Kumar, N. and Kishore, N. (2014), Protein stabilization and counteraction of denaturing
effect of urea by glycine betaine", Biophysical Chemistry, (189): 16-24.