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The effect of milk constituents and crowding agents on amyloid fibril formation by #-casein Jihua Liu, Francis C. Dehle, Yanqin Liu , Elmira Bahraminejad, Heath Ecroyd, David C. Thorn, and John A. Carver J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04977 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on February 3, 2016

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Page 1 of 34

Journal of Agricultural and Food Chemistry

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The effect of milk constituents and crowding

2

agents on amyloid fibril formation by κ-casein

3

Jihua Liu‡§, Francis C. Dehle§, Yanqin Liu§, Elmira Bahraminejad$, Heath Ecroyd#, David C.

4

Thorn$,*, and John A. Carver$,*

5

‡

Pharmacy College, Jilin University, Changchun, Jilin Province, 130021, China

6

§

Department of Chemistry, School of Physical Sciences, The University of Adelaide, Adelaide,

7

South Australia 5005, Australia

8

#

9

of Wollongong, Wollongong, New South Wales 2522, Australia

School of Biological Sciences and Illawarra Health & Medical Research Institute, University

10

$

11

Territory 2601, Australia

Research School of Chemistry, The Australian National University, Acton, Australian Capital

1

ACS Paragon Plus Environment


1

Page 2 of 34

ABSTRACT

2

When not incorporated into the casein micelle, -casein, a major milk protein, rapidly

3

forms amyloid fibrils at physiological pH and temperature. In this study, the effects of milk

4

components (calcium, lactose, lipids, heparan sulfate) and crowding agents on reduced and

5

carboxymethylated (RCM) -casein fibril formation was investigated using far-UV circular

6

dichroism spectroscopy, thioflavin T binding assays and transmission electron microscopy.

7

Longer-chain phosphatidylcholine lipids, which form the lining of milk ducts and milk fat

8

globules, enhanced RCM-casein fibril formation, irrespective of whether the lipids were in a

9

monomeric or micellar state, whereas shorter-chain phospholipids and triglycerides had little

10

effect. Heparan sulfate, a component of the milk fat globule membrane and catalyst of amyloid

11

deposition in extracellular tissue, had little effect on the kinetics of RCM -casein fibril

12

formation. Major nutritional components such as calcium and lactose also had no significant

13

effect. Macromolecular crowding enhances protein-protein interactions, but in contrast to other

14

fibril-forming species, the extent of RCM -casein fibril formation was reduced by the

15

presence of a variety of crowding agents. These data are consistent with a mechanism of -

16

casein fibril formation in which the rate-determining step is dissociation from the oligomer to

17

give the highly amyloidogenic monomer. We conclude that the interaction of κ-casein with

18

membrane-associated phospholipids along its secretory pathway may contribute to the

19

development of amyloid deposits in mammary tissue. However, the formation of spherical

20

oligomers such as casein micelles is favored over amyloid fibrils in the crowded environment

21

of milk, within which the occurrence of amyloid fibrils is low.

22

KEYWORDS

23 24

κ-casein, amyloid fibril formation, heparan sulfate, phospholipid, triglyceride, lactose, calcium chloride, macromolecular crowding, thioflavin T, non-nucleation dependent 2


Page 3 of 34

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INTRODUCTION

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The structure of amyloid fibrils and their mechanism of formation have generated huge

3

research interest because of the association with diseases such as Alzheimer’s, Parkinson’s,

4

Huntington’s and type II diabetes 1. Protein aggregation, including amyloid fibril formation, is

5

also being increasingly recognised for its role underlying many food processing operations 2, 3,

6

particularly in the formation of aggregate gels 4.

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The caseins (e.g. S1, S2,  and  in cows) are the major proteins in milk. They have a

8

nutritional function for the neonate by providing a rich source of protein and calcium 5. We and

9

others have shown that both isolated - and S2-casein rapidly form amyloid fibrils at

10

physiological temperature and pH 6-8. Fibril formation by - and S2-casein is prevented by the

11

other caseins, S1 and , via molecular chaperone action 7-11, leading to the association of casein

12

proteins to form heterogeneous and dynamic micelles. Casein fibril formation has been

13

suggested to contribute to the development of amyloid-like deposits in bovine mammary tissue

14

within calcified stones termed corpora amylacea (CA) 12. Moreover, up to 15% of the κ-casein

15

in bovine milk is not incorporated into micelles 13. In this study, we have investigated whether

16

other physiologically relevant factors (lipids, heparan sulfate, macromolecular crowding etc.),

17

apart from caseins, influence fibril formation by κ-casein and therefore impact on the formation

18

of CA-associated amyloid.

19

In vivo, amyloid fibrils associate with lipid structures such as membranes and lipid rafts,

20

a process which has been implicated in both the formation and toxicity of amyloid fibrils 14. In

21

vitro studies have shown that the kinetics of fibril formation are altered in the presence of lipids;

22

they may promote or inhibit fibril formation depending on both the protein involved and the

23

properties of the lipid. For example, the binding of lipid inhibits fibril formation by α-synuclein

24

15

, the putative causative agent in Parkinson’s disease, whereas for amyloid β, the peptide 3



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involved in Alzheimer’s disease, polar phospholipids enhance the peptide’s fibril formation 16.

2

Lipids comprise approximately 4% w/v of milk (see Table S1) which are secreted in the form

3

of globules

4

amounts of monoglycerides, diglycerides, cholesterol and phospholipids also present 17.

17

. Triglycerides make up approximately 98% of milk fat globules, with small

5

Another component of milk, which is universally associated with extracellular amyloid

6

deposits, is heparan sulfate (HS) (Table S1). HS is a member of the glycosaminoglycan (GAG)

7

family of polysaccharides and is ubiquitously expressed on the surface of cells, including the

8

mammary epithelium, and is a major component of the extracellular matrix. HS binds to a

9

variety of proteins 18 and promotes amyloid fibril formation 19. HS and phospholipids form part 17, 20

10

of the membrane of milk fat globules and mammary epithelial cells

. On the other hand,

11

the surface of casein micelles is composed primarily of κ-casein. The corollary is that the

12

interaction between phospholipids/HS and κ-casein is highly likely, either via the mammary

13

epithelial lining during its transport through milk ducts or with fat globules in milk. Herein, we

14

tested whether this interaction could alter the propensity of κ-casein to form amyloid fibrils.

15

Casein micelles act as calcium-transporting vehicles to supply young mammals with a

16

highly concentrated yet soluble form of calcium phosphate. Due to the calcium-solubilizing

17

properties of casein micelles, cow’s milk contains up to 0.12% w/v calcium (Table S1). Being

18

a major constituent of milk and due to its close interaction with caseins, we also investigated

19

the effect of calcium on κ-casein fibril formation. Of comparable nutritional importance,

20

approximately 5% w/v of milk is composed of lactose (Table S1), a disaccharide sugar derived

21

from the condensation of glucose and galactose. Several sugars including disaccharides have

22

been shown to inhibit the conversion of proteins to amyloid fibrils 21, 22. Accordingly, we also

23

tested whether lactose at concentrations found in milk would protect κ-casein against fibril

24

formation.

4


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The association behaviour of proteins in solution differs markedly in biological

2

compartments where high concentrations of macromolecules are present 23. These effects are

3

known as macromolecular crowding which can be simulated with the use of artificial crowding

4

agents such as inert polymers. Macromolecular crowding generally favors protein aggregation

5

due to excluded volume effects 24. For example, Munishkina et al. 23 observed that crowding

6

agents accelerated α-synuclein fibrillation in vitro. In this study, we investigated how various

7

macromolecular crowding agents influence fibril formation by κ-casein. The rationale for these

8

experiments is that -casein has an unusual mechanism of fibril formation in which the rate-

9

determining step is the dissociation from its oligomeric state to produce the -casein monomer 25, 26

10

which is highly amyloidogenic

. Thus, we hypothesize that, in the presence of crowding

11

agents, -casein fibril formation will be decreased because of the tendency to favor the

12

oligomeric state under such conditions. It is also physiologically relevant in light of the high

13

concentration of macromolecules in milk (around 120 g/L (Table S1)) and milk ducts.

14

While the interaction between κ-casein and other caseins prevents its fibril formation,

15

the extent to which κ-casein fibril formation is affected by other components of milk, and hence

16

its capacity to form fibrils in its natural environment, was unknown. In this work, this aspect

17

was explored. Furthermore, upon reduction and carboxymethylation (RCM), -casein fibril

18

formation occurs in a highly reproducible manner at 37oC and neutral pH 27, making this a very

19

useful system to investigate the effect of small molecules on fibril formation in general.

20

Accordingly, we investigated the effect of calcium, lactose, lipids, HS and crowding agents on

21

amyloid fibril formation by RCM -casein, and interpreted these results in relation to its unique

22

mechanism of fibril formation.

23

5



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MATERIALS AND METHODS

2

Materials

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Bovine -casein was purchased from Sigma Chemical Co. (St Louis, MO, USA). Prior

4

to use, the -casein was reduced and carboxymethylated (RCM) as described by Schechter et

5

al. 28. The concentration of RCM -casein was determined spectrophotometrically using a Cary

6

5000 UV-visible spectrophotometer (Varian) using an absorption coefficient (A280) of 0.95

7

mL·mg-1·cm-1

8

Phospholipids were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA).

9

Triglycerides, heparan sulfate, dextrans, ficolls, and polyethylene glycols (PEG) were

10

purchased from Sigma Chemical Co. Calcium chloride (CaCl2) and lactose were reagent grade.

11

Lipids were stored in chloroform at –20oC and prepared by evaporating the chloroform

12

under nitrogen. Trace amounts of chloroform were then removed under vacuum for two hours.

13

Phospholipids were dissolved in 50 mM sodium phosphate buffer, pH 7.0, to the desired

14

concentrations. Triglycerides were resuspended in ethanol to the desired concentrations. HS

15

was dissolved in Milli-Q water at 1.0 mg/mL and diluted to the desired concentration when

16

used for experimentation. CaCl2 was dissolved in 50 mM HEPES buffer, pH 7.0.

29

. Uranyl acetate was purchased from Agar Scientific (Stansted, UK).

17

Methods

18

Thioflavin T assay of RCM -casein fibril formation. ThT fluorescence was used to

19

monitor the time-course of fibril formation by RCM -casein. RCM -casein (1 or 3 mg/mL)

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was incubated at 37oC for various length of time in 50 mM sodium phosphate buffer, pH 7.0,

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unless stated otherwise. Due to the insolubility of calcium phosphate, mixtures of RCM κ-

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casein and CaCl2 were incubated in 50 mM HEPES buffer, pH 7.0. Two different methods

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were employed to monitor ThT fluorescence over time, as described previously 8. In the

6


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presence of lactose, HS, and crowding agents, RCM -casein fibril formation was monitored

2

in situ. Here, the protein solutions were incubated in the presence of 10 μM ThT in Greiner

3

black μClear 96-microwell plates (Interpath Services, Australia) using a sample volume of 200

4

μL in each well. Plates were sealed with ThinSeal to prevent evaporation and the fluorescence

5

intensity was measured at 10 min intervals for up to 48 h using a Fluostar Optima plate reader

6

(BMG Labtechnologies, Australia) with a 440/490 nm excitation/emission filter set. In the

7

presence of lipids and CaCl2, due to their effects on ThT fluorescence, RCM -casein fibril

8

formation was monitored via an alternative method whereby the protein solutions were

9

incubated in the absence of ThT and 10 L aliquots were removed at the indicated time points

10

and stored at –20oC. Following the completion of the time-course, fibril samples with lipid

11

were thawed and mixed with 200 l of 10 M ThT in 50 mM glycine-NaOH buffer, pH 9.0, in

12

black Clear 96-microwell plates, and their fluorescence measured using a Fluostar Optima

13

plate reader with a 440/490 nm excitation/emission filter set. Fibril samples with CaCl2 were

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mixed with 1 mL of 10 M ThT in 50 mM glycine-NaOH buffer, pH 9.0, in a cell with a 1 cm

15

path length, and their fluorescence measured using a Chirascan™ (Applied Photophysics,

16

Surrey, UK) with 442/485 nm excitation/emission.

17

Transmission Electron Microscopy.

Samples from the above experiments were

18

prepared for transmission electron microscopy (TEM) by adding 5 L of protein solution to

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Formvar and carbon-coated nickel grids (SPI Supplies, West Chester, PA). The grids were then

20

washed three times with 10 L of Milli-Q water and negatively stained with 10 L of uranyl

21

acetate (2% w/v). Samples were viewed using a Philips CM100 transmission electron

22

microscope (Philips, Eindhoven, The Netherlands).

23

Circular Dichroism Spectroscopy. RCM -casein was incubated at 37oC in the absence

24

and presence of lipids, HS, lactose, CaCl2 and crowding agents as described above. Samples

7



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were diluted to 0.15 mg/mL RCM κ-casein with water, immediately before acquiring the CD

2

spectra. Spectra over a wavelength range of 190-250 nm were acquired at room temperature

3

using either a Chirascan™ (Applied Photophysics, Surrey, UK) or JASCO J-810 (Jasco,

4

Victoria, Canada) spectropolarimeter in a cell with a 0.1 cm path length. The spectra acquired

5

for each agent alone were subtracted from the spectra acquired with protein. Results are

6

expressed as the mean molar ellipticity per residue ([θ], degree.cm2dmol-1).

7

Statistics. All tests of statistical significance, smoothing functions and curve fitting

8

were performed using GraphPad PRISM 6.0.5 (GraphPad Software, San Diego, CA, USA).

9

Initial rates of aggregation were determined from the rate of increase in fluorescence intensity

10

during the first hour of incubation. Time-points shown are the average of triplicate

11

measurements representative of at least two independent experiments.

12

RESULTS

13

Longer chain phospholipids promote RCM κ-casein fibril formation

14

To determine whether triglycerides and phospholipids affect fibril formation, RCM -

15

casein was incubated in the presence and absence of the zwitterionic phospholipids, 1,2-

16

dinonanoyl-sn-glycero-3-phosphocholine

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phosphocholine (D6PC) and 1,2-dipropionyl-sn-glycero-3-phosphocholine (D3PC), and the

18

triglycerides, oleic acid (OT), palmitic acid (OP) and a 2:1 molar ratio of oleic to palmitic acid

19

(OPT), and fibril formation was monitored by ThT fluorescence for 72 hours.

(D9PC),

1,2-dihexanoyl-sn-glycero-3-

20

For other amyloid fibril-forming proteins, zwitterionic phospholipids differentially affect

21

fibril formation; the effect is dependent on whether the particular lipid is above or below its

22

critical micelle concentration (CMC)

23

phospholipids on fibril formation by RCM -casein at concentrations above and below their

30

. Therefore, we endeavored to test the effect of

8


Page 9 of 34


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CMC. D9PC caused a concentration-dependent increase in both the initial rate and final level

2

of ThT fluorescence of RCM -casein (Figure 1A). The increase in rate was linear and was

3

independent of whether D9PC was in its micellar or free state, i.e. above or below a

4

concentration of 0.029 mM, the CMC of D9PC

5

mM (Figure S1A). D6PC increased the initial rate and final ThT fluorescence of RCM -casein

6

in a linear, concentration-dependent manner at concentrations up to its CMC of 10 mM (Figure

7

1B and S1B)

8

fluorescence decreased to a level below that of RCM -casein in the presence of 10 mM D6PC

9

(Figure 1B and S1B). Similarly, the initial rate in the presence of 0.3 mM D9PC was not

10

significantly higher than in the presence of 0.1 mM D9PC (Figure 1A and S1A). However,

11

after 72 hours, the ThT fluorescence in the presence of 30 mM D6PC was comparable to that

12

of RCM -casein in the presence of 10 mM D6PC (Figure 1B). Unlike D9PC and D6PC, D3PC

13

and the triglycerides (OT and PT) had little effect on fibril formation by RCM -casein (Figure

14

1D-F). The exceptionally high CMC of D3PC (> 90 mM) precluded investigation in its micellar

15

form. In control experiments, neither the phospholipids nor the triglycerides had any effect on

16

ThT fluorescence when incubated on their own. D6PC and D9PC had little effect on fibril

17

morphology, as judged by TEM (Figure S2).

30

30

, over a concentration range of 0.01 to 0.1

. At a concentration well above its CMC (30 mM), the initial rate of ThT

18

The effect of the phospholipids on the structure of RCM -casein was investigated.

19

Figure 2 shows the far-UV CD spectrum of RCM -casein in the absence and presence of the

20

phospholipids, at their respective concentrations that affect fibril formation, before and after

21

incubation for 24 hours at 37°C. Prior to incubation, the CD spectrum of RCM κ-casein in the

22

absence and presence of phospholipids is very similar, suggesting that these phospholipids have

23

little effect on the initial conformation of the protein, although there is a minor change in the

24

presence of D9PC (0.3 mM). Upon incubation at 37°C, RCM -casein exhibited a time-

25

dependent increase in -sheet formation as indicated by the decrease in ellipticity at 215 nm 9



Page 10 of 34

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and red shift in the ellipticity minimum over 24 hours, which correlates with the formation of

2

amyloid fibrils 25. As assessed by the change in CD spectra and as anticipated from the ThT

3

data, D9PC had the greatest effect of the three lipids on the formation of -sheet structure in

4

RCM -casein after 24 hours of incubation compared with D6PC, D3PC and RCM -casein

5

alone. As expected from the ThT data, there was little difference in the CD spectra of RCM -

6

casein alone or in the presence of 30 mM D3PC over the time-course of the experiment.

7

Overall, the CD spectra support the ThT fluorescence data showing that 0.3 mM D9PC had the

8

largest effect on accelerating the conversion of RCM -casein’s structure to one more rich in

9

-sheet.

10

Effect of HS, calcium and lactose on RCM κ-casein fibril formation

11

The aggregation of RCM κ-casein with time at 37°C in the presence of 0 to 20 μg/mL

12

HS was also monitored by ThT fluorescence (Figure 3A). Bovine milk contains approximately

13

18 μg/mL HS 31, most of which is associated with the membrane of milk fat globules 20. While

14

there was a marginal increase in the initial rate of increase in fluorescence with HS

15

concentration, the final magnitude of ThT fluorescence was comparable. Thus, in contrast to

16

other fibril-forming systems, the kinetics of RCM κ-casein fibril formation is not significantly

17

affected by HS. Similarly, lactose did not influence the kinetics of fibril formation by RCM κ-

18

casein over the concentration range of 50 to 200 mM lactose (Figure 3C). The lactose

19

concentration in bovine milk is typically ~150 mM 32. The effect of calcium on κ-casein fibril

20

formation was investigated by incubating RCM κ-casein at 37ºC in HEPES buffer, pH 7.0, in

21

the presence of 5 to 30 mM CaCl2. The calcium concentration in bovine milk is, on average,

22

~30 mM 32. Low CaCl2 concentrations (i.e. 5 mM) led to a minor increase in the initial rate of

23

fibril formation compared to RCM κ-casein alone, whereas higher CaCl2 concentrations (i.e.

24

30 mM) appeared to reduce the initial rate, as judged by ThT fluorescence. Overall, CaCl2 did

10


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not significantly affect the kinetics of RCM κ-casein fibril formation. These findings obtained

2

by ThT fluorescence results were supported by TEM and far-UV CD spectra. Essentially

3

identical changes in far-UV CD spectra were observed upon incubation of RCM κ-casein in

4

the absence and presence of 20 μg/mL HS, 200 mM lactose and 30 mM CaCl2 (Figure 2B).

5

Likewise, TEM indicated that HS, lactose and CaCl2 had no significant effect on fibril

6

morphology (Figure S3).

7

Crowding agents inhibit RCM κ-casein fibril formation

8

The effects of different macromolecular crowding agents on RCM κ-casein fibril

9

formation were determined by comparing the rate and extent of fibrillation in the presence of

10

150 mg/mL of crowding agent. All five crowding agents investigated, over an average mass

11

range of 200 to 400,000 Da, decreased the extent of fibril formation to various degrees, as

12

judged by lower ThT fluorescence values at the plateau stage, after 13 hours of incubation

13

(Figure 4). Several of the crowding agents (e.g. PEG 200) also slowed down the rate of RCM

14

-casein fibril formation, whereas Ficoll 400, for example, did not. The inhibition of fibril

15

formation by crowding agents was supported by far-UV CD spectroscopy. Prior to incubation,

16

the secondary structure of RCM κ-casein structure was unchanged in the presence of crowding

17

agents; the decrease in ellipticity below 200 nm in the presence of Ficoll 400 is due to the

18

residual background absorption of this crowding agent (Figure 2C). Importantly, there was a

19

reduction in the amount of β-sheet formed after 24 hours in the presence of 450 mM PEG 200

20

and, to a lesser extent, 450 µM Ficoll 400, as indicated by the greater ellipticity at 215 nm and

21

blue shift in the ellipticity minimum compared with RCM κ-casein alone (Figure 2C). TEM

22

suggested that the morphology of the fibrils was not significantly altered when formed in the

23

presence of crowding agents (Figure S3), however, dense, amorphous aggregates were present

24

in electron micrographs of RCM κ-casein in the presence of 450 µM Ficoll 400 (Figure S3).

25

This is consistent with the ThT data, further supporting the general conclusion that crowding 11



Page 12 of 34

1

agents inhibit fibril formation by RCM κ-casein. The concentration dependence of PEG 200

2

and Ficoll 400 on RCM -casein aggregation was investigated. Figure 5A shows the effects on

3

RCM κ-casein fibril formation of varying PEG 200 concentration. Over the concentration range

4

from 0 to 375 mM PEG 200, there was a linear decrease in the initial rate of aggregation and

5

extent of fibril formation of RCM -casein (Figure 5B). Ficoll 400 behaved similarly over the

6

concentration range from 0 to 300 µM, although only the extent of fibril formation was reduced

7

(Figure 6A) and the inhibition was to a lesser extent than in the presence of PEG 200 (Figure

8

5A). The initial rate of aggregation was uniform over the concentration range of 0 to 450 µM

9

Ficoll 400 (Figure 6B). At higher concentrations of crowding agents (e.g. 450 mM PEG 200

10

or 375 µM Ficoll 400), the effect on the rate and/or extent of fibril formation leveled off which

11

may be due to an increase in the solution viscosity which negates excluded volume effects, as

12

has been observed in other fibril-forming systems 23.

13

DISCUSSION

14

Calcified, proteinaceous deposits (i.e. CA) containing amyloid fibrils have been 12

15

described in the mammary glands of various species

. An increasing body of evidence

16

suggests that mammary CA-associated fibrils originate from casein proteins that are highly

17

amyloidogenic under physiologically relevant conditions, namely κ- and αs2-casein. We have

18

previously shown that fibril formation by bovine κ- and αs2-casein is potently inhibited by other

19

caseins (i.e. αs1 and β) 7, 8, 11, with this interaction leading to the formation of casein micelles.

20

κ-Casein fibril formation is further inhibited by extensive intermolecular disulfide bonding 26

21

maintained by the oxidizing environment of the mammary luminal space and milk.

22

Notwithstanding, the development of amyloid deposits within mammary tissue and its

23

association with casein proteins suggest that other physiologically relevant factors should be

24

considered and/or that under certain conditions the mechanisms responsible for ensuring

25

proteostasis within the mammary gland are compromised. Milk, and the mammary ducts 12


Page 13 of 34


1

leading to its secretion, are rich in a variety of molecules. In this study, we investigated the

2

effects of non-protein milk components (calcium, lactose, phospholipids, trigylcerides and HS)

3

on the aggregation of RCM κ-casein. We also monitored RCM κ-casein aggregation in the

4

presence of inert crowding agents, in order to mimic the crowded environment of milk.

5

Milk fat globules consist of an apolar lipid core, which is composed primarily of 33

6

triglycerides, surrounded by a layer of cholesterol, phospholipids and proteoglycans

7

further one third of the phospholipid in milk is found in the aqueous phase 17. Previous research

8

has shown that lipids have variable effects on fibril formation. They can accelerate the

9

formation of the cross β-sheet structure that is characteristic of amyloid fibrils (see 34 for review)

10

or lead to an α-helical structure, which can either act as an intermediate to enhance β-sheet

11

structure and fibril formation 35, 36 or remain a stable structure to inhibit fibril formation 30. In

12

this study, D6PC and D9PC enhanced RCM κ-casein fibril formation, but D3PC had little

13

effect. In particular, D9PC caused a concentration-dependent increase in both the initial rate

14

and the degree of RCM -casein fibril formation irrespective of whether it was in a monomeric

15

or micellar state. In contrast to the effects on other proteins, these lipids had little effect on

16

either the overall secondary structure of either the protein prior to incubation or the morphology

17

of the resulting fibrils, e.g. to make them form straight fibrils as occurs with apolipoprotein C-

18

II

19

forming RCM -casein, which is influenced by their acyl chain length, i.e. the relative

20

hydrophobicity of the phospholipids whereby the longer-chain, more hydrophobic ones,

21

accelerate RCM κ-casein fibril formation. The CD data are consistent with this conclusion in

22

implying an interaction of D6PC and D9PC with the aggregating RCM -casein. However, this

23

interaction is not dependent on whether the lipid is a monomeric or micellar species, in contrast

24

to apolipoprotein C-II fibril formation for which aggregation is promoted only by the

25

monomeric form of lipids such as D6PC but is inhibited by the micellar species

30

. A

. Thus, these three phospholipids have different mechanistic interactions with fibril-

30

. The 13



Page 14 of 34

1

triglycerides OT and PT had little effect on fibril formation by RCM -casein. These

2

triglycerides differ from D6PC and D9PC in terms of their higher acyl chain number and length,

3

and due to their lack of a hydrophilic phosphorylcholine group. Thus, the amphiphilic and

4

zwitterionic properties of D6PC and D9PC are also important for their interaction with κ-casein

5

and/or promotion of fibril formation.

6

Phospholipids are a minor component of milk compared to triglycerides, so the

7

enhancement of fibril formation by phospholipids is probably not of much relevance in milk

8

itself but may be applicable in the mammary gland to the interaction between -casein, which

9

covers the surface of the casein micelle, and membrane phospholipids, for example in milk

10

ducts. The apical (lumen-facing) membrane of mammary epithelial cells is composed of a

11

phospholipid bilayer that functions to prevent leakage of milk components from the alveolar

12

lumen. Cellular injury and disruption of the membrane as caused by mastitis (inflammation of

13

mammary tissue) could lead to increased κ-casein/phospholipid interaction and be a potential

14

trigger of amyloid formation. Indeed, CA development has been associated with mastitis

15

(reviewed by Nickerson et al.

16

fibril formation by s2-casein which has comparable fibril-forming propensity to -casein 7 and

17

has been isolated in a degraded form from CA-related amyloid deposits 12.

37

). Potentially, phospholipids would have a similar effect on

18

HS is associated with the amyloid deposits in a number of extracellular amyloidoses 19.

19

It has been proposed that HS, and the closely related heparin, facilitate amyloid fibril formation

20

in vivo via a scaffold-based mechanism, whereby they act as structural templates for self-

21

assembly 38, 39. Indeed, HS and heparin significantly shorten the lag phase of fibril formation

22

of numerous proteins in vitro (see 40 for review). Interestingly, in the case of immunoglobulin

23

light-chain variable domain, the kinetics of fibril formation is not significantly affected by

24

heparin when incubated in the presence of pre-formed fibrils, i.e. seeds 41, implying that GAGs

25

act at the earliest stage of aggregation, namely the nucleation phase, to accelerate fibril 14


Page 15 of 34


1

formation. In the present work, we found little correlation between HS concentration and the

2

magnitude and rate of fibril formation by RCM κ-casein, a finding that is consistent with the

3

unique mechanism of -casein fibril formation.

4

We have reported that κ-casein fibril formation is induced by quaternary structural

5

destabilization associated with dissociation of the monomer from the oligomeric state, which

6

is promoted by disulfide bond reduction

7

fibril formation is dissociation from the oligomer with the monomer being highly

8

amyloidogenic, particularly at higher temperature. The mechanism of aggregation is therefore

9

very different from the standard nucleation-dependent mechanism in that its rate is not limited

10

by the formation of a metastable, prefibrillar species and therefore κ-casein fibril formation has

11

a very short lag phase and cannot be ‘seeded’

12

accelerate amyloid fibril formation of a variety of proteins including -synuclein 42, the major

13

protein present in the Lewy body deposits in Parkinson’s disease. By contrast, in general, κ-

14

casein fibril formation was inhibited in a linear, concentration-dependent manner by the

15

presence of crowding agents such as PEG 200, which is consistent with stabilization of the -

16

casein oligomeric state (i.e. effective reduction or inhibition of dissociation) leading to

17

prevention of fibril formation. The casein micelle accomplishes this in a similar manner, i.e.

18

the high local concentration of the other casein proteins within the micelle stabilizes -casein

19

to prevent its dissociation and therefore the possibility of deleterious cytotoxic fibril formation

20

5, 43-45

21

described (see

22

involve either the formation of homooligomers (e.g. tetramerization of transthyretin

23

interaction with its natural binding partners (e.g. the binding of ataxin-3 with ubiquitin

24

Indeed, fibril formation of s2-casein is prevented and controlled by its intimate association

25

with s1-casein 7.

8, 25, 26

. Thus, the rate-determining step in -casein

25

. Molecular crowding agents substantially

. Many examples of protein oligomerization to regulate fibril formation have been 46

for review). The stabilization of fibril-forming proteins in this manner may 47

) or 46

).

15



Page 16 of 34

1

A well-characterized, non-casein example is insulin which exists as a hexamer that is

2

not prone to fibril formation in contrast to the dissociated monomer which is amyloidogenic 48.

3

Reminiscent of what we observe for RCM κ-casein, Munishkina et al. 42 showed that insulin

4

fibril formation is retarded by crowding agents under conditions favoring its natively associated

5

state. Inhibition of fibril formation by crowding agents was also observed for other natively

6

associated proteins, e.g. core histone proteins 42. Fibril formation by both insulin and histones

7

is promoted, however, by crowding agents under acidic conditions where these proteins are

8

initially monomeric

9

association, amyloid fibrils are not invariably the most favored outcome of this process 49.

42

. Thus, while crowding agents are expected to enhance protein

10

Fibril formation by RCM κ-casein can be divided into three major steps: (i) the

11

dissociation of the amyloidogenic monomer from the micelle-like oligomer, (ii) the formation

12

of prefibrillar nuclei, and (iii) the successive addition of monomer to growing nuclei

13

(elongation) 25, 26. The net effect of a particular crowding agent on fibril formation depends on

14

its effects on each step of this process. Our contention is that crowding agents reduce RCM κ-

15

casein fibril formation by shifting the monomer-oligomer equilibrium towards the oligomeric

16

state and thereby reducing the concentration of amyloidogenic intermediates. Whereas PEG

17

200 decreased both the initial rate and extent of fibril formation by RCM κ-casein, the effect

18

of Ficoll 400 on aggregation rate was negligible. It is conceivable that Ficoll 400 exerts

19

opposing effects on this system, accelerating fibril elongation while limiting the availability of

20

monomeric subunits for incorporation into the fibril. Since PEG and Ficoll have very similar

21

physicochemical properties (e.g. both are hydrophilic, flexible and spherical), differences in

22

their effects on protein association are likely related to differences in their size, i.e.

23

hydrodynamic radii (~3 Å for PEG 200 and ~80 Å for Ficoll 400 49).

24

Caseins in general are high-affinity calcium-binding proteins and their interaction with

25

calcium is central to their function, their interactions with each other, and their micellar 16


Page 17 of 34


1

structure. Caseins bind calcium via highly phosphorylated sequences called phosphate centers

2

present in αs1, αs2-, and β-casein 5. Calcium binding has the effect of neutralizing the negatively

3

charged phosphoseryl residues and leads to a loss of electrostatic repulsion and, as calcium

4

concentration is increased, these ‘calcium-sensitive’ caseins precipitate from solution. The

5

remaining κ-casein, while typically containing one to three phosphoseryl residues

6

distinct calcium-binding phosphate centers and therefore does not interact as strongly with

7

calcium as other caseins. This is consistent with our finding that CaCl2 has little impact on the

8

kinetics of RCM κ-casein fibril formation.

29

, lacks

9

Amyloid fibril formation generally originates from partially unfolded protein states that

10

expose substantially more surface area prone to intermolecular association e.g. hydrophobic

11

patches. The addition of sugars to protein solutions has the tendency of increasing the number

12

of water molecules that make up the shell around the protein (i.e. preferential hydration),

13

thereby making the increase in surface hydrophobicity upon denaturation a thermodynamically

14

unfavorable process. Owing to their ability to stabilize the more folded, less aggregation-prone

15

states of proteins, several sugars have been shown to inhibit fibril formation. For example,

16

fibril formation by an amyloidogenic mutant of light-chain protein Wil is delayed in the

17

presence of 0.5 M glucose, sucrose or trehalose

18

formation by RCM κ-casein. The absence of a stabilizing effect may be related to the

19

conformational properties of RCM κ-casein, i.e. it is an intrinsically disordered protein that,

20

owing to the presence of proline- and glutamine-rich sequences, is highly hydrated in its

21

partially unfolded state 50.

21

. In this study, lactose did not affect fibril

22

In summary, fibril formation by RCM κ-casein was promoted by longer-chain

23

phospholipids but inhibited by crowding agents. Calcium, lactose, HS, triglycerides and short-

24

chain phospholipids had no major effect. Crowding agents likely act as inhibitors by stabilizing

25

the oligomeric state of κ-casein at the expense of the fibril-forming pathway. The association 17



Page 18 of 34

1

of κ-casein with other caseins to form micelles performs a similar role to prevent the formation

2

of amyloid fibrils in milk. In the mammary gland, the interaction of caseins with lipid

3

membranes along their secretory pathway (from epithelial cells to milk) may contribute to the

4

development of CA-associated amyloid.

5

ABBREVIATIONS

6

A.U., arbitrary units; CA, corpora amylacea; CMC, critical micelle concentration; DTT, 1,4-

7

dithiothreitol; GAG, glycosaminoglycan; HS, heparan sulfate; OT, oleic acid; OP, palmitic

8

acid; OPT, 2:1 molar ratio of oleic to palmitic acid; PC, phosphocholine; PEG, polyethylene

9

glycol; RCM, reduced and carboxymethylated; RFU, relative fluorescence units; TEM,

10

transmission electron microscopy; ThT, thioflavin T.

11

ACKNOWLEDGEMENT

12

We thank Dr Michael Griffin (University of Melbourne) for his advice regarding lipid purchase

13

and handling. We thank Dr Carl Holt (University of Glasgow) for helpful discussions.

14

AUTHOR INFORMATION

15

Corresponding authors

16

*E-mail: [email protected]. E-mail: [email protected]. Phone: +61-2-6215-9748.

17

Fax: +61-2-6125-0750.

18

Funding

19

This work was supported by grants from the Australian National Health and Medical Research

20

Council and the Australian Research Council. Jihua Liu was supported by the China

21

Scholarship Council - University of Adelaide Joint Postgraduate Scholarship Program.

18


Page 19 of 34


1

Notes

2

The authors declare no competing financial interests.

3

ASSOCIATED CONTENT

4

Approximate values for components in bovine milk (Table 1). Linear fits of initial rate of fibril

5

formation as a function of phospholipid concentration (Figure S1). TEM images of RCM κ-

6

casein fibrils formed in the absence and presence of phospholipid (Figure S2). TEM images of

7

RCM κ-casein fibrils formed in the absence and presence of HS, lactose, CaCl2 and crowding

8

agents (Figure S3). The Supporting Information is available free of charge on the ACS

9

Publications website.

10

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8

FIGURE CAPTIONS

9

Figure 1. Effect of phospholipids and triglycerides on fibril formation by RCM -casein.

10

Aggregation curves monitored by ThT fluorescence show RCM -casein (3 mg/ml) in 50

11

mM sodium phosphate buffer, pH 7.0, incubated at 37oC in the absence or presence of (A)

12

D9PC, (B) D6PC, (C) D3PC, (D) OT, (E) PT and (F) OPT. Time-points shown are the

13

average of triplicate measurements representative of at least two independent experiments.

14

Error bars represent the standard deviation of the mean.

15

Figure 2. Effect of phospholipids, HS, lactose, CaCl2 and crowding agents on far-UV CD

16

spectra of RCM κ-casein before and after fibril formation. RCM κ-casein in 50 mM sodium

17

phosphate buffer, pH 7.0, was incubated at 37°C for 0 hours (continuous line) and 24 hours

18

(dashed line) in the absence (blue) and presence of (A) phospholipids; 0.3 mM D9PC

19

(brown), 30 mM D6PC (pink) and 30 mM D3PC (green), (B) HS (brown), lactose (pink) and

20

CaCl2 (green), and (C) crowding agents; 450 mM PEG 200 (pink) and 450 µM Ficoll 400

21

(green). Samples were diluted with water to 0.15 mg/ml RCM κ-casein prior to analysis.

22

Figure 3. Effect of HS, lactose and CaCl2 on RCM κ-casein fibril formation. Aggregation

23

curves monitored by ThT fluorescence show RCM -casein (1 mg/ml) in 50 mM sodium

Kuznetsova, I. M.; Turoverov, K. K.; Uversky, V. N., What macromolecular crowding

Thorn, D. C.; Ecroyd, H.; Carver, J. A.; Holt, C., Casein structures in the context of

25



Page 26 of 34

1

phosphate or (for the effect of CaCl2) 50 mM HEPES buffer, pH 7.0, incubated at 37oC in the

2

absence or presence of (A) HS, (B) lactose and (C) CaCl2. The ThT assay was performed in

3

situ for the effects of HS and lactose and ex situ for the effect of CaCl2 as described in the

4

Materials and Methods. Time-points shown are the average of triplicate measurements

5

representative of at least two independent experiments. Error bars represent the standard

6

deviation of the mean.

7

Figure 4. Effect of crowding agents on fibril formation by RCM -casein in the presence of

8

crowding agents. Aggregation curves monitored by ThT fluorescence show RCM -casein (3

9

mg/ml) in 50 mM sodium phosphate buffer, pH 7.0, incubated at 37oC in the absence or

10

presence of 150 mg/mL of each of Dextran 70000, Dextran 50000, Ficoll 400, PEG 3350 and

11

PEG 200. Time-points shown are the average of triplicate measurements representative of at

12

least two independent experiments.

13

Figure 5. Concentration-dependent effects of the crowding agent PEG 200 on fibril formation

14

by RCM -casein. (A) Aggregation curves monitored by ThT fluorescence of RCM -casein

15

(1 mg/ml) in 50 mM sodium phosphate buffer, pH 7.0, incubated at 37oC with concentrations

16

of PEG 200 ranging from 0 to 450 mM. (B) Dependence of the initial rate and maximum

17

fluorescence of fibril formation by RCM -casein as a function of PEG 200 concentration.

18

Time-points shown are the average of triplicate measurements representative of at least two

19

independent experiments. Error bars represent the standard deviation of the mean.

20

Figure 6. Concentration-dependent effects of the crowding agent Ficoll 400 on fibril formation

21

by RCM -casein. (A) Aggregation curves monitored by ThT fluorescence of RCM -casein

22

(1 mg/ml) in 50 mM sodium phosphate buffer, pH 7.0, incubated at 37oC with concentrations

23

of Ficoll 400 ranging from 0 to 450 µM. (B) Dependence of the initial rate and maximum

24

fluorescence of fibril formation by RCM -casein as a function of Ficoll 400 concentration. 26


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1

Time-points shown are the average of triplicate measurements representative of at least two

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independent experiments. Error bars represent the standard deviation of the mean.

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FIGURES

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Figure 1.

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1 2

Figure 2.

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1 2

Figure 3.

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1 2

Figure 4.

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1 2

Figure 5.

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1 2

Figure 6.

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FOR TABLE OF CONTENTS USE ONLY

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The effect of milk constituents and crowding

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agents on amyloid fibril formation by κ-casein

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Jihua Liu‡§, Francis C. Dehle§, Yanqin Liu§, Elmira Bahraminejad$, Heath Ecroyd#, David C.

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Thorn$,*, and John A. Carver$,*

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The Effect of Milk Constituents and Crowding ... - ACS Publications

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