Effect of natural biopolymers on amyloid fibril formation and morphology

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Abstract

Amyloid fibrils are associated with the pathogenesis of protein misfolding diseases such as Alzheimer’s disease. These fibrils typically exhibit different morphologies when grown in vitro, and this has been known to affect their biological properties and cytotoxicity. The formation kinetics and resultant morphology of fibrils formed from the model proteins Bovine Insulin and Hen Egg White Lysozyme have been measured. We show that the presence of gum arabic and pectin during fibril formation cause the amyloid fibrils formed to associate into higher order fibrillar aggregates. It is postulated that the carbohydrates act as a template to promote inter-fibril association, resulting in larger, thicker fibrils. This observation provides some insight into the differences in growing amyloid fibrils in vitro in the absence or presence of other high molecular weight compounds. Furthermore, these findings suggest a method of tailoring fibril structure for applications in nanotechnology and bio-template applications.

Introduction

Amyloid fibrils are self-assemblies of misfolded proteins [1] that are associated with a number of protein conformational disorders [2], [3], [4], [5]. These insoluble protein aggregates have a characteristic cross-β structure [2] and are resistant to proteolysis [6]. While amyloids of the same protein often form rather consistently sized structures, the morphology and properties of these fibrils can vary depending on the incubation conditions such as temperature [7], salt concentration [7], [8], [9] and shearing [10], [11], [12]. For example, increasing salt concentration can reduce the lag time of fibril formation. However, very high salt concentrations can cause the formation of disordered aggregates instead [7]. Although the extent of how different fibril structures affect cellular and biological functions is not fully understood, it is known that variations in fibril morphology have a profound impact on amyloid cytotoxicity. This effect is evident in the observation that different Aβ fibril morphologies have different levels of cytotoxicity to SH-SY5Y cells [10]. An in-depth understanding of how these protein aggregates grow in the presence of other seemingly inert and non-interacting biomolecules, such as carbohydrates, is important as the environment that amyloid fibrils grow in vitro can significantly affect the kinetics and morphology of the fibrils formed [13].

It is known that molecular crowding can significantly affect the kinetics of amyloid fibril formation [14], [15], [16]. Lipids can alter the kinetics of insulin fibril formation and cause unwinding of the amyloid fibrils formed [17]. Lipids can also interact with amyloid fibrils and cause disassociation of mature fibrils into oligomers and protofilaments in amyloid beta [18]. Nucleic acids have also been found to induce the formation of amyloids in prion proteins [19].

Carbohydrates such as glucose and mannose can also affect the kinetics and morphology of amyloid fibrils by replacing water in hydrogen bonding sites [13]. The presence of small monosaccharaides or disaccharides have been shown to cause longer fibrils to form [13]. Furthermore, the sulfated polysaccharide Chondroitin sulphate B tends to cause the formation of larger smooth fibrils by acting as a template for fibril formation [20]. In light of this, the study of amyloid fibril formation in the presence of large carbohydrates can be used to provide insight into how similar molecules affect the morphology of the fibrils formed in the crowded in vivo environment.

Amyloid fibrils are an attractive option to use as templates for creating various nano- structures, due to their regular structures and ability to self-assemble under mild solution conditions [21]. Amyloid fibrils can also be used in many nano-applications, such as improving the efficiency of solar cells [22]. Furthermore, the ability for amyloid fibrils to take on different morphologies in different incubation conditions has useful nanotechnological applications, as the same protein can be used to make nano-materials of different structures. This would simplify the manufacture of variations of the same nano-structures, For example, the manufacture of nano-wires of different diameters could be simplified by altering the concentrations of carbohydrates during the incubation of the amyloid templates.

Many commercially used food additives such as gum arabic and pectin contain large polysaccharide-based compounds with emulsification properties. One example is gum arabic (GA), a common stabilizer in the food industry. GA consists of 88.4% by weight of 3.8 × 105 Da polysaccharide arabinogalactan, 10.4% arabinogalactan protein (AGP) that has a Mw of 1.45 × 106 Da, and 1.24% of non-AGP glycoprotein that has a Mw of 2.5 × 105 Da. The AGP component of GA is believed to be responsible for the emulsification capabilities of GA [23], [24].

Pectin is a large branched carbohydrate that is commonly used in the food industry as a gelling agent in various foods such as jams [25]. Unlike GA, the emulsification capabilities of pectin are more complex and less understood, as limited quantities of pectin are adsorbed to the surface of oil droplets in an emulsion [26]. For instance the emulsification capability of citrus pectin is less than that of beet pectin and this is believed to be due to a lower acetylation of the citrus pectin [26]. Additionally, the proteins that are normally associated with pectin are believed to strongly contribute to the emulsification capabilities of the pectin, in a similar manner to that in GA [26]. Some plant pectin have more protein content than others, for instance apple pectin has a less protein content than apricot pectin [27]. This affects the emulsification capabilities of the pectin, as although both apple and apricot pectin have emulsifying properties, apple pectin is a less effective emulsifier than apricot pectin [27]. Furthermore, in an acidic medium, pectin was found to be able to bind to casein micelles and can cause depletion flocculation of the micelles [28]. The molecular weights of pectin from the same source tends to vary significantly, for instance the molecular weight of apple pectin varies from 3387 Da to 1877 Da depending on the method of extraction [27]. Both pectin and GA are well studied, commonly available food additives with some emulsifying properties [27], [29], and hence are good candidates to investigate how large polysaccharides affect amyloid self-assembly. Although neither GA nor pectin is absorbed by the body, they are still good candidates for use to simulate the effect of molecular crowding of large branched polysaccharide groups on amyloid fibrils, as similar large polysaccharides containing compounds, such as proteoglycans, are common in vivo.

In this study, BI (bovine insulin) and HEWL (hen egg white lysozyme) were chosen as the two model amyloid-forming protein systems to study the interactions between the biopolymers and amyloid fibril formation. These systems were chosen as they are very well characterized, commercially available protein systems that can form amyloid fibrils reliably in vitro [30], [31], [32], [33], [34], [35], [36]. Furthermore, BI and HEWL are both medically significant, as insulin is known to form amyloids in injection sites of diabetic patients [37] and in the lungs of insulin inhaler users [38]. Lysozyme amyloids are related to systemic amyloidosis in humans [39]. Finally, BI and HEWL are also different and unrelated protein systems, and HEWL is a much larger protein than BI. Thus, this would allow the investigation of the effects of these biopolymers on two different proteins to determine if the effects of these biopolymers on amyloid fibril formation are shared between different amyloid-forming proteins. The biopolymers AGP and PE were chosen due to their amphiphilic nature.

Section snippets

Materials

Bovine insulin (lot number 0001434060), thioflavin T (lot number 043K3506), gum arabic (Lot number 112F-0323), hen egg white lysozyme (lot number 096K1237) and apple pectin (lot number 14H0985) were obtained from Sigma-Aldrich. Arabinogalactan-protein was purified from GA via size exclusion chromatography (SEC) using Waters Ultrahydrogel™ 500 and 2500 columns or using a Sephacryl™ 400 column. The purity of the recovered AGP was verified using SEC. All other reagents were of analytical grade. A

Results

At pH 1.9, BI denatures at 60 °C [45], hence in the incubation conditions used (60 °C, pH 1.5), BI would be expected to be denatured. Although HEWL is fully denatured in a pH 1.5 solution at 55 °C, significant fibril formation only occurs above 55 °C [32]. 70 °C was used as the incubation temperature for HEWL as HEWL fibrils formed at lower temperatures tend to be mixed with non-fibrillar aggregates [32].

As HEWL (14.3 kDa molecular weight [46]) is significantly larger than BI (5.8 kDa molecular

Amyloids in pectin and gum arabic

BI and HEWL amyloid fibrils grown in the presence of pectin or gum arabic resulted in the aggregation and self-assembly of the fibrils into larger fibril structures as seen in Fig. 2, Fig. 3, Fig. 4. The presence of thick BI and HEWL amyloid fibrils shows that GA, AGP and pectin can induce the formation of thicker fibrils in different protein systems. This in turn suggests that the interaction of GA, AGP and pectin with amyloids is non-specific and takes advantage of the generic chemical

Conclusion

It is observed that amyloid fibrils grown in the presence of the carbohydrate rich additives pectin, AGP and GA appear to intertwine into larger and thicker amyloid fibrils. It is believed that the additives bind to and facilitate the aggregation of the fibrils into larger structures, possibly by acting as a template for the large structures or by reducing the electrostatic repulsion of the proteins. Additionally, the presence of larger fibrils resulted in a higher ThT fluorescence intensity

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