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Poly-g-Glutamic Acid a Substitute of Salivary Protein Statherin?

Byline: Zeeshan Qamar, Zubaidah Binti Haji Abdul Rahim, Hooi Pin Chew and Tayyaba Fatima

Summary: The modus operandi of salivary proteins in reducing the kinetics of enamel dissolution during simulated caries challenges is thought to be associated with interaction of glutamic acid residues with human teeth surfaces. Japanese traditional food stuff 'natto' is rich with chain of repeating glutamic acid residues linked by g-peptide bond and hence, named poly-g-glutamic acid (PGGA). It is a naturally occurring polypeptide and may therefore perform similar caries inhibitory functions as statherin.

Keywords: Salivary protein, Caries, Poly- g-glutamic acid, Statherin

Introduction

Saliva, a typical clear complex bio-fluid baths the tissues in the oral cavity. It is a mixture of various glandular and non-glandular secretions. Salivary glands are the main source of glandular secretions, whereas crevicular fluids are known as non-glandular secretions. Crevicular fluid is rich in oral microorganisms and host cells [1].

There are various classifications of salivary glands; the most common is according to their size in which they are classified as major and minor salivary glands. Major glands include parotid, submandibular and sublingual glands. Whereas minor salivary glands are ductless and scattered on the mucosal surface of the oral cavity[2].

Normal pH of saliva ranges between 6.0 and 7.0. The pH can vary from 5.3 to 7.8 and depending on the salivary flow rate, increasing at low flow rate and decreasing at peak flow rate[3].

The consistency of saliva can vary that is watery, thick, gluey or foamy depending on the amount and types of proteins that are present [1]. On an average the flow rate of unstimulated saliva in healthy individuals varies between 1.0 to 1.5 L/day. In this condition the major contribution of saliva is by the submandibular (65%) mixed with 80% serous and 20% mucous content. Parotid glands contribute approximately 20% (purely serous) whereas sublingual 7-8% (purely mucous). The minor salivary glands become the source of almost 10% saliva.

The proportion of the contribution by the different salivary glands may vary significantly in stimulated saliva; in which about 50% is from the parotids [4]. The secretion of stimulated saliva is enhanced when the glands are subjected to mechanical, psychic, gustatory, olfactory, or pharmacological stimulus; representing 80-90% of the daily whole saliva.

Many other factors affect the salivary flow rate, such as preceded stimulus, circadian and circannual rhythms, level of hydration, body location, optical effect, the gland volume and the medications [5].

Composition of Saliva

Salivary secretion is mainly composed of 99% water, whereas the remaining 1% comprises of the organic and inorganic compounds. Secretions produced by major glands are rich in inorganic contents, whereas for the minor salivary glands have a higher organic content[3]. Na+, K+, Cl- and HCO3- are the most common ions in saliva. Ca2+, PO43-, F-, CNS-, Mg2+, SO42- and I- are also present in saliva [6]. The HCO3- determines the pH and buffering capacity of the saliva [3].

Salivary secretion is also rich in proteins, enzymes, mucins, urea, ammonia and immune substances [3]. Immune substances include antibody such as IgA (similar to serum protein); enzymes such as amylase, lysozyme, peroxidase, kallikrein,acid phosphatase and glygoproteins (mucins). Proteins having low molecular weight such as statherin, tyrosine-rich protein, proline-rich proteins (phosphorylated and non-glycosylated), and histatins(histidine-rich protein) are also present in saliva [6].

Salivary secretion is typically hypotonic with plasma. However, saliva has a capability under physiological controlled mechanisms to become isotonic or hypertonic [6]. Hypotonicity of saliva plays a vital role in the detection of salty taste by the taste buds independent of sodium content. It has a significant role in the expansion and hydration of mucin glycoprotiens protecting the oral tissues [3].

In this article the salivary protein of key interest statherin having proficient role in oral environment will be discussed in comparison and contrast to naturally occurring food containing poly- g-glutamic acid.

Statherin

A phospho-peptide consisting of 43 amino acids residues is most commonly found in the saliva [7, 8]. It has unusual characteristics. It has an acidic peptide structure, secreted by different salivary glands typically by major salivary glands. It has a high content of proline, tyrosine and glutamic acid. The glutamic acid is a similar residue found in the poly-g-glutamic acid.

Statherin has an affinity for the phosphate and calcium minerals such as in hydroxyapatite (HAp) [9]. In view of its strong affinity for the surfaces of HAp, there is a major involvement of statherin as a salivary protein in the formation of layers on the tooth surface, the so-called "pellicles"[10].

It is also thought that the statherin can take part in the transportation of the minerals particularly calcium and phosphate [11], in its secretion from salivary glands, thus contribute in transporting ions at the mucosal level of the surface.

Tooth enamel integrity is maintained by the protein film which acts as a boundary on the surface [12]. Bacterial colonization specifically is easily possible due to the typical interaction between the salivary proteins present in the pellicle and the bacterial surfaces during initial stages of plaque formation [13].

Structure of Statherin

Statherin is a polypeptide that has been found in the secretion of parotid and submandibular glands [14]. It has been confirmed by Long et al. (2001) using solid-state NMR studies that the active part involved in statherin binding and preventing HAp precipitation is the N-terminus, whereas the transportable with no affinity to bind hap is the middle- and the C-terminus. [15, 16]. Naganagowda et al. (1998) assumed that this may take place because of the N-terminus occupying almost all of the negative charge. It is believed that the N-terminus of statherin is responsible for 'anchoring' to the HAp surface by structural changes to an alpha-helix form leading to adsorption, even though additional lengths of the molecule may be implicated in adsorption stabilization [17-19].

Functions of Statherin

Statherin organizes the Ca2+ in the mouth by inhibiting HAp crystal nucleation and salivary calcium phosphate salts precipitation [20-23]. It has been found that statherin is the first protein that binds to cleaned enamel [21, 23]. Furthermore, statherin is also involved in inhibiting unwanted precipitation of calcium phosphate from the supersaturated saliva. It is also playing a major role in the formation pellicle layer and provides protection for the teeth [21, 24]. It has been shown that the elevated levels of statherin at the air interface of the biofilm in the mouth leads to collecting and clearing of bacteria to the stomach rather than by surface adhesion [20, 25].

It is also believed that statherin compete with glycoproteins of high molecular weight (GPHMW) to adhere on the tooth surface. Hence, the GPHMW-binding cariogenic bacteria such as streptococcus mutans will be inhibited from adhering to the tooth surface [20, 26]. According to Leito et al. (2009) statherin can be considered as an oral defence against fungi as it enhances candida albicans's hyphae transition to yeast [20, 27].

Also, the lubrication properties of statherin (together with other proteins present in the pellicle) depend on the protein structure following adsorption onto the HAp, making the role of statherin more significant as compared to other proteins [24, 28].

It has been reported by Chin et al. (1993) and Wikiel et al. (1994) that at pH 6.0 and 7.0, statherin and the N-terminus of statherin-like pentapeptides inhibit the dissolution of HAp. However, these experiments were carried out under strictly controlled environments simulating carious and/or erosive challenges. Hence, if these effects may or may not take place in the more complex oral cavity is questionable [21, 29].

Large conformational changes have been seen in protein upon adsorption onto solid surfaces that influence their biological activity significantly [30-32]. This is seen mainly in proteins that act as a substrate during interaction with surfaces where they unfold upon adsorption [31, 33]. Unfolding associated adsorption has revealed that statherin has the ability to prevent calcium phosphate crystallization by binding to the nuclei of the early crystals and inhibiting crystal growth by adsorption on the nucleated crystals [12, 34]. Studies using isothermal titration calorimetry and equilibrium adsorption isotherm, indicated that most of the protein adsorbs by a process that is thermo-neutral and only a small part of the protein adsorbs with detectable heat [28].

Additionally, a study showed that the calcium concentration in a multilayer complex salivary film is 500 times more than calcium concentration in saliva. Electrophoresis of salivary proteins of different individuals shows presence of other salivary proteins in fewer quantities in the salivary films with variability between individuals and types of saliva. The occurrence of statherins in saliva films is considered not important for the creation and maintenance of these films as they were detached by washing [25].

Poly-g-glutamic acid

Poly-g-glutamic acid on the other hand, is a naturally occurring homo polyamide; made of D- and L- forms of glutamic acid units which are classically connected by the amide linkages. The amide linkages are formed between the a-amino group of one glutamic residue and the g-carboxyl group of the adjacent glutamic residue as shown in Fig. 1. This type of linkage has a strong resistance against enzymes known as proteases (enzyme cleaving the amide bonds) [35].

Poly-g-glutamic acid is hydrophilic and negatively charged at pH above 2.2. It is a poly- amino-acid which is microbially synthesized by different strains of bacillus [36, 37]. Ivanovics (1937) discovered poly-g-glutamic acid as a component of the bacillus anthracis capsule [38]. Bovarnick (1942) reported that poly-g-glutamic acid is accumulated as an end product on fermentation in a culture broth of Bacillus subtilis [39]. Sawamura (1913) was the first to report that a Japanese traditional food 'natto', is a mixture of poly-g-glutamic acid and fructans formed by the fermentation of soya beans[40].

Various other bacillus species have also been reported for having the same capability to produce poly-g-glutamic acid in their respective growth mediums, as a consequential of fermentation [41-43]. Poly-g-glutamic acid exists in various forms; broadly it can be classified as a free acid form and a salt form. The salt form of poly-g-glutamic acid contains Na+, or Mg2+, or K+, or NH4+ or Ca2+.

The characteristics and chemical structure of the acidic or salt forms of poly-g-glutamic acid can be determined by the FT-IR. Depending on the environmental changes poly-g-glutamic acid can exhibit in 5 different types of conformational changes. These modifications are known as a-helix, b-sheet, helix to random coil transition, random coil and enveloped aggregate.[42, 44].

Poly-g-glutamic acid being a constituent of traditional food product is edible, non-toxic to human and the environment. It has a high molecular weight and not easily biodegradable [45].

Most importantly it has non immunogenic reactions, probably linked to the degradation of poly- g-glutamic acid into glutamic acid residues [46].

Molecular Weight of Poly-g-glutamic acid

Poly-g-glutamic acid when produced synthetically more often has a molecular weight below 10,000 KDa; thus limiting its utility. However the one produced by bacteria has a molecular weight >10,000 KDa [35]. It is also easily degradable into lesser molecular weight as needed for its specific application [35].

Production of Poly-g-glutamic acid

Poly-g-glutamic acid has a diverse history for being known since last 75 years, but yet the mechanism and major substrates involved in its production are still unclear. Studies have been carried out in order to determine the conditions, nutritional requirements to improve cell growth and develop variations in molecular weight. Thus, ultimately it was suggested that for production of poly-g-glutamic acid, the nourishment varies according to the type of strain.

Therefore, based on the nutritional obligation, poly-g-glutamic acid producing bacterial strains are divided into two groups; one which is dependent on L-glutamic acid as a carbon source and the other is not [43, 45, 47].

Bacterial strains using carbon as a source enhance the production of poly-g-glutamic acid in the presence of glycerol (other source of carbon), glucose, sucrose and citric acid when added in the medium. It is presumed that L-glutamic acid acts as an activator for the enzymatic system of poly-g- glutamic acid [48].

Bacillus licheniformis (ATCC strain 9945a) and Bacillus subtilis are among the strains which are most commonly involved in the production of poly-g- glutamic acid. Leonard (1958) optimized medium e for Bacillus licheniformis (ATCC strain 9945a) in order to produce poly-g-glutamic acid [49]. Bacillus subtilis, a key strain to produce 'natto' is nutrient specific and is reliant on the biotin or vitamin solution.

Chemical Structural Characteristics of poly-g- glutamic acid

The glutamic residue, major building block of poly-g-glutamic acid, comprises of three functional groups which can be illustrated in the sequence of their chemical activity a -NH, a - COOH and g - COOH. The hydrogen dissociation constants for these are: pKa(=pK1)=2.13-2.2, pKg(=pK2)=4.25-4.32, and pK3=9.7-9.95 [50]. During the polymerization (chemically catalysed) of glutamic acid, a-peptide bonds develop between active groups a-COOH and a-NH2 producing a-poly glutamic acid as an end product.

During the process of submerged fermentations, L-glutamic acids are largely racemized to D-glutamic acids. Later on, both the D- and L- glutamic acids are co-polymerized via formation of g-peptide bonds between less reactive g- COOH and a-NH2 resulting in the formation of g- (D,L)-poly glutamic acid as an end product.

Microbial Resistance

a-peptide bond is commonly found in the protein structures. It can be hydrolysed by most proteases. The g-peptide bond of poly-g-glutamic acid can only be hydrolysed by rare but naturally occurring protease, g-glutamyl transpeptidase [51]. None of the other proteases have an ability to hydrolyze the bond of poly-g-glutamic acid. Thus the poly-g-glutamic acid has potent resistance against microbial attack.

Stability of Poly-g-Glutamic Acid

According to Ho et al (2006), on thermal analysis of poly-g-glutamic acid it was determined that hydrated water is 10%, dehydration temperature is 109degC, the melting point (Tm) is 160degc and the decomposition temperature (Td) is 340degc [50].

Capability of Poly-g-Glutamic Acid to Bind Metals

The most important point related to the chemical structure of poly-g-glutamic acid is that it has the capability to bind with different metals [50]. The important metals required are bio-available, most vital among those includes Ca2+ and Mg2+ binding via ionic complex mechanism. The complexes formed are known as calcium poly-g-glutamate and magnesium poly-g-glutamate. These bonds are of vital importance being stable coordinate ionic complexes [50].

Applications of Poly-g-Glutamic Acid

Poly-g-glutamic acid is found to have various implications in different fields such as a thickener particularly in the paints and cosmetic products. It is also being used as a humectant, bitterness revealing agent in edible products, biopolymer flocculants, heavy metal absorber and cryoprotectant [52]. It has a very important role in cancer therapy being a drug carrier with a capability of sustained release of regime.[45, 53, 54]

Aspects of Poly-g-Glutamic Acid Acting as a Substitute for Statherin

In this article taking a note from the above discussion it can be proposed that poly-g-glutamic acid being highly viscous, may have an ability to coat the tooth enamel by forming a protective layer. The free a-COOH group in poly-g-glutamic acid molecule may promote binding of coating to the enamel surface; this will lead in protecting as well as inhibiting the dissolution of the enamel HAp at the time of critical pH similar to that of statherin binding with its terminal on the tooth surface [55].

On the other hand, the anionic a-COOH group in each of the glutamic acid residue has a capability to react and bind to the cationic entity of the other molecule or biopolymer, or can behave or remain as a free carboxylic acid. Therefore, poly-g- glutamic acid can dissolve particularly Ca and Mg compounds to form stable ionic complexes. In supersaturated conditions of the oral cavity, various free ions can bind to poly-g-glutamic acid, particularly the free Ca2+ which may bind and promote remineralization even at lower pH, more than that occurring in the presence of statherin. Poly- g-glutamic acid also has a capability to resist the microbial attacks by the oral micro-flora which cause dental caries (streptococcus mutans) and can also resist from being hydrolysed in the presence of proteases.

As it has a proficiency to bind and form complexes with free ionic calcium, thus, it will play an important role in remineralizing incipient carious lesion than the deep cavities.

Importantly, it has been reported that poly-g- glutamic acid remains stable and hydrated at room temperature (decomposes at high grade temperature) [56]. Property of being hydrated can play a vital role in keeping the oral cavity moist similar to saliva, hence it can be helpful for the patients suffering from dry mouth syndrome and other salivary gland related pathologies.

It has also been suggested that poly-g- glutamic acid has an important nutritional value having a competency to deliver naturally available calcium and magnesium for absorption, which will aid in promoting bone formation and also in reducing the onset of osteoporosis in elderly stage of life [50].

Conclusion

Based on the discussion above, it can be concluded that poly-g-glutamic acid has a potential to provide a new era of interest in developing economical as well as environmental valuable mouthwash and artificial saliva which is protein by nature, having a competence to inhibit dissolution and promote remineralization of the tooth enamel. It makes it valuable as a substitute to synthetically produced statherin being cost effective and naturally produced. Most importantly being viscous and edible (biologically safe), it can be used as an artificial saliva particularly for non-affording patients with salivary gland disorders or undergoing radiation therapy or taking medications such as diuretics (being readily soluble in water and biodegradable).

It is stable at a neutral pH and non-toxic for human even at high dosage as compared to compounds like fluoride salts and other proteins (which require particular storage temperature in order to maintain their effectiveness) used in commercially available mouthwashes.

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Author:Qamar, Zeeshan; Rahim, Zubaidah Binti Haji Abdul; Hooi Pin Chew; Fatima, Tayyaba
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Aug 31, 2016
Words:4955
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