Do Salivary Proteins Play a Role in Tasting Bitter Substances? - ACS

Chapter 14

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Do Salivary Proteins Play a Role in Tasting Bitter Substances? Guy H. Carpenter* Salivary Research, Flr 17, Tower wing, Kings College London Dental Institute, London, U.K. *E-mail: [email protected].

The detection of bitterness occurs via well characterized receptors located in taste buds on the tongue surface. However, genetic variations in these receptors do not account for all the variations between subjects in their perception of bitter substances, which suggests there may be a role for salivary proteins or other factors in their detection. Several groups have shown associations of bitterness perception with carbonic anhydrase 6, proline-rich proteins and cystatins either by GWAS studies or proteomic studies, suggesting they potentially play a role. Some preliminary data is shown by the authors which provides further evidence that salivary proteins may play a role but the responsible protein is not determined, which may reflect the multifunctionality of salivary proteins.

Introduction Generally saliva is considered to have a rather inert role in taste. In contrast to the anti-microbial properties of salivary proteins, in which nearly every salivary protein has some anti-microbial efficacy, very few proteins have been implicated in mediating taste (1). Probably the best known is gustin, later identified as carbonic anhydrase 6, for which there is some evidence by association. The reasons why so few proteins appear to have a role in taste are probably manifold, but a major reason could be the multifunctionality of salivary proteins. This term denotes that one protein can perform many roles but also that other proteins can substitute for another protein if absent. This makes it rather difficult to identify © 2015 American Chemical Society In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


functions if certain subjects lack, or have low levels, of one particular protein, which commonly occurs through alternative splicing as salivary proteins are well known for being highly polymorphic (2). Amylase illustrates this rather well. It is the single most abundant protein in saliva and has well-known starch hydrolytic abilities (3) which relates to its function in the perception of sugar/ starch. One study has related the gene-copy number of amylase to different populations of humans (agricultural vs arctic) based on the amount of starch in their diet (4) and furthermore to levels of obesity (5). However, these studies examine gene-copy number rather than actual protein levels (for which the correlation is not as strong), which suggests these are inherited traits rather than causal effects. There are many subjects who lack amylase and yet have normal taste detection thresholds (a commonly used indicator of taste). A more causal effect of salivary amylase has been implicated in the modification of the physical properties of food by hydrolyzing starch polymers in potato fries (chips) (6) and starch-thickened drinks (7) during in-mouth processing. However, most dietary starch is hydrolysed by pancreatic amylase in the gastro-intestinal tract. Thus perhaps it is more useful to consider the broader context of tasting foods in terms of their mastication and breakdown as well as the transport of tastants to taste receptors located at the taste buds. Although the receptors on taste cells within taste buds on the tongue are now well characterized not all the variation in subject to subject perception of bitterness is fully understood. Some preliminary data is presented in this chapter to suggest that salivary proteins facilitate the transport of bitter substances to the taste bud-located taste receptors.

Saliva Composition Saliva is secreted by the major salivary glands including the parotid, submandibular and sublingual as well as the minor glands. The major glands are located remotely from the mouth and therefore require long ducts to deliver the saliva into the oral cavity. In contrast, the minor salivary glands are located within the oral mucosa and have short ducts to the surface. Nearly all salivary secretion is regulated by autonomic nerves (8), as is evidenced by the cessation of secretion when subjects are unconscious. Salivary secretion is described as having two rates, resting (or unstimulated) and a faster stimulated rate. The faster rate is stimulated by taste, chewing and olfactory stimuli being released from food in the mouth. The resting flow rate is dependent on nerve signals from the higher centres of the brain and thus follows a diurnal variation in flow rate and is lowest when we are asleep. Although Pavlov’s work on dogs implied that humans can become conditioned to food cues to salivate, this is not unequivocally supported by other studies (9). Or at least thinking about food cannot induce a sustained secretion above resting flow rate, whereas taste, chewing and smell can. Since salivary secretion is a reflex most salivary secretion is not influenced by the type of stimuli that elicits it, which appears to be true for secretion from a single gland such as the parotid. However some studies have shown there are differences in certain proteins, such as secretory IgA due to differences in 184 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

the flow rate (10). Larger differences in protein concentration between types of stimuli (chewing, taste and smell) have been detected when whole mouth saliva was analyzed, most likely due to differential activation of each salivary gland (9). More detailed analysis using proteomic methods has shown other differences in levels of salivary proteins following stimulation by different tastes (11).


Salivary Proteins Many of the proteins in saliva are highly specialized proteins with unusual amino acid compositions, which has driven their initial naming. Hence the most abundant class proteins are the proline-rich proteins (PRPs). Although only encoded by six genes (2) there are over 30 different isoforms with varying size, overall charge and degree and type (N- or O- linked) of glycosylation. Many of the PRPs have O-linked glycans (12) and some also have N-linked glycans (13), whilst the majority are also sialylated. These glycans are important for their binding and agglutination of bacteria in the mouth (14). A cysteine-rich protein, named cystatin, forms another large group of proteins, whilst histidine-rich histatins are also present in large amounts, and both have anti-microbial activites (15). Another abundant group of proteins are the mucins. In saliva Mucin-5b and Mucin-7 are the two main species whilst Mucin 1 is present on epithelial cells that regularly slough (desqamate) into saliva. The emergence of proteomics has revealed that there are over a thousand proteins in saliva (16), although most are at lower amounts than those just mentioned. Other proteins which are of particular relevance to this chapter are Carbonic anhydrase 6, which reversibly catalyses the conversion of carbonic acid to water and carbon dioxide and is a major buffer of pH within saliva, Statherin, a surface active protein which maintains calcium homeostasis in the mouth, and Albumin as the blood-derived protein. Although most protein in saliva is the product of salivary gland synthesis, a small portion of salivary proteins is derived from the blood circulation. In addition to leakage via gingival crevicular fluid through the periodontium surrounding teeth, there is also a small leakage of albumin within the glands and into saliva (as shown by the presence of albumin in parotid saliva).

Salivary Peptides, Free Amino Acids, and Other Metabolites Due to the high bacterial load in saliva, the breakdown of proteins is probably to be expected but it is not as random as might be anticipated and indeed shows some consistency-suggesting order (17). Furthermore some proteins are cleaved even before they are excreted into the oral cavity (18); that is, the protein precursor is cleaved already in the salivary gland cell, or at the cell membrane to yield two new proteins. This process seems particularly prevalent for PRPs but is also a feature of transport of IgA across the epithelial cell by the polymeric immunoglobulin receptor (19). In addition to the peptides in saliva there are also free amino acids, presumably either derived from the complete breakdown 185 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


of proteins by proteases or released from serum (20). The scientific progress of metabolomics has also contributed to its impact on taste detection (21). A largely under-investigated area, though, is the effect of bacteria and other microflora on taste. At the two extremes are the effect of reduced bacteria following antibiotic use (22) and bacterial overgrowth (halitosis) where there are clear effects on taste. How bacterial biofilms growing on the fissured tongue affects taste is largely unknown. Saliva contains a large number and variety of bacteria, over half of which cannot be cultured (23), which makes studying them problematic.

The Structure of the Tongue The taste buds are mostly located on the tongue in three main areas: the front, sides and back of the tongue. These are associated with visible bumps on the tongue known as the fungiform, foliate and the circumvallate papillae. In addition there are the filiform papillae – these are tough keratinized ridges on the tongue, particularly noticeable in domestic cats, that provide the roughness of the tongue but have no taste buds associated with them. The most obvious papillae are the red dotted fungiform papillae on the tip of the tongue. In animals each of these may have one or more taste buds (24) but this is quite variable between human subjects (25). The taste maps of the tongue often reproduced in textbooks are now largely discounted – there is abundant evidence to show most areas of the tongue are able to detect most tastes (26). By far the most abundant taste buds are located on the posterior part of the tongue around the foliate and circumvallate papillae. These taste buds are located within crypts on the tongue (see Figure 1) that are filled with saliva produced by serous minor glands also known as the von Ebner’s glands. These glands secrete very small amounts of saliva but govern the environment around taste buds within the crypts. These glands are not well studied due to the difficulties of collecting the tiny amounts of saliva but do have some interesting taste-related molecules (27). Lingual lipase is one such product of the von Ebner’s glands. Although it has been suggested that this enzyme may digest fat in the mouth and facilitate its detection, the very small amounts secreted from von Ebner’s glands in the mouth makes it highly unlikely that it can digest fat from foods in time to detect their taste (28). Instead it probably functions to clear the tongue of any deposited fat layer, as happens following the ingestion of an oil (29). Much progress has been made in characterizing the different channels responsible for the detection of the basic tastes by taste bud cells (30). Salt taste is transmitted by sodium and possibly potassium channels located on the apical surface of taste bud cells and signal to afferent nerves via ATP (adenosine triphosphate) molecules whereas sour taste (which are protons) is detected by a separate channel (31). Receptors for bitter tastes (32) and glutamate have also been determined (33). Now that specific receptors have been cloned more studies are examining the confounding factors of taste receptors, such as age (34). The detection of fat by taste cells is still debated. In rodent models a likely receptor was found (CD36), although its functional expression in humans has yet to be 186 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


shown and emerging evidence points towards long chain fatty acids as being the key tastants – whereas CD36 detects the short chain fatty acids (35). Although free fatty acids may be the tastants within fatty foods, they do not seem to evoke a fatty taste but instead induce a bitter or rough (astringent) sensation (35). It is widely assumed that the preference for fatty foods relates to their improved mouthfeel as a result of increased lubrication (36).

Figure 1. Haematoxylin & Eosin stained section of human tongue dorsum showing taste buds in the epithelium and von Ebner’s salivary glands in the deeper dermis layers. Courtesy of Professor Peter Morgan, King’s college London, U.K.

Perception of Bitterness and Its Relation to Salivary Proteins Although most of the 25 or so bitterness receptors have been cloned the perception of bitterness still seems to be complex, implicating the role of other (unknown) factors. PROP (propylthiouracil) is a useful example which has 187 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


previously been linked to the human tatse receptor TAS2R38 (37). More recent studies have used a GWAS (genome wide association study) approach. These studies are impressive for the immense size, with over 4000 participants in one such study which demonstrated that TAS2R38 variations did not explain all the perceived variation in PROP tasting (38). Other GWAS studies have contrasted PROP with caffeine, another well known bitter molecule, to show they act through different receptors (39) and may be associated with some salivary protein polymorphisms probably the same basic proline-rich proteins described previously (40). A commonly described candidate salivary protein is carbonic anhydrase 6, previously known as gustin (41). This was originally implicated in taste as it was deficient in some patients with altered taste (42). This enzyme mostly functions in the mouth to moderate or neutralize acids by catalyzing the addition of protons with bicarbonate ions to form carbon dioxide and water via carbonic acid (43). Although levels of carbonic anhydrase 6 are associated with supertasters of bitter compounds (44) and it has been suggested that carbonic anhydrase 6 acts as a trophic factor for taste buds on fungiform papillae (45), it is unlikely these relationships are causal. The best evidence comes from the carbonic anhydrase 6 knockout mouse (46), which showed no changes in taste bud number on fungiform papillae and only a very slight decrease in liking of bitter substances. However, even this study is not definitive as the multifunctionality of salivary proteins, mentioned earlier, may overcome for a loss of carbonic anhydrase 6 by some other salivary proteins. The association of carbonic anhydrase 6 with taste sensitivities may stem from its variable upregulation by neural signals. Certainly it is well documented that most salivary protein expression and secretion are tightly regulated by neural signals (8). Principally these signals come from sensory nerves in the mouth via the brain stem, as described earlier. It might even be possible that salivary gland cells have bitter receptors and upregulate salivary proteins in response to serum levels of caffeine as shown in vivo in rats (47) and in human immortalized salivary gland cells (48). However, the in vitro study using immortalized salivary gland cells upregulated cystatin in response to caffeine (48). Work from the same groups have shown changes in bitter perception linked to amylase and albumin (49), zinc-alpha2 glycoprotein (50) and calgranulin (11) in addition to carbonic anhydrase 6. Certainly there is a clear role for carbonic anhydrase in the detection of carbon dioxide in fizzy drinks although this identifies four (membrane bound) rather than six (secreted form) as the main candidate (51). Whether a single protein is involved seems unlikely as other proteins such as albumin (52) or even amino acids such as arginine (53) also interact with bitter substances and are both present in saliva. Hence it seems likely that changes in the salivary protein profile can influence tasting of bitterness, but differentiating which protein is important will be difficult. It is most likely that the heterogeneous nature of bitter tastants (54) has resulted in a large selection of taste receptors and thus it is possible that several salivary proteins may exert an influence in transporting the bitter substance to the receptors.

188 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 2. Mean ± SD (n=3, single subject) parotid salivary flows (ml/min) in response to the same solution of caffeine (50 mM in water) given repeatedly with 5 min rests between each 1 min period of tastant (dark green bars with yellow trend line). In a second series (light green bars) the mouth was washed out with water before test 2 (Post washout). The third test (Recovery) was the same as the first series. The post washout flow rate was significantly lower than without a water washout (p< 0.05, student’s t-test) suggesting that the water washout has reduced the tasting of the caffeine solution.

Preliminary Evidence for the Role of Salivary Proteins Previously we used a simple method to show that salivary proteins attached to the mucosa (termed the mucosal pellicle) were also involved in polyphenolinduced astringency (55). The method used was to wash the mouth out with copious quantities of water which removed most of saliva which forms a thin film on all surfaces of the mouth (56). Instead of reducing astringency this procedure increased the apparent astringency of a solution of black tea. Previously the theory of how astringency occurred was via the interaction and subsequent precipitation of salivary proline-rich proteins and histatins by the polyphenols, destroying the lubricating layer of the mouth either by disrupting PRPs attached to the mucosa or by creating nano-particulates (57). These particulates were thought to act like sand creating roughness by their presence. However, several pieces of evidence point to another mechanism. Firstly, in a detailed study we could not find any proline-rich proteins or histatins attached to oral epithelial cells (58). In fact the mucosal pellicle is composed mostly of salivary mucins (Mucin- 5B and Mucin7), Secretory IgA and carbonic anhydrase 6. The second piece of evidence is that polyphenols also interact with mucins (59). Our ideas of how astringency occurs have been modified to include the mucosal pellicle (60) by the use of a 189 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


simple washout experiment. Similar methods have been used in rats to show the role of saliva in taste (61). We therefore used the same technique (water washout) to determine if salivary proteins mediate the perception of bitterness and obtained some consistently variable results. This sounds like an oxymoron but in one subject a water washout between tests of caffeine consistently caused a significant (p

Do Salivary Proteins Play a Role in Tasting Bitter Substances? - ACS

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