Tannins are polyphenols that occur widespread in plant-based food. They are considered to be part of the plant defense system against environmental stressors. Tannins have a number of effects on animals, including growth-rate depression and inhibition of digestive enzymes. Tannins also have an effect on humans: They are, for example, the cause of byssinosis, a condition that is due to exposure to airborne tannin. Their biological effect is related to the great efficiency by which tannins precipitate proteins, an interaction that occurs by hydrophobic forces and hydrogen bonding. Two groups of salivary proteins, proline-rich proteins and histatins, are highly effective precipitators of tannin, and there is evidence that at least proline-rich proteins act as a first line of defense against tannins, perhaps by precipitating tannins in food and preventing their absorption from the alimentary canal. Proline plays an important role in the interaction of proline-rich proteins with tannins. In contrast, it is primarily basic residues that are responsible for the binding of histatins to tannin. The high concentration of tannin-binding proteins in human saliva may be related to the fruit and vegetable diet of human ancestors.

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Saliva contains a complex mixture of proteins and peptides as well as fragments derived from these molecules. By RP 1 -HPLC-ESI-MS analysis of the acidic soluble fraction of human whole saliva we have identified in the chromatographic pattern more than 120 different proteins and naturally occurring peptides (1-6). Their characterization was performed by a variety of mass spectrometric techniques coupled with different enzymatic treatments and amino acid sequencing. The proteins and naturally occurring peptides belong to families of well characterized salivary proteins including Histatins, Statherin, acidic and basic proline-rich proteins (aPRP and bPRP), Cystatins, and Defensins (1-6). Two-dimensional gel electrophoresis has also been used by other researchers for analysis of salivary proteins and peptides, but this technique is not well suited for identification of small peptides as illustrated by the difficulty in identifying Histatins and the majority of bPRPs and bPRP fragments (7-9). However, knowledge of salivary proteins and peptides as well as their naturally occurFrom the ‡Dipartimento di Scienze Applicate ai Biosistemi, Università di Cagliari,

Proline-rich proteins are major components of parotid and submandibular saliva in humans as well as other animals. They can be divided into acidic, basic and glycosylated proteins. The primary structure of the acidic proline-rich proteins is unique and shows that the proteins do not belong to any known family of proteins. The proline-rich proteins are apparently synthesized the acinar cells of the salivary glands and their phenotypic expression is under complex genetic control. The acidic proline-rich proteins will bind calcium with a strength which indicates that they may be important in maintaining the concentration of ionic calcium in saliva. Moreover they can inhibit formation of hydroxyapatite, whereby growth of hydroxyapatite crystals on the tooth surface in vivo may be avoided. Both of these activities as well as the binding site for hydroxyapatite are located in the N-terminal proline-poor part of the protein. Little is known about the functions of the glycosylated and basic proline-rich proteins.

Dietary tannins are polyphenols that are effectively precipitated by salivary histatins (Hsts), a novel family of tannin binding proteins. Epigallocatechin gallate (EGCG), a flavan-3-ol ester related to condensed tannins (polymerized products of flavan-3-ols), and pentagalloyl glucose (PGG), a hydrolyzable tannin, were used to evaluate the molecular nature of Hst-polyphenol interaction. NMR demonstrated that Hst5, a representative Hst, bound to EGCG in a hydrophobic manner via basic and aromatic residues. In contrast, proline plays a dominant role in polyphenol binding to other tannin precipitating proteins. The role of basic and aromatic amino acids in EGCG binding was investigated using a series of modified Hsts in each of which one type of amino acid was substituted by Ala. EGCG bound to all modified Hsts, but the binding was diminished. Optimal EGCG binding also depended on the primary structure, as a polypeptide with randomised Hst5 sequence showed significantly diminished interaction with EGCG. Soluble EGCG/Hst5 complexes containing up to seven molecules of EGCG per mol of Hst5 had a 1-mM dissociation constant. In contrast to EGCG, PGG formed small soluble complexes with Hst5 consisting of only one molecule each of PGG and Hst5, as demonstrated by analytical ultracentrifugation. These complexes became insoluble upon binding of additional molecules of PGG. Diminished PGG binding was seen to a peptide with a Hst5 randomized sequence showing the importance of the primary structure. Hsts may serve to form insoluble complexes with tannins thereby preventing their absorption from the intestines and potentially harmful biological effects. In contrast the much weaker interaction with EGCG may allow its uptake into the organism and exploitation of its antioxidant effect.Keywords: histatin; tannin; saliva; epigallocatechin gallate; pentagalloyl glucose.Tannins are plant derived polyphenolic compounds that are commonly found in foods such as grains, legumes, fruits and beverages [1,2]. Based on their chemical structure, tannins are divided into hydrolyzable and condensed tannins [3]. Hydrolyzable tannins, also known as tannic acids, consist of a polyhydric alcohol such as glucose to which molecules of gallic or hexahydroxydiphenic acid are linked by ester bonds [3]. Condensed tannins are structurally more complex, being polymers of the flavonoids flavan-3-ols [3]. A characteristic property of tannins is their ability to precipitate proteins from aqueous solution. Many studies have demonstrated the detrimental effects of tannins in animals including decreased digestibility of dietary protein and decreased utilization of mineral in food [4,5]. Condensed tannin is also considered to be a causative agent of acute pulmonary inflammation in cotton-mill and grainelevator workers [6,7]. In addition, tannic acid has been shown to be hepatotoxic [8], and several fatalities in humans have been shown to result from the absorption of tannic acid incorporated in barium enemas used in colonic radiological examinations [9]. On the o...

Tannins have a number of detrimental biological effects and these include interference with normal growth and metabolism if they are present in the feed of various animals. Proline-rich proteins (PRPs) in saliva have been shown to provide protection against tannin, but little is known about the mechanism of protection and interaction of other salivary proteins with tannin. To identify tannin-binding human salivary proteins, parotid and submandibular/sublingual saliva samples were adsorbed with tannin. PRPs, and in particular a group of low-M(r) proteins, were readily precipitated by tannin. The low-M(r) proteins were purified from parotid saliva and demonstrated to be histatins, a family of well-characterized histidine-rich salivary proteins. The ability of synthetic histatin 5, as well as an acidic PRP (PRP-1) and gelatin to precipitate quebracho condensed tannin and tannic acid was determined. At pH 7.4 histatin 5 was the most effective precipitant of both condensed tannin and tannic acid and it also precipitated the largest amount of condensed tannin at pH 3.0, but the smallest amount of tannic acid at that pH. In contrast PRP-1 showed a greater ability to precipitate both condensed tannin and tannic acid at pH 3.0 than at pH 7.4. Under most circumstances histatin 5 was therefore more effective in precipitating tannins than proteins with high proline content which generally have been recognized as strong precipitants of tannin. Pre-incubation of tannic acid with alpha-amylase inhibited the enzyme, but addition of histatin 5 or the acidic PRP PIF-s protected amylase from inhibition by tannin. Similarly salivary proteins may protect other biological activities in the digestive tract from inhibition by dietary tannin.

Human glandular salivary secretions contain several acidic proline-rich phosphoproteins (PRPs). These proteins have important biological functions related to providing a protective environment for the teeth, and appear to possess other activities associated with modulation of adhesion of bacteria to oral surfaces. These functions and activities depend on the primary structures of the PRPs. Previously determined amino acid sequences of two 150-residue molecules, PRP-1 and PRP-2, and two related 106-residue proteins, PRP-3 and PRP-4, indicated that residue 4 was Asn in PRP-1 and PRP-3, and Asp in PRP-2 and PRP-4, and position 50 was Asn in all four proteins. Recent data from cDNA sequence studies and further structural studies, however, showed that the previously proposed sequences cannot be completely correct. The present work has shown that the protein previously designated as PRP-1 actually consisted of two positional isomers, PIF-s, which has Asn and Asp at positions 4 and 50 respectively, and authentic PRP-1, which has the reverse arrangement. The same isomerism is present in the smaller proteins, PIF-f and PRP-3. Since the isomeric pairs have identical compositions and charges, their presence was not previously detected. Also, by using a more highly purified preparation, it has been found that position 50 in PRP-2 and PRP-4 is Asp, rather than Asn previously reported. These new findings for the six PRPs define their complete primary structures, which are now consistent with those proposed for PRP-1 and PIF-s from cDNA data, and are also consistent with the chromatographic and electrophoretic behaviours of the six PRPs and their derived peptides. These corrected structures are important for understanding the biological functions and activities of these unusual proteins.

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