Anders Bennick

Interaction of Plant Polyphenols with Salivary Proteins

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.

Interaction of tannin with human salivary proline-rich proteins.

Tannins are polyphenolic compounds, widely distributed in plant-based foods, which have harmful effects on animals including humans. Salivary proline-rich proteins (PRPs) may act as a defence against tannins by forming complexes with them and thereby preventing their interaction with other biological compounds and absorption from the intestinal canal. The aim here was to compare the ability of members of the family of human PRPs to form insoluble complexes with tannin and to assess the stability of such complexes under conditions similar to those in the alimentary tract. Basic PRPs (BPRPs), which have no other known biological functions, were very effective in forming insoluble complexes with both condensed tannin and tannic acid. Practically no tannin bound to acidic PRPs (APRPs) and glycosylated PRPs (GPRPs), suggesting that tannin in the diet would not affect their biological activities. There were only small differences in the tannin-precipitating ability of various BPRPs of different sizes or sequences, indicating that, although there is considerable phenotypic variation of PRPs, it is not likely to cause marked individual variation in tannin-binding ability. Tryptic digestion of an APRP led to a marked increase in tannin binding to the resulting proline-rich peptides, supporting observations in other studies that there may be an interaction between the proline-poor N-terminal and the proline-rich C-terminal regions in native APRPs, which inhibits the biological activities of the proteins. Deglycosylation of a GPRP also led to a dramatic increase in tannin-binding ability, showing that the carbohydrate side-chains prevent binding of tannin. Most of the condensed tannin-PRP complexes remained insoluble under conditions similar to those in the stomach and small intestine, supporting the proposal that PRPs act as a defence against tannin.

The role of human salivary acidic proline-rich proteins in the formation of acquired dental pellicle in vivo and their fate after adsorption to the human enamel surface

The pellicles formed on fragments of human dental enamel worn on a palatal appliance for 1 min to 24 h were removed by acid extraction and the total amounts of protein and of acidic proline-rich proteins were determined. The percentage of total extracted protein constituted by the proline-rich proteins increased during the first hour of formation to about 37 per cent. Little difference was seen in total proline-rich protein between pellicles formed in a 1 and a 24-h period, but there was a gradual degradation of the proteins. There was no preferential retention of the N- or C-terminal parts of the proteins. Extracts of old acquired dental pellicle contained less than 0.1 per cent proline-rich proteins and pellicles more than 24 h old showed degradation of the adsorbed proline-rich proteins; there is no indication that the N-terminal part which contains the known biological activities was retained to a greater extent than the C-terminal part.

Trafficking and Postsecretory Events Responsible for the Formation of Secreted Human Salivary Peptides A Proteomics Approach

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and “S type” Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.

Collagenolytic activity in oral tissues

Abstract Live and frozen-thawed explants of guinea pig oral tissue and human gingival tissue were assayed for collagenolytic activity in tissue cultures containing a nutrient medium in a gel of reconstituted collagen fibrils. Live gingival, periodontal and palatal tissues, from young, but not adult, guinea pigs, could all break down the collagen gel; however, collagenolysis was not seen with frozen-thawed explants. This is probably due to extensive tissue remodelling in young animals. Healthy, adult, human tissue did not cause any collagen breakdown but a varying amount was seen with inflamed gingival tissue. While only live, chronically inflamed tissue had collagenolytic ability both live and frozen-thawed explants from patients with acute necrotic gingivitis could break down the collagen gel. In the latter type of inflammation it appears that collagenolytic activity was already present when the tissue was removed from the mouth, whereas in chronic inflammations, tissue culturing was necessary to enhance the amount of collagenolytic factor. An association between collagenolytic ability and the presence of connective tissue is possible, but the possibility that epithelium could be important is not ruled out.

Encoding of human basic and glycosylated proline-rich proteins by the PRB gene complex and proteolytic processing of their precursor proteins

Proline-rich proteins (PRPs) constitute a family of about 20 members in human saliva that are encoded by six genes. Assignment of genomic DNA coding regions is complicated because of the occurrence of many alleles and the great similarity of amino acid sequences of PRPs. To overcome these problems, the nucleotide sequences of the genes encoding basic and glycosylated PRPs from one person were determined and then aligned with her previously determined protein sequences. This, together with additional protein data, has also resolved various discrepancies between corresponding protein and DNA sequences. For the first time in one person it is now possible to account for all the regions in the PRB genes encoding basic and glycosylated PRPs, and the primary structures of all secreted basic and glycosylated PRPs have been determined. Each gene encodes a precursor protein that subsequently undergoes proteolytic cleavage, thereby giving rise to the secreted proteins. The results have allowed identification of all the proteolytic cleavage sites in the precursor proteins, which all conform to a consensus cleavage site for furin. To evaluate if furin is responsible for the precursor protein cleavages, a recombinant precursor protein was synthesized by in vitro transcription translation of a PRB1 allele. The protein was shown to be correctly cleaved by furin, giving rise to the expected secreted proteins.

Isolation and characterization of six proteins from rabbit parotid saliva belonging to a unique family of proline-rich proteins

Proline-rich proteins are major components of salivary secretion from humans non-human primates, rats, hamsters and rabbits. They are also synthesized in mice in response to chronic stimulation by beta agonists. This study to provide an understanding of the structural and genetic relationships within these families of proteins to determine the possible function of the proline-rich proteins. Rabbit parotid saliva was collected and proline-rich proteins were affinity purified using goat antibodies to human proline-rich proteins. Purification was achieved by repeated cation exchange chromatography on a Mono S column a Fast Protein Liquid Chromatography system. Six basic proline-rich proteins were purified. The apparent molecular weights were between 75,000 and 125,000, based on their mobilities in sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Glycine, glutamine (and glutamate) and proline accounted for 79-87% of total amino acids in all proteins, but proline was present in smaller amounts (17-21%) than in proline-rich proteins from other species. All proteins were glycosylated but not phosphorylated. Circular dichroism of two proline-rich proteins, MS7A and MS5B, indicated the absence of secondary structure. The N-terminal sequences of three proteins electro-eluted after preparative gel electrophoresis were determined. A high degree of similarity was found in various regions of mouse, rat, monkey and human proline-rich proteins. Rabbits thus synthesize constitutively a family of proteins that are immununologically and structurally related to proline-rich proteins other species.

Demonstration of proline-rich proteins in rabbit parotid saliva and partial characterization of some of the proteins

Rabbit parotid saliva was collected by cannulation of the secretory duct in anaesthetized animals. Proteins which cross-react with antibodies to human acidic proline-rich proteins were demonstrated in the secretion and fractionated by chromatography on an immunosorbent and CM32-cellulose. At least 5 proline-rich proteins were identified with molecular weights ranging from 19,000 to 61,000 as determined by sodium dodecylsulphate acrylamide gel electrophoresis. The major immunoreactive components were basic proteins with similar size and charge properties. In two of the proteins, proline, glycine and glutamic acid or glutamine accounted for 84 or 99 per cent of all residues. In contrast to proline-rich proteins from other species, proline constituted only 13 or 17 per cent of total amino acids.

Purification and characterization of subunits of a high molecular weight human salivary mucin

A high molecular-weight mucin was purified from human submandibular-sublingual saliva. The purity of the mucin preparation was demonstrated by the absence of other salivary proteins, by antibody reactivity and by gel electrophoresis. After reduction with mercaptoethanol a putative link component with approximate Mr 150,000 and a glycoprotein component of higher Mr could be detected by gel electrophoresis. These subunits were subsequently purified and they showed distinct differences in their amino acid compositions, demonstrating that the mucin consisted of two different subunits. The link had a number of similarities with the link component of intestinal mucin and a parotid agglutinin and has previously been shown to cross-react with antiserum to link component from intestinal mucin. Salivary and intestinal mucins may therefore have similar subunit structure.

Purification and characterization of a rabbit salivary protein, a potent inhibitor of crystal growth of calcium phosphate salts

Human saliva is supersaturated with respect to basic calcium phosphate salts but is stabilized by specific macromolecules that inhibit calcium phosphate precipitation. One of the families of inhibitory proteins in human and monkey saliva is the acidic proline-rich proteins. The purpose of this study was to isolate and characterize inhibitors of calcium phosphate precipitation from rabbit parotid saliva. Saliva was fractionated by immunoaffinity chromatography and anion exchange chromatography. Individual fractions were assayed for their ability to inhibit calcium phosphate crystal growth and the fraction associated with the inhibition was purified by repeated anion exchange chromatography, preparative gel electrophoresis and electroelution. A major (APRP) and two minor proteins (AM1, AM2) that were inhibitory were purified. APRP is an acidic proline-rich phospho-glycoprotein and a very potent inhibitor of secondary crystal growth of calcium phosphate as it was active at a concentration of 2 x 10(-8) M in a standard assay. The N-terminal sequence of one APRP was EYENLDGSLAATQNDDD?Q and a clostripain fragment of APRP had the following N-terminal sequence PQHRPPRPGGH-????SPPP?GN???PPP. Although the N-terminal segment of APRP does not resemble that of proline-rich proteins, alignment of the clostripain fragment with the repeat region of such proteins from rat, mouse, monkey and man revealed a high degree of similarity, indicating a structural relationship with the proline-rich protein family.