Salivary proline-rich protein polymorphisms in Chinese, Malays and Indians in Singapore

Salivary proline-rich protein (PRP) polymorphism, PRH1, PRH2, Ps, Pm (PmF), PmS and Gl, were investigated in three ethnic groups in Singapore: Chinese, Malays and Indians. The phenotype and gene frequencies were presented and comparison with other ethnic groups was made. The As protein, which was recently found in Japanese but not in Caucasians as a new allelic product of the PRH1 locus, was also observed in Chinese and Malays but not in Indians. Another allelic product (Ps4) of Ps protein polymorphism was found in Malays but not in Chinese and Indians. The results indicate the usefulness of salivary PRP polymorphism as markers in population genetic studies.     

Salivary peroxidase (SAPX): Genetic modification and relationship to the proline-rich (Pr) and acidic (Pa) proteins

There is genetic polymorphism of the peroxidase of human saliva, but not of leukocytes. The phenotypes are determined by autosomal inheritance, the phenotype of fast mobility (SAPX 1) being determined by homozygosity for a recessive gene (SAPX ¹) and the phenotypes of slow mobility (SAPX 2 and SAPX 3) being determined by two different genes, SAPX ² and SAPX ³, with completely dominant expression at the same locus. The phenotypes are modified by varying degrees of endogenous proteolysis. The SAPX 2 and SAPX 3 types appear to be genetically controlled modifications of SAPX 1 rather than different primary gene products, because of their completely dominant inheritance, their larger molecular size compared to SAPX 1, and their dissociation with 2-mercaptoethanol to give SAPX 1. The acidic protein type Pa 1 is always found in association with SAPX 2, and an uncommon variant type Pa 2 is associated with SAPX 3. The most likely hypothesis is that the genes Pa ¹ and Pa ² produce products which modify the SAPX 1 type. When the Pa type is Pa 0, the SAPX phenotype is SAPX 1. Since 2-mercaptoethanol can dissociate the Pa 1 protein into a probable monomeric form, and can dissociate SAPX 2 and SAPX 3 to give SAPX 1, it is probable that Pa 1 and Pa 2 monomers complex with SAPX 1 through disulfide bonds to give SAPX 2 or SAPX 3 types. The frequencies of the genes determining the SAPX types are the same as those for Pa: SAPX ¹ and Pa ⁰=0.787, SAPX ² and Pa ¹=0.208, SAPX ³ and Pa ²=0.005.This study was supported by a grant from the National Institutes of Dental Research (2-RO1-DE-03658-11).     

Finding new posttranslational modifications in salivary proline-rich proteins

Proline-rich proteins (PRPs) are the most complex family of salivary peptides with distinct isoforms and PTMs. Up to date, only the serine phosphorylation at positions 8, 17, and 22 have been experimentally observed on acidic PRP (aPRPs), and at position 8 on basic PRP1 and 2. The presence of a glucoronyl group at Ser17 was also noticed on aPRP. The main goal of this study was to identify new PTMs and distinct isoforms of salivary PRPs using LC-MALDI-TOF/TOF. Through the salivary peptidome characterization of 20 different subjects from Control, Diabetic, and Head and Neck Cancer groups, it was possible to identify the following species: (i) N-glycosylation sites: two in basic proline-rich protein 2 (bPRP2), one in bPRP3 and one in bPRP4; (ii) O-glycosylation sites: two in bPRP2 and one in aPRP; (iii) other terminal monosaccharide sites: six in bPRP1, two in bPRP2 and two in bPRP3; (iv) other modifications such as N-terminal pyro-Glu (two in bPRP1, six in bPRP2, eight in bPRP3 and nine in bPRP4); (v) phosphorylation in serine, three in bPRP1, one in bPRP2, one in bPRP3 and one in aPRP1; (vi) bPRP1 (allele S, allele M and variant CP5) and bPRP4 (allele M). In summary, salivary peptidome data analysis allowed the identification of 45 new PRP-modified residues, mainly due to glycosylation, phosphorylation and conversion of Gln to pyro-Glu. Moreover, comparing all subject groups, it was noticed a predominance of N-acetyl hexosamine modification on bPRPs in the Head and Neck Cancer patients.     

Secondary structure prediction of human salivary proline-rich proteins

Conformations associated with secondary structure in human salivary proline-rich proteins A (PRPA), C (PRPC), P-D and P-E were predicted by analysis of their respective hydrophobicity profiles by computer programming. Structurally, PRPA and PRPC would present a globular head and a tail that consists of type 3(10) polyproline helices. P-D and P-E would be fibrilar molecules with helical zones of the polyproline 3(10) type. Alternatively for PRPA and PRPC, the head and tail would form one globular domain with the tail folding upon itself at places where random coils occur.     

New proline-rich proteins in isolated insect Z-discs.

1. Protoveratrine A increased the release of gamma-amino[3H]butyrate from small slices of rat cerebral cortex. This effect increased with increasing protoveratrine concentration, reaching a maximum at 100 microM. 2. Removal of Ca2+ from the superfusing medium did not change the increase in release due to 10 microM-protoveratrine; however, the Ca2+ antagonists, compound D-600, La3+, Mn2+, Mg2+ and also high Ca2+ concentration inhibited the effect of the alkaloid, as did procaine. 3. Protoveratrine A increased the uptake of 22Na+ into the slices with a similar dose-response curve to that found for gamma-aminobutyrate release. For the most part, the substances that inhibited protoveratrine-stimulated gamma-aminobutyrate release also inhibited 22Na+ uptake, although the correlation was not perfect. 4. Although extracellular Ca2+ is not required for protoveratrine-induced gamma-aminobutyrate release, an increase in Na+ influx that is susceptible to inhibition by some Ca2+ antagonists does appear to be associated with this phenomenon. However, the possibility remains that changes in the free intracellular Ca2+ concentration may be important for transmitter release induced by depolarizing veratrum alkaloids.     

Differential expression of proline-rich proteins in rabbit salivary glands.

Salivary glands synthesize and secrete an unusual family of proline-rich proteins (PRPs) that can be broadly divided into acidic and basic PRPs. We studied the tissue-specific expression of these proteins in rabbits, using antibodies to rabbit acidic and basic PRPs as well as antibodies and cDNA probes to human PRPs. By immunoblotting, in vitro translation, and Northern blotting, basic PRPs could be readily detected in the parotid gland but were absent in other salivary glands. In contrast, synthesis in vitro of acidic PRPs was detected in parotid, sublingual, and submandibular glands. Ultrastructural localization with immunogold showed heavy labeling with antibodies to acidic PRPs of secretory granules of parotid acinar cells and sublingual serous demilune cells. Less intense labeling occurred in the seromucous acinar cells of the submandibular gland. With antibodies to basic PRPs, the labeling of the parotid gland was similar to that observed with antibodies to acidic PRPs, but there was only weak labeling of granules of a few sublingual demilune cells, and no labeling of the submandibular gland. These results demonstrate a variable pattern of distribution of acidic and basic PRPs in rabbit salivary glands. These animals are therefore well suited for study of differential tissue expression of PRPs.     

Determination of the post-translational modifications of salivary acidic proline-rich proteins

Human salivary acidic proline-rich proteins were analyzed by electrospray-ion trap mass spectrometry and by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. All acidic-PRP isoforms share a common N-terminal region, which contains a pyroglutamic acid residue at the N-terminus, and two phosphorylation sites on Ser 8 and 22. At the same time, HPLC-MS spectra revealed isoforms of PRP-1 and PRP-3 having a different number of phosphoserine residues, namely, a mono-phosphorylated form of PRP-1 and PRP-3 and a tri-phosphorylated form of PRP-1. The analysis of the masses of tryptic digests suggested that the third phosphate residue should be located on Ser 17. Another protein with a mass of 30,923 amu was detected along the HPLC pattern and MS data of its tryptic digest suggested that it corresponds to the dimer of Pa, the isoform of PRP-1 with a substitution Arg-Cys at 103 position. Finally, structural identification is pending for another post-translational modification of acidic-PRP that provides an increase of 111-114 amu.     

Identification of the Pm and PmS human parotid salivary proteins as basic proline-rich proteins

Amino acid composition and electrophoretic mobility suggest that two polymorphic proteins in human parotid saliva, Pm and PmS, are basic proline-rich proteins. Comparison of two basic proline-rich proteins previously isolated by D. L. Kauffman and P. J. Keller (1979) [Arch. Oral. Biol. 24: 249], IB-6 and IB-9, with PmS and Pm demonstrated corresponding electrophoretic mobilities on cationic polyacrylamide slab gels. Further, the amino acid compositions of IB-9 and Pm were found to be similar. Although differences in amino acid composition and carbohydrate content were noted, such differences could be accounted for, suggesting that IB-9 and Pm are identical.     

Salivary gland proteins alterations in the diabetic milieu

Journal of Molecular Histology - Salivary glands are considered the chief exocrine glands of the mouth and physiologically contribute to the maintenance of the homeostasis of the oral cavity. They...     

Repetitive proline-rich proteins in the extracellular matrix of the plant cell

Several classes of hydroxyproline-rich proteins have been found in the cell walls of plants. Monomeric forms of the proteins can be solubilized from the walls, but the majority of the proteins are insolubilized by as yet unidentified crosslinks. The proteins have repeated sequences and are often rich in basic amino acids. The repeat proline-rich region may be serving to generate an elongated rod-like structure while the regularly spaced lysines probably interact with the acidic pectins. Several of the genes coding for these proteins have been isolated, and their expression has been found in some cases to be tissue specific and in others inducible by hormones and various types of stress.     

Different isoforms and post-translational modifications of human salivary acidic proline-rich proteins

The human salivary acidic proline-rich proteins (aPRPs) complex was investigated by different chromatographic and mass spectrometric approaches and the main aPRPs, namely PRP-1, PRP-2 and PIF-s (15,515 amu), Db-s (17,632 amu) and Pa (15,462 amu) proteins, were detected. All these isoforms are phosphorylated at Ser-8 and Ser-22 and have a pyroglutamic moiety at the N-terminus. Apart from Pa, all the other aPRPs undergo a proteolytic cleavage at Arg-106 residue (Arg-127 in Db-s protein), that generates the small PC peptide (4371 amu) and PRP-3, PRP-4, PIF-f (11,162 amu) and Db-f (13,280 amu) proteins, all of which were detected. With regard to the Pa protein, the main form detected was the dimeric derivative (Pa 2-mer, 30,922 amu) originated by a disulfide bond involving Cys-103 residue. Besides these known isoforms, several previously undetected aPRP derivatives were found (in minor amounts): (i) the triphosphorylated derivatives of PRP-1/PRP-2/PIF-s and Db-s, showing the additional phosphate group at Ser-17; (ii) the mono-phosphorylated forms at either Ser-22 or Ser-8 of PRP-1/PRP-2/PIF-s, PRP-3/PRP-4/PIF-f, Db-s and Db-f; (iii) a nonphosphorylated form of PRP-3/PRP-4/PIF-f; (iv) the triphosphorylated and diphosphorylated forms of Pa 2-mer. Moreover, minor quantities of PRP-3/PRP-4/PIF-f lacking the C-terminal Arg (11,006 amu), and of Pa 2-mer lacking the C-terminal Gln (30,793 amu) were found. By this approach the different phenotypes of PRH1 locus in 59 different subjects were characterized.     

Purification of proline-rich proteins from parotid glands of isoproterenol-treated rats.

Prolonged isoproterenol treatment of rats is known to cause hypertrophy and hyperplasia of the parotid glands. Our results show that a dramatic increase in the synthesis or accumulation in the parotid glands of a series of proteins rich in proline also occurs with isoproterenol treatment. After 10 days of treatment (5 mg of isoproterenol/day) these proline-rich proteins (PRPs) comprise more than 50% of the total soluble proteins in parotid gland homogenates. The PRPs are rapidly labeled in vivo by a single intraperitoneal injection of [3H]proline with maximum incorporation occurring at about 3. More than 90% of the [3h]proline found in parotid gland homogenates is incorporated into PRPs with less than 1% of the radioactivity in alpha-amylase. Tritium incorporated into PRPs was isolated as [3H]proline after acid hydrolysis. One acidic and six basic 3H-labeled PRPs were isolated from the 100,000 x g supernatant fraction of parotid gland homogenates by Sephadex G-100 and ion exchange chromatography. The six basic proteins accounted for about 90% of the total PRPs isolated.     

Genetic polymorphism of human salivary proline-rich proteins: Further genetic analysis

Electrophoresis of concentrated parotid saliva on slab polyacrylamide gels negatively stained with 3,3-dimethoxybenzidine and hydrogen peroxide (DMB stain) showed nine phenotypes among the proline-rich proteins. These phenotypes are the expression of four autosomal codominant alleles. Gene frequencies are, for Caucasians, Pr ¹=0.640, Pr ¹=0.005, Pr ²=0.080, Pr ²=0.275; for Negroes, Pr ¹=0.700, Pr ¹=0.050, Pr ²=0.080, Pr ²=0.170; for Chinese, Pr ¹=0.770, Pr ¹=0, Pr ²=0, Pr ²=0.230. The presence or absence of another pair of proteins giving the same negative staining is inherited as an autosomal dominant trait (Db). Homozygous Db + + and heterozygous Db + – individuals could not be distinguished. The genetic determinant (Db) for this pair of proteins is either closely linked to or part of the Pr locus. Gene frequencies are, for Caucasians, Db ⁺=0.12, Db ^–=0.88; for Negroes, Db ⁺=0.56, Db ^–=0.44; for Chinese, Db ⁺=0.07, Db ^–=0.93.This study was supported by a grant from the National Institutes of Dental Research (9-R01-DE-03685-08) and in part by grant GM 15422 from the National Institutes of Health.     

Characterization of cDNAs for stylar transmitting tissue-specific proline-rich proteins in tobacco

The pistil of flowers is a specialized organ which contains the female gametophytes and provides the structures necessary for pollination and fertilization. Pollen deposited on the stigmatic surface of a compatible plant germinates a pollen tube which penetrates the stigmatic papillae and grows intercellularly through the style towards the ovules in the ovary. Pollen tube growth is largely restricted to the transmitting tissue in the style. Therefore the stylar transmitting tissue is extremely important for the migration of the pollen cell towards the ovary. We have isolated two related cDNAs, transmitting tissue-specific (TTS)-1 and TTS-2, derived from two proline-rich protein (PRP)-encoding mRNAs that accumulate specifically in the transmitting tissue of tobacco. The deduced PRP sequences share similarities with proline-rich cell wall glycoproteins found in a variety of plants. TTS-1 and TTS-2 mRNAs are induced in very young floral buds, accumulate most abundantly during the later stages of flower development when style elongation is the most rapid, and remain at relatively high levels at anthesis. These mRNAs become undetectable in maturing green fruits. In situ hybridization shows that TTS-1 and TTS-2 mRNA accumulation is restricted to the transmitting tissue of the style. The possible roles that these transmitting tissue-specific PRPs may play in maintaining the structural integrity of the style or in the function of this organ is discussed.     

Salivary proteins as predictors and controls for oral health

We will provide a translational view of using the recent technological advances in dental research for predicting, monitoring, and preventing the development of oral diseases by investigating the diagnostic and therapeutic role of salivary proteins. New analytical state-of-the-art technologies such as mass spectrometry and atomic force microscopy have revolutionized the field of oral biology. These novel technologies open avenues for a comprehensive characterization of the salivary proteins followed by the evaluation of the physiological functions which could make possible in a near future the development of a new series of synthetic protein for therapeutic propose able to prevent global oral diseases such as periodontal disease and dental caries, the two most prevalent oral diseases in the World.     

Immunological comparison of proline-rich proteins from human and primate parotid secretion.

Antisera raised in response to proline-rich proteins purified from parotid secretions of man and the primate Macaca fascicularis were employed to investigate the interrelationships of these proteins by immunodiffusion, immunoelectrophoresis and the combined use of disc gel acrylamide electrophoresis with radial immunodiffusion. The major human proline-rich proteins, PRP I, PRP II, PRP III and PRP IV as well as several minor proline-rich proteins cross-react with antiserum to PRP I or PRP III. Similarly primate parotid saliva contains several components cross-reacting with antiserum directed against a purified primate proline-rich protein, MPRP. Antiserum to PRP I or PRP III cross-reacted with MPRP and primate parotid saliva protein, whereas antiserum to MPRP cross-reacted only with human parotid saliva protein and not with the isolated human proline-rich proteins. The immunological relationships of these salivary proline-rich proteins within and between species suggest their origin from a common precursor molecule.     

Electron microscopic immunocytochemical localization of proline-rich proteins in normal mouse parotid salivary glands

Summary Rabbit polyclonal antibodies against isoproterenol-induced mouse proline-rich proteins (PRPs) were used to localize PRPs in the parotid salivary glands of normal adult BALB/c mice. The antibodies recognized both acidic-type and basic-type PRPs. Immunoblotting experiments revealed that the glands contained an acidic-type and a basic-type PRP. Parotid gland tissue was fixed with Karnosky's fixative and embedded in Lowicryl resin at low temperature. PRPs were localized at the electron microscope level using an indirect post-embedding staining technique with protein A-gold. The secretion granules of the acinar cells were strongly labelled. Pre-absorption of the antibody with purified acidic-type and basic-type PRPs indicated that the basic-type PRP is mainly located at the periphery of the granules but that the acidic-type PRP is more evenly distributed within the granules. Pre-absorption of the antibody with -amylase did not affect the staining pattern, suggesting minimal cross-reactivity. PRPs were also detected within the rough endoplasmic reticulum and the Golgi apparatus of acinar cells, within the granules of the proacinar cells and in the lumena of the ducts, but not within the intercalated or striated duct cell granules.     

Localization of repetitive proline-rich proteins in the extracellular matrix of pea root nodules

Summary Early responses of legume roots toRhizobium inoculation include new cell wall synthesis and induction of some putative wall protein genes. Although the predicted amino acid sequences of several early nodulins indicate that they encode proline-rich proteins (PRPs), the proteins have been neither isolated nor has their presence been demonstrated in cell walls. We have used polyclonal antibodies against PRP2 from soybean to identify and localize proline-rich proteins in pea nodules. On immunoblots, several PRPs were detected, ranging from less than 20 kDa to 110 kDa. Immunocytochemistry revealed that tissues of the vascular cylinder contained abundant PRPs, particularly in the secondary cell walls of xylem elements and phloem fibers. PRPs were also found within the primary wall of the nodule endodermis and within Casparian strips of the vascular endodermis. Of symbiotic importance, PRPs were a prominent component of the infection thread matrix in newly infected root cells and in nodules. PRPs were also secreted by cells in the uninfected nodule parenchyma, where they were found occluding intercellular spaces outside the middle lamella. Despite structural conservation among members of this class of cell wall proteins, PRPs were targeted to distinct layers of the extracellular matrix dependent upon cell type, and may thus play separate roles in the biology of plant cells. The putative functions and the potential for interactions between PRPs and other wall polymers are discussed.Abbreviations DTT dithiothreitol - EDTA ethylenediamine tetraacetate - GRP glycine-rich protein - PCR polymerase chain reaction - PGA polygalacturonic acid - PMSF phenylmethylsulfonyl fluoride - PRP proline-rich protein - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - Tris tris(hydroxylmethyl) aminomethane - Tween 20 polyoxyethylene sorbitan monolaurate Dedicated to the memory of Professor John G. Torrey