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.     

Proteomic analysis of salivary acidic proline-rich proteins in human preterm and at-term newborns

A 1 year follow-up investigation of salivary acidic proline-rich proteins (aPRPs) in preterm and at-term newborns using HPLC-ESI-IT-MS showed that (i) this class of proteins is constitutive rather than inducible, as it is still found in the oral cavity of preterm newborns from 180 days of postconception age (PCA); (ii) the expression of PRH-2 locus anticipates that of PRH-1, since Db isoforms are expressed some months after the PRP-1 and PRP-2 isoforms. The evaluation of the relative abundances of the different aPRPs isoforms and derivatives (differently phosphorylated and cleaved) as a function of PCA showed that (iii) the proteolytic enzymes generating truncated isoforms are also constitutive because they are fully active since 180 days of PCA; (iv) the kinase involved in aPRP phosphorylation is not fully mature in preterm newborns, but its activity increases with PCA, synchronizing with that of at-term newborns and reaching the adult levels at about 500-600 days of PCA, in concomitance with the beginning of deciduous dentition.     

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.     

The nature of the hydroxyapatite-binding site in salivary acidic proline-rich proteins.

Protein A and C, which are major components of the acidic proline-rich proteins in human saliva, were digested, before or after adsorption to hydroxyapatite, with alkaline phosphatase, trypsin, thermolysin and a proteinase preparation from salivary sediment. The results demonstrate that the binding site is located in the proline-poor N-terminal part of the protein, possibly between residues 3 and 25. Phosphoserine is necessary for maximal adsorption of the proteins to hydroxyapatite. When proteins A and C are adsorbed to hydroxyapatite before proteolytic digestion there is a protection of some of the susceptible bonds in the N-terminal part of the proteins and a gradual removal of the proline-rich C-terminal part. Thermolysin can cleave susceptible bonds in the part of the protein that remains bound to hydroxyapatite, but at least some of the resulting peptides are retained on the mineral. Since the ability of the proteins to inhibit hydroxyapatite formation and to bind calcium is located in the N-terminal proline-poor part, it is possible that these activities are retained after proteolytic digestion of the adsorbed proteins.     

New allelic product of the PRH1 locus coding for salivary acidic proline-rich proteins

A new polymorphic acidic proline-rich protein (As) was found in human parotid saliva by SDS and basic polyacrylamide gel electrophoresis. The phenotypic relationships and family studies support the hypothesis that the As protein is another allelic product of the PRH1 locus. The As protein could not be discriminated from the parotid isoelectric focusing (PIf) protein by isoelectric focusing gel electrophoresis due to similar migration of the two proteins. In order to determine salivary PRH1 phenotypes it is necessary to use SDS or basic gel electrophoresis in addition to the isoelectric focusing gel electrophoresis. The As protein was not found in Caucasians. The allele frequencies of the PRH1 locus in Japanese were PRH1 (double-band protein) = 0.035, PRH1(2) (acidic protein) = 0.193, PRH1(4) (PIf) = 0.751, and PRH1(5) (As) = 0.021.     

PRB1, PRB2, and PRB4 coded polymorphisms among human salivary concanavalin-A binding, II-1, and Po proline-rich proteins.

Six closely linked PRP (proline-rich protein) genes code for many salivary PRPs that show frequent length and null variants. From determined protein sequences and DNA sequence analysis of variant alleles, we here report the coding and molecular basis for Con (concanavalin A-binding) and Po (parotid "o") protein polymorphisms. The Con1 glycoprotein is encoded in exon 3 of a PRB2 allele (PRB2L CON1+) with a potential N-linked glycosylation site. Because of a probable gene conversion encompassing > or = 684 bp of DNA, the "PRB2-like" Con2 glycoprotein is encoded in exon 3 of a PRB1 allele (PRB1M CON2+) with a potential glycosylation site. The PmF protein is also encoded in the PRB1M CON2+ allele, thus explaining the previously reported association between Con2 and PmF proteins. A PRB2L CON1 allele contains a single nt missense change [TCT(Ser)-->CCT (Pro)] that abolishes the potential N-linked glycosylation site (NKS-->NKP) in the Con1 protein, and this explains the Con- type. The Po protein and a glycoprotein (II-1) are encoded in the PRB4 gene, and both proteins are absent in the presence of a mutation in the PRB4M PO- allele that contains a single nt change (G--C) at the +1 invariant position of the intron 3 5'donor splice site. The genetically determined absence of the II-1 glycoprotein leads to altered in vitro binding of Streptococcus sanguis 10556 to salivary proteins, which suggests a biological consequence for null mutations of the PRB4 gene.     

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.     

Phosphopeptides derived from human salivary acidic proline-rich proteins. Biological activities and concentration in saliva.

Human saliva contains a large number of phosphopeptides derived by cleavage of acidic proline-rich proteins (APRPs). These peptides were purified by column chromatography and they constituted 0.5% of APRPs in parotid saliva, but 11% of APRPs in saliva expectorated from the mouth (whole saliva), indicating that there is considerable cleavage of APRPs after secretion from the gland. Similarly to APRP, the phosphopeptides bind Ca2+, but they accounted for only 4% of protein-bound Ca2+ in whole saliva. APRPs as well as the phosphopeptides inhibited formation of hydroxyapatite, but, whereas 19-20 micrograms of APRP was needed for 50% inhibition, only 0.7-3.3 micrograms of purified peptides was needed for the same degree of activity, and the phosphopeptides accounted for 18% of total inhibitory activity in whole saliva. All phosphopeptides adsorbed on hydroxyapatite in vitro, and adsorption of phosphopeptides on tooth surfaces in vivo could also be demonstrated, indicating that they would be able to inhibit unwanted mineral formation on the tooth surface in vivo. Degradation of APRPs after secretion therefore does not lead to a loss of their biological activities.     

Genetic studies of human acidic salivary protein (Pa).

The phenotypic expression of a dominantly inherited human salivary acidic protein (Pa) has been described in acid-urea starch and in Tris-borate acrylamide gel systems. Estimates of the Pa+ allelic frequencies in American Caucasians, American blacks, and Orientals are .21, .14, and .42, respectively. The genetic and biochemical similarities to another series of proline-rich salivary proteins, Pr, and to a pair of similarly staining salivary proteins, Db (double band), are evaluated. It is concluded that either one locus or two (or three) tightly linked loci are viable explanations for this polymorphic system(s). It is suggested that the three factors, Pa, Pr, and Db, be treated as separate loci to allow clarification of their genetic relationships.     

Cellular phosphorylation of an acidic proline-rich protein, PRP1, a secreted salivary phosphoprotein

Phosphorylation of many secreted salivary proteins is necessary for their biological functions. Identification of the kinase, which is responsible for in vivo phosphorylation, is complicated, because several of the protein phosphorylation sites conform both to the recognition sequence of casein kinase 2 (CK2) and Golgi kinase (G-CK), which both are found in the secretory pathway. This study was undertaken to determine the kinase recognition sequence in a secreted proline-rich salivary protein, PRP1, and thereby identify the responsible kinase. This was done by transfecting a human submandibular cell line, HSG, and a kidney cell line, HEK293, with expression vectors encoding wild-type or mutated PRP1. It was shown that phosphorylation occurred only at the same sites, Ser8 and 22, as in PRP1 purified from saliva. Phosphorylation at either site did not depend on the other site being phosphorylated. The sequence surrounding Ser8 has characteristics of both CK2 and G-CK recognition sequences, but destruction of the CK2 recognition site had no effect on phosphorylation, whereas no phosphorylation occurred if the G-CK recognition sequence was altered. The sequence surrounding Ser22 did not conform to any known kinase recognition sites. If Ser22 was mutated to Thr, no phosphorylation was seen, and a cluster of negatively charged residues at positions 27-29 was identified as part of the enzyme recognition site. Ser22 may be phosphorylated by a G-CK that recognizes an atypical substrate sequence or by a novel kinase. No difference in phosphorylation was seen between undifferentiated and differentiated HSG cells.     

Inheritance of the human salivary proline-rich proteins: A reinterpretation in terms of six loci forming two subfamilies

DNA studies suggest that six loci control the synthesis of human salivary proline-rich proteins (PRPs). Genes at two of these loci (proposed names, PRH1 and PRH2) contain regions that strongly hybridize to a probe made from a cDNA in which sites for the restriction enzyme HaeIII occur repeatedly; they code for the acidic PRPs. Genes at the remaining four loci (PRB1, PRB2, PRB3, and PRB4) contain regions that strongly hybridize to a probe with repeated BstN1 sites; they probably code for the basic and glycosylated PRPs. In contrast to these data suggesting six loci forming two gene subfamilies, studies of protein polymorphisms and families have led to the postulation of 13 loci with 11 common null alleles. The discrepancy in the number of loci is partly resolved by the hypothesis that the three acidic PRPs, Db, Pa, and PIF, are coded for by alleles at one of the HaeIII-type loci rather than by three discrete loci.This work was supported by National Institutes of Health Grants DEO 3658-19 (E.A.) and GM 20069 (O.S.). This is paper No. 2774 from the Laboratory of Genetics, University of Wisconsin—Madison.     

PRBI gene variants coding for length and null polymorphisms among human salivary Ps, PmF, PmS, and Pe proline-rich proteins (PRPs).

Six closely linked PRP (proline-rich protein) genes code for salivary PRPs that show frequent length and null polymorphisms. We report assignment of Ps proteins to the PRB1 gene, the derived primary structures of Ps 1 and Ps 2 proteins, and the molecular basis for some null alleles among PRB1-coded PRPs (Ps, PmF, PmS, and Pe). The derived primary structures of Ps 1 and Ps 2 proteins were determined by sequencing exon 3 of the different-length PRB1M (medium) and PRB1L (large) copies from subject C.J. with the Ps 1-2 phenotype. The PRB1L copy (coding for Ps 2) contained three additional tandem repeats within the Ps coding region, and the different-length Ps 1 and Ps 2 proteins can be explained on this basis. The molecular basis for the Ps 0 and the Pe- phenotypes was determined in another individual (M.V.O., a PRB2/1 fusion-gene heterozygote) with a single PRB1L copy. A premature stop mutation (CGA [Arg]-->TGA [stop]) occurred at residue 61 in the Ps-coding region. The identical mutation was found in the PRB1L and PRB1/2S (small) copies of a second individual (E.A.) with reduced Pe protein and the Ps 0 phenotype. This individual is a PRB1/2 fusion-gene heterozygote (Azen et al. 1992) with probably three mutated PRB1 copies (PRB1L-PRB1L-PRB1/2S). DNA sequences of the postulated crossover region of the PRB1/2S fusion-gene copy supported the postulated crossover. The PmF- and PmS- phenotypes in the three subjects were due to both the stop mutation and the lack of suitable proteolytic cleavage sites in the PRB1-coded precursor proteins.     

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.     

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.     

Basic proline-rich proteins from human parotid saliva: complete covalent structures of proteins IB-1 and IB-6

The complete amino acid sequences of two basic proline-rich proteins, IB-1 and IB-6, from human parotid saliva have been determined. Fragments for sequence analysis were obtained by enzymatic digestions. The proteins have molecular weights of 9571 (IB-1) and 11,530 (IB-6) and contain 34 and 39 mol % proline, respectively. IB-1 and IB-6 contain an identical sequence of 54 residues except for an alanine in position 52 of IB-6, where IB-1 has proline. An unusually high number of repeated sequences occurs in both molecules. IB-1 has a blocked amino-terminal residue, pyroglutamic acid, and also contains one phosphoserine residue in position 8. The relationship of these proteins to the basic proline-rich protein IB-9 [Kauffman, D., Wong, R., Bennick, A., & Keller, P. (1982) Biochemistry 21, 6558-6562] and to other salivary proline-rich proteins is discussed.     

Allelic variants of acidic proline-rich proteins observed in Japanese,Chinese, and Malays

Three new variants of acidic proline-rich proteins (At, Au, Aw) were found in human parotid saliva by isoelectric focusing and basic gel electrophoresis. Electrophoretic comparison of the purified proteins and their tryptic peptides suggested minor charge and size differences from other acidic PRPs. Genetic and biochemical studies indicate that the At and Aw proteins are allelic products of thePRH1 locus. Gene frequencies of the At productive allele (PRH1 ⁶) in Japanese, Chinese, and Malays were 0.008, 0.012, and 0.004, respectively. The Au protein was also found in Japanese (2 in 746 samples), Chinese (1 in 215 samples), and Malays (1 in 220 samples), however, the Aw protein was found only in one Japanese (n=746). These three proteins were not found in 106 Indian subjects.This study was supported in part by a grant from International Scientific Research Program of the Ministry of Education, Science and Culture of Japan, No. 62043071 (representative: K.S.).     

Complete covalent structure of a proline-rich phosphoprotein, PRP-2, an inhibitor of calcium phosphate crystal growth from human parotid saliva

Human salivary secretions contain many proteins in which proline forms an unusually large fraction of the amino-acid residues present, typically from 20% to over 40%. These proteins are also unusually rich in glycine and glutamine, generally account for over half the total protein in saliva, and include acidic, basic and glycosylated molecules. The functions of most of these are not clearly defined. One group, however, the acidic proline-rich phosphoproteins (PRP), have been shown to be potent inhibitors of secondary precipitation (crystal growth) of calcium phosphate salts. Acting together with a salivary protein inhibitor of primary precipitation of calcium phosphates, statherin, the PRP stabilize saliva which is supersaturated with respect to the calcium phosphate salts which form dental enamel. These inhibitory activities act to provide a protective, reparative, but stable environment for dental enamel, which is important for maintaining the health of the teeth. The PRP are a complex group of phosphoproteins which include four major and at least eight minor members. The primary structures of three of the major proteins have been determined. These are PRP-1, also designated Protein-C, PRP-3, also designated Protein-A (17), and PRP-4. The designations PRP-1,-2,-3 and -4 will be used here. The purpose of this paper is to report the complete primary structure of PRP-2 as a further step towards establishing the structural basis of the biological activity of the PRP, and clarifying the genetic and biosynthetic relationships of these closely related proteins.     

Linkage relationships and multipoint mapping of the human parotid salivary proteins (Pr, Pa, Db).

Based on data from 76 informative families, linkages between Pa and Pr and between Pr and Db have been established by two-point linkage analysis. In both pairs of loci, there were no significant sex heterogeneity in recombination fractions. Linkage between Pa and Db cannot be established based on two-point analyses, but a significant sex difference in the recombination fraction between Pa and Db was observed. Strong confirming evidence was obtained from three-point analysis to place Pa, Pr, and Db in one linkage group. The most likely order is Pa-Pr-Db, but the relative odds over second order Pr-Pa-Db are small. Haplotype frequencies of Pr, Pa, and Db were obtained based on the phenotypes of the 685 random Caucasians, providing evidence for marked linkage disequilibrium among the three loci.     

Description of a genetic polymorphism of a human proline-rich salivary protein,Pc, and its relationship to other proteins in the salivary protein complex (SPC)

A new polymorphism, Pc, has been identified in human saliva. Two proteins, Pc 1 and Pc 2, are determined by alleles Pc1 and Pc2, respectively, which show autosomal codominant inheritance. No null phenotype has been encountered in 225 randomly collected salivas. The frequencies of the two alleles differ in the Black and White American populations, with Pc1 and Pc2 being 0.670 and 0.330 in the Black (N = 47) and 0.461 and 0.539 in the White (N = 178) populations, respectively. The alleles are in equilibrium in the two populations and segregation analyses (30 families) do not suggest the existence of a null allele in either population. Of seven polymorphic human salivary proteins determined by genes in the salivary protein complex (SPC), Pc phenotypes show association only with Ps phenotypes. Based on that association, our linkage studies, and the biochemical similarities with other SPC proteins, we tentatively conclude that Pc is a member of the SPC, bringing the total number of genes in that group to 13.     

The structure and evolution of the human salivary proline-rich protein gene family

We present the nucleotide sequences of four members of the six-member human salivary prolinerich protein (PRP) gene family. The four genes are PRB1 and PRB2, which encode basic PRPs, and PRB3 and PRB4, which encode glycosylated PRPs. Each PRB gene is approximately 4.0 kb in length and contains four exons, the third of which is entirely composed of 63-bp tandem repeats and encodes the proline-rich portion of the protein products. Exon 3 contains different numbers of tandem repeats in the different PRB genes. Variation in the numbers of these repeats is also responsible for length variations in different alleles of the PRB genes. We have determined a probable evolutionary history of the human PRP gene family by comparing the nucleotide sequences of the six PRP genes. The present-day six PRP loci probably evolved from a single ancestral gene by four sequential gene duplications, leading to six genes that fall into three subsets, each consisting of two genes. During this evolutionary process, multiple rearrangements and gene conversion occurred mainly in the region from the 3 end of IVS2 and the 3 end of exon 3.