Bioactive Peptides Hidden in Human Salivary Proteins

Journal of Oral Biosciences 59 (2017) 71–79

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Journal of Oral Biosciences

journal homepage: www.elsevier.com/locate/job

Review Bioactive peptides hidden in human salivary proteins

Eiichi Saitoh a,n, Masayuki Taniguchi b, Akihito Ochiai b, Tetsuo Kato c, Akane Imai d, Satoko Isemura d a Graduate School of Technology, Niigata Institute of Technology, 1719 Fujihashi, Kashiwazaki, Niigata 945-1195, Japan b Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan c Laboratory of Chemistry, Tokyo Dental College, Tokyo 101-0062, Japan d Department of Dental Hygiene, The Nippon Dental University College at Niigata, Niigata 951-8580, Japan article info abstract

Article history: Background: Extensive peptidomic studies of human saliva have resulted in considerable advances in the ﬁeld Received 28 September 2016 of proteomics. As the next generation in salivary research, a comprehensive understanding of the biological Received in revised form functions of in vivo peptides generated by proteolysis in the oral cavity has been long awaited. A cyclopedic 4 November 2016 functional analysis of salivary peptides may bring promising therapeutic agents and novel clinical applications. Accepted 17 November 2016 Highlight: (1) This review article refers to bioactive peptides hidden in salivary parent proteins. (2) Functions of Available online 5 December 2016 the peptides as anti-microbial, anti-viral, wound-closing, and anti-pain are described. (3) Biological sig- Keywords: niﬁcances of the repeated structures in salivary proline-rich proteins are emphasized. Human salivary proteome Conclusion: Human salivary proteins have the ability to generate bioactive peptides upon proteolytic cleavage. Bioactive peptides in salivary proteins & 2016 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved. Antimicrobial activity Wound-closing factor Anti-cancer activity

Contents

1. Introduction...... 71 2. A gift from the human genome project to salivary research...... 72 2.1. Multiple proteins are produced by one gene ...... 72 2.2. Two gene clusters encoding major salivary proteins ...... 73 2.2.1. The gene cluster on chromosome 4q13.3 ...... 73 2.2.2. The gene cluster on chromosome 12p13.2 ...... 74 3. Bioactive peptides hidden in salivary proteins...... 75 3.1. Bioactive peptides in statherin, histatin 1, and histatin 3 ...... 75 3.2. Bioactive peptides in P-B, P-B1, and PBLP ...... 76 3.3. Bioactive peptides in salivary mucins...... 77 3.4. Bioactive peptides in aPRPs, bPRPs, and gPRPs ...... 77 4. Concluding remarks...... 77 Ethicalapproval...... 77 Conﬂictofinterest...... 77 Acknowledgments...... 78 References...... 78

Abbreviations: N-, amino-; C-, carboxyl-; PRP, proline rich protein; a, acidic; b, basic; g, glycosylated n Corresponding author. E-mail address: [email protected] (E. Saitoh). http://dx.doi.org/10.1016/j.job.2016.11.005 1349-0079/& 2016 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved. 72 E. Saitoh et al. / Journal of Oral Biosciences 59 (2017) 71–79

Fig. 1. Chromosomal localization of the genes encoding salivary (or lacrimal) proteins. Open squares indicate noncoding exon regions. Closed squares denote coding exon regions. Gray squares indicate exons encoding the PRPs-speciﬁc repeating unit G(P)nG(K/R)PQ and its related sequences.

1. Introduction dilution and cleaning, digestion, lubrication, and protection of tooth enamel are granted by parent proteins and occasionally their pro- To develop diagnostic systems based on human salivary protein cessed forms [7]. These biological functions are related not only to components, extensive proteomic and peptidomic studies have been oral health, but also to systemic health. The environment of the oral performed involving whole saliva (WS), parotid saliva (PS), sub- cavity, the “port of entry” of the gastrointestinal tract, is likened to a mandibular/sublingual saliva (SM/SL-S), and exosomes in WS over high-performance and elaborate incubator because the pH and tem- the past decade. Previous studies of the human salivary proteome perature in the cavity and gastrointestinal tract are precisely con- identiﬁed 914 proteins in the PS proteome [1],917intheSM/SL-S trolled. The oral cavity contains culture media rich in nutrients sup- proteome [1], 491 in the parotid exosome [2], 187 in two types of plied by dietary foods, in which notable proteolytic events take place exosomes in WS [3], and 56 in minor salivary gland secretions [4]. on salivary and dietary proteins. The predominate peptide fragments WS proteins in the oral cavity predominantly originate from three present in WS have been reported to be derived from aRRPs, bPRPs, major salivary glands: the parotid, submandibular, and sublingual gPRP,P-B,statherin,andhistatins[8]. Oppenheim et al. [8] have dis- glands. Many proteins have been identiﬁed in WS, including acidic covered unique proteases in WS that cleave preferentially after a (a) proline-rich proteins (PRPs), basic (b) PRPs, glycosylated (g) PRPs, glutamine residue for the tripeptide sequence -KPQ- in aPRPs, bPRPs,

P-B, cystatins, histatins, statherin, mucous glycoproteins, mucin 5 and gPRP. The PRP-specificrepeatingunit,G(P)nG(K/R)PQ, and its (MG1), mucin 7 (MG2), immunoglobulins, amylase, and agglutinin related sequences are excised by proteases. These excised peptides [5]. Multiple variations based on genetic polymorphisms have been display completely different functions than the parent proteins [9,10] observed for each protein [6]. and were of unknown relevance until now. It has been gradually re- According to the NCBI gene database (http://www.ncbi.nlm.nih.gov/ cognized that the peptide fragments created by proteases from oral gene/), salivary proteins, including statherin, histatin 3, histatin 1, P-B1, epithelial cells, bacteria, and a serum-like gingival crevicular transu- P-B, BPLP, and mucin 7, are encoded by a set of clustered genes loca- date, play important roles in both the oral cavity and further down- lized on chromosome 4 [Cytogenetic location: 4q13.3, Genomic stream in the alimentary canal. Despite the identification of more coordinates: 4: 69,995,930-70,482,997; - STATH (encoding statherin) - than 4000 different salivary peptides and protein species [11],phy- HTN3 (alias HIS2; encoding histatin 3) - HTN1 (HIS1;histatin1)-PBI siological functions of salivary peptides are beginning to be under- (SMR3A or PROL5; P-B1 or SMR3A) - PBII (SMR3B or PROL3; P-B or stood and only recently have applications of these peptides been SMR3B) - OPRPN (PROL1; basic proline-rich lacrimal protein BPLP) - examined. MUC7 (mucin 7) -]. Moreover, lacrimal proline-rich protein (LPRP), two In this review article, we summarize the bioactive peptides aPRPs, one bPRP, and three gPRPs are encoded by another gene cluster identified so far that are hidden in major human salivary proteins. [Cytogenetic location: 12p13.2, Genomic coordinates: 12: 10,845,849- 11,501,041; - PRR4 (alias PROL4; encoding LPRP) - PRH1 (encoding aPRP) - PRH2 (aPRP) - PRB3 (gPRP) - PRB4 (gPRP) - PRB1 (bPRP) - PRB2 2. A gift from the human genome project to salivary research (gPRP) -]. The PRH2 geneislocatedintheforwardstrandofchromo- some 4 but the other six genes are in the reverse strand. 2.1. Multiple proteins are produced by one gene Most of the established biological functions of saliva such as antimicrobial properties, wound repairing, pain control, buffering, Brief maps of two gene clusters encoding major salivary proteins E. Saitoh et al. / Journal of Oral Biosciences 59 (2017) 71–79 73

Fig. 2. Summary of biological information for statherin, histatin 3, histatin 4, P-B1, and P-B. Nomenclature of each gene, UniProt/Swiss-Prot number, and amino acid sequence of each protein are present in the NCBI gene and UniProt/Swiss-Prot database. The signal sequence for each protein is shown in italics. The N-terminal and C-terminal amino acid residues for each protein are numbered. Antimicrobial peptide sequence in statherin (P02808) is indicated by a dotted underline. The histatin 5 sequence in histatin 3 (P15516) is indicated by a triple underline. The minimal domain required for wound healing activity in histatin 1 (P15515) is indicated by a solid underline. The opiorphin homolog (QRGPR) in P-B1 (Q99954), P-B (P02814), and P-B variant (Q508X8Q504 8) is indicated by a double underline. The possible N-glycosylation site (N-C-S) in the amino acid sequence of P-B variant is underlined. Sugar chain is indicated by duplex-closed circles. on chromosome 4q13.3 and chromosome 12p13.2 are illustrated in amino acid sequences encoded by the genes (STATH, HTN3, HTN1, Fig. 1 and are based on data resulting from the human genome pro- PBI, and PBII) are given in Fig. 2. In the same manner, Fig. 3 shows ject (http://www.ncbi.nlm.nih.gov/gene/). These achievements re- the amino acid sequences encoded by OPRPN and MUC7. STATH, vealed that alternative splicing causes multiple exon combinations, HTN3, and HTN1 are composed of six exons that are thought to be including coding- and non-coding DNA sequences, thereby many derived from the same common ancestral gene [13]. transcripts can be generated from a single gene. In some cases, an As shown in Fig. 2, multiple transcripts for these genes have been unexpected exon comprised of coding- and/or non-coding sequences identiﬁed and two forms (32 and 22 residues) of mature histatin 3 and located far from the exon in an established gene can be combined. two forms (43 and 33 residues) of mature statherin are thought to be Sometimes the resulting transcript is a protein-coding RNA, and expressed by alternative splicing. In addition, a variant form of histatin sometimes it is not; both coding and non-coding encoding transcripts 3 (27 residues) carrying two mutations (Arg22 - Gln22,Tyr28 - can be generated. In addition, RNA editing can produce a variant termination) has also been frequently identiﬁed in people of African protein with a sequence inconsistent with the DNA sequence of the descent [14]. established gene. In 1979, P-B, a PRP distinct from aPRPs, bPRPs and gPRPs, was discovered in human WS [15] and the corresponding cDNA was 2.2. Two gene clusters encoding major salivary proteins isolated from a human submandibular gland cDNA library [16]. Isemura and Saitoh [17] isolated a genomic clone encoding a 2.2.1. The gene cluster on chromosome 4q13.3 homolog of P-B and its putative translational product was termed Statherin [12] [UniProt/Swiss-Prot (http://www.uniprot.org/., P-B1. The precursor form of P-B1 is composed of 134 amino acid P02808), histatin 3 (P15516, X6RAH8), histatin 1 (P15515), three residues and the signal sequence is composed of 22 residues, PRPs distinct from aPRPs, bPRPs and gPRPs [P-B1 (Q99954), P-B which is identical to the precursor form of mature P-B, as shown (P02814), and BPLP (Q99935, GenBank HC924356.1)], and mucin 7 in Fig. 2. The amino (N-) terminal 61 amino acid residues of P-B1 (Q8TAX7) shown in Figs. 2 and 3 are respectively encoded by the show a 75% sequence homology with P-B (57 residues), when genes STATH, HTN3, HTN1, PBI, PBII, OPRPN, and MUC7. According 4 deleted residues are taken into consideration. Using proteomic to the order of the genomic coordinates demonstrated in Fig. 1, the approaches, it was shown that P-B1 is present in human tears 74 E. Saitoh et al. / Journal of Oral Biosciences 59 (2017) 71–79

Fig. 3. Summary of biological information for BPLP and mucin 7. Nomenclature of each gene, UniProt/Swiss-Prot number, and amino acid sequence of each protein are present in the NCBI gene and UniProt/Swiss-Prot databases. According to patent WO2010/060995 and GenBank HC924356.1, the biological information for the short form of BPLP is given. The signal sequence for each protein is shown in italics. The N-terminal and C-terminal amino acid residues for each protein are numbered. Opiorphin (QRSFR) in BPLP (Q99935 and GenBank HC924356.1) is indicated by a double underline. The possible N-glycosylation sites (N-T-T, N-A-T, N-Q-T, N-I-T and N-S-T) are underlined. The sugar chain is indicated by duplex-closed circles. The N-terminal domain of mucin 7 (Q8TAX7) displaying candidacidal activity is indicated by a solid underline. The 23-mer repetitive motif of mucin 7 displaying antimicrobial activity is indicated by a dotted line with partitions.

[18],SM/SL-S[1] and PS [1].In2000,Isemura[19] reported a recognized as a new intron carrying the unique sequence of splicing corrected DNA sequence of the cDNA encoding P-B and a com- donor and acceptor sites, CC-AC, a non-canonical splice site [22]. plete DNA sequence of the PBII gene for P-B. By Southern blotting Nucleotide positions 1–57 of this putative intron encode and database analysis, it was suggested that P-B is conserved in P39PPPAPYGPYGPGIFPPPPPQP57 in P-B. vertebrates and yeast. Salivary low-molecular weight mucin (mucin 7) is encoded by In 1996, cDNA for basic proline-rich lacrimal protein (BPLP) was the MUC7 gene spanning approximately 10 kb and comprises isolated from a human lacrimal gland cDNA library [20]. BPLP and 3 exons and 2 introns [23], whose exon-intron organization is P-B share some degree of sequence homology with mouse MSG identical to that of PBI, PBII, and OPRPN. The signal sequence of proteins and rat VCS-alpha-1, VCS-beta-1, and submandibular mucin 7 (22 residues) [24] is identical in size to signal sequences apomucin, but do not share sequence homology with aPRPs, of P-B and P-B1, and almost equal in size to that of BPLP (21 re- bPRPs, and gPRPs [19,20]. Northern blot analysis demonstrated sidues). These four proteins also share sequence homology (refer that expression of a 1.05-kb BPLP transcript is higher in the la- to Figs. 2 and 3). crimal gland compared to expression in the submandibular gland. In situ hybridization showed that BPLP is expressed in lacrimal 2.2.2. The gene cluster on chromosome 12p13.2 gland secretory end-piece cells [21]. Seven genes (PRR4, PRH1, PRH2, PRB3, PRB4, PRB1,andPRB2)en- Interestingly, long (227 residues) and short (180 residues, pa- code closely related salivary PRPs [25–29] and LPRP [21],asshownin tent WO2010/060995) forms of mature BPLP with different amino Fig. 1. According to the order of the genomic coordinates indicated in acid sequences in their carboxyl (C-) terminal portions can be Fig. 1, the protein sequences [Q16378, (A0A087WV42, A0A087WYT0, generated from a single gene, “OPRPN”, as shown in Fig. 3.Itis A0A0A0MT3), P02810, and (F5H7C1, Q04118)] encoded by PRR4, PRH1, reasonable to consider that the mRNA encoding the short form of PRH2,andPRB3 are given in Fig. 4. In the same manner, Fig. 5 gives the BPLP is generated by RNA editing (G447 is inserted in exon 3 of protein sequences [(E7EXA8, E9PAL0), (G5E9 6, G3V1R1, G3V1M9), mRNA for Q099935, G446C447-G446G447C448), rather than alter- and P02812] encoded by PRB4, PRB1,andPRB2.Asdemonstratedin native RNA splicing. Ogawa et al. identiﬁed the long form of BPLP Fig. 4, multiple distinct alleles at the PRH1 locus encode aPRPs such as in exosome II in human WS [3]. parotid isoelectric-focusing variant slow (PIF-s: 150 residues, 123rd is Our recent study revealed that the short form of BPLP is certainly arginine), parotid acidic protein (Pa: 150 residues, 123rd is cysteine), present in human whole saliva together with the variant form of P-B and double band slow (Db-s: 171 residues) [28]. Pr (alias Pr1 or PRP-1, (Q504X8Q504 8; 84 residues, see Fig. 2)(datanotshown).Alter- 150 residues), a closely related aPRP, is encoded by a gene located native splicing of P-B pre-mRNA results in mRNA for within the PRH2 locus [25]. Some aPRPs are phosphorylated and are Q504X8Q504 8(http://h-invitational.jp/h-dbas/). The sequence at associated with susceptibility to dental caries [30]. The preproprotein nucleotide positions 124–236 in exon 3 of the PBII gene [19] may be encoded by genes at the remaining four loci (PRB1, PRB2, PRB3,and E. Saitoh et al. / Journal of Oral Biosciences 59 (2017) 71–79 75

Fig. 4. Summary of biological information for proline-rich proteins encoded by the genes PRR4, PRH1, PRH2, and PRB3. Nomenclature of each gene, UniProt/Swiss-Prot number, and amino acid sequence of each protein are present in the NCBI gene and/or UniProt/Swiss-Prot databases. The signal sequence for each protein is shown in italics. The N-terminal and C-terminal amino acid residues for each protein are numbered. The amino acid sequence of proline-rich peptide P-C in the parent proteins (A0A087WV42, A0A087WYT0, A0A0A0MT3, and P02810) is double underlined. The peptide sequence harboring anti-tumoral activity in P-C is indicated by a solid line. The possible N-glycosylation site (N-Q-S) in gPRPs (F5H7C1 and Q04118) is underlined. The sugar chain is indicated by duplex-closed circles.

PRB4) are bPRPs and/or gPRPs, as shown in Figs. 4 and 5.Thepre- formation of harmful deposits in salivary glands and on tooth surfaces proproteins undergo proteolytic processing to generate one or more [32]. Moreover, a peptide (R12FGYGYGPYQPVPEQPLYPQ32)withinsta- mature isoforms such as P-C, P-D (IB-5, Po), P-E (IB-9, PmF), P-F (IB-8b), therin (see Fig. 2) exerts antimicrobial activity against Staphylococcus P-G (IB-7), P-H (IB-4), P-I (IB-6, PmS), IB-1, IB-8a (Pc), IIB-1, and II-B2 aureus. [10]. (Pe), before secretion from the parotid and submandibular/sublingual Histatin 1 is a phosphoprotein and is proposed to be involved in glands [5,30]. formation of the acquired enamel pellicle, a proteinaceous layer The PRR4 gene adjoining the salivary PRPs gene complex en- covering tooth surfaces that forms a barrier over tooth enamel codes LPRP [21]. LPRP is composed of 134 amino acids with a 16- [33]. However, histatins also display multiple physiological func- amino acid signal sequence, and shares 45.5% sequence similarity tions in addition to tooth protection. First, it should be noted that with salivary aPRPs (see Fig. 4). Southern blot analysis showed that histatins 1 and 3 display candidacidal and bactericidal activity [33] LPRP is a single-copy gene. LPRP expression is detectable in the (see Fig. 2). Second, histatins 1 and 2 (lacking the N-terminal 11 human submandibular, von Ebners, sublingual, and parotid glands. residues of histatin 1) were reported to be major wound-closing Furthermore, LPRP expression was reported in the acinar cells of factors in human saliva [34,35]. the lacrimal gland. In the submandibular gland, expression of PRR4 Wounds in the mouth are repaired faster and with less scar for- and PRH1 genes is localized to the serous acini and demilunes [21]. mation and inflammation than wounds on the skin. Saliva is thought to be essential for superior oral wound healing, but specificme- chanisms have not yet been clarified. Histatins were found to enhance 3. Bioactive peptides hidden in salivary proteins reepithelialization in a human full-skin wound model that closely mimics normal skin. In this model, histatins did not stimulate pro- 3.1. Bioactive peptides in statherin, histatin 1, and histatin 3 liferation, but did induce cell spreading, migration, and two key in- itiating steps in reepithelialization. Cellular activation by histatin was Statherin is a tyrosine-rich acidic salivary peptide and an unusually shown to require a G-protein-coupled receptor that activates the potent inhibitor of calcium phosphate precipitation [31]. Like salivary ERK1/2 pathway. Using a stepwise truncation method, it was de- phosphoproteins, aPRPs, statherin binds calcium and inhibits crystal monstrated that the minimal domain S20HREFPFYGDYGS32 of histatin growth. Statherin and phosphorylated aPRPs are known to prevent the 1(seeFig. 2) is required for biological activity [35]. 76 E. Saitoh et al. / Journal of Oral Biosciences 59 (2017) 71–79

Fig. 5. Summary of biological information for proline-rich proteins encoded by the genes PRB4, PRB1, and PRB2. Nomenclature of each gene, UniProt/Swiss-Prot number, and amino acid sequence of each protein are present in the NCBI gene and UniProt/Swiss-Prot databases. The signal sequence for each protein is shown in italics. The N-terminal and C-terminal amino acid residues for each protein are numbered. The anti-cancer peptide P1932 sequence in bPRPs (G5E9X6G5E9 6, G3V1R1, and G3V1M9), and gPRP (P02812) is indicated by a double underline. The possible N-glycosylation sites (N-Q-S and N-K-S) are underlined. The sugar chain is indicated by duplex-closed circles.

Antimicrobial peptides are thought to be multifunctional in Moreover, it should be noted that histatin 5 displays inhibitory innate immunity and wound repair of multicellular organisms. The activity against MMP-9 from the host and Arg-gingipain and Lys- antimicrobial peptide LL-37 enhances both fibroblast migration gingipain from bacteria. Protease inhibitory activity should be and proliferation at a narrow optimal concentration (approxi- emphasized as a new potential biological function of histatin in mately 1 μM). Histatin 2 also enhances fibroblast migration, but the oral cavity [40]. only weakly induces proliferation. At higher concentrations, LL-37 induces cell death, whereas histatin 2 does not. It should be noted 3.2. Bioactive peptides in P-B, P-B1, and PBLP that both peptides do not alter fibroblast-to-myofibroblast differ- entiation. Histatin 2 does not alter interleukin-8 (IL-8) expression In 2006, opiorphin, a QRFSR pentapeptide [41], was discovered or lipopolysaccharide (LPS)-mediated cytokine and chemokine in human saliva. Opiorphin's bioactivity is similar to the activities expression. In contrast, LL-37 induces IL-8 expression but inhibits of rat sialophin (QHNPR) and bovine spinorphin (LVVYPWT). The the LPS-induced immune response. Histatin 2 and LL-37 have only opiorphin family plays the role of a natural antinoceptive mod- a very minor effect on human-neutrophil migration [36]. ulator of opioid-dependent pathways by inhibiting two en- Histatin 5 is a fragment of histatin 3 (see Fig. 2) that exerts anti- kephalin-catabolizing ectoenzymes, human ectoaminopeptidase fungal function through a mechanism other than pore formation [37]. (hAP-N, EC 3.4.11.2) and human neutral ectoendopeptidase (hNEP, This peptide is thought to translocate across the yeast membrane and EC 3.4.24.11) [41]. Opiorphin is equal to the N-terminal five re- target the mitochondria [38]. To study the anticandidal activity of sidues of BPLP (see Fig. 3). Opiorphin concentration was quantified sequencesinhistatin5,Rothsteinet al.[39] chemically synthesized in human plasma, urine, tears, milk, semen, and saliva by Dufour et fragments K5RHHGYKRKFHEKHHSHR22,R12KFHEKHHSHRGYR25 (his- al. [42]. This study indicated that opiorphin is most abundant in 4 15 4 15 4 tatin 9), A KRHHGYKRKFH ,AKRHHGYKRKFH -NH2,AKRHH human tears (220 ng/mL average) and citric acid-stimulated saliva 15 5 15 4 GYKRKFH -NH2 (D-peptide), K RHHGYKRKFH ,andAKRH (163 ng/mL av. for males, 158 ng/mL for females, 60 ng/mL av. for HGYKRKF14, and evaluated their activity (numbering corresponds to six-month pregnant women). Three peptides, opiorphin-QRSFR for histatin 3 residues in Fig. 2). The results indicated that all peptide BPLP, an opiorphin-homolog QRGPR for P-B and P-B1 (see Fig. 2), fragments examined showed anticandidal activity. The peptide and sialophin-QHNPR inhibit hAP-N to the same degree, but only A4KRHHGYKRKFH15 was subsequently proposed as a potential anti- QRSFR exerts inhibitory activity against human hNEP (Patent microbialagentforthetreatmentoforalcandidiasis[39]. W02009/124948). BPLP, P-B, and P-B1 have been proposed as E. Saitoh et al. / Journal of Oral Biosciences 59 (2017) 71–79 77 biomarkers of erectile physiology [43]. regions are frequently found as multiple tandem repeats. The To find a new generation of antibiotics based on the information major tandem repeats in human salivary PRPs of this class are the in the salivary peptidome, Trindade et al. [10] predicted the anti- G(P)nGKPQ sequence and its related homologs (see Figs. 4 and 5). microbial activity of peptides with the AntiMicrobial Peptides Huang et al. [52] observed that the GPPPQGGRPQ peptide binds Website (http://www.bicnirrh.res.in/antimicrobial.) and found that gram-positive Propionibacterium acnes and considerably inhibits five peptides in human salivary P-B exceeded the antimicrobial bacterial growth. This sequence is present in P-C [53], the peptide (AMP) probability of 0.8: (AMP probability 0.887, C-terminal 44 residues of PRH1 and PRH2 as shown in Fig. 4. 36 57 35 R IPPPPAPYGPGIFPPPPPQP ;0.903,GPGRIPPPPPAPYGPGIF Trindade et al. [10] found that this peptide also displays anti- 57 23 45 PPPPPQP ;0.991,FVPPPPPPPYGPGRIPPPPAPY ;0.937, bacterial effects against S. aureus. Moreover, da Costa et al. [54] 33 57 6 G PGRIPPPPPAPYGPGIFPPPPP QP ;0.987,GPYPPGPLAPPQP discovered that a related peptide, GPPPQGGRPQG, interferes with 32 FGPGFVPPPPPPPY ) (see Fig. 2). We chemically synthesized pep- the ability of cell adhesion proteins to stabilize adherens junctions, tides present in PRPs and analyzed their biological activities, in- such as those formed by E-cadherin, leading to apoptosis in breast, cluding antimicrobial activity against several oral pathogenic bac- prostate, colon, osteosarcoma, and bladder cancer cell lines 33 teria. We noticed that a peptide (G PGRIPPPPPAPYGPG (T47-D, PC-3, HT-29, MG63, T-24, respectively). 57 IFPPPPPQP ) in P-B and its related fragments harbored both anti- Recently, Palmerini et al. [55] found that a 20-mer peptide microbial activity and LPS-neutralizing activity (data not shown). We termed P1932, GPPPQGGNKPQGPPPPGKPQ, shows a dose-depen- will describe these results in detail elsewhere. dent antagonistic effect on the cytosolic Ca2þ mobilization in- In addition to bioactivity of peptides in the parent proteins of duced by progesterone in a tongue squamous carcinoma cell line. this class, more attention should be paid to another function of The peptide p1932 can be generated from the proteolytic proces- P-B. It must be emphasized that P-B, P-B1, and BPLP are grouped sing of three bPRPs (G5E9 6, G3V1R1, and G3V1M9) and a gPRP into the P/Q-SCPP (secretory calcium-binding phosphoproteins) (P02819), as shown in Fig. 5. The sequence of p1932 is repeated family. This family is thought to form the tooth surface [44]. several times in both of the above PRPs (see Fig. 5), and this re- petition may bring its average concentration in human saliva to 3.3. Bioactive peptides in salivary mucins approximately 5–10 μM. Radicioni et al. [56] analyzed the cell-penetrating properties of Five secreted gel-forming mucins (mucins 2, 5AC, 5B, 6, and 19), p1932 in a primary gingival fibroblast cell line and in a squamous three secreted non-gel-forming mucins (mucins 7, 8, and 9), ten cancer cell line, and compared the actions of p1932 to its retro- membrane-bound mucins (mucins 1, 3 A, 3B, 4, 11, 12, 13, 16, 17, and inverso form. It was found that both peptides are internalized in 20) and three unclassified mucins (mucins 14, 15, and 18) have been the two cell lines on a time scale of minutes, and that the natural identified [45].Moreover,mucins5B,7,and19havebeenidentified in form was more efficient than the retro-inverso form. Based on the human saliva [46]. Mucin 5B is thought to contribute to the results, it was concluded that the natural peptide might play a role lubricating and viscoelastic properties of WS. Mucins 5B and 7 are reported to inhibit Human Immunodeficiency Virus type 1 (HIV-1) within the cells of the oral mucosa after its secretion and sub- activity based on HIV inhibition assays [47].Incrudesaliva,mucin7is sequent internalization. thought to be highly susceptible to specificproteolysis,thougha Righino et al. [57] explored a possible intracellular target of major part of mucin 5B is more resistant to degradation. peptides in PRPs, p1932 and its retro-inverso form. It was dis- Interestingly, a tandem repeat 23-mer sequence, T145AAPP covered that these peptides interact with SH3 domains of Fyn, TPSATTPAPPSSSAPPE167, in mucin 7 (see Fig. 3) has been shown to Hck, and c-Src, and that all the interacting domains belong to the fi be a potent bactericidal agent for oral pathogens such as Por- Src kinase family. Based on these ndings, it was suggested that phyromonas gingivalis, Aggregatibacter actinomycetemcomitans, p1932 might modulate the signal transduction pathways mediated Streptococcus gordonii, and Streptococcus mutans. [48]. The 23-mer by these kinases. Additionally, it was proposed that p1932 and its retro-inverso form are ideal for application as novel drug delivery motif, TAAPPTP X1 (X1 ¼S, P) ATT X2 (X2 ¼P, Q, L) X3 (X3 ¼A, D) X4 (X4 ¼P, L, S) X5 (X5¼S, A) X6 (X6 ¼S, P) A X7 (X7¼P, Q) E, is re- agents since neither peptide is cytotoxic. peated 6 times in the core protein of mucin 7, as shown in Fig. 3. The N-terminal 49 residues of mucin 7 (see Fig. 3) are known to display candidacidal activity in vitro [50]. A peptide consisting of 4. Concluding remarks the N-terminal 18 residues of mucin 7, R1ERDHELRH 18 RRHHHQSPK , was also proposed to have distinct biological The term “human salivary proteome” refers to the entire set of functions, such as antibacterial activity, since this sequence is proteins present in saliva. However, it is obvious that this term is particularly rich in basic amino acids and is sensitive to proteolytic limited to an understanding of the function of all salivary protei- degradation. Bobeck et al. [49] showed that the 20-mer peptide naceous components. It must be taken into consideration that 30 49 with a net charge of þ7, L AHQKPFIRKSYKCLHKRCR , exerts bioactive peptides can be generated by proteolytic cleavage of broad-spectrum antimicrobial activity. Interestingly, it was also known salivary proteins. Similar findings were obtained for other 38 49 found that the 12-mer peptide (R KSYKCLHKRCR ) with a net protein species such as rice proteins [58,59]. charge of þ6 exhibits an amphipathic helical conformation. This peptide was found to retain stronger antifungal activity compared to the antifungal activity of the 20-mer. Cysteine residues, which Ethical approval play a critical role in bacterial binding, were found to be un- necessary for fungicidal activity. Based on the experimental data, Ethical approval was given by Niigata Institute of Technology this 12-mer peptide was proposed as a potential novel antifungal ethics committee reference numbers 2015-001 and 2016-001. therapeutic agent [50].

3.4. Bioactive peptides in aPRPs, bPRPs, and gPRPs Conﬂict of interest Proline-rich protein regions are widely found in both prokar- yotes and eukaryotes [51]. It should be noted that proline-rich The authors declare no conﬂicts of interest. 78 E. Saitoh et al. / Journal of Oral Biosciences 59 (2017) 71–79

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