Detection in human saliva of different statherin and PB fragments and derivatives

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Statherin is a multifunctional polypeptide specific of human saliva involved in oral calcium homeostasis, phosphate buffering and formation of protein networks. Salivary P-B peptide is usually included into the basic proline-rich protein family but
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  R ESEARCH A RTICLE Detection in human saliva of different statherin andP-B fragments and derivatives Rosanna Inzitari  1, 2  , Tiziana Cabras  3  , Diana Valeria Rossetti  1, 2  , Chiara Fanali  1, 2  ,Alberto Vitali  4  , Mariagiuseppina Pellegrini  3  , Gaetano Paludetti  5  , Armando Manni  6  ,Bruno Giardina  1, 2 ,4  , Irene Messana  3  and Massimo Castagnola  1, 2, 4  1 Istituto di Biochimica e Biochimica Clinica, Università Cattolica, Rome, Italy 2 Istituto Scientifico Internazionale (ISI) per la Ricerca sulla Fertilità e l’Infertilità Umana – Paolo VI, Rome, Italy 3 Dipartimento di Scienze Applicate ai Biosistemi, Università di Cagliari, Monserrato Campus, Cagliari, Italy 4 Istituto di Chimica per il Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy 5 Istituto di Clinica Otorinolaringoiatrica, Università Cattolica, Rome, Italy 6 Istituto di Clinica Odontoiatrica, Università Cattolica, Rome, Italy Statherin is a multifunctional polypeptide specific of human saliva involved in oral calciumhomeostasis, phosphate buffering and formation of protein networks. Salivary P-B peptide isusually included into the basic proline-rich protein family but it shows some similarities withstatherin and its specific biological role is still undefined. In this study, various fragments andderivatives of statherin and P-B peptide were consistently detected by RP-HPLC ESI-IT MS in23 samples of human saliva.They were: statherin mono-and non-phosphorylated, statherin Des-Phe 43 (statherin SV1), statherin Des-Thr 42 ,Phe 43 , statherin Des-Asp 1 , statherin Des 6–15 (statherinSV2), statherin Des 1–9 , statherin Des 1–10 , statherin Des 1–13 and P-B Des 1–5 . Statherin SV3 (sta-therin Des 6–15 , Phe 43 ) was detected only in one sample. Identity of the fragments was confirmedeither by MS/MS experiments or by enzymatic digestion or by Edman sequencing. Detection of the fragments suggests that statherin and P-B peptide are submitted to post-translational pro-teolytic cleavages that are common to other classes of salivary proteins. Received: May 30, 2006Revised: July 27, 2006Accepted: August 15, 2006 Keywords: P-B peptide / Human / Saliva / Statherin6370  Proteomics   2006,  6,  6370–6379 1 Introduction Several proteomic studies recently carried out on humansaliva [1–7] evidenced the complexity of this bio-fluid.Indeed, according to the most up-dated inventories, hu-man salivary peptides and proteins encompass more than1000 different components [7]. Almost certainly, no singlemodern analytical technique could allow an omni-com-prehensive analysis of all the human salivary proteincomponents. A possible approach to face the problem isto focus on the components of a specific group of salivaryproteins. Indeed, the majority of the salivary proteins canbe classified according to their chemico-physical proper-ties in distinct families, which are: proline-rich proteins[subdivided in acidic (aPRPs), basic (bPRPs) and basicglycosylated (G-PRPs)], cystatins, histatins and statherin.These peptides and small proteins with very specificsequences account for more than 75% in weight of thetotal salivary protein content. The remainder principallyconsists of salivary amylase (about 20%), mucins,enzymes and calcium-binding proteins [1–7]. Correspondence:  Professor Massimo Castagnola, Istituto di Bio-chimica eBiochimica Clinica, Facoltàdi Medicina, UniversitàCat-tolica, Largo F. Vito, 00168, Rome, Italy E-mail:  massimo.castagnola@icrm.cnr.it Fax: 1 39-06-305-3598 Abbreviations:aPRPs , acidicPRPs;  bPRPs ,basicPRPs;  PRPs ,pro-line-rich proteins;  SM/SL , submandibular/sublingual;  SV1–4 ,statherin variant 1–4;  XIC , extracted ion currentDOI 10.1002/pmic.200600395 © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  www.proteomics-journal.com  Proteomics   2006,  6,  6370–6379  Clinical Applications  6371 HPLC coupled to multi-dimensional IT MS is particular-ly suitable for the analysis of peptides and relatively smallproteins, as it provides complementary information in com-parison with 2-DE coupled to MALDI-TOF MS. Amylases,mucins and other high-molecular weight proteins that pre-cipitate under acidic conditions, cannot be analyzed byHPLC-ESI-IT-MS. Nonetheless, the HPLC-MS profile of theacidic soluble fraction of whole human saliva shows hun-dreds of mass values. By exploiting the high analyticalpotential of HPLC-MS, we were able to identify severalknown salivary components belonging to the cystatins, his-tatins, acidic and basic PRPs classes, as well as to character-ize new members of these families [8–11]. Moreover, in spiteof the great individual variability (especially with regard tothe bPRPs class), many fragments of acidic PRPs, basicPRPs and histatin 3, most likely srcinated during the secre-tion process by shared post-translational fragmentationpathways were consistently detected [9–11]. The principalobserved cleavage occurs at the level of the sequence R(X) 0/2/4 R that is recognized by furin-like pro-protein convertase of the kexin-subtilisin family [10, 12]. Often, but not always, thefirst cleavage is followed by the removal of the C-terminalarginine by the action of a carboxypeptidase, probably of either CPE or CPZ class [13–15].Statherin is a very specific multifunctional salivary pep-tide that contributes to the mineral dynamics of tooth sur-face. In fact, statherin has great affinity for calcium phos-phate minerals, such as hydroxyapatite [16], because it in-hibits supersaturated solutions of calcium phosphateminerals from precipitation and crystal growth [17, 18].Therefore, it maintains the supersaturated state of calciumin human saliva, inhibiting tooth surfaces from mineralaccretions and contributing, together with other salivaryproteins, to the stabilization of the acquired enamel. Inaddition, statherin contributes to the bacterial colonization[19] and acts as a boundary lubricant on the enamel surface[20]. The specific role of P-B peptide is still completelyobscure [21, 22]. Both statherin and P-B show in the struc-ture convertase consensus sequences. Therefore, objective of this study was to search for possiblefragments deriving fromthe parent peptides, among the many unassigned massvalues detected in human saliva by HPLC-ESI-IT-MS. 2 Materials and methods 2.1 Reagents and instrumentation All analytical grade chemicals and reagents were purchasedfrom Farmitalia-Carlo Erba (Milan, Italy), Merck (Darmstadt,Germany), and Sigma Aldrich (St. Louis, MI, USA). TheHPLC-ESI-MS was a ThermoFinnigan (San Jose, CA, USA)apparatus. Surveyor HPLC system was equipped with a PDADetector and connected to the mass spectrometer XcaliburLCQ Deca XP Plus ThermoFinnigan with a Tsplitter. Themass spectrometer was equipped with an electrospray ionsource. The chromatographic column was a Vydac(Hesperia, CA, USA) C 8  column with 5- m m particle diameter(column dimensions 150 6 2.1 mm). Peptide sequence wasdetermined with a Procise 610A Protein Sequencer (Applera,Foster City, CA, USA). 2.2 Sample collection and treatment Whole saliva samples were collected from 23 normal adultvolunteers between 2 and 4 p.m. with a soft plastic aspirator.Parotid secretion (about 0.2–0.3 mL;  n  = 3) was collected bymeans of a Lashley cup [23]. Submandibular/sublingual(SM/SL) secretion (about 0.2–0.3 mL;  n  = 4) was collectedwith a small aspirator placed at the exit of the gland duct. Anacidic solution (0.2% TFA) was immediately added to eachsalivary sample in 1:1 v/v ratio at 4 7 C and the solution wasimmediately centrifuged at 10000 6  g   for 5 min (4 7 C). Theacidic supernatant was separated from the precipitate andimmediately analyzed with HPLC-MS apparatus or stored at 2 80 7 C. 2.3 Protein isolation and molecular weightdetermination with HPLC-ESI MS Protein identification was performed using RP-HPLC-ESIMS. The following solutions were used for the RP chroma-tography: (eluent A) 0.056% aqueous TFA and (eluent B)0.05% TFA in ACN-water 80/20. Proteins were eluted usinga linear gradient from 0 to 55% in 40 min, at a flow rate of 0.30 mL/min. The mass spectra, in the positive ion mode,were collected every 3 ms. MS spray voltage was 4.50 kVandthe capillary temperature was 220 7 C.Extracted ion-current (XIC) strategy was used to searchfor the different  m/z  values related to possible fragmentsderiving from statherin, SV2 and P-B. The structures of some fragments (see Section 3) were confirmed by MS/MSanalyses, usually performed on the triple-charged ion. MS/MS experiments were performed by detecting parent ionswith a peak width of 2–4  m/z  value and at 40% of the max-imum activation amplitude. 2.4 Purification and enzymatic digestion of salivarypeptides Statherin was purified by using the method of Flora  et al.  [24]with the following modifications. About 20 mL of the acidicsolution obtained from the whole saliva (see Section 2.2)were diluted 1:1 with 0.5 mmol/L zinc chloride. pH of thesolution was brought up to 9.0 by adding 0.5 mol/L NaOHand the solution was stored on ice for 20 min. Afterwards,the suspension was centrifuged at 12 500 6  g   for 20 min at4 7 C. Precipitate was washed with distilled water and dis-solved in 1 mol/L HCl. Solution was dialyzed overnight in30 mmol/L acetate buffer, 10 mmol/L EDTA, pH 5.7 andthen lyophilized. Lyophilized samples were dissolved inabout 0.5 mL of 0.2% aqueous TFA and pure statherin was © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  www.proteomics-journal.com  6372  R. Inzitari  et al. Proteomics   2006,  6,  6370–6379 isolated by RP-HPLC under the conditions described in Sec-tion 2.3. Purity of statherin was checked with HPLC-ESI-MS.P-B peptide and derivatives were isolated from the acidicsoluble fraction of whole saliva by RP-HPLC under the con-ditions described in Section 2.3.About 5 nanomoles of pure statherin were dissolved in280  m L of 70 mmol/L sodium acetate buffer, pH 5.5,10 mmol/L EDTA, and digested at 37 7 C with 0.01 EU of car-boxypeptidase Y (Pierce Biotechnology, Rockford, IL) dis-solved in 20  m L of the same buffer. The same procedure wasapplied to digest statherin with 0.01 EU of aminopeptida-se M (Pierce Biotechnology), but 0.1 mol/L sodium phos-phate, pH 7.2, was used as buffer. Dephosphorylation wasperformed on about 3 nanomoles of pure statherin incu-bated with about 1 EU of calf intestinal alkaline phosphatase(Roche-Boehringer, Mannheim, Germany) in 0.3 mL of TrisHCL buffer 50 mmol/L, pH 8.0. At established times, 50  m Lof the different reaction mixtures were mixed to 0.2% TFAand analyzed with HPLC-ESI-MSaccording to the proceduredescribed in the previous sections. 2.5 Data analysis Deconvolution of averaged ESI-MS spectra was auto-matically performed by using Bioworks Browser softwareprovided with the Deca XP instrument or MagTran 1.0 soft-ware [25]. Experimental mass values of statherin, P-Bpeptideand their derivatives were compared with average theoreticalmass values using PeptideMass and FindPept programs,available at the Swiss-Prot Data Bank (us.expasy.org/tools),where statherin and P-B peptide are coded as P02808 andP02814, respectively. Identifications were consideredpositivewhen differences between theoretical and experimentalmass values were less than to 100 ppm. Experimental MS/MS spectra were compared with theoretical MS/MS spectragenerated by using the MS-product program, available at theProtein Prospector site (prospector.ucsf.edu/). The fragmention tolerance was 0.2 Da. 3 Results In the RP-HPLC-ESI-IT-MS profile of the acidic soluble frac-tion of whole human saliva the different salivary proteinclasses are roughly organized in clusters according to theirstructural similarity. The elution ranges of the principalfamilies of detectable human salivary peptides/proteins areshown in the chromatographic profile (TIC) of Fig. 1C. Fig-ure 1C also reports the enlargement of the TIC profile regis-tered between 25 and 30 min (D), with the peaks of statherinand P-B peptide, the ESI spectra recorded by the MS instru-ment in the samechromatographic interval (E and F) and themass values obtained after deconvolution of the ESI spectra(G and H). The experimental mass value of 5380.0 6 0.5 Dadetermined for statherin is in perfect agreement with theaveraged theoretical value (M av. theor. = 5379.7 Da) com-puted for the peptide di-phosphorylated on Ser 2 and 3 [26].The experimental mass value of 5792.9 6 0.5 Da determinedfor P-B peptide is in perfect agreement with the theoreticalvalue computed for the peptide containing N-terminal pyro-glutamic acid (M av. theor. = 5792.7 Da) as demonstrated byIsemura  et al.  [27]. Identities of statherin and P-B peptidewere confirmed by Edman sequencing of the purified pep-tides and/or of their tryptic fragments. Statherin and P-Bpeptidesweredetectedinallanalyzedsalivarysample(N=23;Table 1).Different isoforms of statherin have been already detec-ted in human saliva by other authors [28]. The variant call-ed SV1 (Statherin variant 1) corresponds to statherin miss-ing the C-terminal Phe residue (Statherin Des-Phe 43 ). Thevariant SV2, showing the deletion of the 6–15 internal resi-dues with respect to staherin (Statherin Des 6–15 ), is srci-nated by an alternative splicing that excludes an exon of 30 nucleotides [29]. SV3 corresponds to the SV2 variantlacking the C-terminal Phe residue (Statherin Des 6–15 ,Phe 43 ).Statherin missing the N-terminal Asp (Statherin Des-Asp 1 )has been recently detected in human saliva by Vitorino andcolleagues [30]. We were able to detect all these variants andfragments in the chromatographic profile by applying theXIC strategy. As an example, Fig. 2 shows the results of theXIC search for SV1, SV2, SV3 and statherin Des-Asp 1 iso-forms. SV1, SV2 and statherin Des-Asp 1 were detected in al-most all the analyzed samples, whereas SV3 was detected inonly one of the 23 analyzed samples (Table 1).Other statherin derivatives were detected in addition tothe above reported isoforms (Table 1). They were statherinmono- and non-phosphorylated, with an average mass of 5299.9  6  0.5 Da (theor. 5299.7 Da) and 5220.0  6  0.5 Da(theor. 5219.7 Da), respectively, and statherin without theThr-Phe C-terminal residues (Statherin-Des-Thr 42 ,Phe 43 ),with M. av. of 5131.2 6 0.5 Da (theor. 5131.5 Da).The structure of statherin shows three convertase con-sensus sequences at positions 10–13 (..RIGR ; F..), 9–10(..LRR ; I..) and 6–9 (..KFLR ; R..). By adopting the XIC strat-egy, we were able to detect the fragments deriving from allthe possible cleavages. They correspond to statherin Des 1–9 (statherin fragment 10–43), statherin Des 1–10 (statherinfragment 11–43) and statherin Des 1–13 (statherin frag-ment 14–43). The fragments were detected in all the humansalivary samples analyzed (Table 1). The same strategy wasapplied to detect the P-B fragment 6–57 (P-B Des 1–5 ) srci-nated by the cleavage at the convertase consensussequence 2–5 ( , QRPGR ; G..). In addition, this P-B frag-ment was detected in significant amounts in all the samplesanalyzed (Table 1).All the fragments detected in this study are reported inFig. 3 together with the structural relationship with the par-ent peptides. It is possible to name the statherin fragmentsin different ways depending on the various existing nomen-clatures. The statherin variant 1–4 names are taken fromother authors’ definitions [28]. According to the internationalpeptide nomenclature, the name of truncated peptides © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  www.proteomics-journal.com  Proteomics   2006,  6,  6370–6379  Clinical Applications  6373 Figure 1.  Typical RP-HPLC pro-file of the acidic soluble fractionof human whole saliva. UV pro-file detected at 214 (A) and276 nm (B). TIC profile (C)showing the elution ranges of the principal classes of humansalivary proteins/peptides.aPRPs, bPRPs, G-PRPs: acidic,basic and glycosylated (basic)proline-rich proteins; HSA: hu-man serum albumin; P-B: sali-vary P-B peptide; defen.: defen-sins; cyst: cystatins. (D) Enlarge-ment of the TIC profile between25.00–30.00 min showing theionic current peaks of statherinand P-B peptide. (E) and (F) ESI-IT mass spectra; (G) and (H):results of deconvolution of themass spectra. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  www.proteomics-journal.com  6374  R. Inzitari  et al. Proteomics   2006,  6,  6370–6379 Figure 2.  XIC (Extracted ion current) strategies for the search of statherin Des-Phe 43 (or SV1 isoform), sta-therin Des-Asp 1 , statherin Des 6–15 (or SV2 isoform) and statherin Des 6–15, Phe 43 (or SV3 isoform), respec-tively. Table 1.  Fragments of statherin and P-B peptides found in this study (see Fig. 3 for the structures) Fragment Theor. av.M (Da)Experim.Av. M (Da)Appr. El.time (min)Freq. wholesalivaFreq. parot.salivaFreq. SM/ SL salivaStatherin 5379.7 5380.0 6 0.5 28.0 23/23 3/3 4/4Statherin mono-phosph. 5299.7 5299.9 6 0.5 27.5 23/23 3/3 4/4Statherin non-phosph. 5219.7 5220.0 6 0.5 27.3 9/23 0/3 2/4SV1 (statherin Des-Phe 43 ) 5232.5 5232.4 6 0.5 26.6 23/23 3/3 4/4Statherin Des-Thr 42 ,Phe 43 5131.4 5131.2 6 0.5 26.8 23/23 3/3 4/4Statherin Des-Asp 1 5264.6 5264.7 6 0.5 27.6 23/23 3/3 4/4SV2 (statherin Des 6–15 ) 4148.2 4148.0 6 0.4 25.5 21/23 3/3 3/4SV3 (SV2 Des-Phe 43 ) 4001.0 4001.0 6 0.4 23.9 1/23 0/3 0/4Statherin Des 1–9 4127.6 4127.8 6 0.4 26.2 23/23 3/3 4/4Statherin Des 1–10 3971.4 3971.3 6 0.4 26.7 23/23 3/3 4/4Statherin Des 1–13 3645.0 3645.0 6 0.4 27.0 23/23 3/3 4/4P-B peptide 5792.7 5792.9 6 0.5 28.4 23/23 3/3 4/4P-B Des 1–5 5215.1 5215.0 6 0.5 28.9 23/23 3/3 4/4 should indicate the sequence number of missing residuespreceded by the Des- prefix. The different options for nam-ing peptide fragments are reported in Fig. 3.In this study, whole saliva collections were performed inorder to minimize proteolytic cleavages that may occur dueto the presence of exogenous oral proteinases. However, inorder to exclude that the fragments and derivatives of sta-therin and P-B peptide derive from oral flora degradation wehave searched the fragments also in salivary secretion col-lected directly from parotid and SM/SL gland ducts by XICstrategies. Except SV3, which shows a low frequency inwhole saliva, all the other fragments and derivatives were © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  www.proteomics-journal.com
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