Tyrosine Polysulfation of Human Salivary Histatin 1. A Post

Institute of Otorhinolaryngology, Catholic University of Rome, Rome, Italy. Received February 8, 2007. Histatin 1 (His-1) derivatives showing serial m...
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Tyrosine Polysulfation of Human Salivary Histatin 1. A Post-Translational Modification Specific of the Submandibular Gland Tiziana Cabras,† Chiara Fanali,‡ Joana A. Monteiro,‡,§ Francisco Amado,§ Rosanna Inzitari,‡ Claudia Desiderio,‡ Emanuele Scarano,| Bruno Giardina,‡ Massimo Castagnola,‡ and Irene Messana*,† Department of Sciences Applied to Biosystems, Cagliari University, Cagliari, Italy, Institute of Biochemistry and Clinical Biochemistry, Institute for the Molecular Recognition, CNR, International Scientific Institute “Paolo VI” (ISI), Catholic University, Rome, Italy, Department of Chemistry, University of Aveiro, Aveiro, Portugal, and Institute of Otorhinolaryngology, Catholic University of Rome, Rome, Italy Received February 8, 2007

Histatin 1 (His-1) derivatives showing serial mass increases of 80.0 ( 0.1 Da were detected in human saliva by HPLC-ESI-MS. The same derivatives were also found in granules of submandibular glands and secretions of submandibular/sublingual glands, but not in granules and secretions of parotid glands. Only one phosphate group was present in His-1 and its derivatives, since treatment with alkaline phosphatase provided an 80.0 Da mass decrease. His-1 derivatives were almost completely transformed into His-1 by treatment with 1 M HCl at 100 °C, suggesting the presence of O-sulfotyrosine, which is more labile than phospho-Tyr to acidic hydrolysis. CE-MS analysis of pronase extensive digestion of derivatives confirmed the presence of sulfotyrosine. Derivatives were digested by trypsin, proteinase K, and protease V-8 and analyzed by different MS strategies. The results allowed locating sulfation on the last four tyrosines (Tyr 27, 30, 34, and 36). This study is the first report of the gland-specific sulfation of a salivary phosphopeptide in vivo. Keywords: human saliva • histatin 1 • sulfated peptides • parotid glands • submandibular/sublingual glands • HPLCESI-MS • MALDI-TOF-MS

Introduction Histatins are low molecular weight histidine-rich peptides specific of human saliva secreted by major and minor salivary glands.1,2 The parent histatins, namely, histatin 1 (His-1) and histatin 3 (His-3) (see Figure 1 for their sequence), are coded by HTN1 and HTN2 genes, located on chromosome 4q13.3 Histatin 3 is submitted to a presecretory fragmentation,4 and some of its fragments, mainly His-5 (histatin3 1/24), show powerful antifungal and antimicrobial properties. Conversely, His-1, a peptide of 38 amino acid residues, phosphorylated on Ser 2, largely sharing its sequence with His-3 (Figure 1), is not submitted to fragmentation, probably due to the lack the proprotein convertase consensus sequence present in His-1. In this study the detection and characterization of several tyrosine-sulfated derivatives of histatin 1 are described. Tyrosine sulfation is a widespread post-translational modification (PTM)5 implicated in the intracellular trafficking of secreted peptides,6 in optimal biological activity of a number of proteins and peptides,7 in the modulation of extracellular protein-protein interactions,5 in the modulation of several * To whom correspondence should be addressed. Phone: +39 070 6754520. Fax: +39 070 6754523. E-mail: [email protected]. † Cagliari University. ‡ Catholic University. § University of Aveiro. | Catholic University of Rome.

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receptors,8 and in the promotion of proteolytic processing of some peptide hormones.9 Sulfation of tyrosines occurs by the transfer of sulfate from the universal sulfate donor 3′-phosphoadenosine 5′-phosphosulfate to the hydroxyl group of tyrosine residues. Until now two different tyrosylprotein sulfotransferases (TPST1 and TPST2) have been identified,10,11 and all mammalian cell types and cell lines studied to date express both TPSTs. Queries for tyrosine sulfation in the Swiss-Prot Data Bank in December 2006 provided 305 entries. However, the number of reported human tyrosine O-sulfated peptides/proteins is actually 37, because the majority of the entries in the data bank corresponded to the same protein of different organisms. Up to now accurate information concerning the structural modification of proteins and peptides connected to tyrosine Osulfation is lacking, and how these modifications can modulate protein function is not well understood. However, the most accepted biological role is the modulation of protein-protein interactions of secreted and membrane-bound proteins.5,12 It has also been reported that multiple sulfations of phosphoproteins is a PTM event involved in the maturation of rat bone sialoprotein II and osteospondin.13 Interestingly, mice null for TPST-1 (tyrosylprotein sulfotransferase 1) showed a ∼5% lower average body weight than wild-type animals.14 In the present paper we report for the first time that sulfation occurs on a salivary phosphopeptide in vivo and interestingly 10.1021/pr0700706 CCC: $37.00

 2007 American Chemical Society

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Figure 1. Sequences of histatin 1 (top) and histatin 3 (bottom).

histatin 1 sulfo derivatives were specifically detected in submandibular glands.

Experimental Section Reagents. O-Sulfotyrosine and O-phosphotyrosine were purchased from Bachem (Bubendorf, Switzerland). Calf intestinal alkaline phosphatase was purchased from BoehringerMannheim Biochemicals (Indianapolis, IN). Pronase, proteinase K, immobilized Staphylococcus aureus V-8 protease, and immobilized trypsin were purchased from Pierce Biotechnology (Rockford, IL). MALDI peptide calibration standards and R-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Bruker Daltonics (Bremen, Germany). All general chemicals were purchased from Farmitalia-Carlo Erba (Milan, Italy), Merck (Damstadt, Germany), and Sigma-Aldrich (St. Louis, MO). Apparatus. The HPLC-ESI-MS instrument was a ThermoFinnigan (San Jose, CA) apparatus. The Surveyor HPLC system was equipped with a PDA (photodiode array) detector and connected by a T splitter to the electrospray ionization/ ion trap mass spectrometer LCQ Deca XP Plus (ThermoFinnigan). The chromatographic column was a Vydac (Hesperia, CA) C8 with a 5 µm particle diameter (column dimensions 150 × 2.1 mm). The matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometer was an Autoflex apparatus from Bruker Daltonics. The semipreparative HPLC instrument was a Surveyor Plus LC system (ThermoFinnigan) equipped with a Surveyor Pump Plus and a Surveyor PDA Plus detector. The chromatographic column was a Hypersil BDS (Agilent Technologies, Santa Clara, CA) C18 (column dimensions 100 × 4.6 mm). The capillary electrophoresis (CE) apparatus was an Agilent Technologies HP3DCE instrument equipped with a diode array UV detector. The separation was performed using a fused-silica uncoated capillary of 50 µm i.d. and 375 µm o.d. (Composite Metal Services, Hallow, Worcs, U.K.). The CE apparatus was coupled to an Esquire 3000 plus ion trap mass spectrometer (Bruker Daltonics) via a coaxial sheet liquid ESI interface (Agilent Technologies). Sample Collection. Whole saliva was collected from normal adult volunteers between 2:00 and 4:00 p.m. by a soft plastic aspirator. Selected saliva from parotid was collected either by a Lashley’s cup or by small plastic aspirators at the duct exit. Saliva from submandibular glands was collected by a small aspirator at the duct exit. This method of collection did not allow exclusion of contaminations from sublingual secretion. The samples were immediately added to aqueous TFA (0.2%) in a 1:1 (v/v) proportion in an ice bath. The solution was centrifuged at 10000g for 5 min (4 °C), and the acidic supernatant was either immediately analyzed by HPLC-MS apparatus or stored at -80 °C.

Parotid and submandibular gland specimens were obtained by surgically preserving a resection from informed donors affected by salivary gland neoplasia. The experimental protocol was approved by the Ethical Committee of the Faculty of Medicine of the Catholic University. Pieces of surgically removed glands considered eligible on the basis of the extemporaneous histopathological analysis were used to obtain enriched secretory granule preparations. The pieces obtained were immediately used to make enriched granule preparations. Preparation of Salivary Granules and Their Extracts. Granules were isolated according to the procedure of Silva and co-workers15 with some modifications. The salivary gland (1.2 ( 0.2 g, N ) 6, parotid tissue; 0.7 ( 0.4 g, N ) 4, submandibular tissue) was washed with a 0.9% NaCl aqueous solution to eliminate blood, minced into small pieces by using a scalpel, and then homogenized in a glass potter with a Teflon pestle as a 5% (w/v) suspension in 340 mM sucrose, 0.5 mM EDTA, 10 mM HEPES, pH 7.4, at room temperature. To remove fibrous connective tissue and insoluble particles, the homogenate was filtered through four layers of clean coarse gauze in the homogenizing medium and then centrifuged at 500g for 10 min at 4 °C. The supernatant was submitted to further centrifugation at 2500g for 15 min at 4 °C, and the pellet, corresponding to the crude fraction of secretory granules, as verified by scanning electron microscopy (S4000 FEG SEM instrument, Hitachi, Tokyo, Japan), was solubilized in 700 µL of 0.2% trifluoroacetic acid (TFA). The solution was centrifuged at 8000g for 10 min, and CHCl3 was added to the supernatant (1:1, v/v) to remove lipidic components. The aqueous phase was directly used for RP-HPLC-ESI-IT-MS analyses. RP-HPLC-ESI-MS Analysis. Salivary proteins were separated by RP-HPLC-ESI-MS using the following solutions: (eluent A) 0.056% aqueous TFA and (eluent B) 0.050% TFA in acetonitrile/water, 80:20 (v/v). The applied gradient was linear from 0% to 55% in 40 min, at a flow rate of 0.30 mL/min. A T splitter addressed a flow rate of 0.20 mL/min toward the diode array detector and a flow rate of 0.10 mL/min toward the ESI source. The mass spectra were collected in the positive ion mode every 3 ms in the range of 300-2000 m/z values. The MS spray voltage was 4.50 kV, and the capillary temperature was 220 °C. Enriched Preparations of Histatin 1 Derivatives. Histatin 1 and its derivatives were partially purified by precipitation following a modification of the method of Flora et al.16 Different salivary samples were treated with TFA, as described above, and the acidic solution (about 400 mL) was diluted 1:1 with 0.5 mM zinc chloride, the pH of solution brought up to 9.0 by adding 0.5 M NaOH, and the solution stored on ice for 20 min. Then the suspension was centrifuged at 15000g for 20 min at 4 °C. The precipitate was washed with distilled water and dissolved in 3 M HCl. The solution was dialyzed overnight in 30 mM acetate buffer, 10 mM EDTA, pH 5.7, and then lyophilized. The lyophilized samples were submitted to semiJournal of Proteome Research • Vol. 6, No. 7, 2007 2473

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Figure 2. Enlargement of a typical RP-HPLC-ESI-MS profile of the acidic soluble fraction of whole human saliva (19-24 min elution range): (a) TIC profile; (b) UV profile at 276 nm; (c-i) extracted ion current (XIC) profiles obtained by searching the tetra- and tricharged ions of histatin 2, histatin 1, His-1 + 80.0 Da, His-1 + 160.0 Da, His-1 + 240.0 Da, His-1 + 320.0 Da, and His-1 + 400.0 Da, respectively. The other two panels on the right show the ESI spectrum collected in the range 20.28-21.66 min (average of 55 spectra, top) and the corresponding deconvolution spectrum (bottom).

preparative liquid chromatography to separate and collect histatin 1 derivatives using the following eluents: 0.056% aqueous TFA (eluent A) and 0.05% TFA in acetonitrile/water, 80:20 (eluent B). The applied gradient was from 0% to 10% B in 1 min and from 10% to 37% B in 35 min, at a flow rate of 0.9 mL/min. Five fractions enriched with His-1 and its derivatives were collected. The composition of the fractions was checked by HPLC-ESI-MS. The approximate total quantity of proteins present in the enriched derivative preparations was in the range 0.2-1.2 mg. CE-ESI-MS Analysis. The stock solution of sulfotyrosine was 1 mg/mL in water; that of phosphotyrosine was 0.5 mg/mL in 0.05 M NaOH. Diluted and mixed solutions were prepared from the stock solutions. Separation of the two amino acids was carried out in the cathodic mode (30 kV). The samples were injected at the anodic inlet of the capillary by applying a pressure of 50 mbar for 4 s. The electrophoretic runs were carried out at 25 °C in 20 mM ammonium acetate buffer, pH 8. Before each run the capillary was equilibrated for 1 min with water and for 2 min with the running buffer. The CE apparatus was coupled to the mass spectrometer through a coaxial sheath-flow interface. The sheath liquid was 0.5% (v/v) ammonium hydroxide in 70% methanol, and it was delivered by an external syringe pump (Cole Palmer, Vernon Hills, IL) at a flow rate of 180 µL/h. The capillary voltage was 2474

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maintained at 3.5 kV. The nebulizing gas pressure and drying gas flow were set at 5 psi and 4 L/min, respectively. The drying gas temperature was 300 °C. The mass spectrometer was operating in the negative ion mode. Spectra were collected in MRM (multireaction monitoring) mode. The ions were isolated using a width of (4.0 m/z and fragmented with a set amplitude value of 0.6. The spectrometer was scanned from 50 m/z to 400 m/z using 100 ms as the maximum accumulation time. Enzymatic Dephosphorylation. Submandibular gland secretions and the enriched preparation of histatin 1 derivatives (EDPs) were submitted to digestion as follows: (a) EDPs (ca. 50 µg) were dissolved in 100 µL of 0.2 M Tris-HCl (pH 8.6), and 40 µL of calf intestinal alkaline phosphatase (1 EU/µL) was added; (b) 200 µL of submandibular gland secretion was added to 40 µL of 1% aqueous TFA. The solution was centrifuged at 8000g and the supernatant added to 110 µL of 0.2 M Tris-HCl (pH 8.6) and 50 µL of calf intestinal alkaline phosphatase solution (1 EU/ µL). Both incubations were carried out at 37 °C, and after 30 min the solutions were centrifuged at 8000g for 5 min. The solution was immediately analyzed by RP-HPLC-ESI-MS. Acidic Hydrolysis. Desulfation of submandibular gland secretions and the enriched preparation of histatin 1 derivatives (EDPs) was performed according to a modification of the method of Ecarot-Charriet et al.:13 (a) 100 µL of submandibular

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Figure 4. Relative abundances of His-1 and His-2 and their sulfo derivatives calculated on the basis of the area of the XIC peaks detected in enriched derivative preparations before any treatment (middle panel), after alkaline phosphatase hydrolysis (upper panel), and after acidic hydrolysis (lower panel). Figure 3. RP-HPLC-ESI-MS profiles of enriched granule preparations from the submandibular gland. (a) TIC profile; (b-h) XIC peaks obtained by searching the tetra- and tricharged ions of histatin 2, histatin 1, His-1 + 80.0 Da, His-1 + 160.0 Da, His-1 + 240.0 Da, His-1 + 320.0 Da, and His-1 + 400.0 Da (not detected). Relative abundances of His-1 and His-2 and their derivatives, calculated on the basis of the area of the XIC peaks, are reported in the bottom panel.

gland secretion was added to 100 µL of 2 M HCl. The solution was centrifuged and the pellet discharged; (b) EDPs (ca. 30 µg) were dissolved in 20 µL of TFA, and 20 µL of 2 M HCl was added. Both samples were incubated at 100 °C for 6 min. At the end of incubation, the samples were cooled in ice and the solutions immediately analyzed by RP-HPLC-ESI-MS. Pronase Digestion of EDPs. The enriched preparation of histatin 1 derivatives (ca. 50 µg) was dissolved in 200 µL of 0.2 M Tris-HCl buffer (pH 8.6), and 2 units of pronase (powder) was added. Digestion was carried out at 37 °C, and after 24 h the solution was frozen and stored at -80 °C until CE-ESIMS analysis. Proteolytic Digestion of EDPs. Enriched preparations of histatin 1 derivatives were digested with trypsin, proteinase K, and protease V-8 from S. aureus. Trypsin digestion was carried out at 37 °C under stirring for 5 h by adding 10 units of immobilized trypsin to ca. 50 µg of lyophilized EDPs dissolved in 250 µL of 0.5 M ammonium hydrogen carbonate (pH 8.0). At the end of incubation, the solution was centrifuged at 8000g for 5 min and the supernatant lyophilized and stored at -20 °C. Proteinase K digestion was carried out at 56 °C under

stirring for 3 h by adding 50 µg/mL proteinase K (>30 units/ mg) to ca. 50 µg of lyophilized EDPs dissolved in 260 µL of 50 mM Tris-HCl, 5 mM CaCl2, pH 7.5. Digestion was stopped by adding phenylmethanesulfonyl fluoride (final concentration 5 mM). The solution was lyophilized and stored at -20 °C. Protease V-8 digestion was carried out at 37 °C under stirring for 7 h by adding 40 µL of protease V-8 (35-60 units/mL) to ca. 50 µg of lyophilized EDPs dissolved in 160 µL of 100 mM sodium phosphate solution, pH 7.8. At the end of incubation, the solution was centrifuged at 8000g for 5 min and the supernatant lyophilized and stored at -20 °C. MALDI-TOF and HPLC-ESI-MS/MS. Lyophilized samples of digested EDPs were dissolved in 0.2% TFA and analyzed by RP-HPLC-ESI-MS/MS. For MALDI-TOF-MS analysis, lyophilized samples were dissolved in 0.1% aqueous TFA and the solution was treated with a C-18 ZipTip micropipet to remove salts. The desalted solution was mixed 1:1 (v/v) with a solution of CHCA, prepared in acetonitrile/water (50:50, v/v) containing 0.1% TFA. Aliquots of 1 µL of the mixture were spotted on the stainless steel target of the MALDI instrument. The calibration was performed using peptide calibration standards (angiotensin I and II, substance P, and bombesin, m/z range 1000-3150 Da). Positive and negative MALDI spectra were acquired in either linear or reflectron mode with a pulsed nitrogen laser (337 nm). In linear mode an acceleration voltage of 20 kV, a detector gain voltage of 1300 V, a pulsed ion extraction time of 350 ns, and a laser frequency of 5 Hz were applied. In the reflectron mode an acceleration voltage of 19 kV, a detector gain voltage of 1400 V, a pulsed ion extraction time of 150 ns, and a laser frequency of 5 Hz were applied. Mass spectra were acquired over the mass Journal of Proteome Research • Vol. 6, No. 7, 2007 2475

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Figure 5. CE-MS/MS electropherogram (MRM mode; for details see the Experimental Section) of the O-sulfotyrosine standard (upper panels) and the pronase digestion mixture obtained from enriched derivative preparations (bottom panels).

range of 700-6000 Da with a low-mass cutoff of 500 Da, and about 400 scans were averaged for each spectrum. The sequence of some peptides was confirmed by RPHPLC-ESI-MS/MS analysis performed on doubly or triply charged ions detected with a peak width of 2-4 m/z value by using 40% of the maximum activation amplitude. Data Treatment. Deconvolution of averaged ESI-MS spectra was automatically performed by using either the Bioworks Browser software provided with the Deca XP instrument or MagTran 1.0 software.17 The mass values obtained were compared with average theoretical values using PeptideMass and FindPept programs available at the Swiss-Prot Data Bank (us.expasy.org/tools) where histatin 1 has the code P15515. Experimental MS/MS spectra were manually compared with the theoretical spectra generated by utilizing the MS product program, available at the Protein Prospector Web site (prospector.ucsf.edu/). Peptide identification was considered positive when the differences between the experimental and theoretical values were less than 0.2 m/z.

Results and Discussion Detection of His-1 Derivatives and Characterization of the Modification. In the RP-HPLC-ESI-IT-MS profile of whole saliva from normal subjects several peptides showing a serial mass increase of 80.0 ( 0.1 Da with respect to His-1 were consistently detected just before the peak of His-1 (His-1, 4928.4 2476

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( 0.5 Da; His-1 derivatives, 5008.6 ( 0.5, 5088.6 ( 0.5, 5168.2 ( 0.5, and 5248.4 ( 0.5 Da). The masses were detected in saliva of both males and females. Panel a of Figure 2 shows an enlargement of the chromatographic region where histatin 1 and acidic PRPs elute. Panel b shows the corresponding UV profile revealed at 276 nm, and panels c and d show the extracted ion current peaks of His-2 (nonphosphorylated His1, average mass 4849.2 Da) and His-1 (average mass 4929.2 Da) revealed by exclusively searching the corresponding tri- and tetracharged ions (His-2, 1616.9-1617.9 + 1212.8-1213.8 m/z; His-1, 1643.6-1644.6 + 1232.8-1233.8 m/z). Panels e-i show the results of the search of the tri- and tetracharged ions corresponding to His-1 derivatives showing mass increases of 80.0 Da (e, 1670.2-1671.2 + 1252.8-1253.8 m/z), 160.0 Da (f, 1696.9-1697.9 + 1272.8-1273.8 m/z), 240.0 Da (g, 1723.61724.6 + 1292.8-1293.8 m/z), 320.0 Da (h, 1750.2-1751.2 + 1312.8-1313.8 m/z), and 400.0 Da (i, 1776.8-1777.8 + 1332.81333.8 m/z), respectively. From the figure it is evident that all the searched masses were detected, with the exception of that corresponding to 5329.2 Da (His-1 + 400.0 Da). The serial mass increases of 80.0 Da could be attributed to either multiple phosphorylations or multiple sulfations of the parent peptide. Histatin 1 has three serine residues in its sequence, and one is phosphorylated (Ser 2) (Figure 1). Nonphosphorylated His-1 (average mass 4849.2 Da) is consistently detectable in whole saliva of normal subjects, and according to Baum et al.,18 it is

Polysulfated Derivatives of Human Salivary His-1

Figure 6. Linear MALDI-TOF-MS in positive and negative modes of an enriched derivative preparation showing the m/z values of monocharged His-2 ([M + H]+ theoretical average 4849.2), His-1 ([M + H]+ theoretical average 4929.2), His-1 monosulfated ([M + H]+ theoretical average 5009.3), His-1 bisulfated ([M + H]+ theoretical average 5089.4), and His-1 trisulfated ([M + H]+ theoretical average 5169.4).

called histatin 2 (Figure 1). In this respect, it is worthwhile to recall that other authors named histatin 2 fragment 12-38 of histatin 1.1 (For the different nomenclatures used to name histatins see refs 4 and 19). In the sequence of His-1 five tyrosine residues are also present, which can be targets of PTMs responsible for the observed mass increases. Selected secretions of parotid (Pr) and submandibular/ sublingual (Sm/Sl) glands, as well as granule preparations from Pr or Sm gland tissue, were analyzed by RP-HPLC-ESI-MS, and His-1 derivatives were detected only in selected secretions of Sm/Sl glands and EGP (enriched granule preparation) from Sm glands (Figure 3). Enriched preparations of His-1 and its derivatives (EDPs) obtained by zinc precipitation from large volumes of both whole saliva and selected secretions of Sm/Sl glands were submitted to semipreparative RP-HPLC to isolate pure peptides. However, due to the very close elution (Figure 2) it was not possible to completely purify each derivative, and only mixtures with a variable percentage of the different peptides were obtained, as shown by ESI-MS. Selected secretions of submandibular glands and EDPs were both incubated with intestinal calf alkaline phosphatase. The main phosphopeptides (i.e., PRP-1 and PRP-3, statherin, cystatin S1 and S2, II-2 and IB-1 basic PRP) present in submandibular secretion were completely dephosphorylated after 30 min of incubation, as shown by RP-HPLC-MS analysis (data not reported), indicating that the enzyme was fully active. In the parallel experiment performed on EDPs, His-1 and its derivatives showed a mass decrease of only 80.0 Da in agreement with the presence of a single phosphate group (Figure 4). These results strongly suggested the presence of sulfated

research articles residues in His-1 derivatives. The effective use of phosphatases to evidence phosphorylation of various peptides has already been reported,20 whereas arylsulfatases resulted in limited desulfation of O-sulfotyrosines, even by using large enzyme amounts.21 However, since O-sulfotyrosine is labile to acidic conditions, while O-phosphotyrosine is not,21,22 we treated EDPs with 1 M HCl at 100 °C for 6 min. The acidic hydrolysis provided the disappearance of the peak of His-1 derivatives, and almost only His-1 and His-2 were detected in the RPHPLC-MS profile (Figure 4), strongly supporting the presence of O-sulfotyrosine residues in His-1 derivatives. The increased percentage of His-2 after acidic hydrolysis (Figure 4) suggests that phosphorylation of Ser 2 is not a structural requisite for O-tyrosine sulfation. To obtain a direct proof of the presence of sulfo-Tyr, EDPs were extensively digested with pronase at pH 8.6, and the digestion products were analyzed by CE-MS. The results compared with those obtained using standards of O-sulfotyrosine and O-phosphotyrosine indicated that sulfo-Tyr, but not phospho-Tyr, was present in the digestion mixture (Figure 5). It has been reported that small peptides containing sulfotyrosine can be differentiated from peptides containing phosphotyrosine by differential negative and positive linear MALDITOF-MS experiments.21 Indeed, the experimental mass of sulfoTyr peptides measured by linear positive MALDI-TOF-MS corresponds to that of the desulfated peptide, due to an ion source decay which triggers the neutral loss of SO3. On the contrary, in negative MALDI spectra sulfate modification is retained and the experimental mass corresponds to that of the sulfopeptide. This different behavior is not observed with phosphotyrosine peptides. It is also reported that the differential analysis provides satisfactory results only for peptides with a mass lower than 2500-3000 Da.21 In agreement, linear negative MALDI-TOF-MS experiments performed on His-1 and its sulfo derivatives did not provide results different from the positive ones (Figure 6). RP-HPLC-ESI-MS/MS experiments performed on His-1 derivatives revealed a series of 80.0 Da losses, which was in agreement with the number of sulfates present in the peptide, and one 98.0 Da loss, attributed to the loss of H3PO4 from phosphoserine 2. The SIM MS/MS spectrum of the tetrasulfo derivative of His-1 (average mass of 5248.4 ( 0.5 Da) shows the four neutral losses of SO3 from sulfotyrosines and one neutral loss of H3PO4 from phosphoserine residues (Figure 7). The isobaric mass increase deriving both from sulfation and phosphorylation may generate serious analytical problems for the distinction between these two PTMs.22 This analytical challenge is today complicated by the recent detection of sulfation further than tyrosine residues also on serine and threonine residues.23 At this moment, no general and simple analytical schemes for a distinction between the two PTMs can be defined. The detection of sulfotyrosine in the pronase digestion mixtures of EDPs provided direct evidence for this PTM on His-1 derivatives. However, it required the collection of great amounts of EDPs and the high sensitivity of the CEMS apparatus. Any approach for sulfate/phosphate characterization offers different advantages and disadvantage, and a standard method for a definitive discrimination was until now a challenging analytical problem. Localization of the Sulfotyrosines. Different EDPs were digested by trypsin, protease V8, and proteinase K, and the digestion mixtures were analyzed both by RP-HPLC-ESI-MS/ MS and by positive and negative MALDI-TOF-MS. In the Journal of Proteome Research • Vol. 6, No. 7, 2007 2477

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Figure 7. ESI-MS/MS spectrum of the tetracharged ion of tetrasulfated His-1 (m/z 1313.0).

Figure 8. ESI-MS/MS spectrum of the doubly charged ion of the tetrasulfated 17-38 fragment of His-1 (m/z ) 3067.6/2 ) 1533.8; protease V-8 digest).

analysis of 86 fragments sulfation was always detected on the last four tyrosines of His-1, namely, Tyr 27, 30, 34, and 36. HPLC-ESI-IT-MS/MS analysis of fragment 17-38 (protease V-8 digest) containing four sulfated tyrosines is reported in Figure 8. Because the MS/MS experiment was performed on the doubly charged ion (m/z 1533.8), four consecutive neutral losses of 40 Da were observed as the main fragmentation signals. The main loss(es) of SO3 in MS/MS spectra was distinctive for any fragment obtained by protease digestion and bearing O-sulfotyrosine modification(s). Figure 9 shows that the bisulfated, monosulfated, and nonsulfated fragment 17-38 (protease V-8 digest) can be detected in the linear negative MALDI-TOF-MS spectrum, but not in the linear positive mode. On the contrary, ESI-IT-MS/MS experiments in 2478

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the positive mode provided almost a fingerprint of the sulfated and phosphorylated fragments, because the major event detected in sulfated peptides was the loss of SO3 (80.0 Da or a fraction of this value, according to the charge of the fragment), while the major fragmentation of phosphorylated peptides was the loss of H3PO4 (98.0 Da or a fraction of this value, according to the charge of the fragment). The two losses could be detected concurrently in peptides bearing both modifications (Figure 7). Unfortunately, since these losses almost completely suppress the classical fragmentation which originates the y and b series ions, detailed information on the site of modification cannot be obtained. According to this study, the terminal four tyrosine residues at the 27, 30, 34, and 36 positions clustered near the C-terminus

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Figure 9. Linear positive and negative MALDI-TOF-MS spectra of the bisulfated, monosulfated, and nonsulfated 17-38 fragment of His-1 (protease V-8 digest).

of the His-1 sequence are substrates of tyrosylprotein sulfotransferases. If the sequence of His-1 is submitted to Sulfinator (Swiss-Prot), only Tyr 27 is recognized as a probable site of sulfation. Indeed, the sequence surrounding Tyr 27 shows several features required for the recognition, such as the presence of two acidic amino acid residues at positions +2 and -4, only one basic residue at -5 position, and several turninducing amino acid residues (Pro, Ser, Gly, Asn) in the (7 proximity.22,24 In agreement with the temporal sequence of O-sulfation reported for the four tyrosine residues of the CCR5 N-terminal peptide,25 it can be proposed that the O-sulfation of Tyr 27 in His-1 activates the other three Tyr residues, bringing on the following polysulfation. Unfortunately, since MS/MS spectra of the proteolytic peptides containing sulfated tyrosine were characterized by non-well-defined y and b fragmentation patterns, it was not possible to obtain any experimental support for the sulfation process hierarchy. On the other hand, His-3 has three clustered C-terminal Tyr residues embedded in a sequence strictly similar to that of His1, but it is lacking a residue equivalent to Tyr 27. The absence of detection of any sulfated derivative of His-3 strongly supports the hypothesis that sulfation of Tyr 27 is the starting event for the polysulfation of His-1.

Conclusions His-1 derivatives were detected only in selected secretions of Sm/Sl glands and granule preparations of Sm glands. Thus, polysulfation of the phosphopeptide histatin 1 seems to be a PTM event confined to submandibular glands occurring during Golgi transit, since sulfated derivatives were also detected in granule preparations of the gland. In this respect, it should be outlined that a tyrosylprotein sulfotransferase has been specifically detected at the level of the Golgi membranes of rat submandibular glands.26 On the basis of the present finding, it could be hypothesized that histatin 1 may play different roles

in different districts of the oral cavity. However, the gland specificity could also be connected to a noncomplete sulfate removal by arylsulfatase E27 in submandibular glands prior to granule storage, even though tyrosine sulfation is considered by many authors a nonreversible PTM. Interestingly, despite the very high sequence similarity of histatin 1 and histatin 3 (Figure 1), sulfated derivatives of His-3 were not detected, at least at the sensitive level of our analytical approach. Previous studies already highlighted that the two peptides follow completely different PTM pathways. For instance, His-1 is not submitted to proteolytic cleavages, differently from His-3, which generates by a sequential cleavage His-6 (histatin3 1/25) and subsequently His-5 (histatin3 1/24) and other fragments.4 His-1 is phosphorylated on the Ser 2 residue, while His-3 is not, probably due to the lack of the consensus sequence SXE (or SXS(Phos)), recognized by the Golgi kinase involved in serine phosphorylation of salivary peptides. Conclusively, the differences observed up to now in His-1 and His-3 cleavage, phosphorylation and sulfation processes, strongly address distinct roles of the two parent histatins in the oral cavity. Abbreviations: Pr, Parotid; Sm/Sl, submandibular/sublingual; EDP, enriched derivatives preparation; His-1, histatin 1; His-3, histatin 3; CHCA, R-cyano-4-hydroxycinnamic acid; RPHPLC, reversed-phase high-pressure liquid chromatography; ESI-IT, electrospray ionization ion trap; MS, mass spectrometry; SIM, selected ion monitoring; MS/MS, tandem mass spectrometry; TIC, total ion current; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; CE, capillary electrophoresis; MRM, multireaction monitoring; PTM, post-translational modification; EGP, enriched granule preparation; XIC, extracted ion current; PDA, photodiode array.

Acknowledgment. We acknowledge the financial support of Cagliari University, the Catholic University in Rome, the International Scientific Institute “Paolo VI” (ISI), MIUR, the Italian National Council (CNR), and Regione Sardegna, thanks to their programs of scientific research promotion and diffusion. References (1) Oppenheim, F. G.; Xu, T.; McMillian, F. M.; Levitz, S. M.; Diamond, R. D.; Offner, G. D.; Troxler, R. F. J. Biol. Chem. 1988, 263, 7472-7477. (2) Piludu, M.; Lantini, M. S.; Cossu, M.; Piras, M.; Oppenheim, F. G.; Helmerorst E. J.; Siqueira, W.; Hand, A. R. Arch. Oral Biol. 2006, 51, 967-973. (3) Sabatini, L. M.; Ota, T.; Azen, E. A. Mol. Biol. Evol. 1993, 10, 497511. (4) Castagnola, M.; Inzitari, R.; Rossetti, D. V.; Olmi C.; Cabras, T.; Piras, V.; Nicolussi, P.; Sanna, M. T.; Pellegrini, M.; Giardina, B.; Messana, I. J. Biol. Chem. 2004, 279, 41436-41443. (5) Kehoe, J. W.; Bertozzi C. R. Chem. Biol. 2000, 7, R57-R61. (6) Baeuerle, P. A.; Huttner, W. B. J. Cell Biol. 1987, 105, 2655-2664. (7) Hortin, G. L.; Farries, T. C.; Graham, J. P.; Atkinson, J. P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 1338-1342. (8) Farzan, M.; Mirzabekov, T.; Kolchinsky, P.; Wyatt, R.; Cayabyab, M.; Gerard, N. P.; Gerard, C.; Sodroski, J.; Choe, H. Cell 1999, 96, 667-676. (9) Bundgaard, J. R.; Vuust, J.; Rehefeld, J. F. EMBO J. 1995, 14, 30733079. (10) Beisswanger, R.; Corbeil, D.; Vannier, C.; Thiele, C.; Dohrmann, U.; Kelnner, R.; Ashman, K.; Niehrs, C.; Huttner, W.B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11134-11139. (11) Ouyang, Y. B.; Moore, K. L. J. Biol. Chem. 1998, 273, 24770-24774. (12) Woods, A.S.; Wang, H.Y., Jackson, S.N. J. Protein Res. 2007, 6, 1176-82. (13) Ecarot-Charrier, B.; Bouchard, F.; Delloye, C. J. Biol. Chem. 1989, 264, 20049-20053.

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