- Email: [email protected]
In vitro hydroxyapatite adsorbed salivary proteins
BBRC Biochemical and Biophysical Research Communications 320 (2004) 342–346 www.elsevier.com/locate/ybbrc
In vitro hydroxyapatite adsorbed salivary proteins ~o C. Lobo,b Jose Duarte,d Anto nio J. Ferrer-Correia,a Rui Vitorino,a Maria Joa c c Kenneth B. Tomer, Joshua R. Dubin, Pedro M. Domingues,a and Francisco M.L. Amadoa,* a
c
Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal b Instituto Superior de Saude—Norte, Portugal Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, Research Triangle Park, NC 27709, USA d Faculty of Sport Sciences and Physical Education, Porto University, 4200 450 Porto, Portugal Received 12 May 2004 Available online 11 June 2004
Abstract In spite of the present knowledge about saliva components and their respective functions, the mechanism(s) of pellicle and dental plaque formation have hitherto remained obscure. This has prompted recent efforts on in vitro studies using hydroxyapatite (HA) as an enamel model. In the present study salivary proteins adsorbed to HA were extracted with TFA and EDTA and resolved by 2D electrophoresis over a pH range between 3 and 10, digested, and then analysed by MALDI-TOF/TOF mass spectrometry and tandem mass spectrometry. Nineteen different proteins were identified using automated MS and MS/MS data acquisition. Among them, cystatins, amylase, carbonic anhydrase, and calgranulin B, were identified. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Hydroxyapatite; Saliva; Two-dimensional electrophoresis; MALDI-TOF/TOF
Formation of enamel pellicle is the result of a selective adsorption of salivary components to the tooth. Enamel pellicle provides a physical and chemical support as for protective interface between the tooth surface and the oral environment. This interface acts as a selective permeable barrier that regulates mineralization/ demineralization processes and also controls the composition of the microbial flora that forms dental plaque [1–3]. Elucidation of the macromolecular composition of in vivo acquired enamel pellicle has been achieved by using different analytical approaches such as amino acid analysis [1], immunological identification [4], electronmicroscopy [5], and electrophoretic methods applied to proteins adsorbed to HA in vitro, which resulted in the detection of several proteins such as proline-rich proteins (PRPs), statherin, histatins, mucin MG1, S-IgA, * Corresponding author. Fax: +351234370084. E-mail address: [email protected] (F.M.L. Amado).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.05.169
amylase, cystatin SA-I, parotid agglutinin, serum albumin, and carbonic anhydrase VI [2,5–8]. Due to the low amount of protein extractable from the pellicle in vivo, most of the studies have been performed in vitro, replacing enamel with its main component hydroxyapatite [3]. The presence of post-translational modifications, in particular phosphorylations, such as seryl phosphates at residues 8 and 22 in PRPs, at residues 2 and 3 of statherin and at residue 2 of histatin 1, is thought to be a major factor contributing to the high affinity of salivary proteins for hydroxyapatite [5,9]. Cationic proteins such as lysozyme [3] and other salivary components (secretory IgA, parotid agglutinin, and a-amylase) [10,11] are also present. These have been described as pellicle precursors for oral streptococcal attachment to HA. In this way, the aim of our work was the separation and identification of proteins adsorbed onto HA using two-dimensional gel electrophoresis and mass spectrometry (using an MALDI-TOF/TOF instrument).
R. Vitorino et al. / Biochemical and Biophysical Research Communications 320 (2004) 342–346
343
Experimental procedures
Protein identification based on tryptic peptide spectra
Chemicals
Spectra were processed and analysed by the Global Protein Server Workstation (Applied Biosystems, Foster City, CA, USA), which uses internal Mascot software (Matrix Science, UK) for searching the peptide mass fingerprints and MS/MS data. Searches were performed against the NCBI non-redundant protein database. Further confirmation of protein identification was obtained using Protein Prospector (www.prospector.ucsf.edu, from the University of California at San Francisco) and/or Prowl (www.prowl.rockfeller.com, Rockefeller University at New York Universities) software.
All general reagents used were analytical grade quality. Sample preparation Hydroxyapatite adsorption of salivary proteins. Whole human unstimulated saliva (WS) was collected from 20 healthy male donors according to standardised procedures defined by the World Health Organisation (WHO) at 8 a.m. in starvation. After collection, WS was immediately centrifuged at 12000 rpm for 20 min at 4 °C to remove particulate matter. The WS supernatant (1 mL) was added to 5 mg of HA powder (particle diameter of 120 lm; Fluka) according to Jensen et al. [12]. The incubation occurred for 2 h at 37 °C under continuous mixing. At the end time, the resulting suspensions were centrifuged at 10000 rpm for 10 min at 4 °C and the supernatants were discarded. 2D-sample preparation. The pellets resultant from HA and WS supernatant incubation were resuspended in 0.2 M EDTA, pH 7.5, under magnetic stirring, according to Yao et al. [13], and in 0.2% TFA under magnetic stirring, according to St€ oser et al. [14]. Both solutions were dialysed with 1000 mol. wt. cut-off membranes against subsequent changes of 0.1 M NaCl (2), 0.01 M NaCl (2), and distilled water (3) for the first case and for the second case against subsequent changes of TFA (2) and distilled water (2). 2D gel electrophoresis and tryptic digestion Two-dimensional gel electrophoresis (2-DGE) was performed in a horizontal apparatus (IPGphor and Hoefer 600 SE from Amersham– Pharmacia Biotech, Uppsala, Sweden) according to Vitorino et al. [15]. Briefly, 150 lg of protein was applied onto IPG strips (13 cm, pH 3– 10), containing immobilines for pH 3–10, thiourea, CHAPS, and urea according to Rabilloud et al. [17]. After isoelectric focusing, the strip was applied on top of a SDS–PAGE gel (12 by 14 cm, 12.5%) and proteins were separated according to molecular weight. The SDS– PAGE gel was stained using colloidal Coomassie blue. The protein spots were excised with a pipette tip from the gel and transferred to the Investigator ProGest automated digester (Genomic Solutions, Ann Arbor, MI, USA) rack. The gel pieces were washed twice with 25 mM ammonium bicarbonate/50% ACN and dried with nitrogen flow. Twenty-five microlitres of 10 lg/mL trypsin in 50 mM ammonium bicarbonate was added to the dried residue and the samples were incubated overnight at 37 °C with sequence grade modified porcine trypsin.
Results Identification of HA salivary proteins adsorbed using 2D gel electrophoresis and MS In this study, HA was incubated with whole saliva supernatant in individual assays with the 20 participating subjects. After HA incubations with whole saliva supernatant, it was possible to recover more than 90 5% of the adsorbed protein using either EDTA (0.2 M) [13] or TFA (2%) [16] in extraction. Since whole saliva composition is affected by circadian rhythm all saliva samples were collected at the same time (8.00 a.m.). Despite inter-individual variations in whole saliva composition, the 2D gel electrophoresis of the proteins of the extracts resulted in the detection of an average of 75 20 common spots after Coomassie colloidal staining and software spot detection (PDQuest v7.1, Bio-Rad). In Fig. 1, a typical 2-DE representative of the salivary proteins adsorbed onto HA is presented. The 2-DE profiles obtained using either trifluoracetic
MALDI-TOF/TOF for tryptic peptides Tryptic peptides were lyophilised and re-suspended in 10 lL of a 50% acetonitrile/0.1% formic acid solution. The samples were mixed (1:1) with a matrix consisting of a saturated solution of a-cyano-4hydroxycinnamic acid prepared in 50% acetonitrile/0.1% formic acid. Aliquots of samples (0.35 lL) were spotted onto the MALDI sample target plate using a SymBiot XVI Sample Workstation (Applied Biosystems, Foster City, CA, USA). Peptide mass spectra were obtained on a MALDI-TOF/TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Foster City, CA, USA) in the positive ion reflector mode. Two spots were used for signal and parameter optimisation. Spectra were obtained in the mass range between 800 and 4000 Da with ca. 1500 laser shots. A data dependent acquisition method was created to select the two most intense peaks in each sample spot for subsequent MS/MS data acquisition, excluding those from the matrix, due to trypsin autolysis, or acrylamide peaks. Trypsin autolysis peaks were used for internal calibration of the mass spectra, allowing a routine mass accuracy of better than 25 ppm.
Fig. 1. Two-dimensional gel map of an hydroxyapatite incubated saliva sample obtained in pH range between 3 and 10 and 12.5% of SDS–PAGE stained with colloidal Coomassie blue. Spots were identified by mass spectrometry with the exception of spot 17, which has been identified by differential staining (Table 1).
344
R. Vitorino et al. / Biochemical and Biophysical Research Communications 320 (2004) 342–346
acid or EDTA, solubilising solutions were very similar in terms of the number and the intensity of the detected protein spots (data not shown). Using pH focusing between pH 3 and 10, it is possible to observe that the majority of spots are visualised in the acidic zone of the gel. Spots visualised on the gel are also preferentially distributed on the low MW zone, below 30 KDa. Due to their primary sequences, PRPs are difficult to identify by MS using trypsin digestion. Alternatively, PRPs (spot series 18 Fig. 1) were identified using the modified Coomassie staining according to Beeley et al. [17], where PRPs are differentially revealed using methanol. All other proteins were positively identified with MALDI-TOF/TOF MS and MS/MS data. For mass spectrometry analysis, 60 spots from 2-DE gels of solubilised HA saliva samples, stained with colloidal Coomassie blue, were excised and digested with trypsin. Protein identification was performed based on a combination of tryptic peptide molecular weight from MS data and sequence information from MS/MS data. The resulting data were searched against a protein database using an internal Mascot search routine. As can be seen in Table 1, of the 70 spots excised, 48 spots corresponding to 18 different proteins were identified with high statistical reliability. Five of these proteins (cystatins C and D, calgranulin B, prolactin, and Igk/k light chain/VLJ chain) are, for the first time, reported to complex with HA. Other proteins that were identified by this approach include albumin, a-amylase, lysozyme, histatin 1, prolactin, carbonic anhydrase VI, the common cystatins (SN, S, SA-III, C, and D), and several Igs family members. Igk/k light chain/VLJ chain does not represent a single protein, but three different homologue proteins with several isoforms each with small MW and
pI differences, better characterised as a gel protein region [18]. Several spots were assigned as the same protein, which could be attributed to post-translational modifications, isoforms, and/or protein fragments. With the exception of spots 6 and 7, zinc-a-2-glycoprotein, all proteins were identified by the Mascot search routine as intact proteins. The four spots identified as cystatin S, spot series 10 (Fig. 1 and Table 1), presented data mining high scores (230, 160, 104, and 130, respectively). However, cystatin SA-III, Sap 1, cystatin S (acidic form) or cystatin SN were also other possible positive identifications (scores of 223, 220, 200, 168, respectively, for the first spot). According to the literature [19] cystatin S has only two variants (cystatin S1 and S2) that are phosphorylated at ser-3 and at ser-1 for the first variant and at ser-2 for the second. According to Lamkin et al. [9] cystatin SA-III presents high homology with cystatin S, but with different number of phosphorylations, three for cystatin S and four for cystatin SA-III. Taking into account the homology between them, and the obtained peptide masses used for identification, it is possible that some spots located on that region could be attributed to cystatin SA-III.
Discussion Addition of trifluoracetic acid [16] or EDTA [13] allows the solubilisation of HA and of the peptides and proteins adsorbed with high recoveries. On searching the literature for a comparison between the different solutions for extraction of proteins from HA, EDTA was
Table 1 Identified salivary proteins adsorbed to hydroxyapatite Spot
Protein namea
SWISS-PROT
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Poly-Ig receptor (IgA secretory chain) Serum albumin Immunoglobulin a-chain C region (Ig A) Human salivary a-amylase Carbonic anhydrase VI Zinc-a-2 glycoprotein Immunoglobulin k/k light chain VLJ chain Prolactin-inducible protein (prolactin) Cystatin S Cystatin SA-III pellicle precursor (Sap1) Calgranulin B (S100 calcium-binding protein A9) Cystatin SN Lysozyme b2-Microglobulin Histatin 1 Cystatin D Proline-rich proteinsb Cystatin C
P01833 P02768 P01878 P04746 P23280 P25311 AAC16777/AAB51622 P12273 P01036 AAB19889 P06702 P01037 P00695 P07151 P15515 P28325
a
P01034
All samples were identified by mass spectrometry with the exception of b which were identified by selective staining according to Beeley et al. [19].
R. Vitorino et al. / Biochemical and Biophysical Research Communications 320 (2004) 342–346
found to be one of the best HA solubilising agents [13]. TFA was only used by St€ oser et al. [16] but with no indication of protein recovery percentage. Applying the two experimental conditions proposed by these authors, no differences in percentage recovery of the total protein amount adsorbed to HA were observed (more than 90% in both cases). Also, the 2-DE patterns obtained using both solubilising solutions were very similar. This observation indicates that eventual acidic proteolysis promoted by TFA does not occur or is scarce in practical terms. The number of spots observed on the 2-DE HA sample gels (an average of 75) is much smaller than that observed on saliva 2-DE gels, obtained under similar experimental conditions (more than 200) [15]. Moreover, comparison between these samples shows that all the spots visualised on HA gels are also present on saliva sample gels and that the majority of proteins bound to HA are of low molecular weight. Although there is a strong interest in the characterisation of the macromolecular composition of in vivo acquired enamel pellicle, only a few reports have been published. The sum of all proteins identified then in the literature indicates that more than 40 salivary proteins have been reported to interact with HA. Most of the 18 proteins identified in our work by 2-DE were identified for the first time by mass spectrometry and five of these proteins (cystatins C and D, calgranulin B, prolactin, and Igk/k light chain VLJ chain) are reported for the first time to bind to HA. The identified proteins are involved in different oral functions as pellicle precursors, inflammatory, and microbial defence agents, pH buffers, and components associated with remineralisation processes, confirming previous proposals that enamel pellicle is a complex structure with manifold factors controlling tooth integrity. All those proteins have been recently identified in whole saliva by our group [15], and it is remarkable that, generally, HA possesses high selectivity to acidic and phosphorylated low molecular weight proteins. Keller et al. [20] have reported the presence of two groups of a-amylase isoforms in saliva with different molecular sizes, 63,000 and 59,000 Da. Isoforms belonging to the first group contain asparagine-linked sugar chains whereas the second group does not. As can be observed (spot series 4 Fig. 1, Table 1), only the group that contains asparagine-linked sugar chains was identified in HA absorbed proteins. This result suggests that the presence of sugar chains in proteins is important to their attachment to HA or even to acquired enamel pellicle. Comparing whole saliva 2D gel patterns [15] and HA incubated saliva 2D gel patterns, it is possible to observe an enrichment of spots in the cystatin S region (spot series 10). Cystatins are a group of known proteins present on saliva and are involved in the regulation of protein degradation at sites of inflammation or micro-
345
bial infection [21]. The presence of several cystatins (cystatins S, SN, D, C, and SAIII) adsorbed onto HA could be associated with the regulation of bacterial activity in parallel with lysozyme, another protein observed at high levels. Lysozyme has already been identified as a pellicle precursor [22]. Due to the presence of the phosphate groups, cystatins S and SAIII may also be directly associated with the remineralisation processes along with other proteins such as PRPs, histatin 1, and calgranulin B, emphasising their importance in maintenance of the crystal structure of HA. Furthermore, the presence of phosphorylations promotes an increase of the proteins’ acidic properties, and, thus, adsorption to HA and, as reported by Lamkin et al. [9], becoming potential pellicle precursors. Carbonic anhydrase VI (four spots detected) which has been reported to exert its buffer capacity predominantly on pellicle surface Kivel€a et al. [23] could also be associated with the remineralisation process.
Concluding remarks In order to elucidate the macromolecular structure of acquired pellicle, we used a proteomics approach to characterise proteins and peptides adsorbed to HA. By using 2-DE with extended pH range (3–10) and high sensitivity protein identification by MALDI-TOF/TOF, 48 spots from the 70 selected were identified. Among those that were identified are cystatins (S, SA-III, C, and D), lyzosyme, amylase (only glycosylated form), carbonic anhydrase VI, and calgranulin B.
References [1] M. Rykke, T. Sonju, Amino acid composition of acquired enamel pellicle collected in vivo after 2 h and after 24 h, Scand. J. Dent. Res. 99 (6) (1991) 463–469. [2] A. Carlen, A.C. Borjesson, K. Nikdel, J. Olsson, Composition of pellicles formed in vivo on tooth surfaces in different parts of the dentition, and in vitro on hydroxyapatite, Caries Res. 32 (6) (1998) 447–455. [3] U. Lendenmann, J. Grogan, F.G. Oppenheim, Saliva and dental pellicle—a review, Adv. Dent. Res. 14 (2000) 22–83. [4] J. Li, E.J. Helmerhorst, R.B. Corley, L.E. Luus, R.F. Troxler, F.G. Oppenheim, Characterization of the immunologic responses to human in vivo acquired enamel pellicle as a novel means to investigate its composition, Oral Microbiol. Immunol. 18 (3) (2003) 183–191. [5] P. Schupbach, F.G. Oppenheim, U. Lendenmann, M.S. Lamkin, Y. Yao, B. Guggenheim, Eur. J. Oral Sci. 109 (1) (2001) 60–68. [6] E.E. Kousvelari, R.S. Baratz, B. Burke, F.G. Oppenheim, Immunochemical identification and determination of proline-rich proteins in salivary secretions, enamel pellicle, and glandular tissue specimens, J. Dent. Res. 59 (8) (1980) 1430–1438. [7] I. Al-Hashimi, M.J. Levine, Characterization of in vivo salivaryderived enamel pellicle, Arch. Oral Biol. 34 (4) (1989) 289–295.
346
R. Vitorino et al. / Biochemical and Biophysical Research Communications 320 (2004) 342–346
[8] J. Leinonen, J. Kivela, S. Parkkila, A.K. Parkkila, H. Rajaniemi, Salivary carbonic anhydrase isoenzyme VI is located in the human enamel pellicle, Caries Res. 33 (3) (1999) 185–190. [9] M.S. Lamkin, F.G. Oppenheim, Structural features of salivary function, Crit. Rev. Oral Biol. Med. 4 (3–4) (1993) 251–259. [10] R.J. Gibbons, Adherent interactions which may affect microbial ecology in the mouth, J. Dent. Res. 63 (3) (1984) 378–385. [11] F.A. Scannapieco, G.I. Torres, M.J. Levine, Salivary amylase promotes adhesion of oral streptococci to hydroxyapatite, J. Dent. Res. 74 (7) (1995) 1360–1366. [12] J.L. Jensen, M.S. Lamkin, F.G. Oppenheim, Adsorption of human salivary proteins to hydroxyapatite: a comparison between whole saliva and glandular salivary secretions, J. Dent. Res. 71 (9) (1992) 1569–1576. [13] Y. Yao, J. Grogan, M. Zehnder, U. Lendenmann, B. Nam, Z. Wu, C.E. Costello, F.G. Oppenheim, Compositional analysis of human acquired enamel pellicle by mass spectrometry, Arch. Oral Biol. 46 (4) (2001) 293–303. [14] L. Stoser, N. Apitz, U. Keher, Th. Henning, In vitro pellicle formation with stimulated mixed saliva and hydroxyapatite, 6th European Symposium on Saliva, Poster 45, 2002. [15] R. Vitorino, M.J.C. Lobo, A.J. Ferrer-Correia, J.R. Dubin, K.B. Tomer, P.M. Domingues, F.M.L. Amado, Identification of human whole saliva protein components using proteomics, Proteomics 4 (2004) 1109–1115. [16] K.H. Eggen, G. Rolla, Gel filtration, ion exchange chromatography and chemical analysis of macromolecules present in acquired enamel pellicle (2-h-pellicle), Scand. J. Dent. Res. 90 (3) (1982) 182–188.
[17] J.A Beeley, F. Newman, P.H. Wilson, I.C. Shimmin, Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human parotid salivary proteins: comparison of dansylation, Coomassie blue R-250 and silver detection methods, Electrophoresis 17 (3) (1996) 505–506. [18] J.L. Dul, S. Abiel, J. Melnick, Y. Argon, Ig light chains are secreted predominantly as monomers, J. Immunol. 157 (7) (1996) 2969–2975. [19] S. Isemura, E. Saitoh, K. Sanada, K. Minakata, Identification of full-sized forms of salivary (S-type) cystatins (cystatin SN, cystatin SA, cystatin S, and two phosphorylated forms of cystatin S) in human whole saliva and determination of phosphorylation sites of cystatin S, J. Biochem. (Tokyo) 110 (4) (1991) 648– 654. [20] P.J. Keller, D.L Kauffman, B.J. Allan, B.L. Williams, Further studies on the structural differences between the isoenzymes of human parotid –amylase, Biochemistry. 10 (26) (1971) 4867– 74.J. [21] A. Baron, N. Barrett-Vespone, J. Featherstone, Purification of large quantities of human salivary cystatins S, SA and SN: their interactions with the model cysteine protease papain in a noninhibitory mode, Oral Dis. 5 (4) (1999) 344–353. [22] L.M. Tellefson, G.R. Germaine, Adherence of Streptococcus sanguis to hydroxyapatite coated with lysozyme and lysozymesupplemented saliva, Infect Immun. 51 (3) (1986) 750–759. [23] J. Kivela, S. Parkkila, A.K. Parkkila, H. Rajaniemi, A low concentration of carbonic anhydrase isoenzyme VI in whole saliva is associated with caries prevalence, Caries Res. 33 (3) (1999) 178– 184.
In vitro hydroxyapatite adsorbed salivary proteins ~o C. Lobo,b Jose Duarte,d Anto nio J. Ferrer-Correia,a Rui Vitorino,a Maria Joa c c Kenneth B. Tomer, Joshua R. Dubin, Pedro M. Domingues,a and Francisco M.L. Amadoa,* a
c
Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal b Instituto Superior de Saude—Norte, Portugal Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, Research Triangle Park, NC 27709, USA d Faculty of Sport Sciences and Physical Education, Porto University, 4200 450 Porto, Portugal Received 12 May 2004 Available online 11 June 2004
Abstract In spite of the present knowledge about saliva components and their respective functions, the mechanism(s) of pellicle and dental plaque formation have hitherto remained obscure. This has prompted recent efforts on in vitro studies using hydroxyapatite (HA) as an enamel model. In the present study salivary proteins adsorbed to HA were extracted with TFA and EDTA and resolved by 2D electrophoresis over a pH range between 3 and 10, digested, and then analysed by MALDI-TOF/TOF mass spectrometry and tandem mass spectrometry. Nineteen different proteins were identified using automated MS and MS/MS data acquisition. Among them, cystatins, amylase, carbonic anhydrase, and calgranulin B, were identified. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Hydroxyapatite; Saliva; Two-dimensional electrophoresis; MALDI-TOF/TOF
Formation of enamel pellicle is the result of a selective adsorption of salivary components to the tooth. Enamel pellicle provides a physical and chemical support as for protective interface between the tooth surface and the oral environment. This interface acts as a selective permeable barrier that regulates mineralization/ demineralization processes and also controls the composition of the microbial flora that forms dental plaque [1–3]. Elucidation of the macromolecular composition of in vivo acquired enamel pellicle has been achieved by using different analytical approaches such as amino acid analysis [1], immunological identification [4], electronmicroscopy [5], and electrophoretic methods applied to proteins adsorbed to HA in vitro, which resulted in the detection of several proteins such as proline-rich proteins (PRPs), statherin, histatins, mucin MG1, S-IgA, * Corresponding author. Fax: +351234370084. E-mail address: [email protected] (F.M.L. Amado).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.05.169
amylase, cystatin SA-I, parotid agglutinin, serum albumin, and carbonic anhydrase VI [2,5–8]. Due to the low amount of protein extractable from the pellicle in vivo, most of the studies have been performed in vitro, replacing enamel with its main component hydroxyapatite [3]. The presence of post-translational modifications, in particular phosphorylations, such as seryl phosphates at residues 8 and 22 in PRPs, at residues 2 and 3 of statherin and at residue 2 of histatin 1, is thought to be a major factor contributing to the high affinity of salivary proteins for hydroxyapatite [5,9]. Cationic proteins such as lysozyme [3] and other salivary components (secretory IgA, parotid agglutinin, and a-amylase) [10,11] are also present. These have been described as pellicle precursors for oral streptococcal attachment to HA. In this way, the aim of our work was the separation and identification of proteins adsorbed onto HA using two-dimensional gel electrophoresis and mass spectrometry (using an MALDI-TOF/TOF instrument).
R. Vitorino et al. / Biochemical and Biophysical Research Communications 320 (2004) 342–346
343
Experimental procedures
Protein identification based on tryptic peptide spectra
Chemicals
Spectra were processed and analysed by the Global Protein Server Workstation (Applied Biosystems, Foster City, CA, USA), which uses internal Mascot software (Matrix Science, UK) for searching the peptide mass fingerprints and MS/MS data. Searches were performed against the NCBI non-redundant protein database. Further confirmation of protein identification was obtained using Protein Prospector (www.prospector.ucsf.edu, from the University of California at San Francisco) and/or Prowl (www.prowl.rockfeller.com, Rockefeller University at New York Universities) software.
All general reagents used were analytical grade quality. Sample preparation Hydroxyapatite adsorption of salivary proteins. Whole human unstimulated saliva (WS) was collected from 20 healthy male donors according to standardised procedures defined by the World Health Organisation (WHO) at 8 a.m. in starvation. After collection, WS was immediately centrifuged at 12000 rpm for 20 min at 4 °C to remove particulate matter. The WS supernatant (1 mL) was added to 5 mg of HA powder (particle diameter of 120 lm; Fluka) according to Jensen et al. [12]. The incubation occurred for 2 h at 37 °C under continuous mixing. At the end time, the resulting suspensions were centrifuged at 10000 rpm for 10 min at 4 °C and the supernatants were discarded. 2D-sample preparation. The pellets resultant from HA and WS supernatant incubation were resuspended in 0.2 M EDTA, pH 7.5, under magnetic stirring, according to Yao et al. [13], and in 0.2% TFA under magnetic stirring, according to St€ oser et al. [14]. Both solutions were dialysed with 1000 mol. wt. cut-off membranes against subsequent changes of 0.1 M NaCl (2), 0.01 M NaCl (2), and distilled water (3) for the first case and for the second case against subsequent changes of TFA (2) and distilled water (2). 2D gel electrophoresis and tryptic digestion Two-dimensional gel electrophoresis (2-DGE) was performed in a horizontal apparatus (IPGphor and Hoefer 600 SE from Amersham– Pharmacia Biotech, Uppsala, Sweden) according to Vitorino et al. [15]. Briefly, 150 lg of protein was applied onto IPG strips (13 cm, pH 3– 10), containing immobilines for pH 3–10, thiourea, CHAPS, and urea according to Rabilloud et al. [17]. After isoelectric focusing, the strip was applied on top of a SDS–PAGE gel (12 by 14 cm, 12.5%) and proteins were separated according to molecular weight. The SDS– PAGE gel was stained using colloidal Coomassie blue. The protein spots were excised with a pipette tip from the gel and transferred to the Investigator ProGest automated digester (Genomic Solutions, Ann Arbor, MI, USA) rack. The gel pieces were washed twice with 25 mM ammonium bicarbonate/50% ACN and dried with nitrogen flow. Twenty-five microlitres of 10 lg/mL trypsin in 50 mM ammonium bicarbonate was added to the dried residue and the samples were incubated overnight at 37 °C with sequence grade modified porcine trypsin.
Results Identification of HA salivary proteins adsorbed using 2D gel electrophoresis and MS In this study, HA was incubated with whole saliva supernatant in individual assays with the 20 participating subjects. After HA incubations with whole saliva supernatant, it was possible to recover more than 90 5% of the adsorbed protein using either EDTA (0.2 M) [13] or TFA (2%) [16] in extraction. Since whole saliva composition is affected by circadian rhythm all saliva samples were collected at the same time (8.00 a.m.). Despite inter-individual variations in whole saliva composition, the 2D gel electrophoresis of the proteins of the extracts resulted in the detection of an average of 75 20 common spots after Coomassie colloidal staining and software spot detection (PDQuest v7.1, Bio-Rad). In Fig. 1, a typical 2-DE representative of the salivary proteins adsorbed onto HA is presented. The 2-DE profiles obtained using either trifluoracetic
MALDI-TOF/TOF for tryptic peptides Tryptic peptides were lyophilised and re-suspended in 10 lL of a 50% acetonitrile/0.1% formic acid solution. The samples were mixed (1:1) with a matrix consisting of a saturated solution of a-cyano-4hydroxycinnamic acid prepared in 50% acetonitrile/0.1% formic acid. Aliquots of samples (0.35 lL) were spotted onto the MALDI sample target plate using a SymBiot XVI Sample Workstation (Applied Biosystems, Foster City, CA, USA). Peptide mass spectra were obtained on a MALDI-TOF/TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Foster City, CA, USA) in the positive ion reflector mode. Two spots were used for signal and parameter optimisation. Spectra were obtained in the mass range between 800 and 4000 Da with ca. 1500 laser shots. A data dependent acquisition method was created to select the two most intense peaks in each sample spot for subsequent MS/MS data acquisition, excluding those from the matrix, due to trypsin autolysis, or acrylamide peaks. Trypsin autolysis peaks were used for internal calibration of the mass spectra, allowing a routine mass accuracy of better than 25 ppm.
Fig. 1. Two-dimensional gel map of an hydroxyapatite incubated saliva sample obtained in pH range between 3 and 10 and 12.5% of SDS–PAGE stained with colloidal Coomassie blue. Spots were identified by mass spectrometry with the exception of spot 17, which has been identified by differential staining (Table 1).
344
R. Vitorino et al. / Biochemical and Biophysical Research Communications 320 (2004) 342–346
acid or EDTA, solubilising solutions were very similar in terms of the number and the intensity of the detected protein spots (data not shown). Using pH focusing between pH 3 and 10, it is possible to observe that the majority of spots are visualised in the acidic zone of the gel. Spots visualised on the gel are also preferentially distributed on the low MW zone, below 30 KDa. Due to their primary sequences, PRPs are difficult to identify by MS using trypsin digestion. Alternatively, PRPs (spot series 18 Fig. 1) were identified using the modified Coomassie staining according to Beeley et al. [17], where PRPs are differentially revealed using methanol. All other proteins were positively identified with MALDI-TOF/TOF MS and MS/MS data. For mass spectrometry analysis, 60 spots from 2-DE gels of solubilised HA saliva samples, stained with colloidal Coomassie blue, were excised and digested with trypsin. Protein identification was performed based on a combination of tryptic peptide molecular weight from MS data and sequence information from MS/MS data. The resulting data were searched against a protein database using an internal Mascot search routine. As can be seen in Table 1, of the 70 spots excised, 48 spots corresponding to 18 different proteins were identified with high statistical reliability. Five of these proteins (cystatins C and D, calgranulin B, prolactin, and Igk/k light chain/VLJ chain) are, for the first time, reported to complex with HA. Other proteins that were identified by this approach include albumin, a-amylase, lysozyme, histatin 1, prolactin, carbonic anhydrase VI, the common cystatins (SN, S, SA-III, C, and D), and several Igs family members. Igk/k light chain/VLJ chain does not represent a single protein, but three different homologue proteins with several isoforms each with small MW and
pI differences, better characterised as a gel protein region [18]. Several spots were assigned as the same protein, which could be attributed to post-translational modifications, isoforms, and/or protein fragments. With the exception of spots 6 and 7, zinc-a-2-glycoprotein, all proteins were identified by the Mascot search routine as intact proteins. The four spots identified as cystatin S, spot series 10 (Fig. 1 and Table 1), presented data mining high scores (230, 160, 104, and 130, respectively). However, cystatin SA-III, Sap 1, cystatin S (acidic form) or cystatin SN were also other possible positive identifications (scores of 223, 220, 200, 168, respectively, for the first spot). According to the literature [19] cystatin S has only two variants (cystatin S1 and S2) that are phosphorylated at ser-3 and at ser-1 for the first variant and at ser-2 for the second. According to Lamkin et al. [9] cystatin SA-III presents high homology with cystatin S, but with different number of phosphorylations, three for cystatin S and four for cystatin SA-III. Taking into account the homology between them, and the obtained peptide masses used for identification, it is possible that some spots located on that region could be attributed to cystatin SA-III.
Discussion Addition of trifluoracetic acid [16] or EDTA [13] allows the solubilisation of HA and of the peptides and proteins adsorbed with high recoveries. On searching the literature for a comparison between the different solutions for extraction of proteins from HA, EDTA was
Table 1 Identified salivary proteins adsorbed to hydroxyapatite Spot
Protein namea
SWISS-PROT
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Poly-Ig receptor (IgA secretory chain) Serum albumin Immunoglobulin a-chain C region (Ig A) Human salivary a-amylase Carbonic anhydrase VI Zinc-a-2 glycoprotein Immunoglobulin k/k light chain VLJ chain Prolactin-inducible protein (prolactin) Cystatin S Cystatin SA-III pellicle precursor (Sap1) Calgranulin B (S100 calcium-binding protein A9) Cystatin SN Lysozyme b2-Microglobulin Histatin 1 Cystatin D Proline-rich proteinsb Cystatin C
P01833 P02768 P01878 P04746 P23280 P25311 AAC16777/AAB51622 P12273 P01036 AAB19889 P06702 P01037 P00695 P07151 P15515 P28325
a
P01034
All samples were identified by mass spectrometry with the exception of b which were identified by selective staining according to Beeley et al. [19].
R. Vitorino et al. / Biochemical and Biophysical Research Communications 320 (2004) 342–346
found to be one of the best HA solubilising agents [13]. TFA was only used by St€ oser et al. [16] but with no indication of protein recovery percentage. Applying the two experimental conditions proposed by these authors, no differences in percentage recovery of the total protein amount adsorbed to HA were observed (more than 90% in both cases). Also, the 2-DE patterns obtained using both solubilising solutions were very similar. This observation indicates that eventual acidic proteolysis promoted by TFA does not occur or is scarce in practical terms. The number of spots observed on the 2-DE HA sample gels (an average of 75) is much smaller than that observed on saliva 2-DE gels, obtained under similar experimental conditions (more than 200) [15]. Moreover, comparison between these samples shows that all the spots visualised on HA gels are also present on saliva sample gels and that the majority of proteins bound to HA are of low molecular weight. Although there is a strong interest in the characterisation of the macromolecular composition of in vivo acquired enamel pellicle, only a few reports have been published. The sum of all proteins identified then in the literature indicates that more than 40 salivary proteins have been reported to interact with HA. Most of the 18 proteins identified in our work by 2-DE were identified for the first time by mass spectrometry and five of these proteins (cystatins C and D, calgranulin B, prolactin, and Igk/k light chain VLJ chain) are reported for the first time to bind to HA. The identified proteins are involved in different oral functions as pellicle precursors, inflammatory, and microbial defence agents, pH buffers, and components associated with remineralisation processes, confirming previous proposals that enamel pellicle is a complex structure with manifold factors controlling tooth integrity. All those proteins have been recently identified in whole saliva by our group [15], and it is remarkable that, generally, HA possesses high selectivity to acidic and phosphorylated low molecular weight proteins. Keller et al. [20] have reported the presence of two groups of a-amylase isoforms in saliva with different molecular sizes, 63,000 and 59,000 Da. Isoforms belonging to the first group contain asparagine-linked sugar chains whereas the second group does not. As can be observed (spot series 4 Fig. 1, Table 1), only the group that contains asparagine-linked sugar chains was identified in HA absorbed proteins. This result suggests that the presence of sugar chains in proteins is important to their attachment to HA or even to acquired enamel pellicle. Comparing whole saliva 2D gel patterns [15] and HA incubated saliva 2D gel patterns, it is possible to observe an enrichment of spots in the cystatin S region (spot series 10). Cystatins are a group of known proteins present on saliva and are involved in the regulation of protein degradation at sites of inflammation or micro-
345
bial infection [21]. The presence of several cystatins (cystatins S, SN, D, C, and SAIII) adsorbed onto HA could be associated with the regulation of bacterial activity in parallel with lysozyme, another protein observed at high levels. Lysozyme has already been identified as a pellicle precursor [22]. Due to the presence of the phosphate groups, cystatins S and SAIII may also be directly associated with the remineralisation processes along with other proteins such as PRPs, histatin 1, and calgranulin B, emphasising their importance in maintenance of the crystal structure of HA. Furthermore, the presence of phosphorylations promotes an increase of the proteins’ acidic properties, and, thus, adsorption to HA and, as reported by Lamkin et al. [9], becoming potential pellicle precursors. Carbonic anhydrase VI (four spots detected) which has been reported to exert its buffer capacity predominantly on pellicle surface Kivel€a et al. [23] could also be associated with the remineralisation process.
Concluding remarks In order to elucidate the macromolecular structure of acquired pellicle, we used a proteomics approach to characterise proteins and peptides adsorbed to HA. By using 2-DE with extended pH range (3–10) and high sensitivity protein identification by MALDI-TOF/TOF, 48 spots from the 70 selected were identified. Among those that were identified are cystatins (S, SA-III, C, and D), lyzosyme, amylase (only glycosylated form), carbonic anhydrase VI, and calgranulin B.
References [1] M. Rykke, T. Sonju, Amino acid composition of acquired enamel pellicle collected in vivo after 2 h and after 24 h, Scand. J. Dent. Res. 99 (6) (1991) 463–469. [2] A. Carlen, A.C. Borjesson, K. Nikdel, J. Olsson, Composition of pellicles formed in vivo on tooth surfaces in different parts of the dentition, and in vitro on hydroxyapatite, Caries Res. 32 (6) (1998) 447–455. [3] U. Lendenmann, J. Grogan, F.G. Oppenheim, Saliva and dental pellicle—a review, Adv. Dent. Res. 14 (2000) 22–83. [4] J. Li, E.J. Helmerhorst, R.B. Corley, L.E. Luus, R.F. Troxler, F.G. Oppenheim, Characterization of the immunologic responses to human in vivo acquired enamel pellicle as a novel means to investigate its composition, Oral Microbiol. Immunol. 18 (3) (2003) 183–191. [5] P. Schupbach, F.G. Oppenheim, U. Lendenmann, M.S. Lamkin, Y. Yao, B. Guggenheim, Eur. J. Oral Sci. 109 (1) (2001) 60–68. [6] E.E. Kousvelari, R.S. Baratz, B. Burke, F.G. Oppenheim, Immunochemical identification and determination of proline-rich proteins in salivary secretions, enamel pellicle, and glandular tissue specimens, J. Dent. Res. 59 (8) (1980) 1430–1438. [7] I. Al-Hashimi, M.J. Levine, Characterization of in vivo salivaryderived enamel pellicle, Arch. Oral Biol. 34 (4) (1989) 289–295.
346
R. Vitorino et al. / Biochemical and Biophysical Research Communications 320 (2004) 342–346
[8] J. Leinonen, J. Kivela, S. Parkkila, A.K. Parkkila, H. Rajaniemi, Salivary carbonic anhydrase isoenzyme VI is located in the human enamel pellicle, Caries Res. 33 (3) (1999) 185–190. [9] M.S. Lamkin, F.G. Oppenheim, Structural features of salivary function, Crit. Rev. Oral Biol. Med. 4 (3–4) (1993) 251–259. [10] R.J. Gibbons, Adherent interactions which may affect microbial ecology in the mouth, J. Dent. Res. 63 (3) (1984) 378–385. [11] F.A. Scannapieco, G.I. Torres, M.J. Levine, Salivary amylase promotes adhesion of oral streptococci to hydroxyapatite, J. Dent. Res. 74 (7) (1995) 1360–1366. [12] J.L. Jensen, M.S. Lamkin, F.G. Oppenheim, Adsorption of human salivary proteins to hydroxyapatite: a comparison between whole saliva and glandular salivary secretions, J. Dent. Res. 71 (9) (1992) 1569–1576. [13] Y. Yao, J. Grogan, M. Zehnder, U. Lendenmann, B. Nam, Z. Wu, C.E. Costello, F.G. Oppenheim, Compositional analysis of human acquired enamel pellicle by mass spectrometry, Arch. Oral Biol. 46 (4) (2001) 293–303. [14] L. Stoser, N. Apitz, U. Keher, Th. Henning, In vitro pellicle formation with stimulated mixed saliva and hydroxyapatite, 6th European Symposium on Saliva, Poster 45, 2002. [15] R. Vitorino, M.J.C. Lobo, A.J. Ferrer-Correia, J.R. Dubin, K.B. Tomer, P.M. Domingues, F.M.L. Amado, Identification of human whole saliva protein components using proteomics, Proteomics 4 (2004) 1109–1115. [16] K.H. Eggen, G. Rolla, Gel filtration, ion exchange chromatography and chemical analysis of macromolecules present in acquired enamel pellicle (2-h-pellicle), Scand. J. Dent. Res. 90 (3) (1982) 182–188.
[17] J.A Beeley, F. Newman, P.H. Wilson, I.C. Shimmin, Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human parotid salivary proteins: comparison of dansylation, Coomassie blue R-250 and silver detection methods, Electrophoresis 17 (3) (1996) 505–506. [18] J.L. Dul, S. Abiel, J. Melnick, Y. Argon, Ig light chains are secreted predominantly as monomers, J. Immunol. 157 (7) (1996) 2969–2975. [19] S. Isemura, E. Saitoh, K. Sanada, K. Minakata, Identification of full-sized forms of salivary (S-type) cystatins (cystatin SN, cystatin SA, cystatin S, and two phosphorylated forms of cystatin S) in human whole saliva and determination of phosphorylation sites of cystatin S, J. Biochem. (Tokyo) 110 (4) (1991) 648– 654. [20] P.J. Keller, D.L Kauffman, B.J. Allan, B.L. Williams, Further studies on the structural differences between the isoenzymes of human parotid –amylase, Biochemistry. 10 (26) (1971) 4867– 74.J. [21] A. Baron, N. Barrett-Vespone, J. Featherstone, Purification of large quantities of human salivary cystatins S, SA and SN: their interactions with the model cysteine protease papain in a noninhibitory mode, Oral Dis. 5 (4) (1999) 344–353. [22] L.M. Tellefson, G.R. Germaine, Adherence of Streptococcus sanguis to hydroxyapatite coated with lysozyme and lysozymesupplemented saliva, Infect Immun. 51 (3) (1986) 750–759. [23] J. Kivela, S. Parkkila, A.K. Parkkila, H. Rajaniemi, A low concentration of carbonic anhydrase isoenzyme VI in whole saliva is associated with caries prevalence, Caries Res. 33 (3) (1999) 178– 184.