Journal of Dental Research http://jdr.sagepub.com/
Structural Relationship Between Human Salivary Histatins R.F. Troxler, G.D. Offner, T. Xu, J.C. Vanderspek and F.G. Oppenheim J DENT RES 1990 69: 2 DOI: 10.1177/00220345900690010101 The online version of this article can be found at: http://jdr.sagepub.com/content/69/1/2
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://jdr.sagepub.com/content/69/1/2.refs.html
>> Version of Record - Jan 1, 1990 What is This?
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
Structural Relationship Between Human Salivary Histatins R.F. TROXLER" 2, G.D. OFFNER1, T. XU2, J.C. VANDERSPEK', and F.G. OPPENHEIM" 2 'Department of Biochemistry, Boston University School of Medicine, and 2Department of Oral Biology, Goldman School of Graduate Dentistry, Boston, Massachusetts 02118 Histatins are a group of electrophoretically distinct histidine-rich polypeptides with microbicidal activity found in human parotid and submandibular gland secretions. Recently, we have shown that histatins 1, 3, and 5 are homologous proteins that consist of 38, 32, and 24 amino acid residues, respectively, and that these polypeptides kill the pathogenic yeast, Candida albicans. We now describe the isolation and structural characterization of histatins 2, 4, 6, and 7-12, the remaining members of this group ofpolypeptides. Histatin 2 was found to be identical to the carboxyl terminal 26 residues of histatin 1; histatin 4 was found to be identical to the carboxyl terminal 20 residues of histatin 3; and histatin 6 was found to be identical to histatin 5, but contained an additional carboxyl terminal arginine residue. The amino acid sequences of histatins 7-12 formally correspond to residues 12-24, 13-24, 12-25, 13-25, 5-11, and 5-12, respectively, of histatin 3, but could also arise proteolytically from histatin 5 or 6. These results establish, for the first time, the complete structural relationships between all members of this group of microbicidal proteins in human parotid saliva. The relationship of histatins to one another is discussed in the context of their genetic origin, biosynthesis and secretion into the oral cavity, and potential as reagents in anti-candidal studies. J Dent Res 69(1):2-6, January, 1990
plays a role in stabilizing the mineral-solute interactions of the oral fluid that is responsible for maintenance of the surface integrity of enamel (Oppenheim et al., 1986). Partially purified mixtures of histatins were shown to inhibit growth of several strains of Streptococcus mutans (MacKay et al., 1984) and to kill the pathogenic yeast, Candida albicans (Pollock et al., 1984). More recently, we have shown that histatins 1, 3, and 5 kill C. albicans in a dose-dependent manner at physiological concentrations, and that the killing effectiveness observed was inversely proportional to molecular size in the order: histatin 5 > histatin 3 > histatin 1 (Oppenheim et al., 1988). These data provide strong evidence that histatins in parotid and submandibular gland secretions play a major role in the non-immune oral host defense system. It seems likely that because histatin 1 is a phosphoprotein, its major function may be that of a precursor of the acquired enamel pellicle and inhibitor of hydroxylapatite crystal growth, whereas the major function of the non-phosphorylated histatins may be microbicidal in nature. The present work describes the structural relationship among histatins 1, 3, and 5, and previously uncharacterized histatins belonging to this group of salivary proteins.
Introduction. Histatins are a family of small, cationic, histidine-rich proteins secreted by human parotid and submandibular glands. We have isolated and characterized three of these and have shown that they are homologous polypeptides, each containing seven residues of histidine (Oppenheim et al., 1988). The amino terminal 24 residues of all three proteins are identical, except for the substitution of Glu and Arg (residues 4 and 11) in histatin 1 with Ala and Lys, respectively, in histatins 3 and 5. Histatin 1 contains a phosphorylated serine at residue 2, whereas histatins 3 and 5 lack inorganic phosphate. Histatin 1 contains a 9 amino acid insert absent in histatin 3, and histatin 3 contains a 3 amino acid insert absent in histatin 1. Histatin 5 is identical to the amino terminal 24 residues of histatin 3. It was reported in the earlier literature (Azen, 1973) that histatin 3 appears to display a genetic polymorphism, and histatin 1 does not, providing indirect evidence for the origin of these proteins from more than one gene. Histatins have been implicated in several different biological processes important to the maintenance of hard tissues in the oral cavity. Histatin 1 was initially discovered when it was shown that this protein selectively adsorbed to hydroxylapatite and enamel powders (Hay, 1973, 1975), implicating it as a precursor of the acquired enamel pellicle (Mayhall, 1970). The composition of this proteinaceous layer covering tooth surfaces is incompletely understood, but it is known that it forms an important physical barrier between tooth enamel and the oral environment (Hay, 1983). We have shown that histatin 1 inhibits hydroxylapatite crystal formation, which suggests that it Received for publication May 1, 1989 Accepted for publication July 20, 1989 This work was supported by National Institutes of Health Grants DE 05672 and DE 07652. 2
Materials and methods. Histatin isolation. -Human parotid saliva was collected with the aid of a Carlson-Crittendon device, as described previously (Oppenheim et al., 1971). Samples collected in glass tubes on ice from multiple donors were pooled, dialyzed in Spectropor 6 membrane against distilled water at 4°C, lyophilized, taken up in 50 mL of 0.05 mol/L ammonium formate (pH 4.0), and chromatographed on a Bio-Gel P-2 column (2.6 x 86 cm) equilibrated with the same buffer, as described previously (Oppenheim et al., 1986). Fractions containing histatins were pooled, lyophilized, dissolved in 1% acetic acid, and subjected to high-performance liquid chromatography (HPLC) on a Vydac C18 column (4.6 x 15 cm; 5 A, 300-,urm pore size) by use of a Waters reversed-phase HPLC system (RP-HPLC). The column was developed with a complex gradient at a flow rate of 1 mL/min from 100% solvent A (0.1% trifluoroacetic acid in water) to 100% solvent B (80% acetonitrile, 20% water, 0.1% trifluoroacetic acid), with monitoring at 214 and 254 nm. Column eluate was collected in 1.0-mL fractions, and those containing the various histatins were evaporated to dryness in a Savant Speed-Vac, taken up in 1% acetic acid, and re-chromatographed under conditions where the gradient was stopped and run isocratically at 6-10% less solvent B than that at which the polypeptide eluted initially. The resulting purified histatins were evaporated to dryness, dissolved in 1% acetic acid, and subjected to automated Edman degradation. Analytical procedures. -Histatins were examined on 15% slab gels with use of a cationic sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) system described previously (Baum et al., 1976, 1977; Oppenheim et al., 1988). The amino acid compositions of selected histatins and derived peptides were determined with use of a Beckman system 6300 amino acid analyzer following hydrolysis in 6 mol/L HCl for
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
E | 0.2
6
J
0
3
HUMAN SALIVARY HISTATINS
Vol. 69 No. 1
2
3
---A--p----
-
B-------
C-----+---I)---i--'--------*:~~ ~ ~ ~ ~~6
-
E
-4
6
2 40 00_ft00000 : 60 00 0;; 100f f 1 001 etuat f at20n Fractio A-E w pold lypilzd w h and formate, pH 4.0, at a flow rate of 385 200 : 400 0207 600 t:t0 Ai
exam~~~~~~~~~.ined .etecroporeicl~lyl~.L Win thectionicsagesytem.Fractiont Ai lan 0-; a, 100 1g1 lan b, 70 iig fratio Bl lanes a an 10 p-;frcinC Fig. 1-Elution profile of 1 g of human parotid saliva protein from Bio-Gel P-2. The column (2.6 x 86 cm) was developed with 0.05 mol/L ammonium formate, pH 4.0, at a flow rate of 38 mL/h, with continuous monitoring of eluate at 230 nm. Fractions A-E were pooled, lyophilized, weighed, and examined electrophoretically in the cationic slab gel system. Fraction A, lane a, 100 ,ug, lane b, 70 ,ug; fraction B. lanes a and b, 100 ,ug; fraction C, lane a, 100 Vg, lane b, 70 pug; fraction D, lane a, 100 pg, lane b, 70 ,ug; fraction E, lanes a and b, 100 pg. Fraction B contained some salt. Inset at left shows cationic electrophoretograms of parotid (PS) and submandibular (SS) proteins (150 ,ug each). Note that the staining intensity of histatins is comparable in both glandular secretions.
22 h in vacuo at 1 100C. Amino acid sequences were determined on an Applied Biosystems, Inc., 470A gas-phase sequencer equipped with a 120 PTH analyzer.
Results. Electrophoretic analysis of human parotid saliva protein in a cationic PAGE system resulted in a reproducible and characteristic pattern in which the top half of the gel contained an
unresolved mixture of salivary proteins, and the bottom half contained three pairs of bands and, in some cases, one or more bands at the dye front (Fig. 1, inset at left, PS). This observation has been extended to show that the same pattern of bands was present when human submandibular saliva protein was examined in the cationic PAGE system (Fig. 1, inset at left, SS). We have named the proteins corresponding to the three pairs of bands histatins 1 and 2, histatins 3 and 4, histatins 5 and 6, and the most rapidly migrating band, histatin 7 (Oppenheim et at, 1988). Histatin 7 is in fact a mixture of small histatins (histatins 7-12) that has been resolved into six components (see below). The nomenclature used for histatins described in this paper is consistent with the original observations on the electrophoretic mobility of histatins in the cationic PAGE system (Baum et al., 1976, 1977). We have shown that when human parotid saliva proteins are chromatographed on Bio-Gel P-2, histatins as a group are selectively retained (Oppenheim et al., 1988). In view of this anomalous behavior, a more detailed electrophoretic analysis of selected fractions in the histatin peak was performed (Fig.
1). The results showed that fraction A containing the bulk of salivary proteins was devoid of histatins, fraction B contained only traces of histatins 1 and 5, and fractions C, D, and E contained the bulk of histatins and no other salivary proteins. In fractions C-E, it was evident that histatins 1, 5, and 7-12 had a tendency to elute somewhat earlier, whereas histatins 2, 3, 4, and 6 had a tendency to elute somewhat later. Histatins 7-12 were incompletely resolved.
0I-
z
0in (A)
0
15
3045
60
TIME (min) Fig. 2-Separation of histatins by RP-HPLC. The histatin fraction from Bio-Gel P-2 was chromatographed on a Vydac C18 column (4.6 x 150 mm; pore size 5 pum) developed with an acetonitrile gradient (dotted line) at a flow rate of 1.0 mL/min. Fractions containing the partially purified histatins were re-chromatographed under isocratic conditions to yield purified preparations (see Fig. 4) that were used for sequence analysis.
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
TROXLER et aL
4
J Dent Res January 1990
10 30 20 Hi stat in 1: Asp-Pse-Hi s-C Iu-Lvs-Arg-Hi s-Hi s-C Iy-Tyr-Arg-Arg-Lgs-Phe-Hi s-G Iu-Lgs-H is-H Is-Ser-Hi s-Arwg-C Iu-Phe-Pwo-Phe-Tyr-Cig-Asp-Tyr-G- I-Ser-
Arg-Lgs-Phe-HIs-GIu-Lys-His-His-Ser -HIs-Ar -CIu-Phe-Pro-Phe-Tyr -G -Asp-Tyr-CGI-Ser-
Histatin 2: 38 Asn-Tyr -Leu-Tyr-Asp-Asn
Asn-Tyr -Leu-Tuyr-Asp-Asn 20
10
I
30
Hi stat in 3:
Asp-Serw-Hi s-Al a-Lys-Arg-H is-His-G Iy-Tyr-Lgs-Arg-Lgs-Phe-Hi s-Cl u-Lgs-H is-His-Serw-Hi s-Arg-C ig-Tyr-Arg-Serw-Asn-Tgr-Leu-Trw-Asp-Asn
Histatin 4:
Arg-Lys-Phe-His-GIu-Lgs-HIs-His-Ser-His-Arg-GI -Tgr-Arg-Ser-Asn-Tgr-Leu-Tgr-Asp-Asn
Histatin 5:
Asp-Ser-His-A la-Lgs-Arg-His-His-GIy-Tyr-Lgs-Arg-Lgs-Phe-His-Clu-Lys-His-His-Ser-His-Arg-GCI-Tgr
HistatIn 6:
Asp-Ser-His- Ala-Ls-Arg-His-His- CI
-Tr-Lgs-Awg-LAs-Phe-His-CIu-Lys-His-His-Ser-His-Arg-G I-Tgr-Arg
Histatin 7:
Arg-Lgs-Phe-His.CIu-Lgs-His-His-Ser-His-Arg-GIy-Tyr
Histatin 8:
Lgs-Phe-His-Glu-Lgs-His-His-Ser-His-Arg-Clg-Tgr
Histatin 9:
Arg-Lgs-Phe-His-Clu-Lgs-His-His-Ser-Hls-Akg-GlI-Tgr-Arg
Lgs-Phe-His-Clu-Lgs-His-His-Ser-His-A-rg-Gl-Twr--Ag
Histatin 10: Histatin I1:
Lgs-Arg-His-His-Clg-Tyr-Lls-Arg
Histatin 12:
Lgs-Arg-His-His-ClV-Tyr-L-s
Fig. 3-Amino acid sequences of histatins 1-12.
When histatins from Bio-Gel P-2 were subjected to RPHPLC, a characteristic elution pattern of peaks was observed (Fig. 2). This consisted of a group of hydrophilic components eluting between 14-19% solvent B (histatins 7-12), followed by three distinct pairs of peaks, eluting at approximately 2122% (histatins 5, 6), 24-26% (histatins 3, 4), and 32-35% solvent B (histatins 1, 2), with each pair consisting of a larger and smaller component. We have previously shown that the larger components in each pair of peaks are histatins 1, 3, and 5, respectively. All histatins not previously characterized have now been purified to homogeneity by RP-HPLC, and their amino acid sequences determined (Fig. 3). The smaller component in the pair of peaks eluting at 3235% solvent B was designated as histatin 2, because it had an amino acid sequence identical to residues 12-38 of histatin 1. This component apparently arises by a tryptic-like cleavage of the bond between Arg 11 and Arg 12 in histatin 1. The smaller component in the pair of peaks eluting at 24-26% solvent B was designated as histatin 4, because it had an amino acid sequence identical to the carboxyl-terminal 21 residues of histatin 3. This component could arise by a tryptic-like cleavage of the bond between Lys 11 and Arg 12 in histatin 3. The smaller component in the pair of peaks eluting at 21-22% solvent B was designated histatin 6, and was identical to histatin 5, but contained an additional carboxyl-terminal arginine residue. The group of more hydrophilic components eluting from the C1s column between 14-19% solvent B (Fig. 2) were purified to apparent homogeneity by RP-HPLC. Complete separation of partially resolved histatins was achieved by re-chromatog-
raphy under conditions of isocratic elution, as described in "Materials and methods". The effectiveness of this procedure for final purification is illustrated (Fig. 4) for histatins 7-10, which eluted as a fused group of peaks during initial RP-HPLC of the histatin fraction from Bio-Gel P-2. The material in fraction 27 (Fig. 4A), which initially eluted at 17.8% solvent B, was re-chromatographed with isocratic elution at 11.2% solvent B, and was resolved into two components, histatins 9 and 10 (Fig. 4B). The material in fraction 28 (Fig. 4A), which initially eluted at 18.4% solvent B, was re-chromatographed with isocratic elution at 12.4% solvent B, and was also resolved into two components, histatins 7 and 8 (Fig. 4C). The material in fraction 29 (Fig. 4A), which initially eluted at 19% solvent B, was re-chromatographed with isocratic elution at 13% solvent B to yield primarily histatin 7 (Fig. 4D). Histatins 7 and 8 were identical to residues 12-24 and 1324 of histatin 3, respectively, and could be derived by cleavage at Lys 11, Arg 12, and Tyr 24 (or by cleavage of histatin 5 at Lys 11 and Arg 12). The amino acid sequences of histatins 9 and 10 were identical to residues 12-25 and 13-25 of histatin 3, respectively, and could be derived by cleavage at Lys 11, Arg 12, and Arg 25 (or cleavage of histatin 6 at Lys 11 and Arg 12). Histatins 11 and 12 were identical to residues 5-11 and 5-12 of histatin 3, respectively, and could formally be derived from histatins 3, 5, or 6 by cleavage at Ala 4, Lys 11, and Arg 12. It is noteworthy that fragments corresponding to residues 111 of histatins 2 and 4, residues 1-4 of histatins 7-12, and residues 25-32 of histatin 5 and 26-32 of histatin 6 were not found among the components retained on Bio-Gel P-2, despite
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
Vol. 69 No. 1
HUMAN SALIVARY HISTA TINS
5
the fact that all histatins resolved by RP-HPLC were sequenced. It is possible that these fragments eluted with the bulk of salivary proteins in the void volume peak from BioGel P-2 and were present in insufficient quantities to be detected electrophoretically.
Discussion. The present investigation has shown that the family of basic histidine-rich polypeptides, known as histatins, occurs as electrophoretic entities in both parotid and submandibular secretions. While histatins from submandibular secretion were not isolated and characterized as proteins in this investigation, there is little doubt that the pairs of protein bands in the lower half of the cationic slab gels (Fig. 1, inset, SS) are indeed histatins. We have shown in Northern analyses that histatin mRNAs are present in both human parotid and submandibular glands (vanderSpek et al., 1989) and have sequenced cDNAs for histatins 1 and 3 from a human submandibular gland cDNA library (vanderSpek et al., 1990). We also describe, for the first time, the complete amino acid sequence of histatins 2, 4, 6, and 7-12 from parotid secretion. It is noteworthy that with the exception of histatin 2, which is probably a proteolytic fragment of histatin 1, all other histatins could be derived by tryptic-like or chymotryptic-like cleavages of histatin 3. The proteolytic cleavages giving rise to the various histatins appear to be quite specific. First, of the 7 Arg + Lys residues in histatin 1, cleavage appears to occur only at Arg 11 to yield histatin 2. Second, if histatins 4-12 are in fact fragments derived from histatin 3, of the 8 Arg + Lys residues present, cleavage appears to occur only at Lys 11, Arg 12, and Arg 25. Of the amino acid residues potentially susceptible to chymotryptic-like cleavage, such cleavage occurs only at Ala 5 and Tyr 24. Since the electrophoretic patterns of histatins in samples of pooled parotid and submandibular secretion are virtually identical, histatins appear to arise by a series of highly specific proteolytic processing events. The details of the regulatory mechanisms governing cleavage of the parent molecules (histatins 1 and 3) are not known, but the constancy of the electrophoretic pattern reflects biological control at some level. The proteolytic events giving rise to histatins are likely to occur in conjunction with secretion either in the endoplasmic reticulum, in the golgi, in secretary vesicles, or during exocytosis. It is unlikely that cleavage of parent histatins occurs in an uncontrolled manner during transit through the glandular duct system, because the electrophoretic pattern of histatins (and, for that matter, of other major salivary proteins) was essentially unchanged from donor to donor, and random proteolysis after secretion would not be expected to lead to the consistent electrophoretic banding pattern observed. Furthermore, it is highly unlikely that histatins arise by proteolysis during collection of glandular secretion, because samples were collected on ice by the same procedure we used to obtain intact salivary proteins (Oppenheim et al., 1971, 1982, 1986, 1988). Dialysis of pooled saliva protein samples, chromatography on Bio-Gel P-2, and fraction collection of column eluate were performed at 4°C. Histatins do not appear to undergo proteolytic degradation in this chromatographic procedure, because the electrophoretic pattern of histatins is the same before and after chromatography on Bio-Gel P-2 (Fig. 1). RP-HPLC was carried out at room temperature in solvents A and B at pH 2. Histatins are stable in this solvent for one to two months, and known salivary proteases are not active at this acidic pH. It is of interest that another family of small cationic polypeptides, termed defensins, occurs in human (Selsted et al.,
E
w z m
z
m
0
C')
TIME(nin)
Fig. 4-Purification of histatins 7-10 by RP-HPLC with use of isocratic elution. The flow rate was 1 mL/min, 1-mL fractions were collected, and
column eluate was monitored at 214 nm. (A) Partial elution profile of histatins 7-10 under conditions described in the legend to Fig. 2. (B) Rechromatography of the fraction eluting at 27 min (tube 27) in (A). The gradient was stopped at 11.2% solvent B, and the column developed isocratically, yielding purified histatins 9 and 10. (C) Re-chromatography of the fraction eluting at 28 min (tube 28) in (A). The gradient was stopped at 12.4% solvent B, and the column was developed isocratically, yielding purified histatins 7 and 8. (D) Re-chromatography of the fraction eluting at 29 min (tube 29) in (A). The gradient was stopped at 13.0% solvent B, and the column was developed isocratically, yielding purified histatin 7.
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
6
TROXLER et al.
1985), guinea pig (Selsted and Harwig, 1987), and rabbit (Selsted et aL, 1985) neutrophils or macrophages. Defensins appear to play a major role in the non-immune, oxygen-independent microbicidal function of phagocytes (Ganz et aL, 1985). These polypeptides are cysteine- and arginine-rich, range in length from 29-34 amino acid residues, and constitute 5-15% of the protein in azurophilic granules (Rice et al., 1987). While the positions of Cys and Arg are conserved in defensins from different mammalian species, 65% of the residues show little homology (Stanfield et at., 1988). Defensins appear to display a broad spectrum of microbicidal effects against bacteria (Selsted et al., 1984) and fungi, including C. albicans (PattersonDelafield et at., 1980), and have also been shown to inactivate HSV-2, vesicular stomatitis, and influenza viruses (Lehrer et al., 1985). While there is no sequence homology between defensins and salivary histatins, both groups of proteins are small and cationic, and appear to play a key role in the non-immune host defense system. It was recently reported that whole saliva, parotid secretion, and submandibular secretion inhibit infectivity of human immunodeficiency virus 1 (HIV-1) (Fox et al., 1988). This effect appears to be specific for HIV-1, because it is well-known that saliva does not exhibit broad-spectrum antiviral activity (Alter, 1977; Schupfer et at., 1986). The protein or proteins from salivary glands that inhibit infectivity of HIV-1 are not known, but the possible involvement of histatins in this process cannot be excluded. REFERENCES ALTER, H.J. (1977): Transmission of Hepatitis B to Chimpanzees by Hepatitis B Surface Antigen Positive Saliva and Semen, Infect Immun 16:928-933. AZEN, E.A. (1973): Properties of Salivary Basic Proteins Showing Polymorphism, Biochem Genet 9:69-86. BAUM, B.J.; BIRD, J.L.; and LONGTON, R.W. (1977): Polyacrylamide Gel Electrophoresis of Human Salivary Histidine-rich Polypeptides, J Dent Res 56:1115-1118. BAUM, B.J.; BIRD, J.L.; MILLAR, D.B.; and LONGTON, R.W. (1976): Studies on Histidine-rich Polypeptides from Human Parotid Saliva, Arch Biochem Biophys 177:427-436. FOX, P.C.; WOLFF, A.; YEH, C.K.; ATKINSON, J.C.; and BAUM, B.J. (1988): Saliva Inhibits HIV-1 Infectivity, J Am Dent Assoc 116:635-637. GANZ, T.; SELSTED, M.E.; SZLAREK, D.; HARWIG, S.S.L.; DAHER, K.; BAINTON, D.F.; and LEHRER, R.I. (1985): Defensins. Natural Peptide Antibiotics of Human Neutrophils, J Clin Invest 76:1427-1435. HAY, D.I. (1973): The Interaction of Human Parotid Salivary Proteins with Hydroxylapatite, Arch Oral Biol 18:1517-1529. HAY, D.I. (1975): Fractionation of Human Parotid Salivary Proteins and the Isolation of an Histidine-rich Acidic Peptide Which Shows High Affinity for Hydroxyapatite Surfaces, Arch Oral Biol 20:553558. HAY, D.I. (1983): Human Glandular Salivary Proteins: In: Handbook of Experimental Aspects of Oral Biochemistry, E. P. Lazzari, Ed., Boca Raton, FL: CRC Press, pp. 319-335. LEHRER, R.I.; DAHER, K.; GANZ, T.; and SELSTED, M.E. (1985): Direct Inactivation of Viruses by MCP-1 and MCP-2, Natural Peptide Antibiotics from Rabbit Leucocytes, J Virol 54:467-472. MACKAY, B.J.; DENEPITIYA, L.; IACONO, V.J.; KROST, S.P.;
J Dent Res January 1990
and POLLOCK, J.J. (1984): Growth Inhibitory and Bacteriocidal Effects of Human Parotid Salivary Histidine-rich Polypeptides on Streptococcus mutans, Infect Immun 44:695-701. MAYHALL, C.W. (1970): Concerning the Composition and Source of the Acquired Enamel Pellicle of Human Teeth, Arch Oral Biol 15:1327-1341. OPPENHEIM, F.G.; HAY, D.I.; and FRANZBLAU, C. (1971): Proline-rich Proteins from Human Parotid Saliva. I. Isolation and Characterization, Biochemistry 10:4233-4238. OPPENHEIM, F.G.; OFFNER, G.D.; and TROXLER, R.F. (1982): Phosphoproteins in the Parotid Saliva of the Subhuman Primate, Macaca fascicularis. Isolation and Characterization of a Prolinerich Phosphoglycoprotein and Complete Covalent Structure of a Proline-rich Phosphopeptide, J Biol Chem 257:9271-9282. OPPENHEIM, F.G.; XU, T.; McMILLIAN, F.M.; LEVITZ, S.M.; DIAMOND, R.D.; OFFNER, G.D.; and TROXLER, R.F. (1988): Histatins, a Novel Family of Histidine-rich Proteins in Human Parotid Secretion. Isolation, Characterization, Primary Structure and Fungistatic Effects on Candida albicans, JBiol Chem 263:74727477. OPPENHEIM, F.G.; YANG, Y.C.; DIAMOND, R.D.; HYSLOP, D.; OFFNER, G.D.; and TROXLER, R.F. (1986): The Primary Structure and Functional Characterization of the Neutral Histidinerich Polypeptide from Human Parotid Secretion, J Biol Chem 261:1177-1182. PATTERSON-DELAFIELD, J.; MARTINEZ, R.J.; and LEHRER, R.I. (1980): Microbicidal Cationic Proteins in Rabbit Alveolar Macrophages: a Potential Host-defense Mechanism, Infect Immun 30:180-192. POLLOCK, J.J.; DENEPITIYA, L.; MACKAY, B.J.; and IACONO, V.J. (1984): Fungistatic and Fungicidal Activity of Human Parotid Salivary Histidine-rich Polypeptides on Candida albicans, Infect Immun 44:702-707. RICE, W.G.; GANZ, T.; KINKADE, J.M.; SELSTED, M.E.; LEHRER, R.I.; and PARMLEY, R.T. (1987): Defensin-rich Granules of Human Neutrophils, Blood 70:757-765. SCHUPFER, P.C.; MURPHY, J.R.; and BALE, J.F. (1986): Survival of Cytomegalovirus in Paper Diapers and Saliva, Pediatric Infect Dis 5:677-679. SELSTED, M.E.; BROWN, D.M.; DELANGE, R.J.; HARWIG, S.S.L.; and LEHRER, R.I. (1985): Primary Structure of Six Antimicrobial Peptides of Rabbit Peritoneal Neutrophils, JBiol Chem 260:4579-4584. SELSTED, M.E. and HARWIG, S.S.L. (1987): Purification, Primary Structure and Antimicrobial Activities of a Guinea Pig Neutrophil Defensin, Infect Immun 55:2281-2286. SELSTED, M.E.; HARWIG, S.S.L.; GANZ, T.; SCHILLING, J.W.; and LEHRER, R.I. (1985): Primary Structures of Three Human Neutrophil Defensins, J Clin Invest 76:1436-1439. SELSTED, M.E.; SZLAREK, D.; and LEHRER, R.I. (1984): Purification and Antibacterial Activity of Antimicrobial Peptides of Rabbit Granulocytes, Infect Immun 45:150-154. STANFIELD, R.L.; WESTBROOK, E.M.; and SELSTED, M.E. (1988): Characterization of Two Crystal Forms of Human Defensin Neutrophil Cationic Peptide 1, a Naturally Occurring Antimicrobial Peptide of Leucocytes, J Biol Chem 263:5933-5935. VANDERSPEK, J.C.; WYANDT, H.E.; SKARE, J.C.; MILUNSKY, A.; OPPENHEIM, F.G.; and TROXLER, R.F. (1989): Localization of the Genes for Histatins to Human Chromosome 4q13 and Tissue Distribution of the mRNAs, Am J Human Genet (in press). VANDERSPEK, J.C.; OFFNER, G.D.; OPPENHEIM, F.G.; and TROXLER, R.F. (1990): Molecular Cloning of Human Submandibular Histatins, Arch Oral Biol (in press).
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
Structural Relationship Between Human Salivary Histatins R.F. Troxler, G.D. Offner, T. Xu, J.C. Vanderspek and F.G. Oppenheim J DENT RES 1990 69: 2 DOI: 10.1177/00220345900690010101 The online version of this article can be found at: http://jdr.sagepub.com/content/69/1/2
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://jdr.sagepub.com/content/69/1/2.refs.html
>> Version of Record - Jan 1, 1990 What is This?
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
Structural Relationship Between Human Salivary Histatins R.F. TROXLER" 2, G.D. OFFNER1, T. XU2, J.C. VANDERSPEK', and F.G. OPPENHEIM" 2 'Department of Biochemistry, Boston University School of Medicine, and 2Department of Oral Biology, Goldman School of Graduate Dentistry, Boston, Massachusetts 02118 Histatins are a group of electrophoretically distinct histidine-rich polypeptides with microbicidal activity found in human parotid and submandibular gland secretions. Recently, we have shown that histatins 1, 3, and 5 are homologous proteins that consist of 38, 32, and 24 amino acid residues, respectively, and that these polypeptides kill the pathogenic yeast, Candida albicans. We now describe the isolation and structural characterization of histatins 2, 4, 6, and 7-12, the remaining members of this group ofpolypeptides. Histatin 2 was found to be identical to the carboxyl terminal 26 residues of histatin 1; histatin 4 was found to be identical to the carboxyl terminal 20 residues of histatin 3; and histatin 6 was found to be identical to histatin 5, but contained an additional carboxyl terminal arginine residue. The amino acid sequences of histatins 7-12 formally correspond to residues 12-24, 13-24, 12-25, 13-25, 5-11, and 5-12, respectively, of histatin 3, but could also arise proteolytically from histatin 5 or 6. These results establish, for the first time, the complete structural relationships between all members of this group of microbicidal proteins in human parotid saliva. The relationship of histatins to one another is discussed in the context of their genetic origin, biosynthesis and secretion into the oral cavity, and potential as reagents in anti-candidal studies. J Dent Res 69(1):2-6, January, 1990
plays a role in stabilizing the mineral-solute interactions of the oral fluid that is responsible for maintenance of the surface integrity of enamel (Oppenheim et al., 1986). Partially purified mixtures of histatins were shown to inhibit growth of several strains of Streptococcus mutans (MacKay et al., 1984) and to kill the pathogenic yeast, Candida albicans (Pollock et al., 1984). More recently, we have shown that histatins 1, 3, and 5 kill C. albicans in a dose-dependent manner at physiological concentrations, and that the killing effectiveness observed was inversely proportional to molecular size in the order: histatin 5 > histatin 3 > histatin 1 (Oppenheim et al., 1988). These data provide strong evidence that histatins in parotid and submandibular gland secretions play a major role in the non-immune oral host defense system. It seems likely that because histatin 1 is a phosphoprotein, its major function may be that of a precursor of the acquired enamel pellicle and inhibitor of hydroxylapatite crystal growth, whereas the major function of the non-phosphorylated histatins may be microbicidal in nature. The present work describes the structural relationship among histatins 1, 3, and 5, and previously uncharacterized histatins belonging to this group of salivary proteins.
Introduction. Histatins are a family of small, cationic, histidine-rich proteins secreted by human parotid and submandibular glands. We have isolated and characterized three of these and have shown that they are homologous polypeptides, each containing seven residues of histidine (Oppenheim et al., 1988). The amino terminal 24 residues of all three proteins are identical, except for the substitution of Glu and Arg (residues 4 and 11) in histatin 1 with Ala and Lys, respectively, in histatins 3 and 5. Histatin 1 contains a phosphorylated serine at residue 2, whereas histatins 3 and 5 lack inorganic phosphate. Histatin 1 contains a 9 amino acid insert absent in histatin 3, and histatin 3 contains a 3 amino acid insert absent in histatin 1. Histatin 5 is identical to the amino terminal 24 residues of histatin 3. It was reported in the earlier literature (Azen, 1973) that histatin 3 appears to display a genetic polymorphism, and histatin 1 does not, providing indirect evidence for the origin of these proteins from more than one gene. Histatins have been implicated in several different biological processes important to the maintenance of hard tissues in the oral cavity. Histatin 1 was initially discovered when it was shown that this protein selectively adsorbed to hydroxylapatite and enamel powders (Hay, 1973, 1975), implicating it as a precursor of the acquired enamel pellicle (Mayhall, 1970). The composition of this proteinaceous layer covering tooth surfaces is incompletely understood, but it is known that it forms an important physical barrier between tooth enamel and the oral environment (Hay, 1983). We have shown that histatin 1 inhibits hydroxylapatite crystal formation, which suggests that it Received for publication May 1, 1989 Accepted for publication July 20, 1989 This work was supported by National Institutes of Health Grants DE 05672 and DE 07652. 2
Materials and methods. Histatin isolation. -Human parotid saliva was collected with the aid of a Carlson-Crittendon device, as described previously (Oppenheim et al., 1971). Samples collected in glass tubes on ice from multiple donors were pooled, dialyzed in Spectropor 6 membrane against distilled water at 4°C, lyophilized, taken up in 50 mL of 0.05 mol/L ammonium formate (pH 4.0), and chromatographed on a Bio-Gel P-2 column (2.6 x 86 cm) equilibrated with the same buffer, as described previously (Oppenheim et al., 1986). Fractions containing histatins were pooled, lyophilized, dissolved in 1% acetic acid, and subjected to high-performance liquid chromatography (HPLC) on a Vydac C18 column (4.6 x 15 cm; 5 A, 300-,urm pore size) by use of a Waters reversed-phase HPLC system (RP-HPLC). The column was developed with a complex gradient at a flow rate of 1 mL/min from 100% solvent A (0.1% trifluoroacetic acid in water) to 100% solvent B (80% acetonitrile, 20% water, 0.1% trifluoroacetic acid), with monitoring at 214 and 254 nm. Column eluate was collected in 1.0-mL fractions, and those containing the various histatins were evaporated to dryness in a Savant Speed-Vac, taken up in 1% acetic acid, and re-chromatographed under conditions where the gradient was stopped and run isocratically at 6-10% less solvent B than that at which the polypeptide eluted initially. The resulting purified histatins were evaporated to dryness, dissolved in 1% acetic acid, and subjected to automated Edman degradation. Analytical procedures. -Histatins were examined on 15% slab gels with use of a cationic sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) system described previously (Baum et al., 1976, 1977; Oppenheim et al., 1988). The amino acid compositions of selected histatins and derived peptides were determined with use of a Beckman system 6300 amino acid analyzer following hydrolysis in 6 mol/L HCl for
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
E | 0.2
6
J
0
3
HUMAN SALIVARY HISTATINS
Vol. 69 No. 1
2
3
---A--p----
-
B-------
C-----+---I)---i--'--------*:~~ ~ ~ ~ ~~6
-
E
-4
6
2 40 00_ft00000 : 60 00 0;; 100f f 1 001 etuat f at20n Fractio A-E w pold lypilzd w h and formate, pH 4.0, at a flow rate of 385 200 : 400 0207 600 t:t0 Ai
exam~~~~~~~~~.ined .etecroporeicl~lyl~.L Win thectionicsagesytem.Fractiont Ai lan 0-; a, 100 1g1 lan b, 70 iig fratio Bl lanes a an 10 p-;frcinC Fig. 1-Elution profile of 1 g of human parotid saliva protein from Bio-Gel P-2. The column (2.6 x 86 cm) was developed with 0.05 mol/L ammonium formate, pH 4.0, at a flow rate of 38 mL/h, with continuous monitoring of eluate at 230 nm. Fractions A-E were pooled, lyophilized, weighed, and examined electrophoretically in the cationic slab gel system. Fraction A, lane a, 100 ,ug, lane b, 70 ,ug; fraction B. lanes a and b, 100 ,ug; fraction C, lane a, 100 Vg, lane b, 70 pug; fraction D, lane a, 100 pg, lane b, 70 ,ug; fraction E, lanes a and b, 100 pg. Fraction B contained some salt. Inset at left shows cationic electrophoretograms of parotid (PS) and submandibular (SS) proteins (150 ,ug each). Note that the staining intensity of histatins is comparable in both glandular secretions.
22 h in vacuo at 1 100C. Amino acid sequences were determined on an Applied Biosystems, Inc., 470A gas-phase sequencer equipped with a 120 PTH analyzer.
Results. Electrophoretic analysis of human parotid saliva protein in a cationic PAGE system resulted in a reproducible and characteristic pattern in which the top half of the gel contained an
unresolved mixture of salivary proteins, and the bottom half contained three pairs of bands and, in some cases, one or more bands at the dye front (Fig. 1, inset at left, PS). This observation has been extended to show that the same pattern of bands was present when human submandibular saliva protein was examined in the cationic PAGE system (Fig. 1, inset at left, SS). We have named the proteins corresponding to the three pairs of bands histatins 1 and 2, histatins 3 and 4, histatins 5 and 6, and the most rapidly migrating band, histatin 7 (Oppenheim et at, 1988). Histatin 7 is in fact a mixture of small histatins (histatins 7-12) that has been resolved into six components (see below). The nomenclature used for histatins described in this paper is consistent with the original observations on the electrophoretic mobility of histatins in the cationic PAGE system (Baum et al., 1976, 1977). We have shown that when human parotid saliva proteins are chromatographed on Bio-Gel P-2, histatins as a group are selectively retained (Oppenheim et al., 1988). In view of this anomalous behavior, a more detailed electrophoretic analysis of selected fractions in the histatin peak was performed (Fig.
1). The results showed that fraction A containing the bulk of salivary proteins was devoid of histatins, fraction B contained only traces of histatins 1 and 5, and fractions C, D, and E contained the bulk of histatins and no other salivary proteins. In fractions C-E, it was evident that histatins 1, 5, and 7-12 had a tendency to elute somewhat earlier, whereas histatins 2, 3, 4, and 6 had a tendency to elute somewhat later. Histatins 7-12 were incompletely resolved.
0I-
z
0in (A)
0
15
3045
60
TIME (min) Fig. 2-Separation of histatins by RP-HPLC. The histatin fraction from Bio-Gel P-2 was chromatographed on a Vydac C18 column (4.6 x 150 mm; pore size 5 pum) developed with an acetonitrile gradient (dotted line) at a flow rate of 1.0 mL/min. Fractions containing the partially purified histatins were re-chromatographed under isocratic conditions to yield purified preparations (see Fig. 4) that were used for sequence analysis.
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
TROXLER et aL
4
J Dent Res January 1990
10 30 20 Hi stat in 1: Asp-Pse-Hi s-C Iu-Lvs-Arg-Hi s-Hi s-C Iy-Tyr-Arg-Arg-Lgs-Phe-Hi s-G Iu-Lgs-H is-H Is-Ser-Hi s-Arwg-C Iu-Phe-Pwo-Phe-Tyr-Cig-Asp-Tyr-G- I-Ser-
Arg-Lgs-Phe-HIs-GIu-Lys-His-His-Ser -HIs-Ar -CIu-Phe-Pro-Phe-Tyr -G -Asp-Tyr-CGI-Ser-
Histatin 2: 38 Asn-Tyr -Leu-Tyr-Asp-Asn
Asn-Tyr -Leu-Tuyr-Asp-Asn 20
10
I
30
Hi stat in 3:
Asp-Serw-Hi s-Al a-Lys-Arg-H is-His-G Iy-Tyr-Lgs-Arg-Lgs-Phe-Hi s-Cl u-Lgs-H is-His-Serw-Hi s-Arg-C ig-Tyr-Arg-Serw-Asn-Tgr-Leu-Trw-Asp-Asn
Histatin 4:
Arg-Lys-Phe-His-GIu-Lgs-HIs-His-Ser-His-Arg-GI -Tgr-Arg-Ser-Asn-Tgr-Leu-Tgr-Asp-Asn
Histatin 5:
Asp-Ser-His-A la-Lgs-Arg-His-His-GIy-Tyr-Lgs-Arg-Lgs-Phe-His-Clu-Lys-His-His-Ser-His-Arg-GCI-Tgr
HistatIn 6:
Asp-Ser-His- Ala-Ls-Arg-His-His- CI
-Tr-Lgs-Awg-LAs-Phe-His-CIu-Lys-His-His-Ser-His-Arg-G I-Tgr-Arg
Histatin 7:
Arg-Lgs-Phe-His.CIu-Lgs-His-His-Ser-His-Arg-GIy-Tyr
Histatin 8:
Lgs-Phe-His-Glu-Lgs-His-His-Ser-His-Arg-Clg-Tgr
Histatin 9:
Arg-Lgs-Phe-His-Clu-Lgs-His-His-Ser-Hls-Akg-GlI-Tgr-Arg
Lgs-Phe-His-Clu-Lgs-His-His-Ser-His-A-rg-Gl-Twr--Ag
Histatin 10: Histatin I1:
Lgs-Arg-His-His-Clg-Tyr-Lls-Arg
Histatin 12:
Lgs-Arg-His-His-ClV-Tyr-L-s
Fig. 3-Amino acid sequences of histatins 1-12.
When histatins from Bio-Gel P-2 were subjected to RPHPLC, a characteristic elution pattern of peaks was observed (Fig. 2). This consisted of a group of hydrophilic components eluting between 14-19% solvent B (histatins 7-12), followed by three distinct pairs of peaks, eluting at approximately 2122% (histatins 5, 6), 24-26% (histatins 3, 4), and 32-35% solvent B (histatins 1, 2), with each pair consisting of a larger and smaller component. We have previously shown that the larger components in each pair of peaks are histatins 1, 3, and 5, respectively. All histatins not previously characterized have now been purified to homogeneity by RP-HPLC, and their amino acid sequences determined (Fig. 3). The smaller component in the pair of peaks eluting at 3235% solvent B was designated as histatin 2, because it had an amino acid sequence identical to residues 12-38 of histatin 1. This component apparently arises by a tryptic-like cleavage of the bond between Arg 11 and Arg 12 in histatin 1. The smaller component in the pair of peaks eluting at 24-26% solvent B was designated as histatin 4, because it had an amino acid sequence identical to the carboxyl-terminal 21 residues of histatin 3. This component could arise by a tryptic-like cleavage of the bond between Lys 11 and Arg 12 in histatin 3. The smaller component in the pair of peaks eluting at 21-22% solvent B was designated histatin 6, and was identical to histatin 5, but contained an additional carboxyl-terminal arginine residue. The group of more hydrophilic components eluting from the C1s column between 14-19% solvent B (Fig. 2) were purified to apparent homogeneity by RP-HPLC. Complete separation of partially resolved histatins was achieved by re-chromatog-
raphy under conditions of isocratic elution, as described in "Materials and methods". The effectiveness of this procedure for final purification is illustrated (Fig. 4) for histatins 7-10, which eluted as a fused group of peaks during initial RP-HPLC of the histatin fraction from Bio-Gel P-2. The material in fraction 27 (Fig. 4A), which initially eluted at 17.8% solvent B, was re-chromatographed with isocratic elution at 11.2% solvent B, and was resolved into two components, histatins 9 and 10 (Fig. 4B). The material in fraction 28 (Fig. 4A), which initially eluted at 18.4% solvent B, was re-chromatographed with isocratic elution at 12.4% solvent B, and was also resolved into two components, histatins 7 and 8 (Fig. 4C). The material in fraction 29 (Fig. 4A), which initially eluted at 19% solvent B, was re-chromatographed with isocratic elution at 13% solvent B to yield primarily histatin 7 (Fig. 4D). Histatins 7 and 8 were identical to residues 12-24 and 1324 of histatin 3, respectively, and could be derived by cleavage at Lys 11, Arg 12, and Tyr 24 (or by cleavage of histatin 5 at Lys 11 and Arg 12). The amino acid sequences of histatins 9 and 10 were identical to residues 12-25 and 13-25 of histatin 3, respectively, and could be derived by cleavage at Lys 11, Arg 12, and Arg 25 (or cleavage of histatin 6 at Lys 11 and Arg 12). Histatins 11 and 12 were identical to residues 5-11 and 5-12 of histatin 3, respectively, and could formally be derived from histatins 3, 5, or 6 by cleavage at Ala 4, Lys 11, and Arg 12. It is noteworthy that fragments corresponding to residues 111 of histatins 2 and 4, residues 1-4 of histatins 7-12, and residues 25-32 of histatin 5 and 26-32 of histatin 6 were not found among the components retained on Bio-Gel P-2, despite
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
Vol. 69 No. 1
HUMAN SALIVARY HISTA TINS
5
the fact that all histatins resolved by RP-HPLC were sequenced. It is possible that these fragments eluted with the bulk of salivary proteins in the void volume peak from BioGel P-2 and were present in insufficient quantities to be detected electrophoretically.
Discussion. The present investigation has shown that the family of basic histidine-rich polypeptides, known as histatins, occurs as electrophoretic entities in both parotid and submandibular secretions. While histatins from submandibular secretion were not isolated and characterized as proteins in this investigation, there is little doubt that the pairs of protein bands in the lower half of the cationic slab gels (Fig. 1, inset, SS) are indeed histatins. We have shown in Northern analyses that histatin mRNAs are present in both human parotid and submandibular glands (vanderSpek et al., 1989) and have sequenced cDNAs for histatins 1 and 3 from a human submandibular gland cDNA library (vanderSpek et al., 1990). We also describe, for the first time, the complete amino acid sequence of histatins 2, 4, 6, and 7-12 from parotid secretion. It is noteworthy that with the exception of histatin 2, which is probably a proteolytic fragment of histatin 1, all other histatins could be derived by tryptic-like or chymotryptic-like cleavages of histatin 3. The proteolytic cleavages giving rise to the various histatins appear to be quite specific. First, of the 7 Arg + Lys residues in histatin 1, cleavage appears to occur only at Arg 11 to yield histatin 2. Second, if histatins 4-12 are in fact fragments derived from histatin 3, of the 8 Arg + Lys residues present, cleavage appears to occur only at Lys 11, Arg 12, and Arg 25. Of the amino acid residues potentially susceptible to chymotryptic-like cleavage, such cleavage occurs only at Ala 5 and Tyr 24. Since the electrophoretic patterns of histatins in samples of pooled parotid and submandibular secretion are virtually identical, histatins appear to arise by a series of highly specific proteolytic processing events. The details of the regulatory mechanisms governing cleavage of the parent molecules (histatins 1 and 3) are not known, but the constancy of the electrophoretic pattern reflects biological control at some level. The proteolytic events giving rise to histatins are likely to occur in conjunction with secretion either in the endoplasmic reticulum, in the golgi, in secretary vesicles, or during exocytosis. It is unlikely that cleavage of parent histatins occurs in an uncontrolled manner during transit through the glandular duct system, because the electrophoretic pattern of histatins (and, for that matter, of other major salivary proteins) was essentially unchanged from donor to donor, and random proteolysis after secretion would not be expected to lead to the consistent electrophoretic banding pattern observed. Furthermore, it is highly unlikely that histatins arise by proteolysis during collection of glandular secretion, because samples were collected on ice by the same procedure we used to obtain intact salivary proteins (Oppenheim et al., 1971, 1982, 1986, 1988). Dialysis of pooled saliva protein samples, chromatography on Bio-Gel P-2, and fraction collection of column eluate were performed at 4°C. Histatins do not appear to undergo proteolytic degradation in this chromatographic procedure, because the electrophoretic pattern of histatins is the same before and after chromatography on Bio-Gel P-2 (Fig. 1). RP-HPLC was carried out at room temperature in solvents A and B at pH 2. Histatins are stable in this solvent for one to two months, and known salivary proteases are not active at this acidic pH. It is of interest that another family of small cationic polypeptides, termed defensins, occurs in human (Selsted et al.,
E
w z m
z
m
0
C')
TIME(nin)
Fig. 4-Purification of histatins 7-10 by RP-HPLC with use of isocratic elution. The flow rate was 1 mL/min, 1-mL fractions were collected, and
column eluate was monitored at 214 nm. (A) Partial elution profile of histatins 7-10 under conditions described in the legend to Fig. 2. (B) Rechromatography of the fraction eluting at 27 min (tube 27) in (A). The gradient was stopped at 11.2% solvent B, and the column developed isocratically, yielding purified histatins 9 and 10. (C) Re-chromatography of the fraction eluting at 28 min (tube 28) in (A). The gradient was stopped at 12.4% solvent B, and the column was developed isocratically, yielding purified histatins 7 and 8. (D) Re-chromatography of the fraction eluting at 29 min (tube 29) in (A). The gradient was stopped at 13.0% solvent B, and the column was developed isocratically, yielding purified histatin 7.
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
6
TROXLER et al.
1985), guinea pig (Selsted and Harwig, 1987), and rabbit (Selsted et aL, 1985) neutrophils or macrophages. Defensins appear to play a major role in the non-immune, oxygen-independent microbicidal function of phagocytes (Ganz et aL, 1985). These polypeptides are cysteine- and arginine-rich, range in length from 29-34 amino acid residues, and constitute 5-15% of the protein in azurophilic granules (Rice et al., 1987). While the positions of Cys and Arg are conserved in defensins from different mammalian species, 65% of the residues show little homology (Stanfield et at., 1988). Defensins appear to display a broad spectrum of microbicidal effects against bacteria (Selsted et al., 1984) and fungi, including C. albicans (PattersonDelafield et at., 1980), and have also been shown to inactivate HSV-2, vesicular stomatitis, and influenza viruses (Lehrer et al., 1985). While there is no sequence homology between defensins and salivary histatins, both groups of proteins are small and cationic, and appear to play a key role in the non-immune host defense system. It was recently reported that whole saliva, parotid secretion, and submandibular secretion inhibit infectivity of human immunodeficiency virus 1 (HIV-1) (Fox et al., 1988). This effect appears to be specific for HIV-1, because it is well-known that saliva does not exhibit broad-spectrum antiviral activity (Alter, 1977; Schupfer et at., 1986). The protein or proteins from salivary glands that inhibit infectivity of HIV-1 are not known, but the possible involvement of histatins in this process cannot be excluded. REFERENCES ALTER, H.J. (1977): Transmission of Hepatitis B to Chimpanzees by Hepatitis B Surface Antigen Positive Saliva and Semen, Infect Immun 16:928-933. AZEN, E.A. (1973): Properties of Salivary Basic Proteins Showing Polymorphism, Biochem Genet 9:69-86. BAUM, B.J.; BIRD, J.L.; and LONGTON, R.W. (1977): Polyacrylamide Gel Electrophoresis of Human Salivary Histidine-rich Polypeptides, J Dent Res 56:1115-1118. BAUM, B.J.; BIRD, J.L.; MILLAR, D.B.; and LONGTON, R.W. (1976): Studies on Histidine-rich Polypeptides from Human Parotid Saliva, Arch Biochem Biophys 177:427-436. FOX, P.C.; WOLFF, A.; YEH, C.K.; ATKINSON, J.C.; and BAUM, B.J. (1988): Saliva Inhibits HIV-1 Infectivity, J Am Dent Assoc 116:635-637. GANZ, T.; SELSTED, M.E.; SZLAREK, D.; HARWIG, S.S.L.; DAHER, K.; BAINTON, D.F.; and LEHRER, R.I. (1985): Defensins. Natural Peptide Antibiotics of Human Neutrophils, J Clin Invest 76:1427-1435. HAY, D.I. (1973): The Interaction of Human Parotid Salivary Proteins with Hydroxylapatite, Arch Oral Biol 18:1517-1529. HAY, D.I. (1975): Fractionation of Human Parotid Salivary Proteins and the Isolation of an Histidine-rich Acidic Peptide Which Shows High Affinity for Hydroxyapatite Surfaces, Arch Oral Biol 20:553558. HAY, D.I. (1983): Human Glandular Salivary Proteins: In: Handbook of Experimental Aspects of Oral Biochemistry, E. P. Lazzari, Ed., Boca Raton, FL: CRC Press, pp. 319-335. LEHRER, R.I.; DAHER, K.; GANZ, T.; and SELSTED, M.E. (1985): Direct Inactivation of Viruses by MCP-1 and MCP-2, Natural Peptide Antibiotics from Rabbit Leucocytes, J Virol 54:467-472. MACKAY, B.J.; DENEPITIYA, L.; IACONO, V.J.; KROST, S.P.;
J Dent Res January 1990
and POLLOCK, J.J. (1984): Growth Inhibitory and Bacteriocidal Effects of Human Parotid Salivary Histidine-rich Polypeptides on Streptococcus mutans, Infect Immun 44:695-701. MAYHALL, C.W. (1970): Concerning the Composition and Source of the Acquired Enamel Pellicle of Human Teeth, Arch Oral Biol 15:1327-1341. OPPENHEIM, F.G.; HAY, D.I.; and FRANZBLAU, C. (1971): Proline-rich Proteins from Human Parotid Saliva. I. Isolation and Characterization, Biochemistry 10:4233-4238. OPPENHEIM, F.G.; OFFNER, G.D.; and TROXLER, R.F. (1982): Phosphoproteins in the Parotid Saliva of the Subhuman Primate, Macaca fascicularis. Isolation and Characterization of a Prolinerich Phosphoglycoprotein and Complete Covalent Structure of a Proline-rich Phosphopeptide, J Biol Chem 257:9271-9282. OPPENHEIM, F.G.; XU, T.; McMILLIAN, F.M.; LEVITZ, S.M.; DIAMOND, R.D.; OFFNER, G.D.; and TROXLER, R.F. (1988): Histatins, a Novel Family of Histidine-rich Proteins in Human Parotid Secretion. Isolation, Characterization, Primary Structure and Fungistatic Effects on Candida albicans, JBiol Chem 263:74727477. OPPENHEIM, F.G.; YANG, Y.C.; DIAMOND, R.D.; HYSLOP, D.; OFFNER, G.D.; and TROXLER, R.F. (1986): The Primary Structure and Functional Characterization of the Neutral Histidinerich Polypeptide from Human Parotid Secretion, J Biol Chem 261:1177-1182. PATTERSON-DELAFIELD, J.; MARTINEZ, R.J.; and LEHRER, R.I. (1980): Microbicidal Cationic Proteins in Rabbit Alveolar Macrophages: a Potential Host-defense Mechanism, Infect Immun 30:180-192. POLLOCK, J.J.; DENEPITIYA, L.; MACKAY, B.J.; and IACONO, V.J. (1984): Fungistatic and Fungicidal Activity of Human Parotid Salivary Histidine-rich Polypeptides on Candida albicans, Infect Immun 44:702-707. RICE, W.G.; GANZ, T.; KINKADE, J.M.; SELSTED, M.E.; LEHRER, R.I.; and PARMLEY, R.T. (1987): Defensin-rich Granules of Human Neutrophils, Blood 70:757-765. SCHUPFER, P.C.; MURPHY, J.R.; and BALE, J.F. (1986): Survival of Cytomegalovirus in Paper Diapers and Saliva, Pediatric Infect Dis 5:677-679. SELSTED, M.E.; BROWN, D.M.; DELANGE, R.J.; HARWIG, S.S.L.; and LEHRER, R.I. (1985): Primary Structure of Six Antimicrobial Peptides of Rabbit Peritoneal Neutrophils, JBiol Chem 260:4579-4584. SELSTED, M.E. and HARWIG, S.S.L. (1987): Purification, Primary Structure and Antimicrobial Activities of a Guinea Pig Neutrophil Defensin, Infect Immun 55:2281-2286. SELSTED, M.E.; HARWIG, S.S.L.; GANZ, T.; SCHILLING, J.W.; and LEHRER, R.I. (1985): Primary Structures of Three Human Neutrophil Defensins, J Clin Invest 76:1436-1439. SELSTED, M.E.; SZLAREK, D.; and LEHRER, R.I. (1984): Purification and Antibacterial Activity of Antimicrobial Peptides of Rabbit Granulocytes, Infect Immun 45:150-154. STANFIELD, R.L.; WESTBROOK, E.M.; and SELSTED, M.E. (1988): Characterization of Two Crystal Forms of Human Defensin Neutrophil Cationic Peptide 1, a Naturally Occurring Antimicrobial Peptide of Leucocytes, J Biol Chem 263:5933-5935. VANDERSPEK, J.C.; WYANDT, H.E.; SKARE, J.C.; MILUNSKY, A.; OPPENHEIM, F.G.; and TROXLER, R.F. (1989): Localization of the Genes for Histatins to Human Chromosome 4q13 and Tissue Distribution of the mRNAs, Am J Human Genet (in press). VANDERSPEK, J.C.; OFFNER, G.D.; OPPENHEIM, F.G.; and TROXLER, R.F. (1990): Molecular Cloning of Human Submandibular Histatins, Arch Oral Biol (in press).
Downloaded from jdr.sagepub.com at Afyon Kocatepe Universitesi on May 21, 2014 For personal use only. No other uses without permission.
