Vol. 36, No. I, pp. 529-534, 1991 Printed in Great Britain. All rights reserved

0003~9969/91 $3.00 + 0.00 Copyright 0 1991 Pergamon Press plc

Archs oral Bid.

MULTIPLE

FORMS OF STATHERIN IN HUMAN SALIVARY SECRETIONS

J. L. JENSEN,’ M. S. LAMKIN,’ R. F. TROXLER’.’ and F. G. OPPENHEIM’*~* ‘Department of Periodontology and Oral Biology, Boston University School of Graduate Dentistry and ‘Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, U.S.A. (Accepted 9 January 1991) Summary-Sequential chromatography of hydroxyapatite-adsorbed salivary proteins from submandibular/sublingual secretions on Sephadex G-50 and reversed-phase HPLC resulted in the purification of statherin and several statherin variants. Amino acid analysis, Edman degradation and carboxypeptidase digestion of the obtained protein fractions led to the determination of the complete primary structures of statherin SVl, statherin SV2, and statherin SV3. SVl is identical to statherin but lacks the carboxyl-terminal phenylalanine residue. SV2, lacking residues 6-15, is otherwise identical to statherin. SV3 is identical to SV2 but lacks the carboxyl-terminal phenylalanine. These results provide the first evidence for multiple forms of statherin which are probably derived both by post-translational modification and alternative splicing of the statherin gene. Key words: statherin, salivary proteins, phosphoproteins, spectroscopy.

INTRODUCTION Secretions from the major salivary glands contain a number of proteins and peptides that have an un-

usually strong affinity to hydroxyapatite. Unlike most proteins, which can be desorbed from hydroxyapatite by high concentrations of phosphate, these salivary proteins often require dissolution of hydroxyapatite by EDTA and therefore may possibly interact with the enamel surface. This group of salivary proteins includes the acidic proline-rich proteins (Hay et al., 1988; Oppenheim, Hay and Franzblau, 1971), statherin (Schlesinger and Hay, 1977), histatins (Oppenheim et al., 1986, 1988) and cystatins (Al-Hashimi, Dickinson and Levine, 1988). Three of these four groups are families of related proteins arising from different mRNAs or post-translational processing, with statherin being the only protein not known to be polymorphic (Minaguchi and Benneck, 1989). Statherin has been purified: it is a polypeptide consisting of 43 amino acid residues, containing vicinal phosphoserines at positions 2 and 3 and seven residues of tyrosine (Schlesinger and Hay, 1977). It is unique among salivary proteins in its ability to inhibit both spontaneous precipitation of supersaturated solutions of calcium phosphate and crystal growth of seeded crystals in supersaturated solutions of calcium phosphates (Schlesinger and Hay, 1977; Hay, Moreno and Schlesinger, 1979). During our studies of precursors of enamel pellicle protein from human submandibular/sublingual secretions, we observed several components that

*To whom all correspondence should be addressed. Abbreviations: FABMS, fast atom-bombardment mass spectometry; HPLC, high-pressure liquid chromatography; TFA, trifluoracetic acid.

phosphoserine,

fast atom bombardment mass

adsorbed to hydroxyapatite, co-eluted with statherin from gel filtration columns and had a high tyrosine content. Our purpose now was to purify these proteins from the glandular secretions and to elucidate the amino acid sequence of these uncharacterized components. MATERIALS

AND METHODS

Salivary secretion collection Stimulated human submandibular/sublingual secretions were collected over ice (Oppenheim, 1970), and NaN, was added immediately to a final concentration of 0.01%. The secretion was clarified by centrifugation for 20 min at 20,000 x g at 4°C. Adsorption to hydroxyapatite

Clarified secretions were incubated with hydroxyapatite (59.56m2/g) (Monsanto, St Louis, MO) at a mineral: secretion ratio of 5 mg/ml for 2 h at 25°C and centrifuged at 27,500 x g for 15 min at 4°C. The sediment was washed three times with 0.1 M NaCl, pH 7.5, and redissolved in 0.2 M EDTA, pH 7.5 overnight at 25°C. The solution, containing adsorbed proteins, was dialysed sequentially against 0.1 M NaCl, pH 7.5, 10 mM NaCl, pH 7.5, and deionized water using Spectropor tubing (i14, cut-off = lOOO), and lyophilized. Gel filtration chromatography Lyophilized protein from the adsorbed fractions was dissolved in 0.1 M ammonium bicarbonate, pH 8.0, containing 6 M guanidine-HCl (10-20 mg/ ml), and applied to a Sephadex G-50 (superfine) column (1.6 x 94 cm) equilibrated with 0.1 M ammonium bicarbonate, pH 8.0. The linear flow rate was maintained at 6cm/h and 4ml fractions were 529

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collected. The eluate was monitored with a Uvicord S monitor at 230nm; u.v.-absorbing fractions were pooled and lyophilized. Reversed-phase HPLC

One millilitre of 2% acetic acid was added to approx. 1 mg of lyophilized protein separated by gel filtration, to which solid guanidine-HCl was added to totally dissolve the sample. The protein solution was clarified, and fractionated by reversedphase HPLC (LKB Ultropac TSK-ODS, 120T 5 pm, 4.6 x 250 mm; LKB HPLC system). The column was equilibrated with 0.1% TFA (solvent A) and proteins were eluted using gradients of solvent A and solvent B (0.1% TFA in 80% acetonitrile) at a flow rate of 1 ml/min. The following gradient was used to separate the protein components of the statherin-containing fraction: 0% B at 0 min, O-20% B from O-10 min, 2&65% B from 10-65min, 65-100% B from 65-70 min and 100% B from 70-75 min. The eluate was monitored at 230 nm with a Uvicord S detector. When necessary, further purification was achieved by further chromatography on the above column using shallower gradients.

for calibration. The Cs+ gun was operated at 26 kV. All mass spectra were recorded in a JOEL DA5000 data system on a DEC PDPI l/73 computer. Glycerol or glycerol : 3-nitrobenzyl alcohol (1: 1, Vol/Vol) was used as the FAB matrix. RESULTS

Gel filtration of total protein from submandibular/ sublingual secretions and of the proteins from these secretions that adsorbed to hydroxyapatite revealed differences in the amounts of protein present in each peak [Fig. l(a)]. All of the proteins present in peaks D and E had the highest affinity to hydroxyapatite, because they were totally adsorbed; only a fraction of the proteins in peaks A and B was adsorbed and none of the proteins in fraction C. This selectivity of adsorption was further demonstrated by comparing the amino acid compositions of total proteins, adsorbed proteins and the protein present in fraction D (Table 1). The striking increase in tyrosine content resulting from adsorption and gel filtration suggests (a)

Amino acid analysis

08-

Lyophilized protein samples were dissolved in a deionized water, transferred to hydrolysis tubes, and dried in vacua. Samples were hydrolysed with 6 N HCl, 1% phenol in the vapour phase at 108°C for 24 h in a Waters PicoTag work-station. Hydrolysates were dried again and dissolved in sample buffer for analysis on a Beckman System 6300 amino acid analyser.

50.6-

Automated Edman degradation

0.2-

Proteins and peptides were subjected to automated Edman degradation using an Applied Biosystems Model 470-A gas-phase sequencer equipped with a Model 120 Pth-amino acid analyser. Carboxyl terminal amino acid determinations

Statherin SVl was digested with carboxypeptidase A (Boehringer Mannheim Biochemicals, Indianapolis, IN) at a ratio of 1: 50, as described by Ambler (1972) and released amino acids were identified by amino acid analysis.

I 40

60

120

160

041175 II/ I

SW 03

1

Enzymatic digestion of proteins

Trypsin (Worthington Biochemical, Freedhold, NJ) and endoproteinase Glu-C from Staphylococcus aureus (Boehringer Mannheim) were used to generate a limited number of peptide fragments of statherin SVl and SV2, respectively, as described previously (Oppenheim, Offner and Troxler, 1982; Oppenheim et al., 1986). The digestion products were subjected to reversed-phase HPLC using the gradient system described above. Fast atom-bombardment mass spectrometry

FABMS of purified protein was carried out in the first component (MS-l) of a tandem high-resolution mass spectrometer (JEOL HXl lO/HXllO) at a V, of 10 kV and a resolution of 1000. Single scans were acquired at a scan rate of 6000 m/z in 2.5 min with 100-300 Hz filtering. (CsI),Cs+ cluster ions were used

10

20

30

40

Fig. 1. Purification of statherin and statherin variants. (a) Gel filtration of total protein from 12.5ml submandibular/sublingual secretions (-----) and the protein fraction from 12.5 ml of submandibular/sublingual secretion that adsorbed to hydroxyapatite (-). The pooled fractions of peak D are indicated by an arrow. (b) Reversed-phase chromatography of fraction D on a C,, column. Fractions S, WI, SV2 and SV3 refer to statherin and variants SVl, SV2, SV3, respectively. The column was developed with a linear gradient as shown, using a flow rate of 1 ml/min. Solvent A: 0.1% TFA. Sohent B: 0.1% TFA in 80% acetonitrile.

Structure of statherin variants Table 1. Amino acid composition of submandibular/ sublingual secretion (HSMSL), the HSMSL fraction adsorbed to hydroxyapatite (HA) and statherine-containing fraction DC

Amino acid

Residues/ 100 residuks HA-adsorbed fraction

Total HSMSL

Asx Thr Ser Glx Pro Gly Ala Val fcys Met Ile

9.5 3.0 5.5 18.2 15.0 12.3 3.9 4.8 1.0 1.5 2.5 4.6 3.2 3.4 4.9 2.9 4.9

JAI

Tyr Phe Lys His Arg

1.1 1.3 4.2 23.5 18.9 15.0 1.3 2.1 0.0 0.0 1.4 3.6 6.2 3.2 2.8 3.5 5.5

Table 2. Amino acid composition of statherin and statherin variants from submandibular/sublingual secretion Residues/l0 Glx Amino acid

2.1 2.2 3.5 23.9 16.6 9.1 0.0 2.5 0.0 0.0 2.0 4.8 16.5 6.8 2.4 3.6 6.6

GlY Ala Val fcys Met Ile LeU Tyr

Phe Lys His

-4%

SVl

Statherin 1.1 cl)* 0.9 iij 1.1 (2) 10.0 (10) 1.1 (7) 4.0 (4) 0.0 (0) 1.0 (1) 0.0 (0) 0.0 (0) 0.1 (1) 2.0 (2) 6.9 (1) 2.9 (3) 1.0 (1) 0.0 (0) 2.8 (3j

Asx Thr Ser Glx Pro

Sephadex G-50 fraction D

531

-

sv2

sv3

1.1 (11’

1.2 cl)*

1.0 (2) 10.0 (10) 1.1 (1) 3.8 (4) 0.0 (0) 1.0 (1) 0.0 (0) 0.0 (0) 0.8 (1) 1.9 (2) 6.1 (1) 1.8 (2) 0.9 (1) 0.0 (0) 2.6 (3j

1.0 (2) 10.0 (10) 6.8 (7) 2.2 (2) 0.0 (0) 1.0 (1) 0.0 (0) 0.0 (0) 0.0 (0) 1.0 (1) 5.9 (I) 1.0 (1) 0.0 (0) 0.0 (0)

I.0 (ij

1.0 iij

0.0 (oj

1.6 cl)* 0.9 (ij 1.5 (2) 10.0 (10) 6.3 (1) 2.2 (2) 0.0 (0) 0.6 (1) 0.0 (0) 0.0 (0) 0.0 (0) 0.9 (1) 5.8 (I) 0.0 (0) 0.0 (0) 0.0 (0)

0.0 (oj

*cf. Fig. l(a).

*Values in parentheses indicate residues based on the amino acid sequence of statherin (Schlesinger and Hay, 1911), SVl and SV2 (this study), and in the case of SV3 values are based on the partial sequence and amino acid analysis.

that fraction D was enriched with respect to statherin. The material in fraction D was subjected to reversedphase HPLC, which resolved the sample into four clearly separated components designated S, SVl, SV2 and SV3 [Fig. l(b)]. The amino acid compositions of these components were determined and it was found that they all had a high tyrosine

content and were very similar or nearly identical to statherin (Table 2). Automated Edman degradation of component S for 8 cycles revealed a sequence identical to statherin (Table 3). Carboxypeptidase A digestion revealed the sequence -(Gln,Tyr,Thr)-Phe-CO,H [Fig. 2(a)], which is identical to the carboxyl-terminal sequence

Table 3. Edman degradation of statherin and statherin variants Cycle

Statherin

pmol

Asp Ser* Ser* Glu Glu Lys Phe

21 81 9 13 11 4

LeU

13 14 15 16 17 18 19 20 21 22 23 24 25 26 21 28 29

SVl

pm01

Pl

pm01

sv2

pm01

P2

pm01

sv3

pm01

Asp Ser+ Ser* Glu Glu Lys Phe Leu Arg Arg Ile Gly Arg Phe Gly Tyr

213 53 68 65 118 46 51 61 41 93 42 29 83 44 63 33

Phe Gly Tyr Gly Tyr Gly Pro Tyr Gln Pro Val Pro Glu Gln Pro

700 580 538 512 548 468 518 525 481 436 458 432 351 369 310 218 291 219 229 284 195 236 170 219 154 199 233 89 48

Asp Ser* Ser* Glu Glu Tyr Gly Tyr Gly Pro Tyr Gln Pro Val Pro Glu Gln Pro

68 24 34 21 33 15 11 25 25 25 14 14 29 5 33 9 11 18 9 3 19 8

‘Or

851 701 651 583 801 634 592 698 683 689 302 196 192 131 165 115 151 183 123 141 135 159 103 144 114 69 16 14

Asp Ser* Ser* Glu Glu Tyr Gly Tyr

96 59 63 131 111 111 96 121 103 96

I&U Tyr

Pro Gln Pro Tyr Gln Pro Gln Tyr Gln Gln Tyr Thr

LeU ‘M

Pro Gln

*Detected as the dehydro derivative wine indicative of phosphoserine.

GUY

Tyr GUY Pro Tyr Gln pro

Val pro

Glu Gln Pro Leu Tyr Pro Gln Pro Tyr Gln Pro Gln Tyr Gln Gln Tyr Thr Phe

GlY Pro

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5

10

15

Time

20

25

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et al.

0

5

10

15

Time

(min)

20

25

30

(min)

Fig. 2. Carboxypeptidase digestion of statherin and statherin WI. Statherin (a) and SVl (b) were incubated with carboxypeptidase A for up to 30min. Phe (O), Thr (a), Tyr (A) and Gln (a). The amounts shown were normalized using norleucine as an internal standard and corrected using enzyme

blanks at each time point. of statherin (Schlesinger and Hay, 1977). The aminoand carboxyl-terminal sequence determinations, taken together with overall amino acid composition, identified component S as statherin. Similarly, the first 16 residues of SVl were deduced by Edman degradation (Table 3) and were found to be identical to the amino-terminal 16 residues of statherin (Fig. 3). SVl was digested with trypsin, the peptides were resolved by reversed-phase HPLC (data not shown), and sequencing of the largest peptide, designated Pl, showed that it was identical to residues 14-42 of statherin (Table 3). Carboxypeptidase digestion showed that the carboxyl-terminal of SVl was -(Tyr, Gin)-Gin-Tyr-Thr-CO, H [Fig. 2(b)], which confirms that the tryptic peptide was sequenced to completion. Edman degradation of SV2 identified the first 22 residues (Table 3). This sequence was identical to residues l-32 of statherin except that the sequence -Lys-Phe-Leu-Arg-Arg-Be-Gly-Arg-Phe-Gly-, present in statherin, was absent in SV2 (Fig. 3). SV2 was digested with endoproteinase Glu-C from Staph. aureus, the peptides separated by reversed-phase HPLC (data not shown), and the largest peptide, designated P2, was sequenced (Table 3). The amino acid sequence of this 28residue peptide was identical to residues 16-43 of statherin (Schlesinger and Hay, 1977). SV3 was directly sequenced for 10 cycles and found to be identical to SV2 (Table 3). The amino acid composition of SV3 (Table 2) indicated that it contained one fewer residues of phenylalanine than SV2. It was concluded that SV3 was identical to SV2 but lacked the carboxyl-terminal phenylalanine. Statherin

When statherin, SVl, SV2 and SV3 were subjected to Edman degradation, the dehydro derivative of serine was recovered almost exclusively at cycles 2 and 3 indicating the presence of O-substituted serines at these positions. By FABMS, statherin, SVl and SV2 each displayed an m/z peak that was 160 mass units greater than predicted from their respective amino acid sequences (Table 4). It has been reported that (a) human and macaque statherins contain 2 mol phosphate per molecule by standard phosphate assays and (b), that these phosphate groups are covalently linked to serine residues in positions 2 and 3 by virtue of dehydro derivatives of serine being detected at the second and third cycles of Edman degradation (Schlesinger and Hay, 1977; Schlesinger, Hay and Levine, 1989; Oppenheim et al., 1982). Taken together, these data strongly suggest that these newly discovered statherin variants, SVl and SV2 (and most probably SV3), like statherin, contain phosphoserines in positions 2 and 3. Initial characterization of the statherin variants were carried out with submandibular/sublingual secretions from several subjects. The distributions of statherin variants was similar among the five donors evaluated. In addition, parotid secretions were found to contain the same statherin variants in similar distributions among 8 donors. DISCUSSION

We show for the mandibular/sublingual of statherin variants.

first time that human subsecretions contain a family This is significant because

DSSEEKFLRRIGRFGYGYGPYQPVPEQPLYPQPYQPQYQQYQQYTF

SVl

DSSEEKFLRRIGRFGYGYGPYQPVPEQPLYPQPYQPQYQQYT

SV2

DSSEE---

SV3

DSSEE-

- - - --

--YGYGPYQPVPEQPLYPQPYQPQYQQYTF

- - - - - - - - -YGYGPYQPVPEQPLYPQPYQPQYQQYT

Fig. 3. Primary structures of statherin and statherin variants purified by reversed-phase HPLC of fraction D from submandibular/sublingual-derived pelhcle. Sequences have been aligned to maximize homology. Dashes indicate residues missing from SV2 and SV3. Edman degradation of SV3 was carried out for 10 cycles and the remainder of its primary sequence was deduced from its amino acid composition. The serine residues at positions 2 and 3 of each protein are phosphorylated.

Structure of statherin variants Table 4. Molecular-weight determinations statherin variants Mr&,,,* Statherin Statherin SVl Statherin SV Statherin SV3

5380.8 5233.3 4149.3 4002.1

for the

(M + H)o+bscrvcdt 5380.1 5233.1 4150.3 -3

*Molecular weight was calculated from the amino acid sequences assuming phosphoserine at positions 2 and 3. t(M + H)+ was determined by FABMS. $Not determined.

statherin had previously been considered the only low molecular-weight component of salivas with high affinity to hydroxyapatite not to be a member of a multigene family (Minaguchi and Bennick, 1989). The gene for statherin is believed to be a single-copy gene and has been mapped to human chromosome 4qll-13 (Sabatini et al., 1987). Recently, the gene encoding statherin has been sequenced (Sabatini, He and Azen, 1990). It is likely that WI, lacking the carboxyl-terminal phenylalanine, arises through post-translational processing of statherin. The existence of SV2, missing a loresidue internal segment present in statherin, clearly points to a genetic basis for the origin of this polypeptide. SV2 would arise by alternative splicing as one element of the statherin gene, exon 4, consists of 30 nucleotides that code for the 10 amino acid residues missing in SV2. SV3, on the other hand, could also arise through post-translational processing of SV2. The finding that the carboxyl-terminal phenylalanine is absent in both SVl and SV3 indicates the presence of a previously unrecognized carboxypeptidase activity associated with submandibular, sublingual and parotid glands. Statherin is by far the most abundant form in the hydroxyapatite-adsorbed fractions from both glandular secretions. Together, the statherin variants SVl, SV2 and SV3 comprise approx. 30% of the statherin family. Furthermore, it is of interest that the ratios of statherin:SVl and SV2:SV3 are in both cases approx. 3 : 1. This suggests that the variants SVl and SV3 are indeed generated by post-translational removal of the carboxyl-terminal phenylalanine. The estimated relative proportions of statherin and its variants are based on data obtained with the salivary protein fraction that adsorbed to hydroxyapatite. As the amount of protein in fraction D of submandibular/sublingual secretions is the same as that in fraction D of the adsorbed material from these secretions, similar proportions of statherin and its variants can be expected in glandular secretions. The existence of variants of salivary proteins in both parotid and submandibular/sublingual secretions is not uncommon. The six major acidic proline-rich proteins have been observed in both glandular secretions. Of these proline-rich proteins, three variants, PRP- 1, PRP-2 and PIF-s, are products of different mRNAs. The other three variants, PRP -3, PRP-4, and PIF-f, are derived by a common proteolytic process (Hay et al., 1988). Similarly, statherin and SV2 are products of two different transcripts found in each of the major salivary glands.

533

It is suggested that SVl and SV3 are derived by post-translational processing of statherin and SV2. On the other hand, several salivary proteins, including cystatins and mucins, appear to be secreted predominantly if not exclusively by the submandibular and/or sublingual gland. The existence of SV2 and SV3 has relevance to the functional role of statherin in the oral environment. The amino-terminal tryptic peptide of statherin, residues 1-6, is the functionally important domain for the inhibition of secondary precipitation of calcium phosphate crystals (Schlesinger et al., 1987). This peptide contains five out of the six acidic amino acid residues found in statherin, namely an aspartic acid residue, the two vicinal phosphoserine residues, and the two glutamic acid residues. The peptide also lacks all but one basic amino acid, the lysine residue at position 6. The observation that this amino-terminal peptide is four times more effective in inhibiting secondary precipitation raises the possibility that SV2 and SV3 could also be more effective inhibitors of secondary precipitation than statherin because they contain the functionally important acidic residues including the two vicinal phosphoserines, they lack all of the basic amino acids, and are also smaller than intact statherin. This role as an inhibitor of secondary precipitation of calcium phosphate salts would be crucial to maintaining the mineral structure of enamel by preventing further mineralization, as the enamel is in continuous contact with saliva that is supersaturated with respect to calcium phosphate salts. Furthermore, as lysine and arginine residues are totally absent from SV2 and SV3, one could expect these variants to be largely resistant to trypsin-like degradation in the oral environment and, though less abundant that statherin and SVl, possibly of greater biological significance. Acknowledgements-Mass spectral data were provided by the MIT Mass Spectrometry Facility which is supported by NIH Grant No. RR00317 (to Dr Klaus Biemann). This research was supported by NIH Grants Nos DE07652 and DE05672. REFERENCES Al-Hashimi I., Dickinson D. P. and Levine M. J. (1988) Purification, molecular cloning, and sequencing of salivary cystatin SA-I. J. biol. Chem. 263, 9381-9387. Ambler R. P. (1972) Enzymatic hydrolysis with carboxypeptidases. Meth. Enzym. 25, 143-154. Hay D. I., Bennick A., Schlesinger D. H., Minaguchi K., Madapallimattam G. and Schluckebier S. K. (1988) The primary structure of six human salivary acidic prolinerich proteins (PRP-1, PRP-2, PRP-3, PRP4, PIFs, and PIF-f). Biochem. J. 255, 15-21. Hay D. I., Moreno E. C. and Schlesinger D. H. (1979) Phosphoprotein-inhibitors of calcium phosphate precipitation from salivary secretions. Znorg. Perspectives Biol. Med. 2, 271-285.

Minaguchi K. and Bennick A. (1989) Genetics of human salivary proteins. J. dent. Res. 68, 2-15. Oppenheim F. G. (1970) Preliminary observations on the presence and origin of serum albumin in human saliva. Helv. odont. Acta 14, l&17.

Oppenheim F. G., Hay D. I. and Franzblau C. (1971) Proline-rich proteins from human parotid saliva. I. Isolation and partial characterization. Biochemistry 10, 42334238.

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Oppenheim F. G., Offner G. D. and Troxler R. F. (1982) Phosphoproteins in the parotid saliva from the subhuman primate, Macaca fascicularis. Isolation and characterization of a proline-rich phosphoglycoprotein and the complete covalent structure of a proline-rich phosphopeptide. J. biol. Chem. 257, 9271-9282. 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 histidine-rich polypeptide from human parotid secretion. J. biol. Chem. Ml, 1177-1182. Oppenheim F. G., Xu T., McMillan 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 and characterization, primary structure and fungistatic effects on Candida albicans. J. biol. Chem. 263, 7472-7477.

Sabatini L. M., Carlock L. R., Johnson G. W. and Axen E. A. (1987) cDNA cloning and chromosomal localiz-

ation (4qIl-13) of a gene for statherin, a regulator of calcium in saliva. Am. J. hum. Genet. 41, 1048-1060. Sabatini L. M., He Y.-Z. and Azen E. A. (1990) Structure and sequence determination of the gene encoding human salivary statherin. Gene 89, 245-251. Schlesinger D. H. and Hay D. I. (1977) Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva. J. biol. Chem. 252, 1689-1695. Schlesinger D. H., Baku A., Wyssbrod H. R. and Hay D. I. (1987) Chemical synthesis of phosphoseryl-phosphoserine, a partial analogue of human salivary statherin, a potent inhibitor of calcium phosphate precipitation in human saliva. Int. J. Peptide Protein Res. 30, 257-262. Schlesinger D. H., Hay D. I. and Levine D. I. (1989) Complete primary structure of statherin, a potent inhibitor of calcium phosphate precipitation, from the saliva of the monkey, Macaea arctoides. Int. J. Peptide Protein Res. 34, 374-380.