©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Molecular Cloning of a Novel Bone Phosphoprotein Related in Sequence to the Cystatin Family of Thiol Protease Inhibitors (*)

(Received for publication, August 12, 1994; and in revised form, October 26, 1994)

Bo Hu Laura Coulson Bryan Moyer Paul A. Price (§)

From the Department of Biology, University of California, San Diego, La Jolla, California 92093-0322

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We describe here the isolation of a novel non-collagenous protein from the acid demineralization extract of bovine cortical bone. This 24-kDa protein is multiply phosphorylated at serine residues in Ser-X-Glu/Ser(P) sequences, a recognition motif for phosphorylation by the secretory pathway protein kinase, and we have termed this protein secreted phosphoprotein 24 (spp24).

The cDNA structure of spp24 was determined by sequencing cDNA fragments obtained by reverse transcription-polymerase chain reaction, 3`-rapid amplification of cDNA ends, and screening a gt11 cDNA library. This cDNA sequence predicts a 200-residue initial translation product which consists of a 20-residue signal sequence and the 180-residue mature spp24. Northern blot analysis using the spp24 cDNA showed that spp24 mRNA is in liver and bone but not in heart, lung, kidney, or spleen. A search of existing protein sequences revealed that the N-terminal 107 residues of mature spp24 are related in sequence to the cystatin family of thiol protease inhibitors, which suggests that spp24 could function to modulate the thiol protease activities that are known to be involved in bone turnover. Several of the proteins in the cystatin family that are most closely related to spp24 are not only thiol protease inhibitors but are also precursors to peptides with potent biological activity, peptides such as bradykinin and the neutrophil antibiotic peptides. It is therefore possible that the intact form of spp24 found in bone could also be a precursor to a biologically active peptide, a peptide which could coordinate an aspect of bone turnover.


INTRODUCTION

Bone is unusual among the extracellular matrices of vertebrates because it is continuously turned over throughout life. This turnover is mediated by the action of osteoblasts and osteoclasts, and serves to provide calcium derived from bone mineral for the maintenance of serum calcium homeostasis. Proteins secreted by bone cells can therefore have functions which range from the formation of the organic bone matrix and its mineralization to the removal of bone matrix by osteoclastic bone resorption and the coupling of resorption to formation.

In spite of over 20 years of concerted effort, only a few proteins have been isolated and characterized from bone matrix(1) , and relatively little is known about the function of these proteins in bone formation and turnover. The non-collagenous bone matrix-derived proteins whose amino acid sequence is presently known include the vitamin K-dependent proteins bone Gla protein (ossteocalcin) (2, 3) and matrix Gla protein (MGP)(^1)(4, 5) , the RGD-containing putative cell adhesion proteins osteopontin (6) and bone sialoprotein(7) , the small proteoglycans decorin and biglycan(8, 9, 10) , and osteonectin(11, 12) .

The objective of the present study was to identify new bone matrix-derived proteins and to evaluate their possible biological activities in bone formation and turnover. We report here the isolation and molecular cloning of a novel bone matrix protein of 24-kDa molecular mass whose N-terminal 107 residues are related in sequence to the cystatin family of thiol protease inhibitors. This protein is multiply phosphorylated at serine residues in Ser-X-Glu/Ser(P) sequences, the recognition motif for phosphorylation by the secretory pathway protein kinase (13) which has been observed in most secreted phosphoproteins(14) . We have termed this novel bone protein secreted phosphoprotein 24 (spp24).


MATERIALS AND METHODS

Purification of spp24 from Bovine Bone

Non-collagenous proteins were first extracted from 300 g of ground calf bone by demineralization with 3 liters of 10% formic acid for 3 h at 4 °C as described previously(15) . The extracted proteins were then adsorbed to a C(18) matrix, washed with 0.1% trifluoroacetic acid to remove bone mineral, and eluted with 50% acetonitrile in 0.1% trifluoroacetic acid. After freeze drying, the neutral pH-soluble proteins were removed by repeated suspension in 25 ml of 50 mM NH(4)HCO(3) followed by centrifugation until the supernatant A fell below 0.1.

The neutral pH-insoluble pellet was dissolved in 3 ml of 6 M guanidine HCl in 0.1 M Tris, pH 9, and applied to a 2 times 150-cm column of Sephacryl S-100 HR equilibrated with the same buffer at room temperature. The eluant fractions containing spp24 were pooled and further purified using a 4.6 mm times 25-cm C(4) reverse phase HPLC column with a 2-h gradient from 0.1% trifluoroacetic acid in water to 0.1% trifluoroacetic acid in 60% acetonitrile at a flow rate of 1 ml/min.

SDS-polyacrylamide gel electrophoresis was performed under reducing conditions as described elsewhere (15) using 4-20% gradient gels (Novex, San Diego, CA).

Determination of cDNA Sequence

Reverse transcription of RNA and polymerase chain reaction amplification of cDNA (RT-PCR) was used to determine the cDNA sequence of spp24. Based on information from the N-terminal amino acid sequence of purified bovine bone spp24 and of the cyanogen bromide peptide 1, a N-terminal sense degenerate primer (5`-TTT/CCCIGTITAT/CGAT/CTAT/CGA-3`, where I stands for inosine) and an internal antisense degenerate primer (5`-AA/GIATA/GTCICCA/GAAA/GAACAT-3`) were designed (see Table 1). Using an RNA preparation from either bovine bone periosteum (plus adjacent bone scrapings) or bovine liver and a GeneAmp RNA PCR Kit (Perkin-Elmer), a 380-bp cDNA fragment was generated and then amplified by RT-PCR in a Perkin-Elmer DNA thermal cycler. The PCR product was cloned directly into a plasmid vector using the TA Cloning System (Invitrogen, San Diego, CA). Following the enzymatic dideoxy chain termination method, both strands of the plasmid cDNA inserts were sequenced with a Version 2.0 DNA Sequencing Kit (U. S. Biochemical Corp., Cleveland, OH) using a 5% Long Ranger gel (AT Biochem, Malvern, PA). Identical sequences were obtained for this 380-bp fragment from liver and bone. The plasmid cDNA inserts were excised with EcoRI. Due to an internal EcoRI site in the insert, this digestion produced 68-bp and 312-bp cDNA fragments. The 312-bp fragment was employed as a probe for screening of a bovine liver gt11 cDNA library and for Northern blots.



The message from the internal region to the 3`-end was sequenced after generating a PCR fragment using 3`-rapid amplification of cDNA ends (3`-RACE)(16) . A specific internal sense primer (5`-CGCTGCCACTGGTCCTCCAGCTCT-3`) was synthesized which was located 45 bp upstream from the internal antisense degenerate primer. In addition, a unique 23-base oligonucleotide adapter primer linked to a 17-oligo(dt) (5`-ACGCGTCGACCTCGAGATCGATG-(dT)-3`), and the adapter primer (5`-ACGCGTCGACCTCGAGATCGATG-3`) were used for the 3`-RACE system. A 370-bp cDNA fragment was produced by both bovine periosteum and bovine liver total RNA and subsequently cloned and sequenced as described above. Identical sequences were obtained for this 370-bp fragment from bone and liver.

The 5`-end cDNA clone was obtained by screening a bovine liver gt11 cDNA library (Clontech, Palo Alto, CA) with the 312-bp cDNA probe. Bacteriophages were plated and transferred to nitrocellulose filters as described by the Clontech protocol. Two replica filters were lifted from each plate. Following hybridization to the P-labeled bovine 312-bp cDNA probe, eight positive phage plaques were isolated. Two of these contained the 5`-end 380-bp cDNA sequence as determined by PCR with the two degenerate primers. cDNA inserts of these two clones were enzymatically amplified by performing PCR with a specific antisense primer (5`-CAGATAGGGGCTCAGTGACTGGGA-3`) located 73 bp downstream from the N-terminal sense degenerate primer and either a gt11 forward or a gt11 reverse primer (Promega, Madison, WI). A 470-bp PCR product was obtained from one clone and a 300-bp PCR product from the other clone. The 5`-end PCR products were cloned and partially sequenced with TA Cloning System and Version 2.0 DNA Sequencing Kit as above.

Northern Blot Analysis

Bovine steer ankle bones were obtained from Talone's Meat Packers (Escondido, CA). The periosteum and adjacent bone scrapings were removed and frozen in liquid nitrogen. Frozen bovine heart, lung, kidney, liver and spleen were purchased from Monfort Biological (Greeley, CO). Total RNA was isolated from the tissues using guanidine isothiocyanate as previously described (17) and fractionated on 1.4% agarose formaldehyde gels in a MOPS buffer and transferred to a nylon membrane (0.45 µm; Nytran, Schleicher & Schuell). The 312-bp cDNA probe was labeled with [alpha-P]dCTP using a Random Primed DNA Labeling Kit (Boehringer Mannheim) to a specific activity of at least 1 times 10^8 cpm/µg. Hybridization to RNA immobilized on Nytran was typically performed for 17 h at 42 °C with 1-5 times 10^6 cpm DNA probe/ml of 50% formamide solution containing 5 times SSPE (1 times SSPE is 180 mM NaCl, 10 mM NaPO(4) (pH 7.4), and 1 mM EDTA), 5 times Denhardt's solution, and 100 µg/ml salmon sperm DNA after prehybridization at 42 °C in the same solution without the cDNA probe. Filters were washed in 0.5 times SSC (1 times SSC is 180 mM NaCl and 15 mM sodium citrate) containing 0.2% sodium dodecyl sulfate for 10 min at room temperature, followed by 1 h at 55 °C. Autoradiography was performed at -70 °C with Kodak XAR-5 film (Eastman Kodak Co.). RNA size markers (Life Technologies, Inc.) were run on an adjacent gel lane and stained with methylene blue.

Cleavage of spp24 with Cyanogen Bromide and BNPS-Skatole

To cleave spp24 at methionine, 5 mg of purified bovine bone spp24 were dissolved in 1 ml of 70% formic acid, and sufficient cyanogen bromide was added to achieve a final 50-fold molar excess of reagent to protein methionine residues. The reaction vessel was immediately flushed with N(2) and sealed, and the reaction mixture was stirred at room temperature for 16 h. To remove unreacted reagent, the digested protein was diluted 10-fold with water and dried, then dissolved in the same volume of water and redried. To cleave spp24 at tryptophan, 5 mg of purified bovine bone spp24 was dissolved in 567 µl of 60% acetic acid. 25 µl of tyrosine (3.67 mg/ml in 60% acetic acid) were added, and the cleavage reaction was then initiated by the addition of 11 mg of BNPS-Skatole (Pierce). The reaction vessel was wrapped with aluminum foil and stirred at room temperature. After 8 h, a second 25-µl aliquot of tyrosine was added. The digestion proceeded for an additional 16 h and was terminated by extracting the BNPS-Skatole reagent with diethyl ether. The aqueous phase, which contained the BNPS-Skatole peptides, was then freeze-dried. The peptides generated by both cleavage reactions were purified by gel filtration over a 2 times 150-cm Sephacryl S-100 HR column equilibrated with 6 M guanidine HCl in 0.1 M Tris-HCl, pH 9.0.

N-terminal Protein Sequencing

Purified proteins and peptides were transferred to poly(vinylidene fluoride) membranes using a ProSpin device (Applied Biosystems, Foster City, CA) and sequenced using an Applied Biosystems 470 A sequenator equipped with a model 120 on-line HPLC. To determine the location of phosphoserine residues within the serine-rich sequence of spp24, phosphoserine residues were converted to S-ethylcysteine by reaction with ethanethiol as described elsewhere(14, 18) . The percent phosphorylation at each target serine residue was determined from the relative recovery of serine and S-ethylcysteine at that sequence position.


RESULTS

Purification of spp24 from Bovine Bone

Spp24 was initially discovered in the course of developing improved procedures for the isolation of MGP from ground bovine bone sand, and the co-isolation of the two proteins during the first steps of purification is a reflection of the similar properties of spp24 and MGP. Both proteins are released from ground bovine bone sand by demineralization in 10% formic acid. Both proteins are insoluble at neutral pH and can be separated from neutral pH-soluble proteins such as the bone Gla protein by repeated washing of the dried proteins in the acid extract with 50 mM NH(4)HCO(3). The neutral pH-insoluble protein fraction can be solubilized in 6 M guanidine HCl buffer and fractionated by gel filtration over Sephacryl S-100 HR. As can be seen in Fig. 1, the major protein component recovered from the gel filtration step is MGP. The MGP isolated by this procedure is pure by the criterion of SDS-gel electrophoresis in 18% polyacrylamide gels and by N-terminal protein sequencing (data not shown). spp24 is recovered in a single peak centered at fraction 62 and, as judged by SDS-gel electrophoresis, is somewhat contaminated with other proteins (Fig. 1, inset). These protein contaminants can be removed from spp24 by chromatography on a C4 HPLC column using a linear acetonitrile gradient (Fig. 2). The resulting purified spp24 is homogeneous by the criterion of SDS-gel electrophoresis (Fig. 2, inset) and N-terminal protein sequencing (Table 1). To obtain additional sequence data for the construction of degenerate primers for RT-PCR, purified spp24 was cleaved at methionine with cyanogen bromide, and the resulting peptides were purified by filtration over Sephacryl S-100 HR in 6 M guanidine HCl buffer and subjected to N-terminal protein sequencing. The two internal peptides isolated by these procedures yielded single N-terminal sequences (Table 1).


Figure 1: Purification of spp24 and MGP by gel filtration over a Sephacryl S-100 HR column. The proteins extracted from ground bovine bone by demineralization in 10% formic acid were dried and the neutral pH-soluble proteins were removed by washing repeatedly with 50 mM NH(4)HCO(3). The water-insoluble proteins were then dissolved in 6 M guanidine HCl with 0.1 M Tris-HCl at pH 9.0 and loaded onto a 2 times 150 cm Sephacryl S-100 HR column equilibrated with the same buffer at room temperature. Fraction volume, 3 ml. Inset, SDS-polyacrylamide gel electrophoresis of partially purified spp24. Proteins were electrophoresed on a 4-20% gradient gel and stained with Coomassie Brilliant Blue. Lane 1, molecular mass standards; lane 2, 20 µg of the purified spp24 in pooled fractions 61-63. (See ``Materials and Methods'' for details.)




Figure 2: Further purification of spp24 by reverse phase high pressure liquid chromatography. 100 µg of the partially purified spp24 in pooled fractions 61-63 from the gel filtration shown in Fig. 1was loaded directly onto a 4.6 mm times 25-cm C(4) column equilibrated with 0.1% trifluoroacetic acid at room temperature. Bound proteins were subsequently eluted with a 2-h linear gradient to 0.1% trifluoroacetic acid in 60% acetonitrile. Fraction volume, 1.3 ml. Inset, SDS-polyacrylamide gel electrophoresis of purified spp24. Proteins were electrophoresed on a 4-20% gradient gel and stained with Coomassie Brilliant Blue. Lane 1, molecular mass standards; lane 2, 10 µg of the purified spp24 in pooled fractions 24-27.



As can be seen in Fig. 1, an additional protein component is recovered in the gel filtration step in a peak centered at fraction 55. When this fraction was subjected to SDS-gel electrophoresis using 4-20% gradient gels, a single major protein fraction was found of 38-kDa molecular mass (data not shown). When this 38-kDa protein was subsequently transferred from the gel to a poly(vinylidene fluoride) membrane and subjected to N-terminal protein sequencing, a single N-terminal sequence was obtained, YPQNWHHXSDLQHVILDKVGLQKIPKVREKT. A search of the non-redundant data base of the NLM using the BLAST search program (19) revealed this 38-kDa bone protein to be nearly identical in sequence to a recently reported 38-kDa protein isolated from bovine cartilage which has been termed cartilage leucine-rich protein (GenBank accession no. U08018). The identity of this 38-kDa bone matrix protein with the cartilage leucine-rich protein was confirmed by the isolation of a cyanogen bromide peptide whose sequence (XNLVSLHLQHXQIREVAAGAF) is identical to residues 77-97 of the bovine cartilage leucine-rich protein.

Nucleotide Sequence of Complete spp24 cDNA

The cDNA sequence of spp24 is shown in Fig. 3and was determined by RT-PCR, 3`-RACE, and nucleotide screening of a gt11 cDNA library following the strategy outlined in Fig. 4. The coding region of spp24 is terminated by a single TGA triplet at nucleotides 691-693 and a TAA triplet follows at nucleotides 700-702. The 3`-untranslated region consists of 139 nucleotides with a polyadenylation signal (AATAAA) at nucleotides 790-795. The ATG, found at nucleotides 91-93, was considered to be the initiation codon according to the rules for translation initiations described previously (20) . The open reading frame codes for a 200-residue long protein containing a 20-residue transmembrane signal peptide (21) with a potential signal peptidase cleavage site at amino acid residue 20(22) . The N terminus of mature spp24 was identified by N-terminal sequencing (Table 1) and is located 20 amino acids downstream from the presumed initiation methionine. The amino acid sequence of cyanogen bromide peptides 1 and 2 (Table 1) agreed with the deduced cDNA sequence. The deduced mature form of spp24 contains a total of 180 amino acids and has a calculated molecular weight of 20,458, in agreement with the 24-kDa size of spp24 determined by SDS-polyacrylamide gel electrophoresis ( Fig. 1and Fig. 2).


Figure 3: Complete nucleotide sequence of spp24 cDNA and deduced amino acid sequence of the protein. Underlined sequences correspond to those determined by protein sequencing (see Table 1). The cleavage site of the putative signal sequence is indicated by the arrow at residue 20 and the N terminus of mature spp24 begins at residue 21. The stop codons are marked by asterisks. The polyadenylation signal sequence AATAAA is indicated in bold lettering.




Figure 4: Strategy for determining the sequence of spp24 cDNA. (1) A 380-bp cDNA fragment which contains the N-terminal portion of spp24 was obtained using RT-PCR with two degenerate primers. (2) A 370-bp cDNA fragment which contains the 3`-untranslated region of spp24 was generated using 3`-RACE with a specific internal primer. (3) A 312-bp fragment of spp24 was used for gt11 cDNA library screening and Northern blot analysis. (4) A 470-bp cDNA fragment was obtained by screening a bovine liver gt11 cDNA library with the 312-bp cDNA probe and the 3`-region (243 bp) of this fragment was sequenced.



Tissue Distribution of spp24 mRNA

The expression of spp24 mRNA in various bovine tissues was examined by Northern blot analysis of total RNA using the 312-bp cDNA probe. This P-labeled probe revealed a single band at 1000-1100 nucleotides in the bovine periosteum and liver RNA samples (Fig. 5). No spp24 mRNA could be detected in bovine heart, lung, kidney, or spleen. The size of the spp24 mRNA from the Northern blots of bovine liver and periosteum agrees with the length of spp24 cDNA ( Fig. 3and Fig. 4).


Figure 5: Northern blot analysis of spp24 message levels in bovine tissues. RNA was extracted from the indicated bovine tissues and 40 µg of total RNA from each tissue was run on a 1.4% formaldehyde-agarose gel, blotted onto a Nytran membrane, and hybridized with a P-labeled, 312-bp spp24 cDNA fragment (Fig. 4). Lane 1, bone periosteum; lane 2, heart; lane 3, lung; lane 4, kidney; lane 5, spleen; lane 6, liver. The migration positions of molecular size markers in kilobases are indicated on the left.



Identification of Phosphoserine in the Serine-rich Sequence of spp24

As can be seen in Fig. 3, there is a serine-rich sequence between residues 128 and 136 of spp24 which contains several potential sites of serine phosphorylation by the Ser-X-Glu/Ser(P)-specific secretory pathway protein kinase (13) . To evaluate the possible phosphorylation of these serine residues, spp24 was first cleaved at tryptophan 127 using BNPS-Skatole, and the peptide corresponding to residues 128-190 of spp24 was purified by gel filtration over Sephacryl S-100 HR. This peptide was then treated with ethanethiol to convert the putative phosphoserine residues to S-ethylcysteine (14, 18) and subjected to N-terminal protein sequencing. The results of this analysis unambiguously identified the phenylthiohydantoin derivative of S-ethylcysteine at every serine residue within the serine-rich region of spp24. As has been noted for all other phosphoproteins secreted into the extracellular environment of cells (14) , the extent of serine phosphorylation was in each instance partial, ranging from 5% to 83% (Table 2). The presence of phosphoserine in spp24 was confirmed by acid hydrolysis and amino acid analysis (data not shown).




DISCUSSION

We have described the isolation and amino acid sequence of a novel bone phosphoprotein of 24-kDa molecular mass which we have termed secreted phosphoprotein 24 (spp24). The purification procedures developed for the isolation of spp24 are based on its insolubility at neutral pH and its solubilization by 6 M guanidine HCl or by acidic pH. spp24 was first separated by precipitation from the neutral pH-soluble proteins in the acid demineralization extract of bovine bone and then purified to homogeneity by gel filtration over Sephacryl S-100 HR in 6 M guanidine HCl buffer followed by reverse phase HPLC using a C4 matrix in an acidic buffer. Two other neutral pH-insoluble proteins were also recovered from the gel filtration step, MGP and a 38-kDa protein that appears to be identical to a 38-kDa protein recently isolated from bovine cartilage and termed cartilage leucine-rich protein (see above).

To evaluate the possible relationships between spp24 and other known proteins, the complete 200-residue spp24 sequence deduced from its cDNA structure was compared with all presently known protein sequences in the non-redundant data base of the NLM using the BLAST search program (19) . This search revealed the presence of a comparable level of sequence identity between spp24 and cystatin domains 1 and 3 of human kininogen (23) and between spp24 and the precursor to the bovine neutrophil antibiotic peptide bactenecin(24) . The bactenecin precursor and cystatin domains 1 and 3 of kininogen are known to be related in sequence to the cystatin family of thiol protease inhibitors(25, 26) , and spp24 is accordingly compared to two additional members of this family in Fig. 6, porcine cathelin (26) and chicken cystatin(27) . As can be seen by analysis of this figure, the bactenecin precursor and cathelin are more closely related to spp24 than to cystatin domains 1 and 3 of kininogen or to chicken cystatin. Cystatin domains 1 and 3 of kininogen and cystatin are also more closely related to spp24 than to bactenecin precursor or to cathelin. It is therefore probable that spp24 is an evolutionary intermediate which links cathelin, bactenecin precursor, and the closely related precursors to the neutrophil antibiotic peptides Bac5 (28) and indolicidin (29) with the various cystatins and with the cystatin domains of kininogen. The structure of a prototypical cystatin domain has been determined from crystallographic studies of the 108-residue chicken cystatin and is a compact structure with a 5-stranded beta-sheet wrapped around a 5-turn alpha-helix(30) . If, as seems probable, the sequence identities observed between spp24 and chicken cystatin reflect similar polypeptide conformations, it seems likely that the entire 107-residue region of spp24 between the N terminus of the mature protein and the 11-residue phosphoserine-rich sequence is folded into a cystatin-like tertiary structure. Since the 4 cysteine residues in the cystatin domain of spp24 lie at sequence positions known to be involved in disulfide bonds in other members of the cystatin family, it is probable that these 4 cysteine residues are likewise involved in disulfide bonds and that these bonds join Cys-63 with Cys-94 and Cys-87 with Cys-105 in the mature spp24 protein (Fig. 6).


Figure 6: Amino acid sequence homologies between spp24 and porcine cathelin, bovine bactenecin precursor, cystatin domains 1 and 3 of human kininogen, and chicken cystatin. Residue numbers refer to the sequence position in mature spp24. The related sequences are residues 1-92 of cathelin(26) , 24-126 of bactenecin precursor (24) , 23-127 of kininogen (cystatin domain 1)(23) , 268-371 of kininogen (cystatin domain 3)(23) , and 12-116 of chicken cystatin (27) . Identical amino acids are boxed.



Many members of the cystatin family have been shown to potently inhibit thiol proteases such as cathepsins and papain, and it is possible that the ability to inhibit thiol proteases is a feature of most proteins with a cystatin domain. Among the proteins most closely related in sequence to spp24, cathelin, chicken cystatin, and cystatin domain 3 of kininogen have been previously shown to inhibit thiol proteases(25, 26) . Although the bactenecin precursor has not itself been tested for its ability to inhibit thiol proteases, the closely related precursor to the neutrophil antibiotic peptide Bac5 has been reported to potently inhibit cathepsin L(28) . If spp24 is in fact a thiol protease inhibitor, the presence of spp24 in bone suggests that the target thiol protease is also found in bone. Several thiol proteases of the cathepsin family are in fact known to be expressed by bone cells(31) , and there is evidence to suggest that such thiol proteases may be released from osteoclasts to digest collagen and various non-collagenous proteins under the acidic conditions of osteoclast-mediated bone resorption(32) .

A second possible spp24 function is suggested by the observation that the cystatin domains most closely related in sequence to spp24, cystatin domain 3 of kininogen and the cystatin domain of the neutrophil antibiotic precursors, lie to the immediate N terminus of a peptide segment which, when released by protease action, has potent biological activity. Cleavage of kininogen with kallikrein releases bradykinin, a potent vasodilator, cleavage of the bactenecin precursor of neutrophils yields the antibiotic dodecapeptide bactenecin, cleavage of the indolicidin precursor yields the tryptophan-rich antibiotic tridecapeptide indolicidin, and cleavage of the Bac5 precursor yields the 46-residue antibiotic polypeptide Bac5. The common location of these peptides to the immediate C terminus of cystatin domains suggests that the proteolytic cleavages which release the active peptides may involve a common mechanism of substrate recognition that is based in part on the presence of proximal cystatin domain. If an analogous proteolytic cleavage were directed by recognition of the cystatin domain of spp24, the resulting peptide would be derived from the C-terminal 62 residues of spp24, a region which is unrelated in sequence to any known protein. It is important to note that, in spite of the very high number of sequence identities between the cystatin domains of these neutrophil antibiotic precursors, there is no significant level of sequence identity between any of the antibiotic peptides themselves or between these peptides and bradykinin. It seems likely that the sequence of each biologically active peptide is different because the structural demands of the target binding site is itself different in each case.

There are several intriguing similarities between spp24 and fetuin which suggest that the proteins could have similar mechanisms of action in bone. Both proteins are synthesized by liver as well as bone (33) and accumulate in the extracellular matrix of bone. Both proteins have cystatin domains, one for spp24 and two for fetuin(34) . Both proteins contain phosphoserine(35) . Finally, both proteins have an extended C-terminal sequence following the last cystatin domain, a C-terminal sequence which could arguably be a precursor to a biologically active peptide. It is of interest to note that alpha(2)HS glycoprotein, the human analogue of fetuin, circulates in blood as a two-chain molecule (36) and that the cleavages which generate the two-chain form occur within the extended C-terminal sequence that follows the last cystatin domain. The connecting peptide which is removed by those cleavages contains a sequence which could be phosphorylated by the SXE/S(P)-specific secretory pathway protein kinase, the sequence SPSGE (residues 310-314) in the protein (36) .

The identification of phosphoserine residues within the serine-rich sequence of spp24 identifies the protein as the third bone-derived phosphoprotein in which the location of phosphoserine residues has been established, the others being osteopontin (37) and matrix Gla protein (14) . The phosphorylation of serine residues in spp24 follows the recognition motif for serine phosphorylation that has been found in most secreted phosphoproteins, including MGP and osteopontin. All but one of the serine residues phosphorylated in spp24 have the negatively charged side chain of glutamate or phosphoserine in the n + 2 position in the consensus recognition sequence Ser-X-Glu/Ser(P), a substrate recognition pattern first observed in milk caseins and now identified in a wide variety of secreted phosphoproteins. The only phosphorylated serine that does not conform to this recognition motif is serine 130, which has a glycine residue in the n + 2 position in the sequence SSSSGSSSS. It is interesting to note that osteopontin also has a phosphorylated serine which has glycine in the n + 2 position in an analogous sequence, SSGSS(37) . It is possible that the small size of glycine may allow sufficient conformational flexibility to enable the SXE/S(P)-specific secretory pathway protein kinase to phosphorylate serines which have glycine in the n + 2 and phosphoserine in the n + 3 positions.

We have noted previously that phosphoproteins secreted into the extracellular environment of cells are invariably partially phosphorylated at each target serine residue, while those phosphoproteins secreted into milk and saliva are fully phosphorylated (14) . This pattern of partial serine phosphorylation is also seen in spp24 (Table 2). We have speculated that such partial serine phosphorylation may reflect a role for phosphoserine residues in the regulation of phosphoprotein activity by modulation of the SXE/S(P)-specific protein kinase or of a phosphoprotein phosphatase(14) . Many secreted phosphoproteins have phosphoserine residues that are clustered in highly anionic sequences, including MGP and osteopontin. Spp24 follows this pattern, with a potential maximum net negative charge of 18 in an 11 residue span, assuming complete phosphorylation of all serine residues. It seems probable that phosphorylation of serine residues in this region will create sufficient charge repulsion to inhibit formation of any secondary structure and that this sequence could therefore act as an anionic spacer separating the cystatin domain of spp24 from its C-terminal domain. If this model is correct, regulated changes in the extent of serine phosphorylation in this region would provide a mechanism to alter the separation of the cystatin and C-terminal domains of spp24 and thereby modulate the activity of spp24 or its susceptibility to proteolytic cleavage.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grant AR25921. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U08018[GenBank].

§
To whom correspondence should be addressed: Dept. of Biology, 0322, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322. Tel.: 619-534-2120; Fax: 619-534-1492.

(^1)
The abbreviations used are: MGP, matrix Gla protein; spp24, secreted phosphoprotein 24; MOPS, 3-(N-morpholino)propanesulfonic acid; BNPS-Skatole, 3-bromo-3-methyl-2(2-nitrophenylmercapto)-3H-indole; HPLC, high performance liquid chromatograpy; RT, reverse transcription; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Matthew K. Williamson for his generous assistance with protein sequencing.


REFERENCES

  1. Young, M. F., Kerr, J. M., Ibaraki, K., Heegaard, A. M., and Robey, P. G. (1992) Clin. Orthop. Relat. Res. 281, 275-294 [Medline] [Order article via Infotrieve]
  2. Price, P. A., Poser, J. W., and Raman, N. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3374-3375 [Abstract]
  3. Pan, L. C., and Price, P. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6109-6113 [Abstract]
  4. Price, P. A., and Williamson, M. K. (1985) J. Biol. Chem. 260, 14971-14975 [Abstract/Free Full Text]
  5. Price, P. A., Fraser, J. D., and Metz-Virca, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8335-8339 [Abstract]
  6. Oldberg, A., Franzen, A., and Heinegard, D. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8819 [Abstract]
  7. Oldberg, A., Franzen, A., and Heinegard, D. (1988) J. Biol. Chem. 263, 19430-19436 [Abstract/Free Full Text]
  8. Day, A. A., McQuillan, C. I., Termine, J. D., and Young, M. R. (1987) Biochem. J. 248, 801-805 [Medline] [Order article via Infotrieve]
  9. Fisher, L. W., Termine, J. D., and Young, M. F. (1989) J. Biol. Chem. 264, 4571-4576 [Abstract/Free Full Text]
  10. Krusius, T., and Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7683 [Abstract]
  11. Mason, I. J., Murphy, D., Munke, M., Francke, U., Elliott, R. W., and Hogan, B. L. M. (1986) EMBO J. 5, 1831 [Abstract]
  12. Bolander, M. E., Young, M. F., Fisher, L. W., Yamada, Y., and Termine, J. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2919 [Abstract]
  13. Meggio, F., Boulton, A. P., Marchiori, F., Borin, G., Lennon, D. P. W., Calderan, A., and Pinna, L. A. (1988) Eur. J. Biochem. 177, 281-284 [Abstract]
  14. Price, P. A., Rice, J. S., and Williamson, M. K. (1994) Protein Sci. 3, 822-830 [Abstract/Free Full Text]
  15. Hale, J. E., Williamson, M. K., and Price, P. A. (1991) J. Biol. Chem. 266, 21145-21149 [Abstract/Free Full Text]
  16. Frohman, M. A. (1993) Methods Enzymol. 218, 340-356 [Medline] [Order article via Infotrieve]
  17. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 [Medline] [Order article via Infotrieve]
  18. Meyer, H. E., Hoffmann-Posorske, E., and Heilmeyer, L. M. G., Jr. (1991) Methods Enzymol. 201, 169-185 [Medline] [Order article via Infotrieve]
  19. Pearson, W. R., and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2444-2448 [Abstract]
  20. Kozak, M. (1989) J. Cell Biol. 108, 229-241 [Abstract]
  21. Perlman, D., and Halvorson, H. O. (1983) J. Mol. Biol. 167, 391-409 [Medline] [Order article via Infotrieve]
  22. Von Heijne, G. (1983) Eur. J. Biochem. 133, 17-21 [Abstract]
  23. Takagaki, Y., Kitamura, N., and Nakanishi, S. (1985) J. Biol. Chem. 260, 8601-8609 [Abstract/Free Full Text]
  24. Storici, P., Del Sal, G., Schneider, C., and Zanetti, M. (1992) FEBS Lett. 314, 187-190 [CrossRef][Medline] [Order article via Infotrieve]
  25. Salvesen, G., Parkes, C., Abrahamson, M., Grubb, A., and Barrett, A. J. (1986) Biochem. J. 234, 429-434 [Medline] [Order article via Infotrieve]
  26. Ritonja, A., Kopitar, M., Jerala, R., and Turk, V. (1989) FEBS Lett. 255, 211-214 [CrossRef][Medline] [Order article via Infotrieve]
  27. Dieckmann, T., Mitschang, L., Hofmann, M., Kos, J., Turk, V., Auerswald, E. A., Jaenicke, R., and Oschkinat, H. (1993) J. Mol. Biol. 234, 1048-1059 [CrossRef][Medline] [Order article via Infotrieve]
  28. Zanetti, M., Del Sal, G., Storici, P., Schneider, C., and Romeo, D. (1993) J. Biol. Chem. 268, 522-526 [Abstract/Free Full Text]
  29. Del Sal, G., Storici, P., Schneider, C., Romeo, D., and Zanetti, M. (1992) Biochem. Biophys. Res. Commun. 187, 467-472 [Medline] [Order article via Infotrieve]
  30. Bode, W., Engh, R., Musil, D., Thiele, U., Huber, R., Karshikov, A., Brzin, J., Kos, J., and Turk, V. (1988) EMBO J. 7, 2593-2599 [Abstract]
  31. Goto, T., Tsukuba, T., Kiyoshima, T., Nishimura, Y., Kato, K., Yamamoto, K., and Tanaka, T. (1993) Histochemistry 99, 411-414 [Medline] [Order article via Infotrieve]
  32. Delaissé, J.-M., Eeckhout, Y., and Vaes, G. (1980) Biochem. J. 192, 365-368 [Medline] [Order article via Infotrieve]
  33. Ohnishi, T., Nakamura, O., Ozawa, M., Arakaki, N., Muramatsu, T., and Daikuhara, Y. (1993) J. Bone Miner. Res. 8, 367-377 [Medline] [Order article via Infotrieve]
  34. Elzanowski, A., Barker, W. C., Hunt, L. T., and Seibel-Ross, E. (1988) FEBS Lett. 227, 167-170 [CrossRef][Medline] [Order article via Infotrieve]
  35. Akhoundi, C., Amiot, M., Auberger, P., LeCam, A., and Rossi, B. (1994) J. Biol. Chem. 269, 15925-15930 [Abstract/Free Full Text]
  36. Lee, C.-C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4403-4407 [Abstract]
  37. Sorensen, E. S., and Petersen, T. E. (1994) Biochem. Biophys. Res. Commun. 198, 200-205 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.