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Originally published In Press as doi:10.1074/jbc.M310299200 on March 5, 2004

J. Biol. Chem., Vol. 279, Issue 19, 19808-19815, May 7, 2004
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Complete Topographical Distribution of Both the in Vivo and in Vitro Phosphorylation Sites of Bone Sialoprotein and Their Biological Implications*

Erdjan Salih{ddagger} and Rudolf Flückiger

From the Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedic Surgery, Harvard Medical School and Children's Hospital, Boston, Massachusetts 02115

Received for publication, September 16, 2003 , and in revised form, February 13, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone sialoprotein (BSP) is a multifunctional, highly phosphorylated, and glycosylated protein with key roles in biomineralization and tissue remodeling. This work identifies the complete topographical distribution and precise location of both the in vitro and in vivo phosphorylation sites of bovine BSP by a combination of state-of-the-art techniques and approaches. In vitro phosphorylation of native and deglycosylated BSPs by casein kinase II identified seven phosphorylation sites by solid-phase N-terminal peptide sequencing that were within peptides 12–22 (LEDS(P)EENGVFK), 42–62 (FAVQSSSDSS(P)EENGNGDS(P)S(P)EE), 80–91 (EDS(P)DENEDEES(P)E), and 135–145 (EDES(P)DEEEEEE). The in vivo phosphorylation regions and sites were identified by use of a novel thiol reagent, 1-S-mono[14C]carboxymethyldithiothreitol. This approach identified all of the phosphopeptides defined by in vitro phosphorylation, but two additional phosphopeptides were defined at residues, 250–264 (DNGYEIYES(P)ENGDPR), and 282–289 (GYDS(P)YDGQ). Furthermore, use of native BSP and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified several of the above peptides, including an additional phosphopeptide at residues 125–130 (AGAT(P)GK) that was not defined in either of the in vitro and in vivo studies described above. Overall, 7 in vitro and 11 in vivo phosphorylation sites were identified unequivocally, with natural variation in the quantitative extent of phosphorylation at each in vivo phosphorylation site.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence supporting many facets of the observed biological functions of bone sialoprotein (BSP)1 and osteopontin (OPN) has been accumulating, with a wide range of implications in both mineralizing and non-mineralizing tissues. BSP and OPN are the major non-collagenous extracellular matrix (ECM) phosphoproteins of calcified tissues such as bone and cartilage. Their intimate relationship with biomineralization suggests that they play key roles during the development and maintenance of these tissues (1–8). In vitro studies using BSP and OPN indicate that, whereas BSP induces calcium phosphate apatite formation (4–6, 9), OPN lacks this property or is inhibitory (3, 4, 10). The biological functions of BSP and OPN are not limited to mineral deposition, but impact cell behavior such as cell motility, cell adhesion, and bone resorption (10–16). Despite extensive studies, however, the precise roles of these phosphoproteins remain to be clearly defined. Although some in vitro studies indicate that BSP promotes bone resorption and hence participates in bone degradation (17), other studies such as those using glucocorticoids, which increase expression of this protein, suggest its involvement in the anabolic phase of bone remodeling (18). In this laboratory, in vivo implants of BSP-collagen composites in the calvarial critical defect bone repair model (19) and during reparative dentinogenesis (7, 8, 20–22) highlighted the impact of BSP during biomineralization and new bone/dentin formation.

As interest continues and evidence accumulates with regard to the general biological functions of bone phosphoproteins, in particular the covalently bound phosphate groups, the biochemical factors or processes that can affect the state of phosphorylation of these proteins become significant. Hence, the protein kinases that are involved in the phosphorylation of BSP/OPN prior to secretion into the ECM become important regulators of ECM phosphoprotein functions. Previous studies from this laboratory show that microsomal casein kinase II (CKII) and CKII are the predominant enzymes involved during the phosphorylation of bone ECM phosphoproteins OPN and BSP (23–26). Also, the use of a panel of purified protein kinases in in vitro phosphorylation of purified bovine bone OPN and BSP demonstrated that the degree of in vivo naturally occurring phosphorylation of bovine BSP and OPN is ~65 and ~85%, respectively. Not all of the molecules are phosphorylated at each of the potential phosphorylation sites within a given population of molecules isolated from bone of a particular age. Such heterogeneity in phosphorylation can be attained by changes in factors such as the rate of phosphoprotein synthesis, the microsomal CKII activity, the available ATP concentrations (within the intracellular compartment where phosphorylation takes place), and some degree of dephosphorylation during the residence of OPN/BSP in the ECM. The precise sites of phosphorylation in chicken bone OPN clearly show that the extent of phosphorylation at each site varies between 30 and 100%, with an average of ~60% total phosphorylation (27). There were 10 phosphorylation sites identified, but the actual quantitative analysis showed 6.3 mol of phosphoamino acids/mol of chicken OPN. The phosphorylated peptide regions are predominantly recognition sequences for CKII (27), confirming our in vitro studies. Additional studies that utilized the in vivo repair of calvarial bone defects induced to heal by implants of demineralized bone matrix highlighted the intimate interrelationship between accumulation and the rate of accumulation of calcium phosphate, OPN, BSP, and microsomal CKII activity as a function of bone development (28). More recently, direct evidence was provided for the first time in support of the concept of a "natural variation" in the extent of phosphorylation of ECM phosphoproteins during mineralized tissue formation. The extent of phosphorylation of OPN and BSP in the two anatomically distinct in vivo sites (bony and soft tissues) is substantially different (between ~1 and ~14 mol of Ser(P)/mol of OPN or BSP) and varies as a function of time within both implant sites (29). Furthermore, there is a direct and linear relationship between the rate of calcium phosphate deposition and the ratio of Ser(P) BSP to Ser(P) OPN in calvarial implants, clearly demonstrating hidden and important facets of the factors that control mineral deposition and the intimate coupling of the state of phosphorylation of BSP and OPN in this process.

Indeed, the covalently bound phosphates of BSP and OPN have been shown to also affect osteoclast cell attachment properties (15, 30). More recent conflicting studies have utilized different forms of OPN and BSP, and the results suggest that, although phosphorylation is not important for cell attachment, it is necessary for in vitro osteoclast resorption pit formation in dead bone slices (31). The differences in the results (Refs. 15 and 30 versus Ref. 31) were suggested to be due to differences in the protein samples and state of phosphorylation. There is no doubt that such conflicting results will probably continue to arise since these proteins are highly phosphorylated/glycosylated, and as noted above, these moieties are subject to inherent natural variation (29). Phosphorylation does not take place to the same extent at each of the potential sites, leading to heterogeneity within a given population of the protein isolated from a natural source. In vitro phosphorylation can also produce proteins with heterogeneous phosphorylation states or fully phosphorylated forms; but in either case, these may not represent precisely the naturally occurring forms.

The phosphorylation sites and regions of chicken bone OPN (27) and bovine milk OPN (32) and the glycosylation sites of human BSP (33, 34) have been reported. However, similar details are not available for BSP phosphorylation sites. In this study, a combination of state-of-the-art experimental approaches has been utilized to define for the first time the complete topographical distribution of both the in vitro and in vivo phosphorylation sites of bovine bone BSP. These included (i) phosphorylation of native and deglycosylated BSPs by CKII and tryptic peptide mapping, followed by solid-phase N-terminal peptide sequence analyses to define in vitro phosphorylation sites; (ii) use of a novel synthesized radiolabeled thiol agent (1-S-mono[14C]carboxymethyldithiothreitol ([14C]CM-DTT)) to derivatize phosphoserine tryptic peptides of BSP, followed by normal N-terminal peptide sequence analysis to define in vivo phosphorylation sites; and (iii) use of MALDI-TOF-MS to rapidly profile BSP proteolytic peptides.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vitro Phosphorylation of Native BSP by CKII Using [32P]ATP and Identification of the Phosphorylation Sites by Solid-phase N-terminal Peptide Sequencing—Bovine bone BSP was isolated and purified as described previously (26). 200 µg of native BSP was phosphorylated by [32P]ATP (diluted with unlabeled ATP to give a specific activity of 250 mCi/mmol) using 200 ng of CKII (Upstate Biotechnology, Inc., Lake Placid, NY) in 0.5 ml of 0.1 M KH2PO4/Na2HPO4 buffer (pH 7.4) containing 5 mM MgCl2 and 1 mM EGTA for 1 h at room temperature (~22 °C). 32P-Labeled native BSP was dialyzed against 50 mM NH4HCO3 (pH ~8.0) and digested with 2% (w/w) tosylphenylalanyl chloromethyl ketone-treated bovine trypsin (Sigma). The sample was freeze-dried, suspended in 0.2 ml of H2O and 0.1% (v/v) trifluoroacetic acid, and subjected to RP-HPLC on a Vydac C18 column (25 x 0.46 cm). The peptides were eluted by a linear gradient from H2O and 0.1% (v/v) trifluoroacetic acid to 60% CH3CN and 0.55% (v/v) trifluoroacetic acid over 90 min at a flow rate of 0.5 ml/min. The absorbance at 219 nm was recorded continuously, and fractions of 0.5 ml were collected. Aliquots from each fraction were counted for 32P radioactivity, and the radioactive fractions were then separately pooled for each peak, freeze-dried, and rechromatographed. Each purified 32P-labeled peptide was sequenced by automated solid-phase amino acid sequencing both to define the sequence and to identify the precise site(s) of phosphorylation as described previously (27).

Deglycosylation of Bovine Bone BSP, Followed by in Vitro Phosphorylation Using CKII and [32P]ATP and Identification of the Phosphorylation Sites—Native bovine bone BSP was deglycosylated by a combination of glycosidases simultaneously. 100 µg of BSP was incubated in 0.5 ml of 20 mM sodium phosphate buffer (pH 7.2) in the presence of O-glycosidase (2.5 milliunits/10 µg of protein), N-glycosidase (0.4 units/10 µg of protein), and neuraminidase (2 milliunits/10 µg of protein) (Roche Applied Science) overnight at 37 °C. Deglycosylated BSP was then isolated by RP-HPLC using a Vydac C4 column (25 x 0.46 cm), and fractions containing BSP (defined by Western blotting using anti-bovine BSP polyclonal antibody) (28, 29) were pooled and freeze-dried. The sample was then phosphorylated using CKII and [32P]ATP and trypsin-digested, and peptides were separated by RP-HPLC as described above. The 32P-labeled peptides were subjected to solid-phase N-terminal sequence analyses to define simultaneously the peptide sequence identity and the location of the phosphorylated residue(s) as described previously (27). For quantitative evaluation, analyses were performed to calculate accurately the degree of absolute phosphorylation in vivo and in vitro at each site and relative to the others within the whole molecule. Since there is an unknown amount of phosphate loss from the Ser(P) residues in Edman degradation cycles, it is not possible to quantify Ser(P) content upon release of 32P alone in that cycle. One way that this was overcome was to calculate the pmol of 32P-labeled Ser released (from 32P counts and the specific activity of [32P]ATP) plus the pmol of dehydroalanine observed in that cycle. This approximated more closely the actual original Ser(P) content of the peptide since generation of the dehydroalanine from Ser(P) is cumulative during Edman degradation.

Identification of the in Vivo Phosphorylation Regions and Sites of BSP Using a Novel Synthesized Radioactive Thiol Agent, [14C]CM-DTT—To overcome the lack of an efficient and rapid determination of the precise sites of in vivo phosphorylation, a novel radioactive thiol reagent, [14C]CM-DTT, was designed and synthesized in this laboratory. The synthesis and utility of such reagent(s) have been recently described in detail for studies related to phosphoserine- and phosphothreonine-containing proteins using N-terminal peptide sequencing and a MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA) (35, 36).

100 µg of bovine bone BSP was deglycosylated and trypsin-digested for chemical derivatization by [14C]CM-DTT. The tryptic peptides were then incubated with 5 mM [14C]CM-DTT in 0.33 M NaOH for 1 h at 50 °C to derivatize the phosphoserine-containing peptides. The 14C-labeled peptides were isolated/purified by RP-HPLC and N-terminally sequenced under normal sequencing conditions. N-terminal sequencing was carried out by Edman degradation (37) using a Model 477A automated protein sequenator (Applied Biosystems) and Biobrene-treated glass filters (27). To define the peptide sequence and the location of the phosphoserine/phosphothreonine-containing peptides, one-third of the anilinothiazoline products from each Edman degradation step were converted to phenylthiohydantoin-derivatives and analyzed by on-line HPLC (Model 120A, Applied Biosystems), and two-thirds were collected as anilinothiazoline derivatives for 14C counting as described recently (27, 36). Unlike the analysis and quantification of the in vitro 32P-labeled peptides by solid-phase sequencing described above, the quantification of the Ser(P) content during sequencing of the [14C]CM-DTT-derivatized peptides was less complicated. This is because the [14C]CM-DTT-Ser moieties during sequence analysis are stable and the 14C released in that cycle reflects the actual original Ser(P) content. To quantify yields of sequenced peptides, the initial yield (I0) and repetitive yield (R) were calculated by linear regression analysis of the observed yield (M) at each cycle (n): log10(M) = nlog10(R) + log10(I0). The information obtained from such analysis was used in conjunction with the release of 14C radioactivity to quantify the original phosphoserine content/mol of peptide. The expected theoretical observable yield for serine quantity in a given cycle was calculated based on the cycle number, initial yield, and repetitive yield. For example, the tryptic peptide containing residues 42–122 (FAVQSSSDSSEENGNGD...) was sequenced for 12 cycles, with release of 14C radioactivity in cycles 5 and 10 (2020 and 780 dpm, respectively). These counts were for the two-thirds of the total Edman degradation products of each cycle, whereas the phenylthiohydantoin-derivatives for the amino acid analyses were for the one-third. Hence, the total expected observable 14C counts if all of the Edman degradation products were counted are 3030 dpm for cycle 5 and 1170 dpm for cycle 10. The expected observable serine content in cycles 5 and 10 if all of the Edman degradation products were converted to phenylthiohydantoin and analyzed are 222 and 132 pmol, respectively. Using the specific activity of [14C]CM-DTT (10 mCi/mmol), 3030 dpm in cycle 5 corresponds to 137 pmol of [14C]CM-DTT incorporated, reflecting 0.62 mol of 14C incorporated per mol of serine, i.e. 62% of this serine was in the form of phosphoserine. A similar calculation for Ser10 indicated 0.40 mol of 14C incorporated per mol of serine, i.e. 40% of this serine was phosphorylated.

Direct Identification of in Vivo Native Phosphopeptides by MALDI-TOF-MS without 32P Labeling or Chemical Modification by [14C]CM-DTT—Native BSP (5 µg) was trypsin-digested, and the peptide mixture was subjected directly to peptide profiling by MALDI-TOF-MS. The advantages of this approach are the ability to rapidly profile peptides, to pinpoint naturally occurring phosphopeptides, and to sequence and identify each peptide within the mixture without any purification steps. In addition, due to the sensitivity of the instrument, a very small amount of the material is required. MALDI-TOF-MS was performed in this laboratory using the Voyager DE ProTM mass spectrometer (Applied Biosystems). Aliquots of BSP tryptic peptides (~1 pmol) in 1 µl of H2O and 0.1% trifluoroacetic acid were mixed with 1 µl of matrix solution ({alpha}-cyano-4-hydroxycinnamic acid, Applied Biosystems), spotted onto a sample target (stainless steel sample plate), allowed to dry, and loaded into the mass spectrometer for analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Complete in Vitro Phosphorylation Sites of Purified Bovine Bone BSP Using [32P]ATP and CKII—Fig. 1A shows a tryptic peptide map of native BSP phosphorylated using [32P]ATP and CKII. The BSP used in this experiment was native, with both glycosylation and phosphorylation intact. Under these circumstances, 32P labeling occurs at residual phosphorylation sites with recognition sequences for CKII. A number of 32P-labeled peptides were observed within the RP-HPLC profile using a C18 column. Several of the peptides were subjected to rechromatography by RP-HPLC for clarification of the sequences (Fig. 1A, inset). It is worth noting that the absorbance profile of each peak was heightened, with the descending limb never declining to the base line, which is obvious in both Fig. 1A and its inset. This phenomenon is a prominent occurrence in the analysis of phosphoproteins with high levels of glycosylation, as is the case with BSP in this work and as previously reported for OPN (27). To simplify the tryptic peptide map and to remove the uncertainty of possible 32P incorporation in glycosylated side chains, native BSP was first deglycosylated by a combination of glycosidases, followed by phosphorylation and peptide mapping (Fig. 1B). The peptide map of deglycosylated BSP clearly revealed simplification and sharpening of the absorbance peaks, including base-line approximation of the peak limbs, as observed in both Fig. 1B and its inset. Interestingly, however, neither the number of radioactive peaks nor their relative positions and intensities within each profile showed any significant change. This suggests that the peptide sequences and sites for phosphorylation and glycosylation do not overlap and was supported by the quantitative data relating mol of 32P incorporation/mol of native and deglycosylated BSPs (~2.5 mol of phosphate/mol of protein in both cases). Sequence analysis of the phosphorylated peptide regions and sites provided direct evidence consistent with this. Each of the radiolabeled fractions in Fig. 1 (A and B) were N-terminally sequenced using the automated solid-phase sequencing approach. Three peptides were identified as in vitro 32P-labeled peptides: residues 12–22 (LEDS(P)EENGVFK), 42–122 (FAVQSSSDSS(P)EENGNGDS(P)S(P)EEEEEEEETSNEEGNNGGNEDS(P)DENEDEES(P)EA...), and 135–216 (EDES(P)DEEEEEEEE...). The length of the tryptic peptide at residues 42–122 was too long for the positions of all of the potential phosphorylated serines beyond ~12–15 residues to be defined clearly. Hence, a portion of this peptide was N-terminally sequenced up to 12 residues to identify the phosphorylated residues within this section. The remaining peptide was further proteolytically digested by Asp-N endopeptidase, and the shorter radiolabeled peptides were isolated and sequenced. Overall, seven phosphorylation sites were defined within the three peptides. As expected, all of the phosphorylated peptides contain recognition sequences for CKII (SEE, SXE, ESDE, etc.), i.e. the phosphorylated serine residues are flanked by acidic amino acids (Glu or Asp). Fig. 4 summarizes the topographical distribution of phosphorylation sites within the primary amino acid sequence of bovine BSP, and Table I indicates the quantitative extent of phosphorylation (% phosphorylation or mol of phosphate/mol of peptide).



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  		FIG. 1.
Isolation and purification of in vivo and in vitro phosphorylated tryptic peptides of bovine BSP. A, 200 µg of purified native bovine bone BSP was 32P-labeled using [32P]ATP and CKII, followed by trypsin digestion. The peptides were separated by RP-HPLC on a Vydac C18 column (25 x 0.46 cm) using a linear gradient from H2O and 0.1% (v/v) trifluoroacetic acid to 60% CH3CN and 0.055% (v/v) trifluoroacetic acid over 90 min at a flow rate of 0.5 ml/min. Absorbance (Abs) at 219 nm was recorded continuously (—). Fractions of 0.5 ml were collected, and aliquots were counted for 32P radioactivity ( ––– ). Inset, rechromatography of fraction 3 using the same RP-HPLC conditions as described for A. B, 100 µg of deglycosylated BSP was 32P-labeled using [32P]ATP and CKII, followed by trypsin digestion. The peptides were separated by RP-HPLC on a Vydac C18 column as described for A, except that the linear gradient was performed over 60 min at a flow rate of 1.0 ml/min, and 1.0-ml fractions were collected. —, absorbance 219 nm; ––– , 32P radioactivity. Inset, rechromatography of fraction 3 under the same RP-HPLC conditions as described for A. C, 100 µg of deglycosylated BSP was trypsin-digested, and the phosphoserine peptides were 14C-labeled using 5 mM [14C]CM-DTT in 0.33 M NaOH at 50 °C for 1 h. The peptides were separated by RP-HPLC on a Vydac C18 column as described for B.

 



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  		FIG. 4.
Complete topographical distribution of both in vivo and in vitro phosphorylation regions/sites of bovine bone BSP. The sites of phosphorylation were all determined in this laboratory. 32P, in vitro phosphorylation sites identified by solid-phase N-terminal sequence analysis; 14C, in vivo phosphorylation sites identified using base-catalyzed derivatization by [14C]CM-DTT and normal N-terminal sequence analysis; MS, in vivo phosphorylation sites identified by the MALDI-TOF-MS method. The complete primary amino acid sequence was from cDNA for bovine BSP (52). , glycosylation sites reported recently (33).

 


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  		TABLE I
Quantitative extent of phosphorylation of bovine bone BSP peptide regions/sites both in vivo and in vitro defined by [14C]CM-DTT, [32P]ATP labeling, and MALDI-TOF-MS

 
Complete in Vivo Phosphorylation Sites of Purified Bovine BSP Utilizing the Novel Thiol Agent [14C]CM-DTT—Although defining the in vitro phosphorylated peptide regions and sites by a specific protein kinase (CKII) was important, it was of further major interest to establish the in vivo phosphorylated peptides of native bone BSP. Fig. 1C shows the 14C-radiolabeled tryptic peptides of deglycosylated native BSP derivatized by [14C]CM-DTT. The deglycosylation step was performed prior to base-catalyzed derivatization to avoid a reaction of O-glycosyl sites with [14C]CM-DTT, as under base catalysis, such moieties can undergo an elimination reaction similar to Ser(P-)/Thr(P) residues. N-terminal sequence analysis of each of the 14C-radiolabeled tryptic peptides identified five phosphopeptides at residues 12–22 (LEDS(P)EENGVFK), 42–122 (FAVQS(P)SSDSS(P)EENGNGDS(P)S(P)EEEEEEEETSNEEGNNGGNEDS(P)DENEDEES(P)EA...), 135–216 (EDES(P)DEEEEEEEE...), 235–264 (... EIYES(P)ENGDPR), and 272–285 (GYDS(P)YDGQDYYSHQ). Peptides 42–122 and 235–264 were subjected to additional proteolytic digestion with Asp-N endopeptidase to define the precise location of the phosphorylated residue(s). The [14C]CM-DTT-derivatized peptides were sequenced under normal Edman degradation conditions using Biobrene-treated glass filters. Fig. 2 shows the sequence data for the first 12 cycles of the peptide starting with Phe42 obtained under such conditions. Overall, almost all of the peptides that were found to be phosphorylated in vitro by CKII were also found to be phosphorylated in vivo. The exceptions were the two additional peptides 235–264 (... EIYES(P)ENGDPR) and 272–285 (GYDS(P)YDGQDYYSHQ), and an additional phosphorylation site at position 46 of peptide 42–122 was identified. These data indicate that most of the phosphorylation sites of native BSP are partially phosphorylated in vivo and that two sites in the N-terminal domain are either fully phosphorylated or somewhat not recognized by or not accessible to CKII.



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  		FIG. 2.
N-terminal sequence analysis of purified 14C-labeled tryptic phosphopeptides of native BSP. During N-terminal sequencing, one-third of the Edman degradation products were analyzed as phenylthiohydantoin-derivatives, and two-thirds were collected and counted for 14C radioactivity; 2020 and 780 dpm were recovered in cycles 5 and 10, respectively. The initial yield was 250 pmol, and the repetitive yield was 90%. , log10 (pmol of amino acid) at each cycle.

 
Identification of in Vivo Phosphorylation Sites by Direct MALDI-TOF-MS Analysis—Native BSP tryptic peptides were directly analyzed by MALDI-TOF-MS. The phosphorylated peptides were directly identified by the presence of species with a theoretical peptide mass of +80 Da (+1 phosphate group or multiples of). For example, Fig. 3A shows phosphopeptide 12–22 (LEDS(P)EENGVFK), where three different tryptic cleavage forms are clearly identified: species with 1347 mass units (LEDS(P)EENGVFK, 1267 + 80 Da), 1547 mass units (AKLEDS(P)EENGVFK, 1467 + 80 Da), and 1702 mass units (RAKLEDS(P)EENGVFK, 1622 + 80 Da). The original spectrum in Fig. 3A was obtained using the linear mode of the mass spectrometer, which does not distinguish the naturally occurring isotopic masses of the peptide. The spectra shown in the insets of Fig. 3A were, however, obtained in the reflector mode of the instrument, where the spectra show distinct naturally occurring isotopic masses of the peptides, each separated by 1 mass unit (1 Da). Fig. 3B shows a phosphopeptide with 841 mass units (peptide 124–130, KAGAT(P)GKK, 761 + 80 Da) identified by MALDI-TOF-MS. Interestingly, this in vivo phosphorylated peptide was not defined during the evaluation of either the in vitro or in vivo phosphorylation sites described above. It is also the only peptide of BSP that is phosphorylated at a threonine residue (all the others are phosphorylated at serines), and the flanking amino acids indicate a recognition sequence for protein kinase C (AT(P)XK). Hence, it is not surprising that this peptide was not identified during the analysis of in vitro phosphorylated BSP by CKII. The [14C]CM-DTT derivatization approach also did not identify this peptide, most likely due to the slower rate of conversion of Thr(P) to its corresponding dehydrothreonine form, with which [14C]CM-DTT can react. The location of the phosphorylated serine/threonine residue in peptides such as the two discussed above can be easily defined since there is only one potential phosphorylation site (hydroxyamino acid, Ser or Thr) in each. For peptides containing multiple possible serine/threonine phosphorylation sites, it would be necessary to sequence the peptide using the PSD/CID mode of the MALDI-TOF mass spectrometer to perform tandem mass spectrometry to observe precisely the location of the specific phosphorylated residue.



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  		FIG. 3.
Identification by MALDI-TOF-MS of tryptic phosphopeptides derived from native bovine bone BSP. A, three different tryptic cleavage forms of the same phosphopeptide region with sequences 12–22 (LEDS(P)EENGVFK, 1347.5 mass units, 1267 + 80 Da), 10–22 (AKLEDS(P)EENGVFK, 1546.7 mass units, 1467 + 80 Da), and 9–22 (RAKLEDS(P)EENGVFK, 1702 mass units, 1622 + 80 Da). B, phosphopeptide 125–130 (AGAT(P)GK, 841.2 mass units, 761 Da + 80 Da). The observed molecular mass for each peptide includes 80 Da, which represents one phosphate group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study utilized a combination of multiple state-of-the-art approaches, techniques, and reagents to define the complete topographical distribution of both in vitro and in vivo phosphorylation sites and regions of bovine bone BSP. These sites include the complete in vitro phosphorylation regions and specific sites of peptides at residues 12–22 (LES(P)DEENGVFK), residues 42–62 (FAVQSSSDSS(P)EENGNGDS(P)S(P)EE), residues 80–91 (EDS(P)DENEDEES(P)E), and residues 135–141 (EDES(P)DEE). Although these data in their own right are significant, there is no doubt that they may represent only part of the overall possible phosphorylation state/sites of native BSP. This is because in vitro phosphorylation was carried out using a single enzyme (CKII), and this process relies on the presence of partially phosphorylated peptides within the native population of BSP molecules. Hence, identification of the complete in vivo phosphorylation sites and regions of native bovine BSP was pursued. To accomplish this task, novel reagents developed in this laboratory were used. We have very recently described in detail thiol agents such as dithiothreitol and [3H]CM-DTT, used as tools to explore the general phenomenon of phosphorylation in conjunction with instrumentation to perform N-terminal sequencing and MALDI-TOF-MS (35, 36). These approaches led to the identification of in vivo phosphorylation sites, which included all of the phosphopeptides identified by in vitro CKII phosphorylation of BSP (peptides described above). However, two additional phosphopeptides were identified by 14C radiolabeling: those starting with Asp250 (DNGYEIYES(P)ENGDPR), and Gly282 (GYDS(P)YDGQ...). Furthermore, the use of native BSP proteolytic peptides and MALDI-TOF-MS provided confirmation of several identified peptide regions by 32P labeling (in vitro) and [14C]CM-DTT derivatization (in vivo); but in addition, peptide 125–130 (AGAT(P)GK), which was not defined by either of the above approaches, was identified. This phosphopeptide, unlike the others, does not have a recognition sequence for phosphorylation by CKII. Instead, the amino acids flanking the phosphothreonine constitute a recognition site (X(T/S)XK) for phosphorylation by protein kinase C; and indeed, this is the reason why we did not define this site using CKII in our in vitro studies. Importantly, however, our previous in vitro quantitative studies using a panel of protein kinases did highlight the presence of a protein kinase C phosphorylation site in bovine bone BSP (26), the location of which is defined herein. Also, this is the only phosphorylated BSP peptide in which the site of phosphorylation is not a serine residue, but rather threonine, and was not identified by the thiol agent approach because Thr(P) residues undergo base-catalyzed elimination much slower than Ser(P) residues. The identification of in vitro phosphorylation sites (using [32P]ATP and CKII) of native bovine BSP was carried out with samples of native BSP with and without glycosylation. Such an approach was of importance for two reasons: (i) to simplify the tryptic peptide maps because the presence of glycosylation leads to peak broadening and (ii) to eliminate the possibility of 32P incorporation in the glycosylated side chains. Fig. 1 shows the impact of deglycosylation on the peptide profile with clear peak sharpening. Similarly, but for a different reason, the identification of in vivo phosphorylation sites by [14C]CM-DTT derivatization was carried out using deglycosylated native BSP. This was an essential step since, under base-catalyzed conditions, the O-glycosyl sites can undergo an elimination reaction similar to Ser(P)/Thr(P) residues that will generate an unsaturated double bond with which [14C]CM-DTT will react.

Overall, this work provides the most extensive and detailed study to date of the precise location of 11 in vivo and 7 in vitro phosphorylation sites. Fig. 4 summarizes the overall sites of phosphorylation of BSP both in vivo and in vitro as defined in this work and the sites of glycosylation of human BSP, which have been reported recently (33). Interestingly, the glycosylation and phosphorylation sites do not overlap, and the glycosylation sites are concentrated in the mid-portion of the protein, somewhat sandwiched in between the N- and C-terminal phosphorylation sites. Clearly, these structural features raise a series of possible biological functions for such post-translational modifications and how they may couple to provide specific functional consequences. The role of BSP in biomineralization has been implicated by its temporal deposition into the ECM during development of the mineralizing tissues (1, 38–40), its high affinity for hydroxylapatite (41), and its action as a de novo nucleator of hydroxylapatite crystals in vitro (4). The poly-Glu regions have been suggested to be involved in hydroxylapatite binding and nucleation of calcium phosphate crystals (5). Similarly, BSP glycosylation has been shown to also influence hydroxylapatite binding (33). Prior to these developments, the participation of the covalently bound phosphates in ECM phosphoprotein-collagen complexes in calcium phosphate deposition was described (42). The complete topographical distribution and location of the phosphorylation regions/sites of BSP defined herein present a plausible synergistic activity between these moieties both in hydroxylapatite binding and calcium phosphate crystal nucleation. Previous work from this laboratory established the quantitative extent of both in vitro and in vivo phosphorylation of BSP using a panel of purified protein kinases and [32P]ATP (26). In that study, the total degree of phosphorylation was defined as well as the global domains containing such modifications, including the predominant protein kinase (CKII) that participates during phosphorylation of BSP. However, the precise locations of the phosphorylation sites and peptide regions were not identified. The location of one phosphoserine in the N-terminal half of BSP, residues 12–22 (LEDSEENGVFK), was identified by in vitro 32P labeling with CKII and solid-phase N-terminal sequence analysis. A second phosphopeptide was identified at residues 42–122 (FAVQSSSDSSEENGNGDSSEEE...), which contained up to five phosphorylated residues, but the precise locations of these sites within the sequence were not defined at that time (25). Peptide 12–22 was latter identified to be also phosphorylated in vivo (34, 36). In addition, a MALDI-TOF-MS approach was used to predict, based on mass difference, that peptide 130–203 in human bone BSP contains zero to four phosphorylated Ser(P) or Thr(P) residues (34). However, because this peptide contains 14 potential phosphorylation sites (seven Ser and seven Thr residues) at which the four phosphates may be residing, it was not possible to define the precise location of these phosphate groups. Thus, only one phosphorylation site was defined unequivocally from previous studies to date (25, 34, 36), compared with 11 in vivo and 7 in vitro sites in this work.

To gain further insights into the biological implications of the phosphorylation state, we have performed extensive quantitative analysis of the extent of phosphorylation at each site both in vivo and in vitro. These data demonstrate the variation in the state of phosphorylation at each site (Table I). The variable extent of phosphorylation most likely relates to the developmental stage, maturation, and age of bone, which in turn reflect regulatory biological processes, where, at different stages of bone development, BSP is synthesized and secreted with differing degrees of phosphorylation (29). Such a process can be the consequence of a combination of intracellular biological variables, including different protein expression levels of both the protein kinases and BSP prior to secretion into the ECM. Direct experimental evidence for such an occurrence was provided in our previous in vivo studies (28), including, most recently, the differential extent of phosphorylation of BSP and OPN as a function of time during new bone formation in vivo (29). The coupling of the state of phosphorylation of BSP with that of OPN in regulating the rate of calcium phosphate deposition presents a unique biological interrelationship between these two proteins. Other studies have indicated that BSP promotes mineral deposition in cell cultures (9, 43) and differentiation of bone marrow cells to osteoblasts (44). Additional in vivo work using implants of native bone BSP-collagen composites in bone repair (19) and reparative dentinogenesis (7, 8, 20–22) elaborated on the multifunctional capacity of this protein. Clearly, these studies, combined with the results of this work, evoke an interesting question as to what extent phosphate groups on BSP influence in vivo bone and dentin formation/repair.

In addition to extensive post-translational modifications, BSP also contains the integrin receptor-binding amino acid sequence RGD, which enables interactions with bone cells such as osteoclasts through their {alpha}v{beta}3 integrin receptor (15, 16, 45–48). Osteoclasts attach to the ECM through (particularly, {alpha}v{beta}3) integrin cell receptors (12, 49), which mediate not only adhesion, but also signal transduction and hence the regulation of osteoclast function. Studies with BSP and its non-RGD fragments (46–48) suggest that sequences other than RGD are also involved in cell attachment and modulation of osteoclast cell behavior. For example, certain non-RGD sequences from BSP mediate a nuclear calcium response in osteoclasts, first observed with RGD peptides (50). Indeed, in vitro studies indicate that BSP can stimulate bone resorption and, in cell co-cultures, inhibits osteoclast formation (51), whereas osteoclast binding can be influenced by the dephosphorylated form of BSP (15). More recently, it was suggested that BSP in its post-translationally modified form is necessary for in vitro pit formation by osteoclasts seeded on dead bone slices (31). The precise mechanisms by which BSP influences various biological processes in these and other studies including the effects of phosphorylation state still remain to be clarified. Our data from this study provide a number of intriguing fundamental structural properties with respect to phosphorylation of BSP that open new avenues to study and address various observed biological phenomena.

In conclusion, our extensive chemical and biochemical analyses of BSP defined complete in vivo and in vitro phosphorylation sites unequivocally, revealing a series of critical information including quantitative natural variation at each phosphorylation site. These results are of major interest to a wide range of biological studies concerning the role of ECM phosphoproteins in biomineralization and the influence of the state of phosphorylation on cellular activity/behavior both in normal and pathological developments. Thus, the overall results have relevance to broad areas of biological sciences such as pathological soft tissue calcification, i.e. atherosclerosis, kidney stones, breast and prostate tumors, and bone metastasis, in addition to general bone biology.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant RO1 AG17969 (to E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Orthopedic Research, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-247-5181; Fax: 617-236-7149; E-mail: erdjan.salih{at}tch.harvard.edu.

1 The abbreviations used are: BSP, bone sialoprotein; OPN, osteopontin; ECM, extracellular matrix; CKII, casein kinase II; [14C]CM-DTT, 1-S-mono[14C]carboxymethyldithiothreitol; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; RP-HPLC, reverse-phase high performance liquid chromatography. Back


   ACKNOWLEDGMENTS
 
We thank Dr. James Clifton for careful proofreading of the manuscript.



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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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