Y-Carboxyglutamic Acid-Containing Protein from Bovine Bone (Calcified Tissues/Protein Sequence/Vitamin K/Hydroxyproline) PAUL A

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Proc. Natl. Acad. Sci. USA Vol. 73, No. 10, pp. 3374-3375, October 1976 Biochemistry Primary structure of the -y-carboxyglutamic acid-containing protein from bovine bone (calcified tissues/protein sequence/vitamin K/hydroxyproline) PAUL A. PRICE*, JAMES W. POSER, AND NEERJA RAMAN Department of Biology, University of California, San Diego, La Jolla, Calif. 92093 Communicated by Andrew A. Benson, July 2, 1976. Additional information added July 30, 1976

ABSTRACT The amino-acid sequence of the y-carboxy- RESULTS glutamic acid-containing protein of bovine bone is presented. The sequence of 43 of the 49 residues was determined auto- The amino acid sequence of the Gla protein from calf bone matically on the intact protein and on a tryptic peptide. The is shown in Fig. 1. A sequencer analysis of the intact S-ami- remainder was determined by conventional procedures on noethylated protein established the sequence of the first 20 tryptic and chymotryptic peptides. Residue 9 in the sequence residues and also identified additional residues in positions up has been identified as 4-hydroxyproline. The rotein contains to residue 35. Three peptides were isolated from a tryptic hy- three y-carboxyglutamic acid residues, which are located at positions 17,21, and 24. The role of these unusual amino acids drolysate of the protein: one corresponding to residues 1-19 in in the binding interaction between the protein and hydroxy- the sequence, a 24-residue peptide, and a pentapeptide: The apatite crystals is discussed. structure of the 24-residue peptide was determined completely in a second sequencer analysis. The sequencer analysis on the We described previously the isolation from calf bone of a 5700 intact protein provided sufficient overlap to establish the 24- molecular weight protein that contains three y-carboxyglutamic residue peptide as residues 20-43 in the structure. Residues 23 acid (Gla) residues (1). This protein (Gla protein) was extracted and 29 are joined in a disulfide bond, since the protein contains from calf cortical bone upon demineralization in EDTA and no free sulfhydryl groups (1). The tryptic pentapeptide se- accounts for 1-2% of the total protein in calf bone. The same quence was determined by the manual Edman procedure and Gla protein was also found in calf cancellous bone and tooth corresponds to residues 45-49 in the final sequence. Chymo- dentine, and a similar protein was isolated from swordfish tryptic peptides provided the necessary overlap between residue vertebrae and human tibia (1). A Gla-containing protein also 43 in the sequence and the tryptic pentapeptide. Only six has been isolated from chicken tibia (2). The abundance and chymotryptic peptides were found. These had compositions wide distribution of this unusual bone protein suggest that it has that correspond to the following positions in the overall se- an important function in calcified tissues. We have described quence: 1-5, 6-38, 39-42, 43-45, 43-46, and 47-49. The se- two important properties of the Gla protein: its strong binding quence of the peptide 43-46 was determined by the manual to hydroxyapatite crystals and not to amorphous calcium Edman procedure and established the presence of an additional phosphate, and its inhibition of the initial formation of hy- arginine residue between residue 43 and the tryptic penta- droxyapatite crystal nuclei (1). To obtain further insight into peptide. The COOH-terminal sequence was verified by car- the function of this protein, we have undertaken the determi- boxypeptidase y digestion, which quantitatively released amino nation of its amino acid sequence. acids corresponding to positions 45-49 and released approxi- mately 0.5 residue of amino acids from positions 41-44. The MATERIALS AND METHODS only other amino acids recovered in significant quantities were Purified calf bone Gla protein was isolated from calf cortical 0.3 residue of glutamic acid (Glu) and 0.15 residue of glutamine bone as described (1), and was reduced and S-aminoethylated (Gln). The residue molecular weight of this protein is 5700, (3) prior to use. Automatic sequence analysis was performed which can be compared with the molecular weight of 5800 with the Beckman Sequencer (model 890 B). Residues were determined by sedimentation equilibrium. The latter value identified by gas chromatography of the phenylthiohydantoin differs from the previously published molecular weight of 6800 derivatives (4) and by amino acid analysis of the residues re- because the value of Up used in the present calculations, 0.713, leased from phenylthiohydantoin derivatives by hydrolysis in is based on the amino acid composition (7) while previously a HI (5). The Gla protein was subjected to tryptic or chymotryptic ivp of 0.75 was assumed (1). hydrolysis. Peptides were isolated by gel filtration on Sephadex There are six possible sites for Gla residues in the calf bone G-25, by ion exchange chromatography, and by high voltage protein, positions 17, 21, 24, 31, 39, and 40. Since y-carboxy- electrophoresis. The sequences of small tryptic and chymo- glutamic acid is stable under the conditions of alkaline hy- tryptic peptides were determined by manual Edman degra- drolysis of proteins (1, 2), but decarboxylates on acid hydrolysis, dation and subsequent dansylation techniques (6). Carboxy- peptides containing residues 1-19, 20-38, and 39-42 were peptidase y (EC 3.4.12.1) digestion was carried out with 0.3% subjected to alkaline hydrolysis and amino acid analysis. This carboxypeptidase y for 3-15 hr at 25° in 0.1 M pyridine acetate established the presence of one Gla and no Glu residues in buffer, pH 5.5. Amino acid analyses were performed with a peptide 1-19 and two Gla and 1 Glu residues in peptide 20-38. Beckman amino acid analyzer (model 119 B) on acid and al- There were no Gla residues in peptide 39-42. Identification of kaline hydrolysates (1). Amide side chains were identified by Gla at position 17 is based also on the release of one Gla and no chromatography. Glu residues upon exhaustive carboxypeptidase y digestion of thin-layer peptide 1-19, and on the complete absence of any detectable Abbreviations: Gla, -y-carboxyglutamic acid; Gla protein, y-carboxy- spot in the Glu and Gln positions upon thin-layer chromatog- glutamic acid-containing protein. raphy of the phenylthiohydantoin derivative of residue 17 (8). * To whom reprint requests should be addressed. Assignment of Gla to positions 21 and 24 and Glu to position 31 3374 Downloaded by guest on September 28, 2021 Biochemistry: Price et al. Proc. Natl. Acad. Sci. USA 73 (1976) 3375 5 10 15 1 Tyr-Leu-Asp-His-Trp-Leu-Gly-Ala-Hyp-Ala-Pro-Tyr-Pro-Asp-Pro- 16 Leu-Gla -Pro -Lys-Arg-Gla -Val-Cys-Gla -Leu-Asn-Pro-Asp-Cys -Asp- 31 Glu-Leu-Ala-Asp-His-Ile -Gly-Phe-Gln-Glu-Ala-Tyr-Arg-Arg-Phe- 46 Tyr-Gly-Pro-Val FIG 1. Primary structure of the -y-carboxyglutamic acid-containing protein from calf bone.

is based upon the absence of any spots in Glu or Gln positions of hydroxyapatite crystal nuclei (1). These properties suggest for residues 21 and 24 and the appearance of an unambiguous an ability to bind to a particular structure on the hydroxyapatite spot in the Glu position for residue 31 upon thin-layer chro- surface. Three of the five Ca2+ atoms in hydroxyapatite are in matography of the corresponding phenylthiohydantoin de- identical environments at the corners of an equilateral triangle; rivatives. Mild alkaline hydrolysis (5) of 20 nM of phenylthio- the other two Ca2+ atoms are in a different part of the crystal hydantoin-Gla derivatives derived from residues 17, 21, and and have different ligands (15, 16). The presence of three 24 failed to yield any Gla or Glu at 1 nM detection limit. Under separated Gla residues in the calf bone protein leads us to these conditions Glu is recovered from phenylthiohydantoin- suggest that the Gla residues bind selectively to these three Ca2+ Glu in 23% yield (5) and Glu was recovered from the phenyl- atoms on the hydroxyapatite crystal surface. Amorphous cal- thiohydantoin derivative of residue 31. The identification of cium phosphate lacks long-range order (17) and would be un- Gln at position 39 and Glu at position 40 is based on the amino likely to have a similar Ci2+ atom arrangement for Gla protein acid analysis of carboxypeptidase y digests of peptide 39-42 binding. before and after one cycle of the Edman degradation. Other structural features of the Gla protein may provide clues Residue 9 has been identified as 4-hydroxyproline. The to its function. The Gla protein contains four basic residues phenylthiohydantoin derivative of residue 9 and the synthetic grouped into two pairs. We have suggested the possibility that phenylthiohydantoin derivative of 4-hydroxyproline have the the Gla protein could be a signal that a given bone segment has same mass spectrum and both give 4-hydroxyproline upon been demineralized (1). In this context, it is interesting that the hydrolysis in HCI (9). The previous report that this protein same basic residue pairs are found in proinsulin at the positions contains no hydroxyproline (1) was in error. where proteolytic cleavage occurs to give insulin (18). The presence of hydroxyproline in the Gla protein indicates that the DISCUSSION protein is probably synthesized by the same cells that synthesize bone collagen. The Gla protein could be a fragment of pro- The factors governing which glutamic acid residues in a protein collagen released during collagen synthesis. are -y-carboxylated have not yet been determined. There is no sequence homology between the calf bone protein and the We thank Lisa Katter for assistance in preparing bone samples and Gla-containing, amino-terminal regions of either prothrombin Allen Otsuka for help in the sequencer run on the intact protein. We for assistance in the of the (10) or factor X (11, 12). This suggests that specificity of -y- also thank Peter Segrist operation sequencer. carboxylation could be based upon accessibility and not on sequence order. The Gla-containing region of the calf bone 1. Price, P. A., Otsuka, A. S., Poser, J. P., Kristaponis, J. & Raman, protein may be relatively disordered and accessible. There are N. (1976) Proc. Natl. Acad. Sci. USA 73,1447-1451. several proline residues in this section of the sequence, and the 2. Hauschka, P. V., Lian, J. B. & Gallop, P. M. (1975) Proc. Natl. protein behaves physically more like a random coil than a Acad. Sci. USA 72,3925-3929. globular protein, since it emerges from Sephadex G-100 at the 3. Cole, R. D. (1967) in Methods in Enzymology, ed. Hirs, C. H. position expected for a globular protein of 11,500 daltons (1, W. (Academic Press, New York), Vol. II, pp. 315-317. 13), while the true particle weight is 5700. 4. Hermodson, M. A., Ericsson, L. H., Titani, K., Neurath, H. & K. A. Biochemistry 11, 4493-4502. Some conclusions can be drawn tentatively about the Ca2+ Walsh, (1972) 5. Mendez E. & Lai, C. Y. (1975) Anal. Blochem. 68,47-53. binding properties of Gla residues and the likely function of the 6. Bruton, C. J. & Hartley, B. S. (1970) J. Mol. Biol. 52,165-178. three Gla residues in the calf bone protein. In the calf bone 7. Cohn, E. J. & Edsall, J. T. (1943) Proteins, Amino Acids, and protein sequence the three Gla residues are separated by two Peptides (Hafner, New York), pp. 370-381. or three intervening residues, while in prothrombin six of the 8. Summers, M. R., Smythers, G. W. & Oroszlan, S. (1973) Anal. ten Gla residues are in pairs (10). Since we find no high affinity Biochem. 53, 624-628. Ca2+ binding sites in the calf bone Gla proteint, the tight Ca2+ 9. Fietzek, P. P., Rexrodt, F. W., Wendt, P., Stark, M. & Kuhn, K. binding sites found in the Gla-containing NH2 terminal region (1972) Eur. J. Biochem. 30, 163-168. of prothrombin (14) are probably achieved by coordination of 10. Magnussen, S., Sottrup-Jensen, L., Petersen, T. E., Morris, H. R. a Ca2+ atom with the -y-carboxyl groups of two adjacent Gla & Dell, A. (1974) FEBS Lett. 44, 189-193. residues (11). The calf bone Gla protein does bind strongly to 11. Enfield, D. L., Ericsson, L. H., Walsh, K. A., Neurath, H. & Ti- tani, K. (1975) Proc. Natl. Acad. Sci. USA 72, 16-19. hydroxyapatite crystals, and it seems likely that some of the 12. Howard, J. B. & Nelsestuen, G. L. (1975) Proc. Natl. Acad. Sci. binding energy is achieved by the bivalent coordination of Ca2+ USA 72, 1281-1285. atoms at the crystal surface with the two y-carboxyl groups of 13. Atassi, M. & Caruso, D. R. (1968) Biochemistry 7,699-706. a Gla residue. If this hypothesis for the function of Gla residues 14. Nelsestuen, G. L., Broderius, M., Zytokovicz, T. H. & Howard, in the calf bone protein is correct, it is clear that strong Ca2+ J. B. (1975) Biochem. Biophys. Res. Commun. 65, 233-240. binding to Gla residues would competitively inhibit crystal 15. Arnold, P. W. (1950) Trans. Faraday Soc. 46, 1061. binding and would therefore be functionally useless. 16. Carlstrom. D. (1955) Acta Radiol. Suppl. 121. The Gla protein binds to hydroxyapatite and not to amor- 17. Betts, F., Blumenthal, N. C., Posner, A. S., Becker, G. L. & phous calcium phosphate, and it strongly inhibits the formation Lehninger, A. L. (1975) Proc. Natl. Acad.-Sci. USA 72, 2088- 2090. 18. Steiner, D. F., Kemmler, W., Tager, H. S. & Peterson, J. D. (1974) t P. A. Price and J. Kristaponis, submitted for publication. Fed. Proc. 33,2105-2115. Downloaded by guest on September 28, 2021