©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation, Characterization, and Primary Structure of a Calcium-binding 63-kDa Bone Protein (*)

(Received for publication, August 11, 1994; and in revised form, November 18, 1994)

Mikael Wendel Yngve Sommarin Tomas Bergman (1) Dick Heinegård (§)

From the Department of Medical and Physiological Chemistry, University of Lund, P. O. Box 94, S-221 00 Lund and the Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-17177 Stockholm, Sweden

ABSTRACT
INTRODUCTION
MATERIALS METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A novel noncollagenous protein of the mineralized matrix of bovine bone was isolated by ion exchange and gel permeation chromatography. The apparent M(r) of the protein is 63,000 as determined by SDS-polyacrylamide gel electrophoresis. The protein is a rather minor constituent in bone and could not be detected in other connective tissues by enzyme-linked immunosorbent assay of guanidine HCl extracts. The 63-kDa protein was detected in the osteoid and around the osteocytes upon immuno-histochemical staining of bovine compact bone. The sequence of the 63-kDa protein was deduced from cDNA clones isolated from a rat calvaria gt11 expression library. The protein contains two centrally located EF-hand Ca-binding domains. Seven heptad repeats are present indicating the ability of the protein for coiled-coil interactions. Ability to bind calcium was confirmed by Ca binding to protein blotted onto nitrocellulose membrane. The protein was synthesized in calvaria explants as detected by immunoprecipitation of radiolabeled protein from the culture medium. Although the protein can be detected in biochemical amounts in bone only, varying amounts of mRNA for this protein were detected in several rat tissues by RNase protection assay with highest levels in rat calvaria. This extracellular protein corresponds to a mouse protein called nucleobindin.


INTRODUCTION

Bone is composed of a well organized extracellular matrix that contains embedded crystals of hydroxyapatite. The major part, 90%, of the organic matrix is collagenous and consists mainly of type I collagen (for review, see (1) ). The remaining 10% consists of well over 200 other proteins (2, for review, see (3) and (4) ). Many of these proteins originate from plasma or other non-bone sources. Examples are albumin, the alpha(2)HS-glycoprotein(5, 6) , and the 62-kDa protein(7) .

The major bone matrix components produced by the cells in bone are osteonectin(8, 9) , matrix GLA protein(10) , osteocalcin(11) , proteoglycans like decorin and biglycan (for review, see (12) ), bone acidic glycoprotein 75 (BAG 75)(13) , osteopontin (2ar, Spp1, pp69) (14) , bone sialoprotein (BSP)(15, 16) , fibronectin, vitronectin, and thrombospondin(17) .

All these molecules appear to be bound in the mineral matrix. Prevailing data show that they are produced by osteoblastic cells. Several of the proteins have been extensively characterized, but their functions in the bone are still largely unknown. The mineral phase contains additional minor proteins which have not been studied. This is mainly due to the small amount of organic matrix that hampers preparative procedures and limits characterization. In order to increase our understanding of complex processes such as bone formation and bone remodeling it is, however, necessary to isolate and characterize also the minor proteins from bone tissue.

This paper describes the purification and characterization of a 63-kDa protein that represents one of these minor constituents from the mineralized matrix of bovine bone. Its primary structure was determined by isolation and sequencing of cDNA from a rat calvaria library encoding the 63-kDa protein.


MATERIALS METHODS

Extraction

Frozen powderized bovine bone, 1.5 kg, was extracted sequentially with 10 volumes of 4 M guanidine HCl, 50 mM sodium acetate buffer, pH 5.8, and 30 volumes of 4 M guanidine HCl, 50 mM Tris-HCl buffer, pH 7.4, also containing 0.25 M disodium EDTA as described in detail elsewhere(18) . Both extraction solvents were supplemented with proteinase inhibitors and with 5 mMN-ethylmaleimide, the latter included mainly to prevent disulfide exchange(19) . The second extract only, primarily containing components from the mineral phase, was further studied. This extract was concentrated at 4 °C by ultrafiltration over a PM-10 filter (Amicon Corp., Lexington, MA). The material retained was brought into 7 M urea, 0.1 M sodium acetate, 10 mM Tris-HCl buffer, pH 6.0, by diaflow with 10 volumes of the urea solution.

Anion-exchange Chromatography at pH 6 on DEAE-Cellulose

The guanidine HCl-EDTA extract from 1.5 kg of bone, brought into the 7 M urea-Tris buffer (see above) was chromatographed on a DEAE-cellulose (DE52, Whatman Chemicals, Maidstone, Kent, United Kingdom) ion-exchange column as described by Franzén and Heinegård (20

Cation-exchange Chromatography at pH 4 on CM-Cellulose

The flow-through from the DEAE-cellulose chromatography at pH 6, containing the 63-kDa protein, was extensively dialyzed against distilled water and lyophilized. The material was dissolved in 7 M urea, 20 mM sodium acetate, 15 mM sodium chloride, pH 4, and applied to a CM-cellulose ion-exchange column, (5.0 cm times 8.5 cm), which was first eluted at 14 °C with three bed volumes of 7 M urea, 20 mM sodium acetate, 15 mM sodium chloride, pH 4, at a flow rate of 55 ml/h. Bound material was eluted with a gradient (2 times 1000 ml) of 0.015 M to 0.15 M sodium chloride in 7 M urea, 20 mM sodium actetate, pH 4. Fractions of 15 ml were collected and analyzed for protein by their absorbance at 280 nm.

Gel Chromatography

Pool 5 from the CM-cellulose chromatography was transferred by diaflo into 4 M guanidine HCl and applied to tandemly coupled columns of Superose 12 and 6 (HR 10/30, Pharmacia Fine Chemicals, Uppsala, Sweden). Elution was with 4 M guanidine HCl, 20 mM Tris, pH 8, at a flow rate of 0.25 ml/min with 0.5-ml fractions collected.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Gradient polyacrylamide (4-16%) slab gels were cast with a 3% stacking gel and the SDS-buffer system according to Laemmli(21) . Samples were not reduced before electrophoresis. Gels were stained with 0.25% (w/v) Kenacid Blue R (BDH Chemicals, Poole, Dorset, U.K.). Samples were prepared for electrophoresis by ethanol precipitation as described elsewhere(22) .

Antibody Preparation

Antiserum was raised against the 63-kDa protein by immunizing rabbits with the 63-kDa protein (100 µg). Initial immunization was with Freund's complete adjuvant (Difco Laboratories, Detroit, MI), and subsequent boosters after 4 and 8 weeks were with Freund's incomplete adjuvant (Difco Laboratories). Rabbits were bled from the ear vein at 3-week intervals.

Enzyme-linked Immunosorbent Assay (ELISA)(^1)

The assay procedure developed for the cartilage 36-kDa protein (23) was adapted. Thus the 63-kDa protein was coated at 0.1 µg/ml in 4 M guanidine HCl, 50 mM sodium carbonate, pH 10.0, overnight at room temperature to polystyrene ELISA-plates (NUNC immunoplate 1, NUNC, Roskilde, Denmark). Samples and standards to be assayed were dissolved in 0.4% (w/v) SDS and added to an equal volume of antiserum appropriately diluted in 2% (w/v) Triton X-100 to give final concentrations of 0.2% SDS and 1% Triton. Excess SDS was thereby bound in mixed micelles with the Triton. The mixture was then incubated in the coated plates. After rinsing, bound antibodies were detected using a swine anti-rabbit alkaline phosphatase conjugate (Orion Diagnostica, Helsinki, Finland) and p-nitrophenyl phosphate (Sigma) as the substrate.

Preparation of Tissue Samples for Immunoassay

Tissues (1 g wet weight), i.e. liver, trachea, tendon, articular cartilage, nasal septum cartilage, kidney cortex, heart, intestine, muscle cornea, and bone were extracted with 10 volumes of guanidine HCl always containing 10 mM EDTA. The bone tissue was initially preextracted with guanidine HCl not containing EDTA and then extracted a second time with guanidine HCl/EDTA as described above. This extract was dialyzed against guanidine HCl to remove the EDTA. Samples (2 mg) were precipitated with 10 volumes of ethanol, suspended in sodium acetate, and reprecipitated with ethanol. Precipitates were dissolved in 1 ml 0.4% SDS and analyzed by ELISA.

Western Blotting

Samples were electrophoresed and transferred to nitrocellulose and processed for immunodetection essentially as described by Towbin et al.(24) using a 1:200 dilution of affinity purified rabbit anti-bovine 63-kDa protein and a 1:500 dilution of peroxidase-conjugated pig anti-rabbit IgG (DAKO, Denmark). Enzyme activity was determined with diaminobenzidine hydrochloride (Janssen Chemicon, Belgium).

Immunohistochemical Staining

A piece of compact bone (5 mm times 5 mm times 5 mm), from the tibia of a 2-year-old steer was demineralized in 10% EDTA in PBS for 4 weeks. The specimen was quick frozen in liquid N(2) and mounted in O.C.T. Compound (Tissue Tek II, Miles Laboratories, Naperville, IL) on a cryostat holder. Sections (5 µm) were prepared at -22 °C in a cryostat and picked up on gelatin-coated slides. The sections were dried at room temperature for 2 h and subsequently incubated for 10 min in absolute ethanol. Nonspecific binding was blocked by incubating the sections with goat-serum (1:70 in PBS) for 20 min at room temperature in a humidified chamber. The sections were rinsed with PBS-0.01% bovine serum albumin after each incubation step. Unlabeled affinity purified primary antibody against the 63-kDa protein (diluted to 1:300 in PBS-0.01% bovine serum albumin) or the preimmune serum (diluted to 1:300 in PBS-0.01% bovine serum albumin) were added to the sections and left for incubation at 4 °C overnight.

Bound antibodies were detected by incubation with biotinylated second antibody (diluted 1:200) and avidin-peroxidase conjugate using the Vectastain ABC kit (Vector laboratories, Burlingame, CA) following the manual supplied.

Organ Culture

Parietal, frontal, and occipital bones of the skull of 3-4-day old rats were dissected clean, in one piece, under sterile conditions. The preparations were cultured individually in 2 ml of Ham's F-12 medium supplemented to 10% bovine fetal serum, and with penicillin, and streptomycin. After 24 h the medium was changed to Ham's F 12 without serum addition but with [^3H]leucine (50 µCi/ml) added. After a pulse of 6 h, the bones were removed from the medium. The medium was stored frozen until analyses.

Immunoprecipitation of Radiolabeled Proteins

After thawing, the medium was precipitated with ethanol (18) and processed for immunoprecipitation, as described elsewhere(25) . The antiserum to the bovine 63 kDa was used. It reacts well with rat 63-kDa protein on Western blots (data not shown). Immunoglobulins with bound antigen were recovered bound to protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden). Immunoprecipitates were dissolved under reducing conditions (1% mercaptoethanol in starting buffer) and electrophoresed on 4-16% SDS-PAGE gradient gels. Radioactive proteins were visualized by fluorography using salicylate as the scintillator(26) .

cDNA Cloning and Nucleotide Sequence Analysis

A gt11 expression library from rat calvaria (14) was screened with the antiserum against bovine 63-kDa protein. Four clones were selected on the basis of the strength of antibody staining. To confirm the identity of the clones, plaque adsorption of the antiserum to plaque lifts of dense plates was performed. Bound antibodies were desorbed from the nitrocellulose sheets by low pH elution and neutralized by dialysis against PBS. The respective desorbed antisera were tested for ability to detect the 63-kDa protein in protein transfer blots of bovine bone extracts separated on SDS-PAGE. Antibodies adsorbed to a plaque lift from one of the clones strongly stained a band at the position of the 63-kDa protein. Antibodies adsorbed to the other clones showed no staining. The verified clone was subcloned into Bluescript II KS(+) and M13mp18 vectors for dideoxy chain termination sequence analysis. Sequence analysis indicated that this was not a full-length clone. The library was therefore rescreened with a digoxygenin-labeled EcoRI/SacI fragment from the most 5`-end of the original cDNA clone. One clone with a further 222 base pairs including the initiation codon was obtained, subcloned into Bluescript II, and sequenced. The clones were sequenced by double- or single-stranded sequencing from both directions.

CNBr Cleavage and Amino Acid Sequence Determination

Intact bovine 63-kDa protein was used for NH(2)-terminal amino acid sequencing. For internal sequence information the protein was subjected to CNBr cleavage, and fragments were separated by standard reversed-phase high performance liquid chromatography. Two well separated peptide peaks were chosen for sequencing by the Edman procedure in an automatic protein/peptide sequencer (Applied Biosystems model 470A sequencer, Foster City, CA)

Production of Partial Length Recombinant Protein

From the original clone an EcoRI fragment, from position 222 to 2152, was isolated and subcloned into the bacterial expression vector pTrcHisB (Invitrogen). The expressed protein consists of a 44-amino-acid NH(2)-terminal part including a 6 histidine stretch followed by the 63-kDa sequence beginning with a leucine at position +29 in the amino acid sequence. Protein production was induced and recombinant protein isolated by metal chelate affinity chromatography under denaturing conditions according to the manufacturer's instructions.

Assay for Ca Binding

Analysis was performed essentially as described by Maruyama et al.(27).

Analysis of mRNA

Total RNA was isolated essentially as described by Adams et al.(28) . In short, all tissues were homogenized with a Polytron in guanidine isothiocyanate, extracted for 1 h at room temperature, followed by a single low pH phenol/chloroform extraction and subsequent isolation by centrifugation through a CsTFA (Pharmacia, code number 17-0847-02) cushion. Ten µg of total RNA from rat calvaria was separated by formaldehyde-agarose gel electrophoresis, transferred to nitrocellulose, and probed according to standard procedures with a P-labeled 800-base pair EcoRI fragment from the original clone. RNA was similarly isolated from a number of tissues, and 20 µg from each tissue was subjected to RNase protection assay, using an RPA II kit from Ambion, according to the manufacturer's recommendations. The amount of RNA was quantitated by absorbance at 260 nm and by running 10 µg of each sample on a formaldehyde-agarose gel to control that the same amounts were seen in all samples by ethidium bromide staining. For Rnase protection a probe, covering nucleotides 870-1007 of the 63-kDa cDNA, giving a 136-bp protected fragment was used. Protected fragment was detected and quantitated by the use of a Fuji BAS 2000 PhosphorImager.

Sequence Evaluation

Computer analysis of sequence data was performed with the PCGENE program package (Intelligenetics). Similarity searches were performed by the use of the E-mail service at the National Center for Biotechnology Information using the BLASTP program(29) .


RESULTS

Purification of the 63-kDa Protein

Ground bovine diaphyseal bone was preextracted with 4 M guanidine HCl containing protease inhibitors but no EDTA to remove macromolecules in non-mineralized tissues of bone, i.e. those from blood, blood vessels, bone marrow cells, and fibrous tissue. The residue was washed and proteins, including the 63-kDa protein, were extracted under demineralizing conditions by including 0.25 M EDTA in the extraction solvent. Virtually all of the non-collagenous proteins associated with the hydroxyapatite crystals were then released. The guanidine HCl-EDTA extract was brought into 7 M urea, 0.1 M sodium acetate buffer, pH 6, and chromatographed on DEAE-cellulose, chromatogram not shown, described by Franzén and Heinegård(20) . We chose to further purify the 63-kDa protein eluting in the flow-through fraction. The material was brought into 7 M urea, 20 mM sodium acetate, 15 mM sodium chloride, pH 4, and chromatographed on a CM-cellulose column eluted with a gradient of 0.015-0.15 M NaCl, all eluents containing the 7 M urea buffer. Subsequently, SDS-polyacrylamide gel electrophoresis under non-reducing conditions was used to identify the elution position of the protein. The fractions were combined into five pools, Fig. 1. Pool 5 contained predominantly the 63-kDa protein, migrating as a 70-kDa component due to non-reducing conditions, and a 40-kDa protein. The proteins were dissolved in 4 M guanidine HCl, 20 mM Tris, pH 8, and the sample was chromatographed on tandemly coupled columns of Superose 12 and Superose 6. The lower molecular mass component was removed by this purification step resulting in pure 63-kDa protein. The fractions containing the protein were identified by SDS-polyacrylamide gel electrophoresis, and fractions 48-50 were pooled as shown in Fig. 2.


Figure 1: CM-cellulose chromatography pH 4.0. The pool representing material not binding to the DE52 column, was dissolved in 7 M urea, 15 mM sodium chloride, 20 mM sodium acetate, pH 4, and chromatographed on a cation exchange CM-52 column, eluted with a gradient (dashed line) of 0.015-0.15 M NaCl in the urea solution. Samples of fractions were electrophoresed on 4-16% gradient SDS-PAGE gels under non-reducing conditions. The 63-kDa protein, here migrating as a 70-kDa component in these non-reducing conditions, represents one of the predominating components in fraction 98-122 which were pooled as pool 5.




Figure 2: Superose 12/Superose 6 chromatography. Pool 5, from the CM-52 in Fig. 1, was desalted, freeze dried, and dissolved in 4 M guanidine HCl, 20 mM Tris, pH 8, and chromatographed on Superose 6 and Superose 12 (V = 50 ml and V(0) = 15 ml) columns tandemly arranged. Fractions of 0.5 ml were collected. Inset shows SDS-PAGE analysis of fractions under non-reducing conditions where the 63 kDa has a electrophoretic mobility of 70 kDa. The bar shows final pooled fractions (48-50).



Specificity of Antibodies to the 63-kDa Protein

Antibodies were raised against purified bovine bone 63 kDa protein in rabbits. Affinity purified antibodies were prepared, and specificity was tested by Western blotting. Purified protein along with a non-fractionated guanidine HCl-EDTA extract sample were electrophoresed on a gradient SDS-polyacrylamide gel. The gel was electrophoretically transblotted and the membrane probed for positive immunoreactions with the antiserum. Only one prominent immunoreactive band was observed with the guanidine HCl-EDTA extract of bovine bone, Fig. 3B, lane 1. Its migration corresponds to that of the 63-kDa protein.


Figure 3: Indirect immunodetection of electroblots. A bone mineral compartment extract, i.e. with guanidine HCl-EDTA (lane A1) was electrophoresed together with the 63-kDa protein (lane A2), on a sodium dodecyl sulfate 4-12% polyacrylamide gel, under non-reducing conditions. One part of the gel was stained with Coomassie Blue (A). An identical set was electroblotted onto nitrocellulose. Reactive components were detected using an affinity purified rabbit antiserum against the bovine bone 63-kDa protein and a peroxidase-conjugated second antibody, followed by development with substrate color reagent, diaminobenzidine hydrochloride (B). Only one band stained heavily, in the guanidine HCl-EDTA extract (lane B1) This component corresponds to the migration position of the 63-kDa protein. Positive control (lane B2).



Distribution of the 63-kDa Protein between Tissues

The presence of the protein in 4 M guanidine HCl extracts of a number of connective tissues including bone was determined with enzyme-linked immunosorbent assay, Fig. 4. The protein was only detected in extracts of bone. The concentration in bone extracts of the 63-kDa protein was calculated to be 150 µg/g of tissue wet weight. Nevertheless small amounts in select compartments of other tissues cannot be ruled out.


Figure 4: ELISA of the 63-kDa protein in extracts of bovine tissues. A number of bovine tissues were extracted with 10 volumes of 4 M guanidine HCl and precipitated with ethanol. Precipitates were dissolved in SDS, dilutions were prepared, and ELISA performed as described under ``Materials and Methods.'' The left panel shows the inhibition curve obtained with a standard of purified 63-kDa protein. The right panel shows extracts of 10 non-bone tissues (-, see text) tested in the same assay and, as a positive control, an extract of bovine bone is included (dashed line). None of the non-bone tissues gave significant inhibition, showing the absence of the 63 kDa at the level of detection limit (<0.5 µg/mg of original tissue weight).



Immunolocalization

The distribution of the protein in compact, demineralized bone was studied by the indirect immunoperoxidase technique. The 63-kDa protein is localized in the soft tissue in the center of the osteon and in the osteocyte lacuna, Fig. 5. In the mineralized matrix, staining is weaker but is detectable especially along the concentric lamellae of the osteon.


Figure 5: Immunolocalization of the 63-kDa protein in compact bovine bone. Decalcified compact bone from tibia of a 2-year-old steer was sectioned on a cryostat in 5-µm sections. The antiserum against the bovine 63-kDa protein was used for immunostaining. The sections were incubated with antibodies directed against the 63-kDa protein (a and b)or with preimmune serum (c and d) followed by a biotinylated second antibody. The sections were incubated with Vectastain ABC reagent and developed in peroxidase solution. The left panels show compact bovine bone treated with antiserum against the 63-kDa protein, magnification times 100 (a) and magnification times 200 (b). The right panels show the sections treated with preimmune control serum, magnification times 100 (c) and magnification times 200 (d). Strong specific staining for the 63 kDa is seen in the osteoid that covers the walls of the central canal in the osteon as well as in the osteocyte lacuna. The protein has a very restricted tissue distribution in the extracellular matrix.



Organ Culture

Synthesis and secretion of the 63-kDa protein in bone was shown by metabolic labeling with [^3H]leucine in a cultured rat neonatal calvaria and immunoprecipitation of the protein from the medium. The precipitates were electrophoresed under reducing conditions. Two bands were detected by the fluorography, Fig. 6; one migrates at the position of the 63-kDa protein, the other migrates as 280 kDa. The high molecular weight band is a major component in the medium which frequently is found in the precipitates with many different antisera. For instance specific antiserum against BSP also precipitate the 280-kDa component from the same medium (data not shown).


Figure 6: Detection of the 63-kDa protein in the medium of rat calvaria explant cultures. Calvaria of 3-day-old rats were carefully cleaned from surrounding tissues and cultured in Ham's F-12 containing 10% fetal bovine serum. After 24 h the cultures were incubated with Ham's F-12 containing [^3H]leucine for 6 h. The 63-kDa protein immunoprecipitations (A) as well as the whole medium (B) were electrophoresed on 4-16% SDS-Polyacrylamide gels after reduction of disulfide bonds. Radiolabeled proteins were visualized by fluorography.



Isolation and Sequencing of cDNA Clones

Initially, the rat calvaria cDNA library in the gt11 expression vector was screened with a polyclonal antiserum raised against the 63-kDa protein isolated from the mineralized phase of bovine bone. Several positive clones were found in the first screen. These clones were verified to be reactive with antibodies to the 63-kDa protein by adsorbing the antiserum on plaque lifts of dense plates of the different clones. One clone adsorbed antibodies from the rabbit antiserum specific for the 63-kDa protein in immunoblotting experiments. This clone was selected for further analysis. It represented almost the entire coding sequence including the 3`-end of the message since sequence analysis revealed the presence of a poly(A) tail. Sequencing indicated that the original clone did not represent a full-length cDNA. To obtain the full-length cDNA, the library was screened again with a digoxygenin (Boehringer Mannheim)-labeled 202-bp EcoRI/SacI fragment from the 5`-end of the original cDNA clone. A clone containing the full-length cDNA with a further 222 bp of 5`-end sequence was identified, and a 1007 bp 5`-end EcoRI fragment of this clone was subsequently sequenced.

The identity of the clone with isolated protein was further confirmed by amino acid sequencing of the NH(2) terminus (VPLEXXAA) and of two internal CNBr-cleaved fragments (LLKAK; EQRKQQQQ) from the 63-kDa protein isolated from bovine bone. Both internal peptides match perfectly with the sequence deduced from the rat cDNA in positions 88-92 and 371-378, respectively, Fig. 7. The nucleotide sequence predicts a 459-amino-acid protein with a calculated molecular mass of 53,506 Da for the complete protein and 50,919 with the signal peptide removed, Fig. 7. A 25-amino-acid signal sequence could be predicted on the basis of its hydrophobicity and that it conforms well with the -3, -1 rule for predicting signal peptidase cleavage sites(30) . The NH(2)-terminal amino acid sequence obtained does not match perfectly with the predicted NH(2) terminus of the mature protein, but it does confirm the position of the signal peptide cleavage site after the alanin residue at position 25. No sites for N-linked oligosaccharide attachment are present in the sequence.


Figure 7: Nucleotide and deduced amino acid sequence of the 63-kDa protein. The complete nucleotide sequence is shown, with the deduced amino acid sequence shown below in single letter code. Arrow indicates signal peptide cleavage site. Peptide sequence from amino acid sequencing is indicated by double underlined letters below the deduced amino acid sequence. A polyadenylation signal is single underlined.



Presence of EF-hands

Two centrally located EF-hand Ca-binding sites with a 36-amino-acid intervening sequence were detected. Both EF-hands conform well to the consensus sequence for EF-hands(31) , Fig. 8. Both loops are well conserved with the essential sites for binding of the calcium atom coordinates X, Y, Z, and -Z identical to known Ca-binding proteins. In the second loop the commonly found glycine at position 6 is replaced with arginine and the aspartic acid at coordinate -X replaced with a threonine. These replacements have, however, been found in several functional EF-hands and should therefore not impair calcium binding.


Figure 8: Alignment of putative Ca-binding domains in the 63-kDa protein with EF-hand consensus sequence. The EF-hand loop pattern of the 63-kDa protein is aligned to each other and to the consensus sequence for calcium-binding EF-hands. The coordinates for binding of the Ca ion are indicated.



Ability to Bind Ca

The potential calcium binding ability of the 63-kDa protein was confirmed by the use of polyacrylamide gel electrophoresis of the protein and transfer to nitrocellulose(27) . The protein isolated from bovine bone as well as the recombinant, partial protein clearly bound Ca, Fig. 9, panel 2. For positive control, calmodulin was included and migrated as one distinct component that bound calcium, Fig. 9, panel 2a. Neither the negative control CAT construct, nor ovalbumin or collagen bound Ca.


Figure 9: Ca binding to the 63-kDa protein blotted onto nitrocellulose paper. Ten µg of each protein was electrophoresed on a 10% SDS-polyacrylamide gel and stained with Coomassie (panel 1) and transferred to nitrocellulose membrane (panel 2) by passive diffusion. Lanes: a, calmodulin; b, bovine 63-kDa protein; c, recombinant rat 63-kDa protein; d, a control recombinant chloramphenicol acetyltransferase with a 6 histidine region; e, ovalbumin; f collagen I.



Heptad Repeat

Every seventh amino acid from position 321 to 369 is a leucine residue. The protein thus contains seven heptad repeats(32) , aligned in Fig. 10. To form stable coiled-coil homointeractions, amino acids in position a and d of the repeat unit should be apolar. This is not the case for the 63-kDa protein where charged residues are found in several of the d positions. Analysis by non-denaturing polyacrylamide gel electrophoresis of the 63-kDa protein produced in Escherichiacoli did not indicate ability of the protein to self interact (data not shown).


Figure 10: Alignment of the heptad repeats in the 63-kDa protein. The seven consecutive heptad repeats from position 321 to 369 in the protein sequence are aligned. The positions in the heptad consensus sequence are indicated on top.



Nuclear Localization Signal

A potential nuclear localization signal was detected in the 63-kDa protein between residues 173-189, Fig. 7. The bipartite nuclear targeting signal motif, proposed by Dingvall and Laskey(33) , comprising 2 basic amino acids, a spacer region of any 10 amino acids, and a basic cluster of at least 3 basic residues in the five positions after the spacer region. The nuclear localization signal has the following sequence RKEAERKLQEQQRRHRE, where the basic amino acids of the motif are underlined.

RNA Analysis

RNA hybridization analysis of RNA isolated from rat calvaria indicated the presence of a single mRNA species of approximately 2.4 kilobases, Fig. 11A. Messenger RNA for the 63-kDa protein was detected by RNase protection assay in all rat tissues examined, i.e. calvaria, rib cartilage, liver, kidney, spleen, brain, lung, skeletal muscle, and heart muscle, Fig. 11B. Highest levels of mRNA was found in calvaria with approximately half the amount in kidney, liver, and brain.


Figure 11: Analysis of the 63-kDa protein mRNA. A, Northern blot analysis of rat calvaria RNA. A sample of 10 µg of total RNA was analyzed. The positions of 28 S and 18 S ribosomal RNA are indicated by arrows. B, RNase protection analysis of 20 µg of total RNA each from rat tissues. Protected 136-bp fragment is indicated by arrow. Lane: a, nondigested probe; b, brain; c, lung; d, spleen; e, kidney; f, liver; g, rib cartilage; h, sternum; i, calvarial bone.



Identity with Nucleobindin

Similarity searches showed that the protein is identical to the recently cloned protein nucleobindin (34) from mouse and human. The protein is also highly similar to a human protein, NEFA, recently submitted to the EMBL data base, accession number X76732. An alignment of the published amino acid sequences with the rat sequence, Fig. 12, show that the protein is very highly conserved, with only a few substitution and insertions in the COOH-terminal end. The central portion of the protein with the EF-hand and heptad repeat structure is almost identical, with only conservative amino acid substitutions. The NEFA protein is somewhat shorter but appears to be a closely related member of the family.


Figure 12: Alignment of the deduced rat 63-kDa amino acid sequence with mouse nucleobindin, human nucleobindin, and NEFA. The rat 63-kDa primary sequence is aligned against mouse (MMNUC) and human (HSNUC) nucleobindin and NEFA protein (NEFA). The character * indicates that the position is perfectly conserved and bullet indicates a conserved replacement.




DISCUSSION

The isolation and characterization of macromolecular constituents of bone provides important tools for studying bone biology including cell differentiation and growth, cell recognition and attachment, organized matrix production, and modulation of bone resorption processes that regulate calcium homeostasis. We have purified and determined the primary structure of an extracellular protein of 63 kDa from bovine bone. The protein is synthesized and secreted into the medium in calvaria explant cultures, thus representing an extracellular osteoblast product. Immunostaining showed that the protein is present in bone matrix, in the osteoid and in the osteocyte lacuna. The complete primary structure of the rat 63-kDa protein was determined by cDNA cloning. The primary structure indicates a molecular mass of 50,919 Da for the mature protein with the signal peptide removed. This is considerably less than the molecular weight obtained on SDS-PAGE. However, it should be noted that even the bacterially produced fusionprotein, thus devoid of post-translational modifications, also exhibited an aberrant migration on SDS-PAGE with a higher molecular weight than expected. The construct corresponds to a molecular mass of 52,626 but migrates on SDS-PAGE gels at a position corresponding to approximately 62 kDa.

The 63-kDa protein does not contain any potential sites for N-linked oligosaccharide substitution. Attempts to show the presence of O-linked oligosaccharides by digestion of isolated bovine 63-kDa protein with O-glycanases and subsequent SDS-PAGE analysis were negative (results not shown) indicating little or no oligosaccharide substitution. In support no hexosamines were detected upon amino acid analysis (data not shown). Calculations of a theoretical pI from the primary amino acid sequence gives a value of 4.8 indicating that most of this negative charge is likely to be a result of the amino acid composition. A protein motif search for potential phosphorylation sites reports six casein kinase II, three protein kinase C, and one tyrosine kinase phosphorylation site which potentially may be substituted with groups contributing to the negative charge.

The previously not described two EF-hand motifs now found in the central portion of the molecule conform well to the consensus pattern for EF-hands indicating that they are functional and probably contain bound Ca at the concentrations found in the extracellular milieu. It is likely then, that Ca is a necessary component for stabilizing the structure of the protein. In support of a tight calcium binding, the 63-kDa protein isolated from the tissue as well as a recombinant partial protein, containing the EF-hands, bound calcium even in the rather harsh conditions of the assay. In contrast, a CAT construct containing a motif of 6 histidine residues often used for divalent ion chelate affinity purification showed no calcium binding in this assay.

EF-hands in intracellular proteins such as calmodulin are arranged pairwise and exhibit cooperative binding of Ca. Alignment of the amino acid sequence between the pairs of EF-hands in rat calmodulin with the 63-kDa protein sequence between EF-hands shows that inter-EF-hand sequence of the 63 kDa has little similarity to calmodulin with a length of 37 amino acids compared to 24 in calmodulin and multiple amino acid substitutions. It is unusual for extracellular proteins to have two well conserved EF-hands. In other calcium binding extracellular matrix proteins, such as the well studied bone protein osteonectin, only one functional EF-hand is present(8, 9) . The second loop of osteonectin does not conform well to the consensus sequence.

Although mRNA for the 63-kDa protein can be detected in all tissues examined, the protein can only be detected in appreciable amounts in bone. In agreement the highest levels of mRNA were found in calvaria. Another possible explanation for the enrichment of the protein in bone is that the EF-hands mediate binding of the protein to calcium in the hydroxyapatite of the mineral matrix.

The heptad repeats found toward the COOH terminus are not as perfectly conserved as in known coiled-coil regions of the extracellular matrix proteins laminin and thrombospondin(35) . These proteins form three-stranded coiled-coils. The limited conservation of the heptad repeat in the 63-kDa protein suggests that this protein does not self-interact to form multimers. This is supported by the inability of the protein to form multimers on native polyacrylamide gels (data not shown). However, the heptad repeat is reasonably well conserved to suggest that the protein may be capable of coiled-coil interactions with other proteins.

A bipartite nuclear localization signal has been identified in the 63-kDa protein. In a study by Dingvall and Laskey (32) about 50% of the nuclear proteins harbored the motif in the Swiss Prot data base. However, the motif was found in 4.2% of non-nuclear proteins. Presence of the bipartite motif in the 63-kDa protein should therefore be interpreted cautiously.

A similarity search showed that the 63-kDa protein is identical to nucleobindin(33) . This protein was described as an intracellular DNA-binding protein. It was suggested that it is involved in the generation of the autoimmune response to single-stranded and double-stranded DNA in systemic lupus erythromatosis. The protein was isolated from a cell line established from a mouse strain with an inherited disposition for developing an systemic lupus erythromatosis-like condition(36) . The authors find, in vivo and in vitro, that injection of the protein into susceptible mice increases formation of autoantibodies to DNA. It appears, that DNA is bound to the protein during purification perhaps explaining the increase in autoantibody formation after addition of the protein. Kanai et al.(36) find it likely that nucleobindin binds to the DNA by the use of a leucine zipper motif. A heptad repeat is also found in the ``leucine zipper'' proteins giving these proteins the ability to form homo- and heterodimers through coiled-coil interactions(37) . However, to be functional in DNA binding a basic region should be located close to the heptad repeat(38) . In the 63-kDa protein a rather basic region is present from position 171 to 217 in the amino acid sequence. However, this basic region is located on the NH(2)-terminal side of the EF-hands making it unlikely that it would participate in a classical DNA-binding leucine zipper mechanism.

The protein is well conserved between the examined species, mouse/rat and human, indicating an important biological role. The function of the protein in the bone tissue is unknown. One main task in its further characterization is to elucidate its affinity for hydroxyapatite and whether it has a role in maintaining adequate mineralization and/or in the regulation of calcium homeostasis in bone.


FOOTNOTES

*
This work was supported by grants from the Swedish Medical Research Council, Axel och Margaret Ax:son Johnsons Stiftelse, Greta och Johan Kock's Stiftelser, Konung Gustaf V:s 80-års Fond, and the Faculties of Medicine and Odontology, Lund University.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z36277[GenBank].

§
To whom correspondence should be addressed: Dept. of Medical and Physiological Chemistry, University of Lund, P. O. Box 94, S-221 00 Lund, Sweden. Tel.: +46-46-108571; Fax: +46-46-113417.

(^1)
The abbreviations used are: ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).


ACKNOWLEDGEMENTS

The skillful technical assistance of Karin Lindblom, Annika Påhlsson, Viveka Nilsson, and Cecilia Jönsson is gratefully acknowledged.


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