(Received for publication, August 11, 1994; and in revised form, November 18, 1994)
From the
A novel noncollagenous protein of the mineralized matrix of
bovine bone was isolated by ion exchange and gel permeation
chromatography. The apparent M 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.
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
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.
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.
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
= 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).
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).
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).
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 100 (a) and
magnification
200 (b). The right panels show the sections treated with preimmune control serum,
magnification
100 (c) and magnification
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.
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
[H]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.
The identity of the clone with isolated protein was
further confirmed by amino acid sequencing of the NH 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
-terminal amino acid sequence obtained does not match
perfectly with the predicted NH
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.
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.
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.
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.
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.
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 indicates a conserved
replacement.
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-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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z36277[GenBank].