Clinical Science( 1987)7 2 , 399-408
399
EDITORIAL REVIEW
The special proteins of bone tissue
JAMES T. TRIFFITT
MRC Bone Research Laboratoty, Nicffield Department of Orthopaedic Surgety, University of Oxford, Niiffield Orrhopaedic
Centre, Oxford, U.K.
Our bones are dried and our hope is lost
Ezekiel, The Bible, Chapter XXXVII, V. 11
Introduction
The proteins of bone tissue are many and of diverse
character, which is not surprising as the tissue itelf is
very heterogeneous [l].Recently, a great deal of
effort has been applied to the characterization of the
proteins that are found particularly in bone. This is
due largely to the input of research funds into this
area of investigation. Skeletal abnormalities become
more prevalent in aging populations, and osteoporosis, for example, has now reached epidemic
proportions [2].Medical interests are turning, therefore, to disorders of the elderly, particularly with
regard to cardiovascular disease and osteoporosis
[3]. It is politic, therefore, to support skeletal
’ research, and many laboratories especially in Japan
-and the U.S.A. have turned their attention to this
field of study with a resultant information explosion
in this area. In the U.S.A. a major national campaign
(The National Osteoporosis Foundation) has been
launched recently to increase public awareness of
osteoporotic disorders in order to obtain research
funds for investigations designed to combat this
crippling disease.
Bone tissue is obviously different from most connective tissues by its content of calcium phosphate
mineral. The process by which this specialization
occurs requires further understanding, and the
question of the precise mechanisms of the formation
and mineralization of bone are central issues to all
involved in bone research. The homoeostatic control of bone mass and quality are other areas of
active study. The idea that bone contains specific
factors essential to the process of mineralization has
been the ardent belief of many investigators for a
Correspondence: Dr J. T. Triffitt, MRC Bone Research
Laboratory, Nuffield Department of Orthopaedic
Surgery, University of Oxford, Nuffield Orthopaedic
Centre, Headington, Oxford OX3 7LD, U.K.
number of years. Type I collagen is a major constituent of the organic matrix of bone tissue, and defective synthesis of this molecule has a dramatic effect
on the skeleton. Recent work on the molecular
genetics of collagen disorders has been reviewed
recently in an Editorial in this series [4].
The lack of
apparent tissue specificity of collagen, the major
protein species of bone matrix, has focused attention on the minor proteinaceous constituents of the
tissue. This review will concentrate, therefore, on
some of this latter group of substances, which are
now thought not only to be implicated in formative
but also in resorptive mechanisms and controls. The
nature of some of these materials will be described
and the possible functions and some of the changes
seen in their metabolism in various bone disease
states discussed. It must be stressed that apart from
the likely functions of the inductive and growth
factors [S-71 that are present in bone matrices in
minor amounts, and which are outside the scope of
the present review, no proven metabolic or structural functions of the non-collagenous constituents
are known. Much of the work on this topic has concentrated on experimental animal studies and only
relatively recently have direct studies focused on the
problems of human bone disease.
The proteins of bone tissue range from collagen,
which is found in all connective tissues, to the
quantitatively minor but more specific proteins
located mainly, if not solely, in bone tissue as a
result, presumably, of synthesis by the osteoblast.
Furthermore, because of the capacity of the
hydroxyapatite mineral phase of bone to adsorb
polyvalent molecules, especially during its formation, other proteins normally present in the blood
stream, and hence in tissue fluids, are also found
associated with bone tissue. In addition, diffusion of
molecules synthesized during bone matrix formation and osteoblast metabolism, away from the
developing and mineralizing osteoid, occurs and the
value of quantitative changes in blood contents of
some of these bone-related proteins is being
assessed as possible indices of bone formation or
400
J. T Trijiti
osteoblastic activity. It is difficult to determine by
non-invasive methods the metabolic status of bone
tissue and appraisal of conditions of bone disease is
a continuing problem in clinical medicine. The
usual measurements of serum alkaline phosphatase
for bone formation, and urinary hydroxyproline for
bone resorption, are not specific for bone tissue.
More characteristic bone-derived constituents or
their fragments emanating from either sites of bone
formation or sites of bone destruction would, at
least in theory, be more suitable in this regard.
Bone sialoproteins
The pioneering work of Dame Janet Vaughan and
Dr Geoffrey Herring on the non-collagen proteins
of bone in our laboratory in the 1960s began with
an attempt to explain the peculiar location of the
transuranic elements in bone tissue [8]. This work
resulted in the first isolation and description of a
protein that appears to be specifically present in
bone tissue. This was named bone sialoprotein
(BSP) because of its high sialic acid content [9].
Over a period of about a decade the chemical
nature of this molecule was investigated thoroughly
and until a few years ago was the most highly
characterized connective tissue glycoprotein. BSP
was found to have a mol. wt. of about 23000 and
comprised 8-12% (w/w) of the total noncollagenous protein of bovine bone. Carbohydrate
residues make up about 40% by weight of the
molecule and sialic acid accounts for about half of
this value. The amino acid composition shows that
acidic residues, aspartate and glutamate, make up
together 40%of the total amino acid residues. Also
present are 2-4 mol of phosphate/mol of protein,
which further increases the acidic nature of the
polypeptide chain. This results in strong binding at
neutral pH to anion exchangers and to hydroxyapatite, and explains at least some of its presence in
calcified bone tissue. A similar constituent has been
found in the human [lo] and more recently, with
different methods of extraction, two separate
research groups have isolated similar sialic acidrich molecules from developing foetal [ll] and
more mature [ 121 bone. These dissociative methods
are thought less likely to allow autolytic degradation
of extracted macromolecules and indeed the
recently isolated sialoproteins have very similar
chemical characteristics to BSP but exhibit higher
apparent mol. wts. Also, trypsin cleavage of the
larger sialoprotein yields species of similar mol. wt.
to Herring’s original BSP, but the possibility still
exists that these are the natural molecules present in
adult bone and are derived from the higher mol wt.
precursor by lytic processes in vivo. By immunochemical criteria BSP is not present in any other
TABLE1. Representative values for the amounts of
noncollagenous proteins present in compact bone
The amounts given are composite representative
values and are based on amounts found in the compact bone of a number of species, including human,
bovine and rabbit bone.
Content*
(“76wlw)
Sialoprotein
Bone Gla-protein(osteocalcin)
Matrix Gla-protein
Phosphoproteins
Osteonectin
Proteoglycans
Albumin
a,HS-glycoprotein
Others
9
15
2
9
23
4
3
5
30
*As OO/ of total non-collagenous protein.
tissue or organ in vivo, except for its detection at
low levels in dentine. It was originally considered a
calcium binding protein but other possible functions have not been assessed in detail. Sialic acidrich components are found in cell membranes [13],
but the levels of sialoprotein in bone matrix (Table
1 ) are too high for solely a cellular presence and
most of this sialoprotein must reside in an extracellular pool in bone matrix. Its function, therefore,
is unknown, yet its peculiar presence in bone tissue
indicates a unique function in this tissue.
y-Carboxyglutamic acid-containing proteins
The amino acid, y-carboxyglutamic acid (Gla), was
first found in prothrombin and its presence in factors VII, IX and X, and proteins C, S and Z, is now
known to be of paramount importance in the biochemical reactions involved in blood clotting. Particular glutamic acid residues at selected sites on the
polypeptide chain are converted to Gla by addition
of a carboxy-group in post-translational events,
which have a specific requirement for vitamin K
and bicarbonate ion [ 141. The characteristic interaction of calcium ions with Gla residues, which it
was considered could, by analogy with the blood
clotting process, be necessary for mineralization,
led to the survey of bone tissues for the presence
of this unusual amino acid and independently two
groups isolated low mol. wt. constituents containing
this amino acid from different animal species [15,
161. Gla has also been found in several types of
ectopic calcifications.
Bone Gla-protein (osteocalcin)
Over the past ten years, the bone constituent
known as bone Gla-protein (BGP), or osteocalcin
Special proteins of bone
(OC), has been investigated both chemically and
biologically in great detail and recent articles review
the findings [17, 181. Both these names are in
current practice and will be stated together throughout this article to retain impartiality. The recent
proposals accepted by the American Society for
Bone and Mineral Research [ 191 for the nomenclature of non-collagenous proteins, although cumbersome, should serve to resolve this difficulty. This
protein has taken over the robe of BSP and is now
the most highly characterized bone protein. It is
impossible to consider all the information relating
to this constituent here and only the more salient
features will be described.
BGP (OC)is a small (6000 approx.), highly conserved protein found in bone tissues of all vertebrates examined. It is found also in tooth dentine
and is synthesized by cells of the osteoblast and
odontoblast lineage as an intracellular precursor of
mol. wt. about 9000. Before cellular secretion it is
altered to the product BGP (OC)and subsequently
becomes associated with the mineralized matrix of
these tissues, where it is detected shortly after
mineralization commences. Immunolocalization
studies indicate that BGP (OC) is present in osteoblasts, osteocytes and calcified bone matrix [20-231.
Molecular biological studies indicate that there is a
single gene for BGP and that by Northern blot
analysis no other tissue investigated expresses the
BGP (OC)message (P. A. Price, personal communication). In most species studied it is one of the most
abundant non-collagenous proteins (Table 1)
present in bone and makes up to 15% (w/w) of the
non-collagenous protein content. In man, however,
the value is only about 1% and this may indicate a
less important involvement of BGP in human bone
metabolism.
The primary structure of BGP shows a high
degree of conservation of structure during evolution. There are normally three Gla residues with an
associated disulphide bond and over this region
that makes up about one-third of the molecule only
minor changes in amino acid content have occurred
since swordfish evolution diverged from that of
man. There is no homology between the structure
of BGP and the blood clotting factors, indicating
separate evolution of these molecules for functions
in bone and blood tissues. There is, however,
limited homology of nine amino acid residues
proximal to the N-terminus between the propeptides of BGP and the coagulation factors [24].Pan
& Price [24] suggest that this common structural
feature may be involved in targeting these proteins
for post-translational vitamin K-dependent carboxylation.
The strong interaction of BGP (OC) with bone
mineral in vivo or hydroxyapatite in vitro is depen-
401
dent on the presence of unmodified y-carboxyglutamic acid residues in BGP (OC). Consequently,
the defective carboxylation mechanisms induced by
vitamin K deficiency result in the amounts of the
protein in bone in the deficient state being reduced
to 2% of control values seen in experimental
animals [25].No particular defects or abnormalities
in the bone mineralization process or in the structure of bone tissue or its strength have been seen in
these deficient animals. The bone mineral is identical with that of control animals in surface area and
X-ray diffraction pattern. However, when maintained in a vitamin K-deficient state by warfarin
injection for a prolonged period of 8 months, rats
show an abnormal closure of the epiphyses that
does not normally occur in the rat [26]. In these
studies it appeared that this mineralization of the
cartilage plate must have occurred towards the end
of the 8 month treatment period as tibial length was
90% of the control value. More controlled time
studies indicated that this process could be detected
after only 1 week of treatment [27]. The implication
of these investigations is that carboxylated BGP
(OC)in some way is necessary for the prevention of
mineralization in the epiphyseal cartilage growth
plate. If this is so then this must be a unique
property of the carboxylated protein as in these
studies on the vitamin K-deficient state the amount
of synthesized protein per se is not reduced, only its
carboxylation. The lack of influence on the structure or metabolism of bone indicates no requirement for Gla-residues in any function of BGP (OC)
in this tissue, unless the minimal remaining (2%)
carboxylated molecules are sufficient to maintain
these processes. Alternatively, the biriding of this
protein to hydroxyapatite is not of major physiological importance and the function of this protein
or a precursor precedes its adsorption to bone
mineral.
The synthesis of a small, rapidly diffusible protein at sites of bone formation suggests that a proportion may migrate through tissue fluid and be
found in blood. The development of radioimmunoassay methods for these procedures [28-321 has
shown that this is the case and between one-quarter
and one-ninth of the total bone production leaks to
blood in the rat. This is almost entirely derived
from bone, with dentine production giving an
insignificant contribution to the serum values.
Human serum, however, contains only 5-10 ng/ml
[28, 291 compared with 100-300 ng/ml seen in
bovine and rat serum [28,33] and this low level reflects the decreased synthesis by human bone
mentioned earlier. Nevertheless, measurements of
BGP (OC)levels in human sera have been recently
under intense investigation to assess their value as a
clinical parameter for bone synthesis or osteoblast
402
J. T Triffitt
metabolism. Plasma and sera values yield identical
results but in all assays the antibodies do not
distinguish between fully, under- or de-carboxylated BGP (OC).
The difficulty of producing human BGP (OC)for
production of antibodies has been circumvented by
the complete cross-reactivity of this protein with
antibody raised in rabbits against the more easily
prepared bovine protein [28]. Serum BGP (OC) is
identical with that found in bone with respect to its
molecular weight and Gla content, and is solely a
result of cellular synthesis by osteoblasts or their
precursors. Resorption destroys at the very least the
immunoreactivity of the molecule [34], if not the
complete peptide structures. Furthermore, the
clearance from blood is very rapid with a halftime
of a few minutes and most of the BGP (OC) is
cleared by glomerular filtration. It is apparent,
therefore, that the assay of the levels of serum BGP
(OC)could give a specific measurement of the metabolic activity of bone and should be of value in the
diagnosis and treatment of bone diseases. In this
respect it would seem to be a most ideal molecule
for this purpose: almost complete bone specificity;
highly diffusible from the site of production; synthesized by osteoblastic cells; rapid blood clearance; highly sensitive immunochemical detection.
All these characteristics serve to imply that the
moment to moment serum levels should reflect
bone cell activity. Unfortunately, attempting to correlate serum levels with bone cell activity is beset
with problems and clinical interpretation of the
changes observed is difficult.
For instance, not knowing the particular function
of BGP
or differentiation stages of the producing cells in the osteoblast lineage it is impossible
to know exactly to what cellular or extracellular
function or activity the serum measurements of this
protein relate. Furthermore, there is little
knowledge of the changes in anabolic processes
producing osteocalcin, and catabolic processes
removing it, that occur in disease, and blood clearance has not been studied in many cases. In
addition, immunochemical reagents and antibodies
used by investigators in different laboratories are
not of equal purity, sensitivity and specificity, and
this has lead to conflicting results. The challenge,
therefore, is to employ all methods of clinical and
experimental study to determine the relationship of
BGP (OC) production to a particular function or
activity so that the potential value of BGP (OC)
measurements may be realized, for only then will
assessment of its value as a clinical measurement be
determined. To this end, in clinical science accumulation of knowledge on serum BGP (OC)changes in
disease states and their treatments will require of
necessity the correlation with morphometric and
(w-)
other biochemical parameters of disease. In this
type of study with patients with uraemic bone
disease it was found that serum BGP correlated
best with the number of osteoblasts and the volume
of osteoid, consistent with a possible inhibition of
the mineralization process [35]. A circadian rhythm
suggested by one group [36]has not been confirmed
by others. Normal adult human circulatory levels of
BGP (OC)differ between laboratories but generally
fall into two groups: those finding mean values of
5-7 ng/ml [29-31, 371 and those finding higher
mean values of 12-16 ng/ml [32, 38, 391. In the
literature there is some disagreement regarding the
effect of age and sex. Serum values in males have
been reported to be higher [29, 30, 391, lower [37]
or not different [32] from those of females, and
there is also discrepancy concerning an increase of
serum BGP (OC)in adult age.
Generally, serum BGP (OC) is elevated in conditions characterized by increased bone turnover
and correlates with serum alkaline phosphatase [29,
31, 32, 37, 401. In fact, Brown et af. [41] indicate
that it is the only serum parameter that discriminates between high and low turnover osteoporosis
as determined histomorphometrically. The earliest
studies showed serum BGP (OC)was slightly higher
than normal in women with postmenopausal osteoporosis [29], and this correlation is more apparent
when age and kidney function are taken into
account. But in Paget’s disease, where marked
increase of bone turnover occurs, although it is
increased [29, 32, 421, serum BGP (OC) is not a
sensitive marker of bone turnover [43]. BGP (OC)
and alkaline phosphatase levels were halved and the
fall in BGP (OC)could be detected 2 h after administration of calcitonin [40].
High values of serum BGP (OC)are seen in renal
failure, probably indicating an effect on increased
bone formation with secondary hyperparathyroidism together with a reduced clearance of osteocalcin from the circulation by impairment of the
normal catabolism by the kidney tissue. Decreased
values in surgical hypoparathyroidism and increased values in primary hyperparathyroidism
[29, 42, 441 have been observed. In the latter
patients the serum levels fall significantly after parathyroidectomy [40, 441. Decreased levels in malignant hypercalcaemia have been interpreted to
suggest an uncoupling between the increased
resorption and decreased formation in these
patients [44].The alteration in the serum levels seen
in hyperthyroidism and in euthyroid patients [45],
the changes in normal and in growth hormonedeficient children [46] and the observations on bone
histomorphometry in hyperthyroidism and hyperparathyroidism [47] all suggest that serum BGP
(OC)levels are markers of the activities of the bone-
Special proteitts of bone
forming cells in these conditions. There is significant agreement on serum BGP (OC)measurements
in many conditions. But it is obvious that the existing variability between the investigatinglaboratories
in the serum BGP (OC) levels in normals and
diseased patients can only be resolved by the ready
exchange of antibodies and samples, and this must
occur in the near future.
A further aspect that may be related to the possible function of BGP (OC)is the recently recorded
observations that the increase in its synthesis is
modulated by the active metabolite of vitamin
D, 1,25-dihydroxycholecalciferol [ 1,25-(OH),D,].
This effect is observed in vitro with osteosarcoma
cell lines [48] or in vivo after administration of the
metabolite to normal [49] or vitamin D-deficient
[ 181animals, the latter exhibiting decreased levels of
the bone protein [50]. This effect appears to be a
transcriptionally regulated process and in rats the
level of BGP message (specific mRNA) is increased
for a considerable time after primary stimulation
[51]. 1,25-(OH),D, treatment also elevates serum
BGP (OC) in patients with X-linked hypophosphataemia or autosomal recessive vitamin D dependence 1521. This serum change correlated with
histomorphometric studies in the former disease
group which showed increased bone mineralization.
In the osteopenia developing during spaceflight,
BGP (OC)appears to be a sensitive indicator of the
early effects on bone metabolism [53].
Although BGP (OC) levels correlate best with
bone mineralization the protein has been implicated
in controlling the resorption of bone. BGP (OC)
and its degraded fragment and other bone constituents including type I-collagen peptides and
a,HS-glycoprotein (see below) were found to
possess chemotactic activity to populations of
blood monocytes [54-561 and may influence osteoclastic differentiation [55]. Indeed, powdered bone
from warfarin-treated rats when implanted in
normal hosts shows reduced phagocytic removal
compared with implants of normal bone powder
[57]. No evidence in support of this concept is seen
in warfarin-treated rats [25,26], however.
In summary, therefore, the weight of current
evidence suggests that BGP (OC) is linked with
some unknown function of cells of the osteoblast
lineage rather than with resorptive mechanisms,
although this protein or its products may have an
influence on the latter process. The local production of this protein appears to be of some influence
in controlling mineralization.
Matrix Gla-protein (MGP)
Recently, another distinct Gla-containing protein, named Matrix Gla-protein (MGP), has been
403
isolated from bovine bone matrix and extensively
characterized [58, 591. It associates strongly with
fractions containing bone morphogenetic protein
from where it was extracted initially as a by-product
and may be important in packaging this molecule
[58].Although it is extremely insoluble, it is a low
mol. wt. (9961) protein with five Gla residues per
molecule. This molecule accounts for the majority
of the Gla content of newborn rat bone, precedes
the accumulation of BGP, binds to the organic
matrix of bone and parallels the accumulation of
mineral. Its accumulation appears to be less
dependent on Gla residue content than BGP (OC),
however, as 27% normal levels MGP occur in warfarin-treated animals compared with 1-2% normal
levels of BGP (OC) [60]. The amino acid sequence
homology that exists between MGP and BGP (OC)
of various species indicates that these molecules
must have arisen by gene duplication at some time
before swordfish evolution diverged from the other
vertebrates studied [59].
The many observations on the chemical nature
and biological characteristics of MGP and BGP
(OC)indicate critical roles of these molecules in the
formation of calcified tissues. But the lack of major
changes in the skeleton of experimental animals
upon warfarin treatment is difficult to reconcile
with this suggestion. In the foetal wadarin syndrome bone defects with stippled epiphyses and
abnormal calcification occur [61, 621. Recently,
osteoporotic patients with spinal crush fractures or
femoral neck fractures have been found to have
significantly lower levels of circulating vitamin K
than those of age-matched control subjects [63].
These clinical features are consistent with the
important subtle participation of Gla-containing
proteins in bone metabolism and studies to determine the biological role of these special bony molecules will accelerate in the near future.
Proteoglycans
Proteoglycans are general components of connective tissues and are high molecular weight polymeric substances with carbohydrate prosthetic
groups named glycosaminoglycans (GAGS).
Herring [64] demonstrated that at least some of the
GAGS of bovine bone exist as proteoglycans and in
mammalian cortical bone Meyer et al. previously
found that chondroitin-4-sulphate was the only
significant GAG present [65]. Further work showed
that the major bone proteoglycans are much smaller
in size than the major species present in cartilage [9,
661. More recently, Fisher et al. [67] have isolated and characterized the proteoglycans of developing foetal bovine bone. In most species these
404
J. 7: Trifitt
appear to be of similar nature to those seen in
lamellar and woven bone and exist as two classes of
small proteoglycan [68]. The larger, PGI, has a
protein core of molecular weight 38000, with two
attached chondroitin sulphate chains of molecular
weight 40 000, similar to the small cartilage proteoglycan [69].The smaller, PGII, has a protein core of
similar mol. wt. (38000) but of different chemical
character with only one attached chondroitin sulphate chain of mol. wt. 40000. Similar characteristics have been noted by others with PGII-type
proteoglycans being more prevalent in adult bovine
bone [70-721. Antibodies to bone PGII cross-react
with proteoglycans from tendon, sclera, skin and
articular cartilage and the distribution of mRNA
coding PGII has been sekn also in these tissues and
in smooth muscle by hybridization of cDNA probes
[73]. The tissue specificity of the protein of PGII is
therefore not as great as was first envisaged [74]but
subtle differences in GAG content and protease
susceptibility may indicate functional differences
with tissue location (681. As in other tissues proteoglycans may be involved in interactions with
collagen fibrils to affect fibrillogenesis. This possible function in bone must precede mineralization
but proteoglycans have also been shown to have
inhibitory actions on calcification processes [75].
What particular function these molecules have in
bone is not known.
Phosphoproteins
Proteins containing phosphate groups covalently
bound to some of their constituent serine and
threonine residues were first described in chicken
and bovine bone a number of years ago [76]. Since
this time it has been obvious that the strong ionic
interactions of phosphate compounds with calcium ions are likely to have some influence on
mineralization. The potential mechanisms of these
interactions have been amply reviewed by Glimcher
[77, 781. Recently, the isolation and purification to
homogeneity of 15 phosphoprotein components
has been reported, with mol. wts. ranging from
5000 to 150000, and two components apparently
covalently bound to collagen [79].The best characterized phosphoprotein extracted from bone is
osteonectin [80-831. This is a 30000 mol. wt. glycoprotein that has affinity for both collagen and
hydroxyapatite mineral and in vitro causes mineralization of type I collagen [81, 841. It makes up to
2-4% of the total organic matrix and is a major
non-collagenous constituent of bovine and porcine
bone and dentine but is present in only low
quantities in rat mineralized tissues [85]. It is synthesized as a pre-osteonectin, which is processed to
osteonectin by removal of a signal peptide [86].
Human, bovine and porcine bone cells synthesize
osteonectin in tissue culture [87-891 and in vivo
osteonectin is located predominantly in bone and
dentine [81, 901. Even so, osteonectin is produced
by many fibroblasts such as those from skin, sclera,
tendon and periodontal ligament in culture [91, 921
and is found also in vivo in periodontal ligament
and reticular material within the endosteal spaces
[91]. It is possible that culture conditions mhy
stimulate cells to produce osteonectin, but alternatively rapid removal and clearance of osteonectin
synthesized in vivo could explain these findings. A
recent report shows that an identical protein is
found in human platelets [93]. In addition, osteonectin cDNA probes have been isolated and
hybridized to osteonectin message extracted from a
variety of bovine tissues [94].Osteonectin mRNAais
shown to be widespread particularly in tendon as
well as in bone tissue and there appears to be one
copy of the osteonectin gene in the bovine genome
1941.
In two bovine models of human osteogenesis
imperfecta, which is characterized as group of
diseases resulting from collagen gene mutations [4],
bone osteonectin levels have been found to be
either severely depressed [95, 961 or normal [97].
Similar ranges of content have been seen in human
patients [98].These observations indicate that basic
genetic defects may severely alter bone composition
but throw no further light on the function of osteonectin.
Plasma proteins
Albumin and a,HS-glycoprotein were the first
serum proteins to be found in abundance associated
with bone tissue [99]. The finding that a,-acid
glycoprotein and immunoglobulin E are also concentrated [ 1001has not been confirmed [ 1011. Since
even dense compact tissue contains variable
amounts of blood vessels, it is not surprising to find
plasma proteins in extractant solutions and indeed
by using two-dimensional electrophoresis Delmas et
al. [ 1021have shown a large number of plasma proteins present. Plasma proteins are present in extravascular sites of bone also, both in the tissue fluid
and complexed as significant components in the
calcified matrix [103].Albumin makes up about 3%
(w/w) of the non-collagenous matrix of bovine bone
(Table l), and probably accumulates by adsorption
to the mineral phase during the formation of calcified tissue I1041 and remains there until the bone is
resorbed. Plasma proteins generally operate as
osmotic pressure regulators and as transporters of
hormones, ions, metals and other metabolites to
cellular sites, but the function in bone or influence
on metabolic processes in this tissue is unknown.
Special proteins of bone
Plasma a,HS glycoprotein has a mol. wt. of
about 50000, is synthesized by the liver and subsequently accumulated in bone and dentine tissues
[105]. It is extracted in high yield from bone where
it is concentrated 30-300-fold relative to albumin,
depending on the species [l]. The selective concentration of plasma a,HS-glycoprotein in bone and
dentine tissues can be mimicked by precipitation of
calcium phosphate in vitro [99] and the affinity for
bone mineral can explain its concentration from the
tissue fluids. It constitutes one of the major noncollagenous components of the organic matrix
(Table l), and the changes in concentration have
been determined in various bone disorders. Evidence that bone may have an influence on plasma
levels is given by the fact that calculations show that
40% of the liver production goes to bone and is
incorporated in young growing animals [106]. In
Paget’s disease patients the plasma levels are substantially lower than the normal range and show
negative correlations with alkaline phosphatase
activity levels [99]. On treatment with calcitonin or
diphosphonate (EHDP) the plasma a,HSglycoprotein levels increased as alkaline phosphatase levels fell [107]. Ashton & Smith [lo71 could
find no difference in plasma levels with age or sex in
normal adults, but more recently Dickson et al.
[lo81 observed a decrease with age in women and
no age-relation in men, which have higher overall
levels. Variable serum levels were seen in osteogenesis imperfecta patients but urinary excretion of
plasma a,HS-glycoprotein was normal. Studies on
plasma levels and response on treatment of a
number of other bone diseases have proved unrewarding [109]. This is not surprising as plasma
changes are difficult to interpret. They are the end
result of net changes in catabolism and synthesis,
and whether bone tissue plays a significant role in
alterations in blood plasma a,HS-glycoprotein
levels awaits determinaton of the function of this
protein.
Apart from its possible effect on mineralization
processes, a,HS-glycoprotein may be implicated in
bone resorption. It has been reported that a,HSglycoprotein has opsonic properties [ 1101 and
Malone et al. [ l l l ] have shown that a,HSglycoprotein is chemotactic for monocytes to which
the protein binds with low affinity [112j. It enhances
macrophage phagocytic function [ 1131, promotes
the endocytosis of DNA [114] and binds via a
specific receptor to Epstein-Barr virus-transformed lymphocytes [115]. In fact the a,HSglycoprotein receptor may be identical with the
Epstein-Barr virus nuclear antigen [ 1161.The tenuous link between changes in Paget’s disease, which
is possibly the result of a slow virus infection of
bone particularly affecting the osteoclast [117], and
405
plasma a,HS-glycoprotein levels therefore warrants attention.
Conclusions
There are therefore about half a dozen major noncollagenous constituents of bone with an additional
number being detected by two-dimensional electrophoresis [ 1021. Application of the monoclonal
antibody technique to bone [ 1181 will be increasingly valuable in future work in identifying, characterizing and isolating these bone-related proteins.
The changes observed during development and in
disease states [68,119-1211 are likely to be investigated further in bone tissue and any marked plasma
and urine changes noted. It is likely that research on
this topic will accelerate as techniques of molecular
biology are applied to the field of non-collagenous
bone proteins.
Notwithstanding the tremendous amount of
knowledge known about these substances, we still
have no notion of their physiological functions.
Some possible functions of these molecules are
indicated in Table 2 and their value as phenotypic
markers of osteogenic differentiation and clinical
indices of bone function will continue to be
assessed. We may need to await the future possibilities of experimentally creating specific genetic
defects to alter synthesis and metabolism of particular proteins for clues to their actions. This daunting
possibility appears all the more real when it is
TABLE
2. Some possible finctions of major bone noncollagenous proteins
(A) Mineralization functions
( 1 ) Deposition of mineral phase
(a) nucleator
(b) inhibitor
(2) Mineral maturation and crystal growth
(a) promotor
(b) inhibitor
(3) Enzymatic activity
(4) Ion transport
(B) Resorption functions
(1) Chemotactic agents
(2) Recognition factors
(3) Regulators of cell activity
(C) Other functions
(1) Structural
(a) matrix structure and organization
(b) mineral structure and organization
(c) membrane proteins
(2) Morphogenetic controlling factors
(3) Enzymes and enzyme inhibitors
(D) Non-functional: inactive, bone mineral-adsorbed
components
J. T Trifitt
406
realized that there is no proof that any disease
affecting bone discovered to date has as its basis the
defective synthesis or metabolism of a major noncollagenous matrix-related protein. The complexity
of the initiation, development and maintenance of
bone tissue will require increasing attention of
researchers from many disciplines to expose the
secrets of the controls of these processes by bone
cells, and the special involvement of the noncollagenous proteins.
teins in developing molar tooth germs of the rat. Collagen and
Relafed Research, 5 , 17-22.
22. Thavarajah, M., Evans, D.B.. Russell, R.G.G. & Kanis, J.A.
( 1985) lmmunocytochemical demonstration of osteocalcin in
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24. Pan, L.C. & Price, P.A. (1985) The propeptide of rat bone y-
and mineralization, of bone matrix. Philosophical Transactions of
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25. Price, P.A. & Williamson. M.K. (1981) Effects of warfarin on
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