Download Ageing and the aggregating proteoglycans of

337
Clinical Science ( 1 986) 7 1 , 337-344
EDITORIAL RE VIEW
Ageing and the aggregating proteoglycans of human articular
cartilage
P. J. ROUGHLEY
AND
J.
s. MORT
Joint Diseases Laboratory, Shriners Hospital for Crippled Children, Montreal, Quebec, Canada
Introduction
Proteoglycans form an integral component of all
body tissues, though they are most abundant in the
connective tissues, where they play an important
functional role. Proteoglycan structure is not constant, but varies with both the tissue of origin and
the age of the individual. Irrespective of this variation, all proteoglycans share the common feature of
a central core protein to which one or more glycosaminoglycan chains are covalently attached. The
glycosaminoglycan may be chondroitin sulphate,
dermatan sulphate, keratan sulphate or heparan sulphate, depending on the tissue in question. In the
fibrous connective tissues, such as skin, ligament
and tendon, small dermatan sulphate proteoglycans
predominate [ 1,2] and they appear to play an essential role in collagen fibril organization [3]. In
contrast, hyaline cartilage [4-71 and the nucleus pulposus [8, 91 of the intervertebral disc contain large
proteoglycans, bearing both chondroitin sulphate
and keratan sulphate chains, which play an important role in the ability of these tissues to resist compressive loads [lo, 111. Keratan sulphate is also
present in the proteoglycans of the cornea [ 121, and
is essential for maintaining the regular array of collagen fibrils necessary for light transmission [13,
141. Heparan sulphate proteoglycans appear to be
present on all cell surfaces [ 151 and are an integral
component of basement membranes [16, 171. All
these proteoglycans have unique structures that are
intimately associated with their functional role. This
is particularly true of the cartilage proteoglycans,
which are the most complex in structure and have
the most abundant tissue content, sometimes
accounting for as much as 10% of the tissue dry
weight. This review will focus on the structure of the
Correspondence: Dr P. J. Roughley, Joint Diseases
Laboratory, Shriners Hospital for Crippled Children,
1529 Cedar Avenue, Montreal, Quebec, Canada H3G
1A6.
proteoglycans characteristic of cartilage, how their
structure changes with age, the role of proteolytic
enzymes in mediating such changes, and the effect
the changes may have on tissue function.
The structure of the cartilage proteoglycans
Hyaline cartilages appear to contain two types of
proteoglycans: large aggregating proteoglycans,
which have the ability to interact with hyaluronic
acid, and smaller non-aggregating proteoglycans
[ 18-20]. The aggregating proteoglycans are the
most abundant in tissue content, and, as their name
implies, they do not exist in isolation, but rather as
large multimolecular aggregates composed of a central filament of hyaluronic acid to which numerous
proteoglycan subunits are non-covalently attached
[21-231 with each interaction being stabilized by a
link protein [24-261. The proteoglycan subunits
consist of a large core protein bearing many chondroitin sulphate and keratan sulphate chains (Fig.
1).One terminus of the core protein (the hyaluronic
acid-binding region) is devoid of glycosaminoglycan
chains and possesses the specific globular conformation essential for the interaction with hyaluronic
acid [27, 281. The glycosaminoglycan chains are
then dispersed in apparently random groups
throughout the remainder of the core protein [29],
though in many cases keratan sulphate is predominant adjacent to the hyaluronic acid-binding region
[30]. The core protein also contains smaller oligosaccharide substituents, which may be as numerous
as the glycosaminoglycan chains [31,32]. The oligosaccharides are of two types: the most numerous are
0-linked and resemble the linkage region between
keratan sulphate and protein, the remainder are N linked. While the former may be located throughout
the core protein, the latter are situated mainly in the
hyaluronic acid-binding regions. The core protein
may also be substituted with phosphate groups in
the form of phosphoserine [33], and in some
proteoglycans the xylose residue in the linkage
P. J. Roughley and J. S.Mort
338
f-
NEONATE
Chondroitin sulphate (CS)
Glycosaminoglycan
attachment region
--i
ADULT
Hyaluronic acid
binding region
2
NEONATE
<
...........
3*
......
.
I
.
.
.
.
+
"*.-,
,DULT
FIG.1. Changes in the structure of cartilage proteoglycan subunits with age. Proteoglycan subunits are
represented as a central protein core bearing chondroitin sulphate (CS) and keratan sulphate (KS)
chains. With age the subunit size decreases, the
number of CS chains and their size decrease, the
number of KS chains and their size increase, and
the core protein sue decreases and distinct forms
are present (each possessing globular disulphidebridged hyaluronic acid-binding regions). No
attempt is made to represent the precise number or
distribution of CS and KS chains.
region between chondroitin sulphate and protein
may also bear a phosphate group [34]. The functional significance of both the oligosaccharides and
the phosphate is at present unclear. The structural
features described above appear common to the
proteoglycan subunits from the cartilage of all
species and from all anatomical sites studied,
though the precise arrangement of glycosaminoglycan chains varies greatly between the species and
with age in a given species.
B
C
D
B C D
B C
D
FIG.2. Changes in the structure of cartilage link
proteins with age. Link proteins are represented as
a protein backbone with various degrees of glycosylation (>) near the N-terminus and extensive
intramolecular disulphide bridging. In both the
neonate and adult three link proteins are present,
the larger differing in their degree and/or type of
glycosylation, and the smallest being derived from
larger by proteolytic cleavage near the N-terminus
(site A). Further cleavage in the adult also occurs in
all three forms of link protein at identical sites (B, C
and D), though the molecules are held together by
disulphide bridging. The relative abundance of the
various components at different ages changes and is
represented diagrammatically by variation in line
thickness.
While the proteoglycan subunits are of large size,
with molecular weights greater than lo6, the link
proteins are comparatively small with molecular
weights of 40000-50000 [35-381, a size comparable with that of the hyaluronic acid-binding region
of the proteoglycan subunits. Link proteins are
glycoproteins, and exist in a number of isoforms
(Fig. 2) that are separable based on their size and
charge properties [39]. Those from human cartilage
Ageing and cartilage proteoglycans
appear to be the most heterogeneous, being separable into three components by size and at least nine
by charge [38]. The two larger components
probably bear the same protein core and differ in
their degree and/or type of oligosaccharide substitution [40, 411. Much of the oligosaccharide
appears to be at the N-terminus of the protein, and
the smallest of the link proteins appears to be derived from either of the larger by proteolytic
cleavage within this region [42]. Irrespective of the
heterogeneity, all the link proteins are able to stabilize the proteoglycan aggregate towards dissociation [43,44], though it is probable that the different
forms confer differing degrees of stability [45]. It
appears likely that the actual hyduronic acidbinding site on both the link protein and the proteoglycan subunits may share a conserved amino acid
sequence [46], though in most respects the molecules are structurally distinct. In the proteoglycan
aggregate a single link protein stabilizes the interaction between each proteoglycan subunit and the
hyaluronic acid [47,48]. The number of proteoglycan subunits able to interact with a single hyaluronic acid chain is dependent on the length of the
chain and the steric' hindrance between adjacent
subunits [22, 491. The actual binding site for each
subunit comprises a length of only ten monosaccharide units [49], though the spacing between subunits
is often 50 monosaccharides in length.
Age-related changes in proteoglycan structure
The structure of the proteoglycan subunits is not
constant with age (Fig. 1).In the human [50-551,
such age changes are characterized by (a) a decrease in the size and number of chondroitin sulphate chains, together with an increase in
6-sulphation relative to 4-sulphation, (b) an
increase in the size and number of keratan sulphate
chains, and a concomitant decrease in the number
of 0-linked oligosaccharide chains per core protein, and (c) a change in the amino acid composition
of the core protein, though a functional hyaluronic
acid-binding region appears to be a common structural feature at all ages. At present there are no data
available on age-changes relating to the N-linked
oligosaccharide distribution or phosphorylation. As
a consequence of these changes the proteoglycan
subunits in the adult are smaller in size and less
glycosylated, with most of the changes arising in the
glycosaminoglycan attachment region of the core
protein. Similar changes are seen in the proteoglycan subunits from other species [56-601, though the
degree to which the changes progress does vary
between the species. There are three possible
origins for such changes: (a) a variation in core protein gene expression, (b) a variation in the activity of
339
the post-translational enzymes responsible for
glycosylation and sulphation, and (c) proteolytic
modification of the intact proteoglycan after its
secretion into the extracellular matrix. It is likely
that all the above mechanisms are acting during the
normal ageing process, though the evidence for
multiple genes coding for the aggregating proteoglycans is still indirect.
The change in glycosaminoglycan chain length
and the position of sulphation are the best examples
of variation due to the alteration of post-translational enzyme systems, and they undoubtedly contribute to much of the change in glycosaminoglycan
structure and distribution associated with ageing.
This situation is compounded, however, by the
probable expression of multiple genes during
ageing, with each distinct core protein possessing a
separate glycosylation pattern. Evidence for such
genetic heterogeneity is provided by the separation
of chemically and immunologically distinct populations by chromatographic, centrifugal and electrophoretic techniques [61-651. All of the above
changes that are associated with the synthetic
capacity of the chondrocytes are essentially completed by the end of growth [53,66], and are therefore more characteristic of juvenile development
than maturation of the adult.
There are, however, changes that take place
throughout ageing and these are characterized by
degradative changes due to proteolysis. Such
changes undoubtedly contribute to the variation in
proteoglycan subunit size, due to the action of proteinases on the glycosaminoglycan attachment
region of the core protein [5, 6, 671. Proteolysis
would be expected to produce isolated hyaluronic
acid-binding regions as the limit product, which
would presumably remain bound to hyaluronic
acid, and indeed adult human cartilage is characterized by the accumulation of such moieties
[68-701. Further evidence for proteolysis in situ is
seen in the increased abundance of the smallest link
protein with age [71] (Fig. 2). The link protein also
shows a second type of proteolytic cleavage during
ageing [71]. This occurs in the central region of the
molecule at a series of identical sites in all the link
proteins [41]. Such fragmented link proteins are,
however, held in a pseudo-native conformation by
the presence of disulphide bonds, and appear to be
functional in the proteoglycan aggregate. Our preliminary observations indicate that while the synthetic changes occur to similar extents in different
anatomical sites and may thus represent a largely
pre-programmed response of the chondrocyte, the
degradative changes occur to different extents in
different sites. Such changes may reflect the different environments to which individual joints are subjected and the consequences of such differences on
340
P. J. Koughley and J. S.Mort
the activity of the proteolytic enzymes. In this
respect the cartilage of the hip appears to exhibit a
greater degree of degradative change than that of
the knee. In species other than the human, the
degradative changes do not appear to be as pronounced, though this may relate to the longer
human life span, and therefore a greater propensity
to accumulate the products of the degradative process.
Proteoglycan-degrading enzymes produced by
chondrocytes
The most obvious origin of the degradative changes
characteristic of ageing is via the proteolytic enzymes
produced by the chondrocytes themselves. There
are two potential sources for such proteinases: (a)
through the release of lysosomal enzymes, such as
the aspartic proteinase cathepsin D and the cysteine
proteinases cathepsins B, H and L, or (b) through
the activation of the secreted latent metalloproteinases characteristic of connective tissue cells.
Even though lysosomal proteinases have been
localized in the extracellular cartilage matrix
[72-741, it is unlikely that they would have any prolonged activity at extracellular pH levels. In
contrast, the metalloproteinases are ideally suited to
act in the extracellular environment. The chondrocytes of adult human articular cartilage produce at
least two latent metalloproteinases [75-771: one
specific for collagen and the other of general
proteolytic activity capable of degrading proteoglycans. Similar enzymes are produced by a variety of
connective tissue cells from many species [78]. Such
proteoglycan-degrading enzymes have broad pH
profiles, being optimally active around neutrality,
and require Ca2+for activity. They are produced in
a latent form as proenzymes [79, 801 and, in vitro,
can be activated by treatment with proteinases or
organomercurial compounds [811.Under tissue culture conditions normal adult human articular cartilage does not produce such enzymes in large
amounts, though production can be enhanced by
external stimuli [77], such as exposure to the monocyte-derived factor interleukin 1 [82]. While the
enzyme secreted into the tissue culture medium is in
a latent form, the level of this enzyme correlates
well with the release of proteoglycan products [83].
It is therefore not unreasonable to assume that a
proportion of the enzyme is activated in situ,
though not released, and that the action of this
enzyme accounts for the proteoglycan release. Such
an enzyme would appear to be an ideal candidate
for producing the age-related degradative changes
in proteoglycan structure.
When activated, the human cartilage enzyme is
able to degrade both the proteoglycan subunits and
the link proteins in vitro [83a]. The limit digestion
products for the proteoglycan subunits are of a
similar size to those produced by pepsin, with an
estimated ten glycosaminoglycan chains per core
protein fragment [84]. It is interesting to note that
the degradation products produced in organ culture
range in size from almost intact molecules to the
limit product for this enzyme. Few of the glycosaminoglycan-containing products are capable of
interacting with hyaluronic acid, and as the fragments of largest size predominate, it is likely that
the core protein adjacent to the hyaluronic acidbinding region is a preferred site for cleavage. A
similar mechanism has been suggested for the
degradation of bovine nasal cartilage proteoglycan
induced by catabolin [85] (a synovial factor analogous to interleukin 1 [86]). When proteoglycan
aggregates are- subjected to degradation by the
human metalloproteinase in vitro, one observes the
production of hyaluronic acid bearing isolated
hyaluronic acid-binding regions and the smallest
link protein. Such an action is not unique to this
enzyme, but is common to many proteinases [48].
Again, the situation is mirrored in organ culture,
where on exposure of the cartilage to interleukin 1,
one observes an increase in the abundance of the
smallest intact link protein at the expense of the
larger two.
It would therefore appear that the activated form
of the latent proteoglycan-degrading metalloproteinase secreted by the chondrocytes is capable
of producing many of the degradative changes associated with the age changes in proteoglycan structure. For example, it can account for the decrease in
proteoglycan subunit size, the accumulation of isolated hyaluronic acid-binding regions, and the
increased abundance of the smallest link protein.
Perhaps surprisingly, it does not appear to produce
the internal fragmentation of the link protein. It is
possible that this latter type of cleavage may not be
due to proteinase action, but may represent the
result of reaction with free radicals, as has been
postulated for many other age-related changes [87].
Role of proteoglycans in cartilage function
In order to discuss what effect, if any, the age
changes in proteoglycan structure have on articular
cartilage function, it is first necessary to present a
model of how the proteoglycans function in the
extracellular matrix. As a result of their high anionic
charge, proteoglycans have a high affinity for water,
which results in their swelling and occupying large
molecular domains in solution. In the tissue, however, such swelling is impeded by the collagenous
network within the extracellular matrix, and, provid-
341
Ageing and cartilage proteoglycans
ing a sufficientcontent of proteoglycans is present,
an equilibrium is established whereby the swelling
of the proteoglycans is balanced by tensile forces
developed in the collagen network. When the cartilage is subjected to compression, water is extruded
and the proteoglycan concentration at the site of
compression is increased, so increasing its swelling
pressure. On removal of the load this increased
swelling pressure is dissipated by water being
imbibed to restore the equilibrium conditions. In
such a way the cartilage can function as a type of
reversible ‘damper’,which can soften the impact of
the load as it is applied. Thus, for normal cartilage
function, by this model, it is important that (a)the
proteoglycan content of the tissue be high, (b) the
charge density of the proteoglycans be high, and (c)
the proteoglycans be unable to diffuse freely from
the site of compression.
The latter parameter is met by the process of
aggregation, and the apparent interaction of the
hyaluronic acid filaments of the aggregate with the
collagen fibrils [88]. It would appear that during
ageing there is no impairment in the ability of the
proteoglycan subunits to form such aggregates [53],
though whether the generation of fragmented link
proteins [71] with age affects the stability of the
aggregates is at present unknown. However, it is
clear that ageing does result in a lower content of
glycosaminoglycan-rich proteoglycan subunits,
largely due to proteolytic degradation, and if of sufficient magnitude, this may be envisaged as being
detrimental to the tissue, as it would lower the fixed
charge density. Such proteolytic degradation results
in (a) the generation of proteoglycan fragments
bearing glycosaminoglycan chains that are presumably free to diffuse from the cartilage, and (b)
hyaluronic acid filaments bearing the hyaluronic
acid-binding terminus of the proteoglycan subunits
and modified link proteins (Fig. 3). As there are no
well described enzymic systems operating in the
extracellular matrix for degrading the hyaluronic
acid filaments, the latter products might be
expected to be retained in the tissue. This would be
consistent with the accumulation of hyaluronic
acid-binding regions with age [70], and the reported
increase in the hyaluronic acid content of the tissue
[52].Such an accumulation might be envisaged as
occupying space that is therefore no longer available for the localization of new proteoglycan, even
though the chondrocytes may possess the synthetic
capacity for repair.
Thus, while the age-related changes in proteoglycan structure due to synthesis may have little effect
on function, those due to degradation are potentially damaging. Those individuals in whom the
degradative process is most pronounced would be
considered most susceptible to deterioration of tis-
NEONATE
ADULT
Hyaluronic acid
0
Link protein
3
Fragmented link protein
3F
Proteoglycan subunits
FIG.3. Changes in the organization of the cartilage
extracellular matrix with age. Proteoglycan is represented in an aggregated form, comprising central
hyaluronic acid filaments bearing proteoglycan subunits in association with a link protein. In the neonate proteoglycan subunits are large and the link
proteins are intact, whereas in the adult the proteoglycan subunits are smaller and many link proteins
are fragmented. The decrease in proteoglycan size
due to proteolytic cleavage in the glycosaminoglycan attachment region yields molecules ranging in
size from intact subunits to that of just the hyaluronic acid-binding region. The adult also possesses
an increased hyaluronic acid concentration, and
aggregates are represented in closer proximity.
sue function. Prolonged exposure to agents such as
interleukin 1 would be expected to augment the’
age-related changes, and it can be envisaged that a
stage may be reached when loss of function leads to
degeneration of the tissue. In this way, events
characteristic of the normal ageing process may
represent one mechanism through which an osteoarthritic lesion may develop.
342
l? J. Roughley and J. S. Mort
Acknowledgments
we thank the S h h e r s ofNorth America, the Me&
cal Research Council of Canada and the Arthritis
Society of Canada for financial support.
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