Extracellular Matrix -- Neame College of Medicine
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Extracellular Matrix
The
extracellular matrix (ECM) is (obviously!) the material
found around cells. Until recently, this was often
referred to by enigmatic terms such as "ground
substance". However, the ECM has recently received
considerable attention due to its importance in
cell-cell signaling, wound repair, cell adhesion and
tissue function.
The three most obvious forms of ECM are bone,
cartilage, and basement membrane. All
three utilize collagen as a structural scaffold. Bone
utilizes type I collagen (also the major collagen in
skin), cartilage utilizes type II, while basement
membrane utilizes type IV. Collagens I and II form long
fibrillar arrays, while type IV forms a mesh-type
structure, reminiscent of a chain-link fence.
Apart from collagens,
proteoglycans are also major component of the ECM.
In cartilage, aggrecan
is a space-filling proteoglycan, maintaining the tissue
in a hydrated and load-resistant form. In many ECMs,
the small, leucine-rich proteoglycans ("SLRPs") decorin
and fibromodulin, are thought to be part of the
mechanism for controlling the diameter of the fibrillar
collagens. Heparan sulfate proteoglycans are considered
to be a major element in the ability of the basement
membrane in the glomerulus of the kidney to filter
solutes in blood.
The ECM interacts with the surface of the cell. Some of
the more striking interactions exist with the large
glycoprotein, fibronectin. New mechanisms of cell
adhesion are found with considerable frequency, but all
seem to involve cell-surface receptors for molecules
that are found in the space surrounding the cell (pericellular
matrix) which, in turn, interact with molecules in the
territorial matrix. Thus the matrix can exert a
physical force on the cell and supply feedback which is
undoubtedly of importance in controlling tissues shape.
The role of cartilage in the adult is primarily to absorb loads at articulating joints. However, there are cartilages that provide a support role in the nose, ears and in the trachea. All of these cartilages have similar biochemical characteristics and consist primarily of large, hydrated proteoglycan aggregates trapped within a mesh of collagen fibrils. A number of other components aid in maintenance and organization of the matrix, including the leucine-rich proteoglycans (decorin (DS-PGII), biglycan (DS-PGI), fibromodulin , lumican , etc.), cartilage matrix protein (CMP), cartilage oligomeric protein (COMP). The extracellular matrix is not homogeneous, but has differing ratios of its components in differing regions. The elements of the matrix can thus customize it for specific function. For example, the cartilage at the surface of a joint needs to be more resistant to shear forces than cartilage adjacent to bone. Nasal cartilage does not have to resist deforming loads as much as articular cartilage.
The cells in cartilage, chondrocytes, are rounded. Towards the surface of the cartilage they tend to be separate from each other, while adjacent to bone (hypertrophic chondrocytes), they are in columns. While chondrocytes do not adhere to a basement membrane (see Extracellular Matrix), perlecan, a basement membrane component is found around chondrocytes and may play a role in chondrocyte attachment to the matrix.
During development, the majority of the calcified skeleton is preceded by a cartilaginous precursor. This is slowly replaced by bone. During growth, the bones grow in length at the ends and only grow in width in the center. An important feature of correct bone growth is the compressive force placed on the tissue during movement and as result of muscle contraction. It is considered that this plays an important role in correct growth of the cartilaginous precursor of bones and of the continuous remodeling of the adult skeleton. Growth factors (for example bone morphogenetic proteins, or BMPs) are also thought to be important.
Fetal cartilage contains many of the same components as adult cartilage. However, there are a variety of low-molecular proteins which are especially prominent in fetal cartilage and either not as abundant, or virtually absent in adult cartilage. Two of these proteins, chondromodulin (or SCGP) and pleiotrophin , are found only in fetal and newborn cartilage, while a third, PARP , is the N-pro peptide of type XI alpha2 collagen and may be a marker for active collagen type II biosynthesis.
Growth plate cartilage represents another specialist structure. The growth plate, as the name implies, is where the increase in length of bones occurs. There is no growth plate in a human adult limb, and so no further growth in length can occur. There are a number of specialist molecules found in the growth plate, notably collagen type X, chondroadherin, and a member of the C-type lectin family, CLECSF1. How cartilage remodels itself and removes all the cartilage-specific macromolecules and replaces them with bone-specific molecules in a regulated fashion is currently not understood. However, current understanding suggest that mechanical loading is important in repairing damaged cartilage. A report on some research in this area can be found at Stanford University.
Aggrecan
Aggrecan is
the shortened name of the large aggregating chondroitin
sulphate proteoglycan. Aggrecan, which is one of the
most widely studied proteoglycans, is abundant; it
represents up to 10% of the dry weight of cartilage (articular
cartilage is up to 75% water).
There can be some confusion about the use of the term
aggrecan, and to what it refers. Many individual
monomers of aggrecan bind to hyaluronic acid to form an
aggregate, it is the monomer which is termed aggrecan.
These aggregates are comprised of up to 100 monomers
attached to a single chain of hyaluronic acid (HA).
Structure
An aggrecan monomer consists of a protein backbone of 210-250 kDa to which is attached both chondroitin sulphate and keratan sulphate chains. The chains are attached to the central portion of the core protein, chondroitin sulphate chains (100 - 150 per monomer), being located in the C terminal 90%, while the keratan sulphate (30 - 60 per monomer) is preferentially located towards the N terminus.
Aggrecan monomer
Individual aggrecan monomers, up to 100, interact with hyaluronic acid to form an aggregate of very high molecular weight. This interaction involves a globular domain at the N-terminus, termed G1 or the hyaluronic acid binding region (HABR). The interaction is stabilised by a short protein called link protein which interacts with both the HA and G1.
Aggregate
There is considerable sequence homology between link protein and G1, and with G2, a further globular domain close to G1 on the aggrecan core protein, termed G2. Although it is not involved in the binding with either link protein or hyaluronic acid, it shares considerable sequence homology with G1. A function for G2 has not been identified.
A third globular domain, G3, is located at the C-terminus, and has three structural domains: an EGF repeat, a lectin-like sequence, and a region homologous to the complement regulatory protein (CRP) motif. The lectin-like domain appears to be present in all forms of the molecule, while the EGF and CRP like domains are only present in an alternatively spliced variant.
The roles of the various components of G3 have not been determined. The lectin-like sequence of can bind to fucose and galactose.
While aggrecan is
found in cartilage, there are considerable similarities
emerging between it and several other large
proteoglycans. There are homologies with versican and
the hyaluronate receptor CD44.
Versican has a C-terminal lectin like domain and
EGF-like repeats along with a central GAG binding
domain, which unlike aggrecan is glycosylated
exclusively with chondroitin sulphate. There is also
some evidence that Link protein may interact with
Versican
Function
The primary role of aggrecan appears to be a physical one, as it brings about an osmotic swelling and maintains the high levels of hydration in the cartilage extracellular matrix. In this way aggrecan plays a crucial role in the normal function of articular cartilage, which is found at the ends of long bones.
The extracellular matrix of articular cartilage is comprised of fibril forming collagens, aggrecan and many other important molecules. The fibrillar collagens form a network which has a very high tensile strength, and which entraps the aggrecan molecules. The presence on aggrecan of a very large numbers of chondroitin sulphate chains generates an osmotic swelling pressure. It is this which results in the wet weight of articular cartilage being 75% water.
During resting, such as sitting down and reading, the osmotic swelling is at a maximum, and is contained only by the collagen network. However, during loading, such when you stand up or walk, the weight of your body is supported by the cartilagenous ends of the long bones. In this state your weight compresses the cartilage, literally squeezing water out. This continues until the osmotic swelling generates a force equal to the compressive force generated by your weight which it supports. When the load is removed, you sit down again, the compressive force is removed, and the cartilage swells to it's full extent.
This osmotic swelling is brought about by the glycosaminoglycan chains attached to the aggrecan core. As you may imagine anything which compromises the ability if the glycosaminoglycan chains to exert their osmotic swelling effect will have a severe effect upon the functionality of the cartilage.
ISOLATION OF AGGRECAN
Aggrecan can be extracted from diced cartilage into 4M guanidine hydrochloride plus protease inhibitors (50mM sodium acetate, 100mM 6-aminocaproic acid, 10mM EDTA, 5mM benzamidine hydrochloride, pH 6.8). The extract is taken to associative conditions by dialysis against protease inhibitors, and solid CsCl added to achieve a density of 1.6g/ml.
Following density gradient centrifugation at 100,000g for 48 hours the gradient is fractionated. The material with the highest density is termed A1 and it is this which contains the aggrecan. The A1 is almost pure aggregate.
Further purification to generated aggrecan monomers, link protein and hyaluronic acid may be performed by density gradient centrifugation under dissociative conditions.
GENES
Both human (GenBank M55172) and rat (GenBank J03485) cDNAs have been sequenced in full. Partial sequences have been deposited for human (GenBank J05062), bovine (GenBank Y00319, J05028), and chicken. The sequence of the alternatively spliced EGF domain is also available.
Aggrecan Biblography
A biblography of general aggrecan references along with some key primary references.
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Back to the Proteoglycan contents page.
Back to the Proteoglycan / glycosaminoglycan home page.
Last updated Sept 27 1995
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