Collagen is a tough protein that makes up approximately 30% of the proteins within the body and found all over the body in bones, tendons, and ligaments.
It is the main component of connective tissue.
There are over 11 types of collagen characterized by having 19 unique chains. They are divided into three general classes:
- A molecule containing a length greater than 30 nm in the uninterrupted helical chain.
- 301 nm molecules in which the helical chain is interrupted.
- Relatively short molecules in which the helical region may be continuous and uninterrupted.
Types of Collagen
Class 1 (300 nm triple helix)
Type I – Skin, bone, ligament
Type II – Cartilage, disc, eye
Type III – Skin, blood vessels, ligament
Type V – With type I
Type XI – With type I
Class 2 (basement membranes)
Type IV – Basal lamina
Type VII – Epithelial basement membrane
Type VIII – Endothelial basement membrane
Class 3 (short-chain<300 nm molecules)
Type VI – Widespread
Type IX – Cartilage (with type II)
Type X – Hypertrophic cartilage
Type XII – Tendon
Type XIII – Endothelial cells
In group 1, which includes types I, II, III and V, type V was most commonly found in the musculoskeletal system, in arteries, in the cornea, in neuroretinal tissues, in the uterus, and in placental membranes. As well as different structural appearances, they have varying amounts of hydroxylysine, glycosylated hydroxylysine, hydroxyproline, etc.
Type I fibrils serve as a substrate for the deposition of the mineral component. Any alteration in this will produce a weakening of the skeleton. This is particularly seen in osteogenesis imperfecta.
Type I, II and III make up the bulk of the collagen in the body. Type II is restricted to hyaline cartilage, the intervertebral disc, and the vitreous humour of the eye.
Types V and XI are fibrillar collagens.
Type V is also distributed in small amounts (about 3 percent of type I) wherever type I collagen appears, and type XI is similarly distributed with type II collagen.
Hybrid molecules containing chains of both types V and XI collagens may also occur in bone and cartilage.
Type IV is the major structural component of basement membranes. Type X appears exclusively in the calcified cartilage zones of the epiphyseal plate, articular cartilage, and bone fracture callus. Type VI collagen appears in small amounts as filamentous material around cells and between the banded collagen fibrils of most soft connective tissues.
Type VI collagen is enriched in the certain tissue, including the intervertebral disc and the cornea. It appears to act as a structural element between the cells and the matrix of soft connective tissues which can deform.
Increased amounts of type VI collagen have been noted in inflamed skin, in the skin with certain forms of Ehlers-Danlos syndrome, and in the articular cartilage of patients with osteoarthrosis.
The mechanical strength of the common collagens arises from the formation of two or three covalent intermolecular bonds (cross-links) per collagen molecule.
Cross-links based on other pathways can be seen in some connective tissues, such as tendon and muscle. Similarly, ligaments that bear high loads, such as the anterior cruciate ligament of the knee, contain the highest levels of such cross-links.
Other tissues that dissipate or transmit high mechanical force, such as fibrocartilages (knee meniscus, intervertebral disc) are also rich in mature hydroxypyridium, a cross-linking amino acid.
The synovial membrane is rich in type II collagen.
Collagen is the major constituent of bone matrix. Collagen is also the major extracellular protein of the body and comprises some 30 percent today body protein.
A collagen molecule is composed of three, distinct, polypeptide chains wound around each other to form a triple helix.
This triple helical structure results in a long rigid molecule some 300 nm long and 1 nm wide. The triple helix of human collagen can be dissociated at temperatures above 40 degrees C.
One third of the total number of amino acid residues is glycine, and one-fifth proline and its derivative, hydroxyproline. Hydroxyproline is almost solely confined to collagen. In addition, collagen contains hydroxylysine and several oxidized derivatives of lysine (and hydroxylysiine).
The collagen molecule is first synthesized as a precursor, procollagen, which consists of three pro-alfa chains. During biosynthesis the long terminal regions are cleaved by specific proteases to form a collagen molecule consisting of the central (Gly-X-Y) region and flanked by two non-collagenous sequences, or telopeptides, of some 10-30 amino acid residues.
The assembly of the molecules appears to be determined chiefly by the distribution of charged amino acids along the sequence of the three alfa chains, but can be influenced by various substances, as for example, the proteoglycans with which collagen is associated in vivo. The fibrils show an asymmetric cross-striation pattern with a periodicity of 68 nm with each molecule being 4.4 times this repeat period.
The fibers themselves may be formed from bundles of microfibrils, each of which consists of five collagen molecules lying side by side but displaced by the repeat pattern and rolled together to form a long fibril, which in cross-section contains different parts of five collagen molecules (or four molecules and a gap) at any specific level. The physiological control of microfibril formation or of the lateral aggregation of these microfibrils remains unknown.
Ligaments are composed of a complex macro-molecular network with water making up about two thirds of the weight and the fibrillar protein collagen making up the majority of the remaining dry weight.
A normal ligament consists of about 90 percent fibrillar types I collagen with less than 10 percent being type III collagen. Other collagen types are present in smaller quantities.
Five types (II, VI, IX, X and XII) have been identified in epiphyseal cartilage, the most prevalent collagen being type II.
Although type X collagen is thought to be required in cartilage calcification, its relation to other matrix constituents remains unclear.
Articular cartilage contains at least five genetically distinct types of collagen, type II, VI, IX, X, and XI, which cross-link in a polymeric network to form a fibrillar framework of tissue. Type II collagen is a major component of this framework and represents more than 95 percent of all cartilage collagens.
Type X collagen is a minor fibril-forming collagen. It is only present in the hypertrophic zone of growth plate and basal calcified zone of articular cartilage. Type IX collagen is a minor fibril-nor-forming collagen, covalently linked to the surface of type II collagen fibrils.
Type VI collagen was found in the pericellular capsule and matrix around the chondrocytes. Electron microscopy also showed type VI collagen anchored to the chondrocyte membrane at the articular pole, suggesting a dual role of this collagen in the maintenance of chondrocyte integrity and as part of a cell-matrix signaling system.
In normal articular cartilage, matrix molecules are constantly synthesized and degraded by chondrocytes. The rates of synthesis and degradation for different types of molecules vary with aging and exercise.
Collagen in Bone
The skeleton, the vertebral column, and the pelvis are formed by endochondral ossification. Endochondral bone development begins as a condensation of mesenchymal cells derived from mesoderm, which form an extracellualr matrix. The mesenchymal cells surrounding the cartilage become the periosteum. These cartilage cells go through a maturation process that can be visualized in the area of the developing long bone called the growth plate.
Growth plates consist of zones of rapidly proliferating chondrocytes secreting collagens II, IX and XI, maturing and hypertrophic chondrocytes secreting predominantly collagen X. Collagen I is the major extracellular matrix (ECM) molecule of bone.
A summary of the many steps is given
A. Sequence of intracellular biosynthesis
1. Assembly pro-alfa chains (directed by specific mRNAs)
2. Proline hydroxylation
3. Lysine hydroxylation
4. Hydroxylysine glycosylation
5. Disulphide bond formation
6. Triple helix formation
B. Sequence of extracellular biosynthesis
1. Amino terminal extension cleavage
2. Carboxyl terminal extension cleavage
3. Microfibril formation
4. Lysine hydroxylysine terminal NH2 oxidation (Cu-containing lysyl oxidase)
5. Fibril formation
6. Reducible cross-link formation
7. Maturation of cross-links. Growth and reorganization of fibers
Specific enzymes are required for the hydroxylation of certain of the prolines and lysines present in the forming collagen peptide alfa chain. These ferrous iron-containing enzymes require molecular oxygen and alfa-ketoglutarate as additional substrates, and in vitro (and probably in vivo as well) vitamin C as a cofactor.
Hydroxyproline is thus not incorporated directly into the forming collagen amino acid sequence, and free hydroxyproline will not usually be incorporated into collagen; hydroxyproline is thus a specific marker for collagen. Measurement of hydroxyproline levels provides a marker for estimation of collagen or its breakdown products. It is the introduction of hydroxyproline which leads to an amino acid sequence that can form a triple helix at normal body temperature (37 degree C).
There is fine control of collagen biosynthesis at the hydroxylation stage, as the hydroxylase cannot hydroxylate proline residues in triple helical conformation while some hydroxyp-proline is required for the triple helix to form.
Galactose and glucose are then added to some of the hydroxylysine residues, so collagen is a glycoprotein. The formation –S-S- links between the three carboxyl regions of the pro-alfa chains of a collagen molecule probably occurs before the triple helix is formed and may indeed be essential for this process to occur rapidly and efficiently in vivo. The collagen molecule is then secreted, probably via the Golgi apparatus of the forming cell.
Extracellularly, several specific proteases cleave the procollagen molecules to collagen which, in contrast to procollagen, is virtually insoluble in physiological fluids. The levels of these proteases must, therefore, be important in the spatial control of collagen fibril and fiber formation. The collagen molecules are then chemically cross-linked.
The chemistry of cross-linking is not yet fully understood. It is based, however, on the reaction of the lysine and hydroxylysine residues of two collagen molecules lying side by side in a fibril. The terminal additional, amino group of one lysine residue is oxidized by a specific copper-containing amino acid oxidase to yield a reactive aldehyde grouping at the end of the lysine carbon chain.
This active group can then react with the amino group of neighboring unoxidized lysines, to form a chemical cross-link between two collagen molecules. This link is transformed during the maturation of collagen fibers to an unknown, or perhaps new peptide, bonds.
Recent evidence suggests that some cross-linking may even occur intracellularly and that collagen is secreted as packets of 10 or so procollagen molecules. In any event, the order of the various extracellular steps of collagen biosynthesis remains uncertain.
Various chemicals can prevent collagen cross-linking and thus lead to a weak connective tissue. These include the lathyrogens (beta-amino propionitrile), penicillamine and homocysteine accounting for the connecting tissue defects seen in some penicillamine treated patients or those with homocystinuria.
Non-Collagen Bone Proteins
Non-collagenous bone proteins are a heterogeneous group which varies from entrapped serum protein to glycoproteins, which are unique to the bone and which probably play a role in mineralization and are formed exogenously or locally.
Osteocalcin or bone Gla protein (BGP) is the best characterized of the non-collagen bone proteins and it makes up between 10 and 20 percent of them.
It is synthesized by the osteoblast as a 99 amino acid propeptide which is then cleaved to leave a 50 amino acid protein which is secreted.
Osteocalcin is produced only by osteoblasts and a proportion of the newly synthesized protein escapes into the serum. Raised serum levels of osteocalcin have been reported in diseases which are associated with increased bone turnover such as Paget’s disease, renal osteodystrophy, and primary hyperparathyroidism.
This has led to an interest in the measurement of osteocalcin as a biochemical marker of bone formation.
Matrix Gla Protein
As much as 50 percent of the total glutamic acid containing bone proteins are distinct from osteocalcin. The best characterized of these is matrix Gla protein (MGP), a 10,000 Da protein found in association with bone morphogenetic protein (BMP).
A number of phosphoproteins and glycoproteins are found in bone. The phosphate is bound to the protein backbone through serine or threonine amino acid residues. The best characterized of these bone protein is osteonectin. It binds collagen and hydroxyapatite through separate areas of its molecule, is found in relatively large amounts in immature bone and promotes mineralization of collagen. Thus it is possible that osteonectin plays a crucial role in mineralization.
Bone proteoglycan has a small protein core with up to two chondroitin sulphate chains attached. They constitute approximately 10 percent of the non-collagen bone proteins. Their role is unclear.
The sulphated glycosaminoglycans in connective tissue are chondroitin sulphate, keratan sulphate and dermatan sulphate. They are bound at one end to a protein core; e.g., 50-100 chondroitin sulphate chains are attached laterally by the saccharide sequence of neutral sugars to the core protein, giving a molecular weight of about 1-3 X 106. Proteoglycans are particularly vulnerable to proteolytic enzymes, with the whole molecule breaking up when a few peptide bonds are split off.
Some keratan sulphate chains are also Attached to the same protein core. It should be noted that in cartilage the proteoglycan population is heterogeneous, varying in chemical composition and size of their molecules.
Chondoitin sulphate consists of disaccharide units of glucuronic acid and N-acetylgalactosamines with sulfate residues. Keratan sulfate has in its structure galactose instead of glucuronic acid, with varying amounts of sulfate.
The elasticity and resilience of cartilage results from this matrix of proteoglycans, collagen, and water. They have demonstrated how the compressive stiffness of cartilage over short intervals directly correlates to the presence of proteoglycans when measured as glycosaminoglycans.
Because of this, as a load is applied to cartilage there is an increase in the fluid pressure and water is driven out, but the cartilage deforms only slowly.
The proteoglycans have a very significant role in controlling swelling, pressure and the movement of water molecules when cartilage is placed under load.
In cartilage, most proteoglycans are in the form of large aggregates that provide the tissue with its resilience under compressive loads. The basic structure of the cartilage proteoglycan aggregate has been well established by biochemical methods and molecular biological techniques.
It is constructed from glycosaminoglycan chains attached to core protein molecules which are themselves attached to hyaluronic acid under the stabilizing influence of link of protein. Although most recent studies of cartilage proteoglycan have dealt with articular cartilage, such data are not uniformly transposable to the growth plate.
The interaction of link protein with proteoglycan monomer and hyaluronic acid from bovine fetal epiphyseal cartilage was recently characterized. The proteoglycan monomers from this cartilage are almost aggregating monomers. As expected, link protein substantially increased the percentage of aggregating monomers.
Proteoglycan aggregate stability was found to be highly pH-dependent: decreasing the pH from 5 to 4 in the absence of link protein resulted in essentially complete aggregate dissociation. Link protein was protective against much of the pH-induced instability. Optimization of both pH and link protein increased not only the stability of the aggregate but also its size.
In addition to their contribution to the matrix structure, proteoglycans in the growth plate may play a role in mineralization. Focal concentrations of proteoglycan at the sites of mineralization are well documented.
Because the chondroitin sulfate chains of proteoglycans bind calcium and because phosphate can displace this calcium from the proteoglycans, proteoglycan may serve as the medium within which calcium release by ion exchange could raise the Ca X PO¬4 product above the threshold for hydroxyapatite precipitation.
These are glycoproteins containing the sugar N-acetylneuraminic acid (sialic acid). The make up approximately 7.5 percent of the total non-collagen bone proteins and their function is unclear.
These constitute the largest number of non-collagen bone proteins. They include serum albumin and some immunoglobulins. They constitute approximately a quarter of the total non-collagenous protein and their function is unknown.