collagen

Tropocollagen triple helix.

Collagen is the main protein of connective tissue in animals and the most abundant protein in mammals,^[1] making up about 25% to 35% of the whole-body protein content. It is naturally found exclusively in metazoa, including sponges. ^[2] In muscle tissue it serves as a major component of endomysium. Collagen constitutes 1% to 2% of muscle tissue, and accounts for 6% of the weight of strong, tendinous muscles.^[3] The gelatin used in food and industry is derived from the partial hydrolysis of collagen.

Uses

Collagen is one of the long, fibrous structural proteins whose functions are quite different from those of globular proteins such as enzymes. Tough bundles of collagen called collagen fibers are a major component of the extracellular matrix that supports most tissues and gives cells structure from the outside, but collagen is also found inside certain cells. Collagen has great tensile strength, and is the main component of fascia, cartilage, ligaments, tendons, bone and skin. Along with soft keratin, it is responsible for skin strength and elasticity, and its degradation leads to wrinkles that accompany aging. It strengthens blood vessels and plays a role in tissue development. It is present in the cornea and lens of the eye in crystalline form. It is also used in cosmetic surgery and burns surgery. Hydrolyzed collagen can play an important role in weight management, as a protein, it can be advantageously used for its satiating power.^{[citation needed]}

Industrial uses

If collagen is sufficiently denatured, e.g. by heating, the three tropocollagen strands separate partially or completely into globular domains, containing a different secondary structure to the normal collagen polyproline II (PPII), e.g. random coils. This process describes the formation of gelatin, which is used in many foods, including flavored gelatin desserts. Besides food, gelatin has been used in pharmaceutical, cosmetic, and photography industries.^[4] From a nutritional point of view, collagen and gelatin are a poor-quality sole source of protein since they do not contain all the essential amino acids in the proportions that the human body requires—they are not 'complete proteins' (as defined by food science, not that they are partially structured). Manufacturers of collagen-based dietary supplements claim that their products can improve skin and fingernail quality as well as joint health. However, mainstream scientific research has not shown strong evidence to support these claims. Individuals with problems in these areas are more likely to be suffering from some other underlying condition (such as normal aging, dry skin, arthritis etc.) rather than just a protein deficiency.

From the Greek for glue, kolla, the word collagen means "glue producer" and refers to the early process of boiling the skin and sinews of horses and other animals to obtain glue. Collagen adhesive was used by Egyptians about 4,000 years ago, and Native Americans used it in bows about 1,500 years ago. The oldest glue in the world, carbon-dated as more than 8,000 years old, was found to be collagen—used as a protective lining on rope baskets and embroidered fabrics, and to hold utensils together; also in crisscross decorations on human skulls.^[5] Collagen normally converts to gelatin, but survived due to the dry conditions. Animal glues are thermoplastic, softening again upon reheating, and so they are still used in making musical instruments such as fine violins and guitars, which may have to be reopened for repairs—an application incompatible with tough, synthetic plastic adhesives, which are permanent. Animal sinews and skins, including leather, have been used to make useful articles for millennia.

Gelatin-resorcinol-formaldehyde glue (and with formaldehyde replaced by less-toxic pentanedial and ethanedial) has been used to repair experimental incisions in rabbit lungs.^[6]

Medical uses

The cardiac valve rings, the central body and the cardiac skeleton of the heart summarily represent a unique and moving collagen anchor to the fluid mechanics of the heart. Individual valvular leaflets are arguably held in shape by collagen under great extremes of pressure. Calcium deposition within collagen occurs as a natural consequence of aging. These fixed points in an otherwise static display of blood and muscle enable current cardiac imaging technology to arrive at ratios essentially stating blood in Cardiac Input and blood out Cardiac Output. Specified imaging such as Calcium Scoring illustrates the utility of this methodology, especially in an aging patient subject to pathology of the collagen underpinning.

Collagen has been widely used in cosmetic surgery, as a healing aid for burn patients for reconstruction of bone and a wide variety of dental, orthopedic and surgical purposes. Some points of interest are:

1. when used cosmetically, there is a chance of allergic reactions causing prolonged redness; however, this can be virtually eliminated by simple and inconspicuous patch testing prior to cosmetic use, and
2. most medical collagen is derived from young beef cattle (bovine) from certified BSE (Bovine spongiform encephalopathy) free animals. Most manufacturers use donor animals from either "closed herds", or from countries which have never had a reported case of BSE such as Australia, Brazil and New Zealand.
3. porcine (pig) tissue is also widely used for producing collagen sheet for a variety of surgical purposes.
4. alternatives using the patient's own fat, hyaluronic acid or polyacrylamide gel are readily available.

Collagens are widely employed in the construction of artificial skin substitutes used in the management of severe burns. These collagens may be derived from bovine, equine or porcine, and even human, sources and are sometimes used in combination with silicones, glycosaminoglycans, fibroblasts, growth factors and other substances.

Collagen is also sold commercially as a joint mobility supplement^[7]. Because proteins are broken down into amino acids before absorption, there is no reason for orally ingested collagen to affect connective tissue in the body, except through the effect of individual amino acid supplementation.

Recently an alternative to animal-derived collagen has become available. Although expensive, this human collagen, derived from donor cadavers, placentas and aborted fetuses, may minimize the possibility of immune reactions.

Although it cannot be absorbed through the skin, collagen is now being used as a main ingredient for some cosmetic makeup.^[8]

Conformation and structure

Collagen structure is complex. Its conformation can be considered at the monomeric level (individual collagen molecules) and/or at its aggregate level (how the trimers are arranged), i.e. their packing structure (fibrils, networks, etc.—see below)^[9].

History and background

The molecular and packing structures of collagen have eluded scientists for decades; the first evidence that it possess a regular structure at the molecular level was presented in the mid-1930s ^{[10] [11]}. Since that time many prominent scholars, including (but not limited to) Nobel laureate Crick, and Pauling, Rich, Yonath, Brodsky, Berman and Ramachandran concentrated on the conformation of the collagen monomer. Several competing models although correctly dealing with the conformation of each individual peptide chain, gave way to the triple-helical "Madras" model which provided an essentially correct model of the molecule's quaternary structure ^[12][13][14] although this model still required some refinement ^{[15][16][17][18]}. The packing structure of collagen has not been defined to the same degree outside of the fibrillar collagen types, although it has been long known to be hexagonal or quasi-hexagonal ^[19][20][21]. As with its monomeric structure, several conflicting models alleged that either the packing arrangement of collagen molecules is ‘sheet-like’ or microfibrillar.^[22][23] Recently it was confirmed that the microfibrillar structure as described by Fraser, Miller, Wess (amongst others) was closest to the observed structure, although it over-simplified the topological progression of neighboring collagen molecules and hence did not predict the correct conformation of the discontinuous D-periodic pentameric arrangement termed simply: the microfibril^[24]

Molecular structure

The tropocollagen or "collagen molecule" is a subunit of larger collagen aggregates such as fibrils. It is approximately 300 nm long and 1.5 nm in diameter, made up of three polypeptide strands (called alpha chains), each possessing the conformation of a left-handed helix (its name is not to be confused with the commonly occurring alpha helix, a right-handed structure). These three left-handed helices are twisted together into a right-handed coiled coil, a triple helix or "super helix", a cooperative quaternary structure stabilized by numerous hydrogen bonds. With type I collagen and possibly all fibrillar collagens if not all collagens, each triple-helix associates into a right-handed super-super-coil that is referred to as the collagen microfibril. Each microfibril is interdigitated with its neighboring microfibrils to a degree that might suggest that they are individually unstable although within collagen fibrils they are so well ordered as to be crystalline.

A distinctive feature of collagen is the regular arrangement of amino acids in each of the three chains of these collagen subunits. The sequence often follows the pattern Gly-Pro-Y or Gly-X-Hyp, where X and Y may be any of various other amino acid residues. Proline or hydroxyproline constitute about 1/6 of the total sequence. With Glycine accounting for the 1/3 of the sequence, this means that approximately half of the collagen sequence is not glycine, proline or hydroxyproline, a fact often missed due to the distraction of the unusual GXY character of collagen alpha-peptides. This kind of regular repetition and high glycine content is found in only a few other fibrous proteins, such as silk fibroin. 75-80% of silk is (approximately) -Gly-Ala-Gly-Ala- with 10% serine—and elastin is rich in glycine, proline, and alanine (Ala), whose side group is a small, inert methyl group. Such high glycine and regular repetitions are never found in globular proteins save for very short sections of their sequence. Chemically-reactive side groups are not needed in structural proteins as they are in enzymes and transport proteins, however collagen is not quite just a structural protein. Due to its key role in the determination of cell phenotype, cell adhesion, tissue regulation and infrastructure, many sections of its non-proline rich regions have cell or matrix association / regulation roles. The relatively high content of Proline and Hydroxyproline rings, with their geometrically constrained carboxyl and (secondary) amino groups, along with the rich abundance of glycine, accounts for the tendency of the individual polypeptide strands to form left-handed helices spontaneously, without any intrachain hydrogen bonding.

Because glycine is the smallest amino acid with no side-chain, it plays a unique role in fibrous structural proteins. In collagen, Gly is required at every third position because the assembly of the triple helix puts this residue at the interior (axis) of the helix, where there is no space for a larger side group than glycine’s single hydrogen atom. For the same reason, the rings of the Pro and Hyp must point outward. These two amino acids help stabilize the triple helix—Hyp even more so than Pro—a lower concentration of them is required in animals such as fish, whose body temperatures are lower than most warm-blooded animals.

Fibrillar structure

1 The tropocollagen subunits spontaneously self-assemble, with regularly staggered ends, into even larger arrays in the extracellular spaces of tissues^[25][26]. In the fibrillar collagens, the molecules are staggered from each other by about 67nm (a unit that is referred to as ‘D’ and changes depending upon the hydration state of the aggregate). Each D-period contains 4 and a fraction collagen molecules. This is because 300 nm divided by 67 nm does not give an integer (the length of the collagen molecule divided by the stagger distance D). Therefore in each D-period repeat of the microfibril, there is a part containing five molecules in cross-section—called the “overlap” and a part containing only 4 molecules, called the "gap".^[24] The triple-helices are also arranged in a hexagonal or quasi-hexagonal array in cross-section, in both the gap and overlap regions.^[24][27].

There is some covalent crosslinking within the triple helices, and a variable amount of covalent crosslinking between tropocollagen helices forming well organized aggregates (such as fibrils)^[28]. Larger fibrillar bundles are formed with the aid of several different classes of proteins (including different collagen types), glycoproteins and proteoglycans to form the different types of mature tissues from alternate combinations of the same key players^[29]. Collagen's insolubility was a barrier to the study of monomeric collagen until it was found that tropocollagen from young animals can be extracted because it is not yet fully crosslinked. However, advances in microscopy techniques (Electron Microscopy (EM) and Atomic Force Microscopy (AFM)) and X-ray diffraction have enabled researchers to obtain increasingly detailed images of collagen structure in situ. These later advances are particularly important to better understanding the way in which collagen structure affects cell-cell and cell-matrix communication and how tissues are constructed in growth and repair, and changed in development and disease^[30][31].

Collagen fibrils are semi-crystalline aggregates of collagen molecules. Collagen fibers are bundles of fibrils.

Collagen fibrils/aggregates are arranged in different combinations and concentrations in various tissues to provide varying tissue properties. In bone, entire collagen triple helices lie in a parallel, staggered array. 40 nm gaps between the ends of the tropocollagen subunits (approximately equal to the gap region) probably serve as nucleation sites for the deposition of long, hard, fine crystals of the mineral component, which is (approximately) hydroxyapatite, Ca₁₀(PO₄)₆(OH)₂ with some phosphate. It is in this way that certain kinds of cartilage turn into bone. Type I collagen gives bone its tensile strength.

Types and associated disorders

Collagen occurs in many places throughout the body. The 29 types of collagen have thus far been identified and described in literature. Over 90% of the collagen in the body, however, are of type I, II, III, and IV.

• Collagen One: skin, tendon, vascular, ligature, organs, bone (main component of bone)
• Collagen Two: cartilage (main component of cartilage)
• Collagen Three: reticulate (main component of reticular fibers), commonly found alongside type I.
• Collagen Four: forms bases of cell basement membrane
• Collagen Five: Cells surfaces, hair and placenta

Collagen diseases commonly arise from genetic defects that affect the biosynthesis, assembly, postranslational modification, secretion, or other processes in the normal production of collagen.

In addition to the above mentioned disorders, excessive deposition of collagen occurs in Scleroderma.

Staining

In histology, collagen is brightly eosinophilic (pink) in standard H&E slides. The dye methyl violet may be used to stain the collagen in tissue samples.

The dye methyl blue can also be used to stain collagen and immunohistochemical stains are available if required.

The best stain for use in differentiating collagen from other fibers is Masson's trichrome stain.

Synthesis

Action of lysyl oxidase (in French)

Amino acids

Collagen has an unusual amino acid composition and sequence:

• Glycine (Gly) is found at almost every third residue
• Proline (Pro) makes up about 9% of collagen
• Collagen contains two uncommon derivative amino acids not directly inserted during translation. These amino acids are found at specific locations relative to glycine and are modified post-translationally by different enzymes, both of which require vitamin C as a cofactor.
  • Hydroxyproline (Hyp), derived from proline.
  • Hydroxylysine, derived from lysine. Depending on the type of collagen, varying numbers of hydroxylysines have disaccharides attached to them.

Cortisol stimulates degradation of amino acid from skin collagen.^[33]

Collagen I formation

Most collagen forms in a similar manner, but the following process is typical for type I:

1. Inside the cell
   1. Three peptide chains are formed (2 alpha-1 and 1 alpha-2 chain) in ribosomes along the Rough Endoplasmic Reticulum (RER). These peptide chains (known as preprocollagen) have registration peptides on each end; and a signal peptide is also attached to each
   2. Peptide chains are sent into the lumen of the RER
   3. Signal Peptides are cleaved inside the RER and the chains are now known as procollagen
   4. Hydroxylation of lysine and proline amino acids occurs inside the lumen. This process is dependent on Ascorbic Acid (Vitamin C) as a cofactor
   5. Glycosylation of specific hydroxylated amino acid occurs
   6. Triple helical structure is formed inside the RER
   7. Procollagen is shipped to the golgi apparatus, where it is packaged and secreted by exocytosis
2. Outside the cell
   1. Registration peptides are cleaved and tropocollagen is formed by procollagen peptidase.
   2. Multiple tropocollagen molecules form collagen fibrils, and multiple collagen fibrils form into collagen fibers
   3. Collagen is attached to cell membranes via several types of protein, including fibronectin and integrin.

Synthetic pathogenesis

Vitamin C deficiency causes scurvy, a serious and painful disease in which defective collagen prevents the formation of strong connective tissue. Gums deteriorate and bleed, with loss of teeth; skin discolors, and wounds do not heal. Prior to the eighteenth century, this condition was notorious among long duration military, particularly naval, expeditions during which participants were deprived of foods containing Vitamin C. In the human body, a malfunction of the immune system, called an autoimmune disease, results in an immune response in which healthy collagen fibers are systematically destroyed with inflammation of surrounding tissues. The resulting disease processes are called Lupus erythematosus, and rheumatoid arthritis, or collagen tissue disorders.^[34]

Many bacteria and viruses have virulence factors which destroy collagen or interfere with its production.

Fossil record

Because the synthesis of collagen requires a high level of atmospheric oxygen, complex animals may not have been able to evolve until the atmosphere was oxygenic enough for collagen synthesis.^[35] The origin of collagen may have allowed cuticle, shell and muscle formation. However, the preservation of collagen in the fossil record is very scarce.^[36] There is mounting evidence—which remains controversial—that collagen has been preserved in dinosaur specimens dated as long ago as 80 million years ago.^[37]

Also worth of noting are the actinofibrils, collagen fibers present on the wings of pterosaurs.

Art

Julian Voss-Andreae's sculpture Unravelling Collagen (2005), stainless steel, height 11'3" (3.40 m).

Julian Voss-Andreae has created sculptures based on the collagen structure out of bamboo and stainless steel. His piece "Unravelling Collagen" is, according to the artist, a "metaphor for aging and growth"^[38][39].

See also

• Animal glue
• Collagenase, the enzyme involved in collagen breakdown and remodelling. For more on other proteases that target collagen see The Proteolysis Map
• Ehlers-Danlos Syndrome
• Fibrous protein
• Gelatine
• Hypermobility Syndrome
• LOXL1
• LOXL2
• LOXL3
• LOXL4
• Marfan Syndrome
• Osteoid

References

External links

Wikimedia Commons has media related to: Collagen

• The Collagen Protein
• 12 types of collagen
• Database of type I and type III collagen mutations
• Science.dirbix Collagen
• Hydrolyzed Collagen
• Collagen Stability Calculator
• Computer-generated animations of the assembly of Type I and Type IV Collagens
• Integrin-Collagen interface, PMAP (The Proteolysis Map)—animation
• Integrin-Collagen binding model, PMAP (The Proteolysis Map)—animation
• Collagen-Integrin atomic detail, PMAP (The Proteolysis Map)—animation