Collagen

Triple Helix

The structure of collagen eluded scientists for decades. Many prominent scholars, including Nobel laureates like Watson and Crick and Linus Pauling were known to have been working on collagen structure when it was finally discovered. The triple helical structure that is known to be correct in the essentials was proposed by G. N. Ramachandran and Gopinath Kartha in the year 1954. This proposed structure came to be known as the Madras helix.

collagen, fibers, skin, anti aging,

The tropocollagen or "collagen molecule" subunit is a rod about 300 nm long and 1.5 nm in diameter, made up of three polypeptide strands, each of which is a left-handed helix, not to be confused with the commonly occurring alpha helix, which is right-handed. 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. Tropocollagen subunits, produced by the fibroblast, spontaneously self-assemble, with regularly staggered ends, into even larger arrays in the extracellular spaces of tissues. There is some covalent crosslinking within the triple helices, and a variable amount of covalent crosslinking between tropocollagen helices, to form the different types of collagen found in different mature tissues — similar to the situation found with the α-keratins in hair. Collagen's insolubility was a barrier to study until it was found that tropocollagen from young animals can be extracted because it is not yet fully crosslinked.
Collagen fibrils are collagen molecules packed into an organized overlapping bundle. Collagen fibers are bundles of fibrils.

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. Gly-Pro-Hyp occurs frequently. 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. Chemically-reactive side groups are not needed in structural proteins as they are in enzymes and transport proteins. The high content of Proline and Hydroxyproline rings, with their geometrically constrained carboxyl and (secondary) amino groups, 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, 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 thermally stabilize the triple helix — Hyp even more so than Pro — and less of them is required in animals such as fish, whose body temperatures are low.


In bone, entire collagen triple helices lie in a parallel, staggered array. 40 nm gaps between the ends of the tropocollagen subunits 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)2with 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. There are 28 types of collagen described in literature. Over 90% of the collagen in the body, however, are of type I, II, III, and IV.
Collagen One - bone (main component of bone)
Collagen Two - cartilage (main component of cartilage)
Collagen Three - reticulate (main component of reticular fibers)
Collagen Four - floor - forms the basement membrane

Effects of ascorbic acid on collagen matrix formation and osteoblast differentiation in murine MC3T3-E1 cells.

J Bone Miner Res. 1994 Jun;9(6):843-54. Franceschi RT, Iyer BS, Cui Y.
Department of Periodontics, Prevention, and Geriatrics, University of Michigan School of Dentistry and Biological Chemistry, University of Michigan School of Medicine, Ann Arbor.

Treatment of mouse MC3T3-E1 cells with ascorbic acid initiates the formation of a collagenous extracellular matrix and synthesis of several osteoblast-related proteins. We recently showed that ascorbic acid dramatically increases alkaline phosphatase and osteocalcin mRNAs and that this induction is blocked by inhibitors of collagen triple-helix formation (Franceschi and Iyer, J Bone Miner Res 7:235).

In the present study, the relationship between collagen matrix formation and osteoblast-specific gene expression is explored in greater detail. Kinetic studies revealed that ascorbic acid increased proline hydroxylation in the intracellular procollagen pool within 1 h and stimulated the cleavage of type I collagen propeptides beginning at 2.5 h. Mature alpha 1(I) and alpha 2(I) collagen components were first detected at 10 h and continued to increase in both cell layer and culture medium for up to 72 h. Ascorbic acid also increased the rate of procollagen secretion from cell layers to culture medium. The secretion of another matrix protein, fibronectin, was only slightly affected. Alkaline phosphatase or its mRNA was first detected 2-3 days after ascorbic acid addition, but osteocalcin mRNA was not seen until day 6. Two inhibitors of collagen triple-helix formation, ethyl-3,4-dihydroxybenzoate and 3,4-dehydroproline, inhibited procollagen hydroxylation and alkaline phosphatase induction. 3,4-Dehydroproline also inhibited the induction of alkaline phosphatase and osteocalcin mRNAs.

Surprisingly, induction was not blocked if cells were exposed to ascorbic acid before inhibitor addition. Alkaline phosphatase was also partially inhibited if cells were grown in the presence of purified bacterial collagenase. These results indicate that the induction of osteoblast markers by ascorbic acid does not require the continuous hydroxylation and processing of procollagens and suggest that a stable, possibly matrix-associated signal is generated at early times after ascorbic acid addition that allows subsequent induction of osteoblast-related genes.

Benefits of concurrent delivery of hyaluronan and IGF-1 cues to regeneration of crosslinked elastin matrices by adult rat vascular cells.

Kothapalli CR, Ramamurthi A. J Tissue Eng Regen Med. 2008 Mar-Apr;2(2-3):106-16.

Clemson University, Medical University of South Carolina Bioengineering Program, Charleston, SC 29425, USA.

vein, atherosclerosis, smooth muscle cells

Elastin, a major component of vascular matrices, critically determines vascular mechanics and maintains the quiescence of smooth muscle cells (SMCs). Attempts to regenerate elastin in elastin-compromised blood vessels using tissue-engineering approaches is limited by the unavailability of elastogenic cues to upregulate poor elastin output and matrix assembly by adult vascular cells. We previously showed that hyaluronan (HA) elastogenically stimulates aortic SMCs, although these effects are highly specific to HA fragment size.

The elastogenic response of SMCs can also be modulated with growth factors such as insulin-like growth factor (IGF-1). Here, we evaluate the benefits of concurrent delivery of HA fragments (0.76-2000 kDa) and IGF-1 (500 ng/ml) to elastin synthesis, organization and crosslinking. The study outcomes show that, relative to supplement-free cultures, IGF-1 and long-chain HA/large HA fragments, but not HA oligomers, together induce multifold increases in the synthesis of elastin precursors, structural elastin matrix yields and crosslink densities within cell layers, and encourage elastic fibre formation.

These outcomes are not all obtained when either of the cues is provided separately. IGF-1 and large HA fragments (>20 kDa) also together inhibit cell proliferation, a concern in elastin-compromised vessels, where SMC hyperproliferation is common. The results will benefit efforts to provide exogenous or scaffold-based elastogenic cues (IGF-1 + HMW HA/large HA fragments) to enable robust and faithful regeneration of elastin matrix structures in vivo or in vitro. The present outcomes may be used to restore elastin matrix homeostasis in de-elasticized vessels and tissue-engineered constructs that may be grafted as a substitute.