COLLAGEN: A NOT SO SIMPLE PROTEIN

Journal of the Society of Leather Technologists and Chemists, Vol. 82,p. 104.

COLLAGEN: A NOT SO SIMPLE PROTEIN

A. J. BAILEY AND R. G PAUL

Collagen Research Group, University of Bristol, Langford, Bristol, BSI8 7D Y Summary

Collagen is the major protein of animal bodies from simple sponges toHomo sapiensand exists in various forms from skin, tendon and bone to cornea and basement membrane of the capillaries. This biological variation can now be accounted for on the basis of a whole family of genetically distinct collagens. Over the past two decades 19 different collagens have been identified, although the major types are the fibrous types I, II and III and the non-fibrous type IV of basement membrane. They all possess the basic triple helix based on multiple repeats of the simple tri-peptide Gly—X—Y, but this varies in length and forms different supramolecular aggregates to achieve optimum function for particular tissues. The major function of collagen is to provide shape and mechanical strength and the latter is achieved by intermolecular cross-linking of the collagen molecules in the supramolecular aggregate. The monomeric molectiles in the aggregates are stabilised by two different pathways. Initially cross-linking occurs through an enzymic mechanism involving oxidation of specific lysine and hydroxylysine residues providing divalent cross-linking which subsequently matures to multivalent cross-links. As the rate of turnover decreases a non enzymic pathway takes over, which is mediated through the adventitious accretion of glucose. Collagen therefore, unlike other proteins shows considerable changes with age which in turn affect its physical properties. These changes must be taken into account when preparing collagen based products.

All the amino acid side chains project radially from the rod-like triple helix and the quarter-staggered array of the molecules allows highly specific intermolecular cross-linking either naturally, or artificially with bifunctional reagents. Reactions with basic or acid groups can therefore be carefully controlled and

in some cases their location predicted. Synthetic cross-links bind the molecules closer together and increase

intermolecular interactions, thus increasing the shrinkage temperature and resistance to enzymic degradation.

The turnover of collagen is generally slow but in fact can vary from 2/3 days for periodontal ligament to several years for skin and tendon. Mature collagen fibres are highly resistant to enzymes and degradation

is achieved by specific collagenase that can cleave the triple helix at one particular point. The shorter

helical fragments can then unravel and denature to gelatin when other metalloproteinases (MMPs) degrade

it to amino acids. A family of 14 metalloproteinases have been identified along with some specific tissue

inhibitors (TIMPS).

The sharp denaturation temperature of collagen attests to the almost crystalline character of the triple helix and the variation in shrinkage temperature between species is primarily due to the number of hydroxyproline based water hydrogen bridges. The presence of a hydroxyproline deficient thermally labile domain near the carboxy terminus of the molecule initiates the melting process allowing the triple helix to unzip along its length.

Recent studies have demonstrated that collagen is not an inert structural material but interacts with other molecules to control the development of collagenous tissues. Despite the ancient lineage of this ubiquitous protein, collagen is still revealing exciting new scientific features.

Introduction

The biological diversityofcollagenous structures and their varied function is unequalled by any other protein. The variation from bone to skin, through tendon, cornea, capillaries and basement membrane can now be explained to a large extent by the existence of a whole

family of collagens, (for reviews see Comper 1996;

Kielty et al., 1993). Despite this variety the basic structure of the collagen molecule is very simple. The primary sequence is basically a tripeptide repeat,

(Gly-X—Y)ioo_400

where X is often proline and Y sometimes hydroxypro line. Glycine therefore occupies every third residue in a chain which is twisted into a polyproline helix by the pyrrole residues, rather than the more common ct-helix of the globular proteins. The small side-chain of glycine, a simple hydrogen atom, allows these polyproline helices to pack together in close contact and form a

super-Presented at the IULTCS Centenary Conference, London, 11

September 1997 (Heidemann Symposium Fundamental Aspects

of Collagen).

coiled triple helix which is stabilised by intra-helical H-bonds between glycines in adjacent chains (Fig. 1). The molecule is therefore a long rigid rod, which in the fibrous collagen types is 1.5nm by 300nm, which is, equivalent to a hose-pipe 1 inch in diameter and 17 feet long. The larger polar and non-polar amino acid side-chains face radially outwards from the cylindrical helix providing a charge profile along the molecule. It is this

charge profile that spontaneously drives the

self-assembly of the collagen molecules into the quarter-staggered alignment (Hulmes et al., 1973) and results in the classical banded appearance of the collagen fibre. It is the ability of nature to modify this simple structure that is the key to its ubiquitous presence and

Figure 1. Structure and organisation of the collagen molecule-Diagrammatic representation of the molecule showing hydrogen bonds within the triple helix.

over the past decade several new collagens have been identified (Fig. 2). At the present time 19 genetically distinct collagens have been reported. Many of these new collagens do not form banded fibres despite the presence of the typical triple helical structure and assume a variety of different supramolecular aggregates (Fig. 3). This is achieved by minor alterations in the properties of the collagen molecule which include changes in: 1,11, III, V, XI IV VI VII Ix VIII, X

Figure 2. Diagrammatic representation of the domain structures of collagen molecules types LIX.

The triple-helical domains are represented by solid blocks and non-helical regions as lines.

IV

Type II collagen fibril

VIII (X?)

VI

Figure 3. Supramolecular collagen assemblies of collagen types IV, VII, IX, VI and VIII.

(i) the length of the triple helix, (ii) the charge profile along the helix,

(iii) interruptions in the integrity of the triple helix; (iv) the size and shape of the terminal globular

domains;

(v) the cleavage or retention of the latter in the

supramolecular aggregate; and

(vi) variation in the post-translational modifications. The identification of these aggregates and their func tional determination has at last allowed the collagen chemist to begin to account for the biological diversity of collagenous tissues.

Classification of these extremely variable structures is consequently difficult. Some classifications have been based on the genetic structure, others on the molecular weight, or length of the triple helix, others on the fact

that the triple helix is interrupted by non-helical

domains. As more collagens are being characterised the most useful approach is probably to categorise on the basis of the aggregated structure. This allows classifi

cation of the 19 types identified to date into 4

main groups:

(i) Fibrous collagens, e.g. types I, II, III, and (V and XI)

(ii) Non-fibrous collagens, e.g. types IV, VIII and X (iii) Filamentous collagens, e.g. types VI and VII (iv) Fibril associated collagens, e.g. types IX, XII,

XIV and XV. (I) Fibre forming collagens

The fibre forming collagens include types I, II, III, V and XI. Each possesses a 300 nm triple helix from which the N- and C-globular domains have been partially removed prior to aggregation of the molecules

in the quarter-stagger alignment. Cleavage of the

propeptide regions results in the formation of a

tropocollagen molecule comprised of a triple helix with small globular terminal regions (telopeptides). The tropocollagen molecules then assemble into fibres which act as the main framework of the mammalian body. Type I is the major collagen of the skin and tendon, and also the hard calcified tissues such as bone and dentine. Type III collagen forms widely distributed, fine fibres present in high proportions in tissues such as foetal skin and the vascular system where a more flexible framework is required. Collagen type II is the

predominant component of cartilage and vitreous

humour and is expressed transiently in other tissues during embryogenesis. Types V and XI are almost identical in nature and exist as minor components of nearly all tissues composed of fibrillar collagen. Type V

is co-expressed with types I whilst type XI is co

expressed with type II. (ii) Non-fibrous collagens

The most abundant non-fibrous collagen, Type IV, possesses a long triple helix (400 nm) which lacks the charge profile required for the formation of quarter-staggered fibres, and in contrast to the fibrous collagens retains its terminal globular domains in the aggregated structure. The molecules interact in an anti-parallel fashion through their N-terminal regions with a small overlap of 80 nm to form tetramers. The C-terminal globular domains at the end of these extended arms of the tetramer then interact to form a network often

described as a “chicken-wire” structure (Timpi et a!., 1981), Fig. 3. The latter is a simplistic structure and is believed to be more complex by further alignment of this structure to provide some mechanical strength (Yurchenco and Orear, 1994). This network forms the framework of basement membranes, for example the kidney glomeruli, dermal-epidermal junction and capil laries. The open structure is ideal for the incorporation of other component molecules, e.g. laminin, heparan sulphate, entactin and osteonectin.

Type VIII collagen is thought to be one of the major constituents of Descernet’s membrane of the eye, a unique hexagonal network synthesised by corneal endo thelial cells (Labermeier and Kenney, 1983).

Type X is synthesised specifically by hypertrophic chondrocytes in the growth plate of growing long bones possibly forming similar network structures to type VIII

(Kwan et a!., 1991). However, this observation is based

upon in vitro studies and remains to be confirmed in vivo. (iii) Filamentous collagens

Type VI collagen has been shown to form loosely packed filamentous structures which have a repeating

pattern of about 100 nm. The molecules possess short

triple helices of about 100 nm with larger globular regions at both the N- and C-termini. The globular region of one of the three ce-chains has a molecular weight about double that of the other two c,.-chains. Aggregation is complex, initially involving the extensive overlapping of two molecules in an anti-parallel manner, then two of these dimers aggregating to form a stable tetrarner. The tetramers subsequently aggregate longi tudinally through their terminal globular domains to form long filamentous structures (Bruns et a!., 1986), Fig. 3. Type VI fibres are widely distributed as a minor component of many different collagenous tissues, for example, cartilage, skin, tendon and bone, but the function of these fibres has not been elucidated. It has been suggested that they may play a role in the spacing and alignment of other fibres in tissues, for example, the highly organized type I fibres in the cornea.

Type VII collagen forms fibrils made up of two molecules overlapped in an anti-parallel fashion (Fig. 3). Several of these dimers can then combine laterally to form larger fibres visible in the electron microscope. These fibrils attach some basement membranes, e.g. in the placenta and dermal-epidermal junction to small plaques of type IV collagen in the matrix so acting as anchoring fibrils (Keene et al., 1987; Sakai et al., 1986). (iv) Fibril associated collagens

These collagens are found in association with the quarter-staggered fibres of other collagen types and includes the FACIT collagens, i.e. Fibril Associated Collagens with Interrupted Triple Helices. For example, type IX collagen has been shown to be attached to the surface of type II fibres (Vaughan et a!., 1988), Fig. 3, and type XII which has been reported to be attached to type I fibres in some tissues. The function of these collagens is not clear. They are possibly involved in the regulation of fibre diameter or they may be involved in the interaction of fibres with other components of the extra-cellular matrix. Iii this context it is interesting to note that type IX could be considered as a proteoglycan since the ri-2 (LX) chain acts as a core protein for a chondroitin side-chain. Two further collagens, types

XIV and XV have been identified and tentatively assigned to this group of collagens.

Until recently all of the different types of collagen described above were thought to be reasonably tissue specific. However, more detailed analyses has shown there to be considerable overlap, particularly in respect

of the association with minor collagens.

Metabolism of Collagen Synthesis

Collagen biosynthesis follows the same pattern of protein synthesis to that of other proteins on free ribosomes but differs in the extensive post-translational modification of nascent os-chains by enzymatic reactions. At least twelve different enzymes are involved in these modifications and their reactions have been extensively reviewed (Kivirikko and Myllyla, 1984). Initial associ ation of the three c-chairis is through the disuiphide bonded C-terminal, following which spontaneous propa gation of triple helix occurs. The triple helical molecule

is then secreted into the extracellular space through the

Golgi apparatus where it is believed to be packaged into secretory vesicles. Further post-translational modi

fications then take place extracellularly. Separate

enzymes remove the N and the C-propeptides from the fibrous collagens. The removal of these pro-peptides does not seem to be obligatory, since partial processing

of fibrous collagen occurs in which the N pro-peptide

is retained, and in the case of type IV collagen both the

pro-peptides are retained. Fibrillogenesis

Processed collagen monomers then undergo a self-assembly into the various supramolecular aggregates. The in ‘ivo self-assembly process is a complex process and thought to be governed by systematic cleavage of pro-peptides, the different proportions of collagen types and by the presence of other extracellular matrix components. Consequently, the route by which fibril logenesis occurs is unclear, although, a number of models based upon in vitro observations have been proposed (Veis and George, 1994). In the case of fibrous collagens the monomers aggregate into quarter-staggered ordered fibrils ranging from 50 to 1000 nm in diameter with an axial periodicity of 67 nm readily discernible in the electron microscope. It has been suggested that fibre forming molecules may be packaged

in vesicles within the cell prior to secretion via

invaginations in the cell wall (Treistad and Hayashi, 1979).

Degradation

The tight triple helical structure of the collagen molecule makes it very resistant to enzymes. However, specific enzymes are available for the necessary turnover and remodelling of the collagen fibres. Collagenases are capable of cleaving the collagen molecule across the three chains of the helix at a specific point 3/4 of the distance from the N-terminus (Fig. 4). Similar cleavages have been shown to take place with type II and type III collagens. The cleaved fragments possess a lower thermal denaturation temperature and the denatured fragments are then readily cleaved by other proteinases, primarily gelatinases. An additional mode of attack on

Collagenase

3/4

1/4

Cathepsins

Denaturation

3/4

_1/4

Amino Acids

I

Denaturation

Figure4. Enzymatic degradation of collagen molecules by collagenase and cathepsins.

Collagenase cleaves the molecule at a specific Site 3/4 along the triple helix. Cathepsins cleave the molecule at the telopeptide releasing the molecule from the intact fibre. Cleaved fragments then denature and are digested further by other proteinases, primarily the gelatinases.

the intact fibre is via the acid cathepsins, which cleave the collagen at the telopeptide thus releasing the molecule from the fibre. This single molecule denatures and is then digested by the gelatinases (Fig. 4). The regulation of these enzymes is achieved at the gene level but also by specific tissue inhibitors TIMPS (Murphy and Reynolds, 1993; Werb et al., 1989).

Denaturation of Collagen

The highly crystalline structure of collagen results in a specific melting point, the molecule melting at 39°C and the aggregated fibre at 67°C. We have recently shown that denaturation is governed by an irreversible rate process rather than the equilibrium process as previously thought (Miles ci a!., 1995). The stability of the molecule depends upon a high content of hydroxy proline residues which forms stabilising H-bonds via water molecules with the peptide backbone and interes tingly we have identified a thermally labile domain which is deficient in hydroxyproline residues. This domain is located in the gap region of the fibre near the C-terminus making the gap zone less stable than the rest of the fibre. Consequently, it may be more susceptible to attack by gelatinases following collagenase and cathepsin activity due to its increased ability to

initiate unwinding of the helix, the collagenase cleavage site being at the N-terminus and the cross-link site at the C-terminus of the domain. The cross-link site located at the end of this domain would also account for the increase in thermal stability with increasing cross-linking with age (see below).

Strength of Collagen Fibres

The strength of the collagen fibres and therefore of the dermis, is determined by cross-links between the molecules making up the fibrils and between the fibrils making up the fibres. The chemistry of these cross-links has now been elucidated and provides an explanation of the high mechanical strength of the collagen fibre. These cross-links are covalent bonds formed between the individual molecules wihin the fibre at highly specific points governed by the precise alignment of the fibre. In the absence of these cross-links the fibre has no mechanical strength whatsoever, demonstrating that they are crucial for the optimal functioning of collagen fibres as a framework structure in the body (Bailey et a!., 1974).

Collagen cross-links are formed as a result of the oxidation of specific lysyl and hydroxylysyl residues in the telopeptide regions by the enzyme lysyl oxidase.

The binding site for the enzyme is the sequence Hyl-Gly--His—Arg within the helix, which because of the end-overlap arrangement of the molecules in the fibres, is opposite the single lysine or hydroxylysine in the telopeptide. The resulting oxidative deamination of the lysine forms an aldehyde which spontaneously reacts with a hydroxylysine residue to form a divalent, aldimine cross-link between two molecules, i.e. dehydro-hydroxy lysinonorleucine (Fig. 5). If the aldehyde formed is hydroxylysine derived from the aldimine bond formed undergoes a spontaneous Amadori rearrangement to give the keto-imine cross-link, hydroxylysino-keto-nor leucine (Fig. 5). These two divalent cross-links polymerise the molecules in a head to tail fashion and confer strength to the fibre. However, the divalent cross-links

are only intermediates and with time are converted to multivalent cross-links capable of linking several mol ecules. For such a reaction to occur the molecules would have to be in register. We have therefore proposed that cross-linking takes place in two stages. Firstly, longitudi nal cross-linking of the end-overlapped molecules, and secondly by interaction of these cross-links between two microfibrils in register (Bailey et al., 1980), Fig. 5. Several of these multivalent structures have now been characterised but not all have been confirmed as cross-links. The aldimine bond is stabilised by the addition of histidine across the double bond of the Schiff base to give histidino-hydroxylysinorleucine (HHL), Fig. 5. This trivalent crosslink may link three molecules, but it

is possible that the histidine involved is present in the

H

histidino-hydroxylysinorleucine

hydroxylysino-keto-norleucine

Figure 5. Structure and location of collagen crosslinks.

Reducible aldimine and keto-imine crosslinks present in immature fibres spontaneously form mature crosslinks linking microfibrils in register.

Dehydro-hydroxylysinorleucine

enzyme binding sequence of the initial crosslink. in which case it would only link two molecules. Reaction of the keto-imine crosslink with another hydroxylysine aldehyde results in the formation of the ring compound

pyridinoline which again may link two or three

molecules (Robins and Duncan. 1983). A derivative of pyridinoline derived from lysine rather than hydroxylys me in the iysyi oxidase binding site has also been identified, but in most tissues levels are only about 1/10th that of hydroxylysyl-pyridinoline. Other candi dates have been proposed but not yet confirmed.

The cross-linking of skin collagen varies with the

physiological age of the animal (Fig. 6) and the

conversion of divalent to trivalent cross-links certainly accounts for the steadily increasing tensile strength of collagen fibres with increasing age. Foetal skin is stabilised by hydroxylysino-keto-norleucine but post natally the major cross-link is the intermediate aldimine dehydro-hydroxylysinonorleucine. With increasing age the latter is converted to the stable trivalent cross-link HHL. Pyridinoline is only present at low concentrations in adult bovine skin, approximately 1/10th that of HHL.

The Nature of Collagen Fibres in the Dermis

We can now consider the genetic types of collagen distributed in the dermis, their distribution in relation to fibre bundle packing, together with the differences in the intermolecular cross-linking of the individual fibres, and the interaction with the proteoglycans. These parameters are clearly of importance in determining the type of processing and the ultimate properties of the finished leather.

Fine type III fibres are usually present in tissues that require flexibility, e.g. growing skin. During the develop ment of the dermis there is a high proportion of type III collagen fibres, about 40%, but this decreases rapidly post-natally. As the animal grows these type III fibres are gradually replaced by progressively larger and stronger type I fibres and only account for about l5% of collagen fibres at maturation. Nearing maturity growth slows and the fibres throughout the reticular

layer possess a fairly uniform diameter which is

maintained throughout life. The grain layer also contains a number of very small diameter fibres in which the

collagen molecules have retained their N-terminal

-10

AGE (Months)

Figure6. Change in crosslink content in bovine skin with age. The aldimine and keto-imine crosslinks are detected in their reduced forms, hydroxylysinonorleucine (HLNL) and dihydroxylysinorleucine (DH LN L) respectively.

propeptide extensions as demonstrated by immunofluo rescence staining of the N-propeptide. In contrast, the C-terminal propeptide has been completely removed by the C-propeptidase (Fleischmajer et a!., 1990). Whether the fibres are inhibited from growing larger by the steric hindrance of the large propeptides, or whether they are in a transition state before removal of the propeptides and subsequent growth is not yet clear. Certainly they do not continue to grow whilst retaining the N propeptide since this would result in the formation of fibres with star shaped cross-sections as seen in the dermatosparactic calf skin (Bailey and Lapiere, 1973). The p-N-type I collagen is believed to be involved in growth of the fibril up to about 20—30 nm whereupon the N-propeptide is cleaved thus allowing aggregation with other type I fibrils. On the other hand the p-N-type III collagen is retained throughout the growth of the fibril. What effect procedures such as liming during the processing of leather has upon these unusual fibres has not as yet been studied.

Evidence has accumulated in recent years indicating

that the fibres of the dermis are actually co-polymers of

type I together with minor amounts of collagen types III, V and VI, and possibly XII. Type III has been shown by immunohistochemical studies to be present in the type I fibres regardless of the diameter. Its presence has been confirmed by the identification of a cross-link between type I and type III o-chains. Recently, a double immunolabelling study of disrupted adult dermal fibres using antibodies to both type I and type III collagen has been carried out in an attempt to determine the distribution of the various types. The large type I fibres were found to possess type III on the periphery of the fibres (Fleischmajer et a!., 1990). Whether they are part of the hexagonal packing of the type I molecules, or exist as separate fibrils on the surface is not clear. Type XII collagen has also been reported to exist on the surface of type I fibres in foetal tendon, but whether they are retained through to maturity of the tissue is not known (Keene et a!., 1991). The presence of the type III and type XII collagens on the surface poses the question as to their function, i.e. are they regulating the fibril growth, controlling ultimate fibre diameter, or facilitating interaction of the fibre with other compo nents of the matrix such as fibronectin or proteoglycan. The mechanism of growth and regulation of fibre diameter is an important unsolved question in collagen biochemistry.

The nature of the collagens in the bundle sheath and their resistance to liming and enzymatic attack deter mines the “opening up” of the structure and hence plays a major role in determining the final quality of the leather. Both type III and type V collagen fibres appear to be present in these bundle sheaths. Type VI collagen is distributed throughout the grain layer and the corium (Keene et a!., 1988). The function of these filamentous type VI structures has not been determined but it has been suggested that their position between the thicker type I fibres indicates a role in maintaining the alignment of the fibres in the tissue. This proposal

is based primarily on the large number of cell binding

sequences (Arg—Gly—Asp or RGD) in the short triple helical region of type VI, and its widespread distribution. The type VI collagen fibre does not possess the stabilising covalent lysine-derived cross-links, but are polymerised by disulphide bonds. Consequently, they are likely to be damaged by the liming process. If type

C 0 U 0 0 20 40 60 80

VI does indeed play a role in aligning the fibres, then its removal from a hide containing a high proportion of this collagen could lead to an opening up of the structure and concomitant decrease in leather quality.

Of similar importance to the type of collagen present in determining the stability of the hide is the nature and extent of the inter-molecular cross-linking within the fibres. Reducible aldimine bonds are readily cleaved by the changes in pH, leading to swelling, whereas a high

proportion of the mature cross-links prevents swelling

and opening-up of the fibre bundles and consequently the penetration of the tanning solutions. A more vigorous treatment is therefore required for these mature skins and results in a reduction in quality as side-chains and peptide bonds are cleaved.

Collagen is relatively inert to chemical and enzymic attack under physiological conditions but to increase its resistance to external wear both the mechanical strength and resistance to deterioration must be increased by both

cross-linking and reduction of the water content.

Considerable variation in the stiffness can be achieved by the type of intermolecular cross-link introduced. The most commonly used cross-linking agents used are metal salts such as chrome, organic aldehydes such as glutar aldehyde or vegetable tannins. The mineral tanning agents cross-link through co-ordination bonds with the carboxyl side chains of glutamic and aspartic acids. These complexes are stable and can increase the shrinkage temperature by as much as 30°C. On the other hand the organic aldehydes form covalent cross-links by reacting with the c-amino group of the lysine side-chain. For

example, glutaraldehyde stabilises the collagen and

increases the shrinkage temperature by about 15°C. Vegetable tannins, that is, high molecular weight polyhyd roxy compounds form inter-molecular H-bonds and salt links. The shrinkage temperature is little affected, but the collagen is hydrothermally more stable since the tannins effectively block the ingress of water.

An understanding of the structure of collagen fibres in the dermis can be invaluable in increased efficiency

of the processing of leather. The precise parallel

alignment of the molecules in the fibre and the radially disposed reactive side-chains readily allow specific cross-linking between the acid and basic groups on one molecule with those on an adjacent molecule along the

length of the fibre, thereby stabilising it against

mechanical and chemical insults. Further, an appreci ation of the age of the hide and its inherent cross-linking is of some importance in determining the extent of increased cross-linking.

Collagen is perfectly structured to act as a protective covering for all animals and these same features are ideally suited for additional processing as an accessory for man.

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