Dept.

of Periodontology
New Horizon Dental College & Research Institute

Seminar on:
Collagen

Guided by: Presented by:

Dr. P.V. Sunil Reddy (Professor & Head) Dr. Javeria Khan
Dr. Shailendra S. Chaturvedi (Reader) (P.G. Student)
Dr. Pramod V. (Reader)
Dr. Hiroj Bagde (Senior Lecturer)
Dr. Abhilasha Singh (Senior Lecturer)
 CONTENTS:

1. Introduction
2. Components of Extra-cellular matrix
3. Structure and Biochemistry of collagen
4. Genetic organization
5. Biosynthesis of collagen
6. Types of Collagen
7. Properties of Collagen
8. Collagen in periodontium
9. Functional adaptation of collagen in periodontium
10. Degradation & Remodeling of collagen
11. Diseases of collagen related to periodontium
12. Conclusion
13. References.
 INTRODUCTION:
 The extracellular matrix (ECM) is an intricate network composed of an array of multi-
domain macromolecules organized in a cell/tissue-specific manner.
 Components of the ECM link together to form a structurally stable composite,
contributing to the mechanical properties of tissues. The ECM is also a reservoir of
growth factors and bioactive molecules.
 It is a highly dynamic entity that is of vital importance, determining and controlling the
most fundamental behaviors and characteristics of cells such as proliferation, adhesion,
migration, polarity, differentiation, and apoptosis.

 Components of ECM:
1.Collagen: It is a family of proteins which provide structural support to the multi-
cellular organism. It is the main component of tissues such as fibrous tissue, bone,
cartilage, heart valves, cornea, basement membrane, etc. Provides tensile strength.

2.Adhesive Glycoprotein: It comprises of fibronectin, tenascin/cytotactin and

thrombospndin. These glycoproteins bind other cells of ECM. These are synthesized
either by liver cells (plasma fibronectin), fibroblasts (tissue fibronectin & tenacin) or
alpha granules of platelets (thrombospondin)

3.Basement Membrane: It is a periodic acid-schiff (PAS) positive amorphous structures

that lie underneath epithelia of different organs & endothelial cells. It consists of type
IV collagen & laminin.

4.Elastic Fibres: Elastin imparts elasticity to tissue subjected to repeated stretch, such as
vascular vessels and the lung. It is degraded by elastases eg. during inflammation &
emphysema.

5.Proteoglycans: Proteoglycans consist of a core protein to which glycosaminoglycan

(GAG) side chains are attached. GAGs are linear, anionic polysaccharides made up of
repeating disaccharide units. There are four groups of GAGs: hyaluronic acid, keratan
sulfate; chondroitin/dermatan sulfate; and heparan sulfate, including heparin.
 COLLAGEN
 The word collagen, which was first used in 1865, originated from the Greek language
and means “glue-maker.”
 Collagen is also the oldest known protein. While less than 40 years ago, only one type
of collagen was known, today 29 types of collagens and collagen-like proteins have
been investigated.
 In mammals, every tissue contains collagen, making it the most abundant protein and
the most important component of the body structurally and functionally.
 It has ubiquitous distribution throughout the human body. It is involved in all growth
and remodeling processes, and because of its very specific structural, chemical, and
biological characteristics, it may be readily studied from the viewpoints of
morphology, biosynthesis, degradation, and geographic distribution as a function of
time and physiologic change.
 STRUCTURE AND BIOCHEMISTRY OF COLLAGEN:
 The most defining feature of collagen is its structure, which gives it stability,
functional ability, strength, and physical characteristics.
 It has three parallel polypeptide strands which coil about each other with one residue
stagger to form a right-handed triple helix. This elegant structural motif is the same
for all of the 29 collagens types.
 Astbury and Bell in 1942 were the first to describe it.
 In 1955, this structure was refined by Rich and Crick and by North and coworkers to
become the triple helical structure accepted today.
 Collagen is composed of three chains each of which is more than 1400 amino acids
in length that are wound together in a tight triple helix.
 This robust structure is formed by a repeating sequence of three amino acids, i.e.
glycine, proline and hydroxyproline.

 Each polypeptide chain in type I collagen contains 1056 amino acid residues, over
90% of which are in the form of the repeating tri-residue Motif (Gly-x-y), where x is
often proline and y is often hydroxylproline. Together, these aminoacids account for
over 20% of all residues. It is this high proportion of amino acids, coupled with the
presence of glycine at every third residue, which confers the characteristic
conformation to collagen molecules
 Water present in the structure plays a role in maintaining the conformation of the
native collagen molecule. The individual water bridges form and break within a few
picoseconds to keep the imino-poor region from unwinding.
  However, the water molecules that form a single hydrogen bond to the backbone
actually destabilize the triple helix, as they merely provide thermal energy to the
structure.
 In addition, a hydration shell, which covers the entire triple helix, serves as an
extensive cylinder of hydration surrounding the triple helix, and Hyp residues seem
to act as “keystones” supporting the connection between the water network and the
peptide molecules.
 The hydration shell is a biological lubricant in collagen self-assembly and determines
the inter-helix distance. Without the hydration shell, collagen molecules would
aggregate by forming non-specific bonds, resulting in kinetically trapped amorphous
states.

 GENETIC ORGANIZATION:
1.Review by Chu & Prockop demonstrates that the primary sequence of aminoacids in the
fibrous collagen is directly related to their genetic organization.
2.The a1 (1) chains of type I collagen are encoded by 41 small exons, most of which
comprise of 54 bp and code for 6 (GLY-X-Y) repeats.
3.The remainder either code for two or three (GLY-X-Y) 6 repeats or contain a 9 bp
deletion, corresponding to one (GLY-X-Y).
4.This evidence suggest that fibrillar collagens are evolved from an ancestral molecule
based upon the repetition of a (GLY-X-Y) six folds repeat and those modern collagens
are the result of gene duplication.
 BIOSYNTHESIS OF COLLAGEN:
 There are more than 20 collagen types found in the body, out of which type I is
found in most abundance. These collagen types are encoded by genes.
 The presence of a soluble precursor form of collagen was predicted many years
before its ultimate isolation and characterization.
 Studies by Fessler & Smith have found the precursor to be “procollagen” synthesized
from fibroblasts of tissue explants in vitro.
 Procollagens have a molecular weight which is up to 50% greater than that of the
final tropocollagen molecule. These are due to the peptide extensions on the C & N
termini and are called propeptides which are non-helical.
 The triple helical structure of collagen is made up of 3 very long protein chains each
of which is referred to as “α” chains
 Two of the α chains are identical & is called as α1 chain, while the slightly different
third chain is called as α2 chain.
 The triple helical structure made of these chains is called as “collagen monomer”

Collagen monomer

 Most of the collagens has triple helical structure except a few with a slightly different
amino acid composition, performing other specific functions. Eg.
1. Type II collagen - functions as shock absorber because of its association with the
ground substance or proteoglycans.
2. Type III collagen – important component of blood vessel wall & skin providing
flexibility & plays an important role in wound healing.
3. Type IV collagen – important component of basement membrane and basal
lamina functioning as filtration system.
 I. Synthesis of procollagen: Procollagen biosynthesis begins at the ribosomes on the
rough endoplasmic reticullum (RER) and involves extensive co-translation & post¬
translational modifications which are controlled by 9 to 10 different enzymes
II. Hydroxylation of proline and lysine: Hydroxylation of PRO & LYS residues is
apparently initiated as a co-transitional event that occurs on the nascent α-chains
during chain elongation at the ribosome Hydroxylation is catalysed by three
hydroxylase enzymes with specific target sequences. All the hydroxylases require
Fe2+, a-ketc glutarate, O2 and ascorbic acid. The reaction produces Co2+ and
succinate. The role of ascorbic acid is indirect; it probably plays a part in the
regeneration of Fe2+ following uncoupling althe a-ketoglutarate decarboxylation
reaction. The role of hydroxyproline and hydroxylysine in helix stability and
intermolecular cross-linkage is an important one and failure of this mechanism (eg. In
vit-c. deficiency) results in a range of pathologies (Bailey, et al.,), including scurvy,
hydroxylysine-deficiency disease, lathyrism & Menkes‟ Kinky hair syndrome.
III. Glycosylation of hydroxylysine and Asparagine: Collagen is a glycoprotein and the
addition of a-linked carbohydrate to some hydroxylysine residues in the triple helical
domain also occurs at the co-translational and posttranslational levels. The amount of
glycosylation varies with tissue and age, but essentially two carbohydrate residues are
involved. The reactions are catalysed by hydroxylysyl galactosyl transferase and
galactosyl hydorxylysyl glucosyl transferase respectively, utilizing the activated
(UDP) sugars in the presence of bivalent cations (preferably M2 +). The significance
of these sugars is not yet fully understood although their role in fibril organization is
suggested.
IV. Helix formation:
1. Triple helix formation is initiated via the association of the three c-terminal
propeptides. A role for the signal peptide in the assembly of two a1 chains and one
a2 chain at a common site at the rough endoplasmic reticulum membrane has been
suggested, chain alignment begins by non-covalent (hydrophobic) interaction at
the c-terminal propeptide.
2. The c-terminal end of the triple helical domain consists of a highly conserved
stretch of 3-10 repeating GLY-PRO-HYP motifs that stabilize the propagating
helix. This alignment is further stabilized by interchain disulphide bond formation
in the propeptide domain. The enzyme protein, disulphide isomerase, which has
 wide substrate specificity, is required for correct cis/trans conformational
alignment and re arrangement of (incorrect) disulphide bridges.
3. Subsequent further folding of the collagen triple helix proceeds rapidly after the
initial stabilization of the c-terminal propeptide, which is the rate¬ determining
step for helix formation.
4. The fully associated trimeric procollagen molecule is then exported via the golgi
apparatus in the classical secretory pathway. Once outside the cell, in the
extracellular matrix, further processing occurs by endopeptidase activity which
cleaves the N- Emd C- propeptides leaving the tropocollagen molecules which can
then form aggregate.
5. Removal of the propeptidases is achieved via at least two specific endopeptidases,
procollagen-c-proteinase and procollagen-N-proteinase, which cleave at the C- &
N termini respectively. These are both enzymes of matrix metalloproteinases
class.
6. There is also some evidence to suggest that at least in case of type I procollagen,
degradation may proceed via an intracellular route. Suggested that fibroblasts may
produce collagen in excess of normal requirements, a fraction of which is broken
down intracellularly before becoming incorporated into fibrils. A role for this
route in rapidly meeting increased demand for collagen has been suggested.
 PROPERTIES & TYPES OF COLLAGEN:
1. Collagen which is the most abundant protein in the animal kingdom, is
now understood to comprise a large family of related but genetically
distinct proteins.
2. Collagens are secreted primarily but not exclusively by fibroblasts,
though many other cell types have also been shown to produce collagen
(eg. Epithelial cells).
3. Although some 28 different collagen types have now been described, all
share to greater or lesser degree common features by basic molecular
structure and homology of condensed amino acid sequence.
4. This is the collagen triple helix or „super helix‟, which arises because of
the primary sequence of the component polypeptide chains (known as „α‟
chains)
5. The collagen superfamily comprises 28 members numbered with Roman
numerals in vertebrates (I–XXVIII) (Table 1). A novel epidermal
collagen has been called collagen XXIX, but the COL29A1 gene was
shown to be identical to the COL6A5 gene, and the a1 (XXIX) chain
corresponds to the a5(VI) chain. The common structural feature of
collagens is the presence of a triple helix that can range from most of
their structure (96% for collagen I) to less than 10% (collagen XII). As
described in the following discussion, the diversity of the collagen family
is further increased by the existence of several a chains, several molecular
isoforms and supramolecular structures for a single collagen type, and the
use of alternative promoters and alternative splicing.
6. Collagens can be subdivided into subfamilies based on their
supramolecular assemblies: fibrils, beaded filaments, anchoring fibrils,
and networks (Fig. 2).Most collagen fibrils are comprised of several
collagen types and are called heterotypic. Collagen fibrils are made of
collagens II, XI, and IX or of collagens II and III in cartilage, of collagens
I and III in skin, and of collagens I and V in cornea. Furthermore,
collagen fibrils can be considered as macromolecular alloys of collagens
and non-collagenous proteins or proteoglycans. Indeed, small leucinerich
proteoglycans regulate fibrillogenesis, as do collagens V and XIV and
could also influence collagen cross-linking. Fibronectin and integrins
could act as organizers, and collagens Vand XI as nucleators for
fibrillogenesis of collagens I and II.
 COLLAGEN DEGRADATION:
 Matrix metalloproteinases (MMPs) are zinc dependent endopeptidases
belonging to the metzincin superfamily. They participate in
physiological (development and tissue repair) and pathological
(tumorigenesis and metastasis) processes.
 Fibril-forming collagens I, II, and III are cleaved by MMP-1
(interstitial collagenase), MMP-8 (neutrophil collagenase), MMP-13
(collagenase 3) that generate three quarter and one-quarter sized
fragments, and by membrane-anchored MMP-14. MMP-2 is also able
to cleave collagen.
 Collagen II is a preferential substrate of MMP-13, whereas collagens I
and III are preferentially cleaved by MMP-1 and MMP-8. Denatured
collagens and collagen IV are degraded by MMP-2 and MMP-9 (also
known as 72-kDa and 92-kDa gelatinases respectively).
 In contrast to the [a1 (I)]2a2(I) heterotrimer of collagen I, the [a2(I)]3
homotrimer is not degraded by mammalian collagenases. This is
because of resistance of the homotrimer to local triple helix unwinding
byMMP-1 because it has higher triple helix stability near the MMP
cleavage site.
 MMPs also contribute to the release of bioactive fragments or
matricryptins such as endostatin and tumstatin from full-length
collagens. Another group of enzymes, collectively called sheddases
(Murphy 2009) releases the ectodomain of membrane collagens as
soluble forms.
 COLLAGENASE INHIBITORS
 Some of the most interesting findings in recent years have been the
discoveries of potent specific natural inhibitors of MPS.'~-~*S tudies
have shown that inhibitors from various tissue sources including cell and
tissue culture media, plasma, amniotic fluid, vitreous humour and
cartilage extracts are all closely related. We named this type of inhibitor
TIMP (tissue inhibitor of metalloproteinases) and first purified it from
human amniotic fluid:23 it is a glycoprotein of M, 28,000 and combines
essentially irreversibly with active MPs.
 Apart from TIMP, the serum proteinase inhibitor a,-macroglobulin is the
only other known collagenase inhibitor. Due to its large M, (780 K), the
latter is probably confined to sites close to blood vessels, although it may
be synthesized by some connective tissue cells. It has been observed that
most cells, especially connective tissue cells, synthesize TIMP
constitutively and we proposed that this is a fail-safe mechanism'~ to
'mop-up' the low levels of MPs which may be produced by resting cells.
 Little is known about the control of the levels of TIMP, although
production is elevated in cells stimulated to produce MPs by IL 1 or
phorbol esters, and by treatment with corticosteroids. We have been able
to localise TIMP in cells stimulated by phorbol esters using
immunocytochemical methods. Also we have found that cells can
simultaneously produce both TIMP and latent MPs and hence we have
concluded that the activation of latent MPs and the subsequent
interactions of active enzymes with either substrates or TIMP must all be
extracellular events. MPs will only be active at sites where they are
secreted and activated in greater amounts than TIMP, because active
species are rapidly sequestered by the inhibitor.15 Enzyme-inhibitor
complexes cannot be reactivated to any significant extent but the fate of
enzyme-TIMP complexes has not yet been determined; they may be
phagocytosed like protease nexin-thrombin complexes. Much of our
biological work is now centred on the hypothesis that tissue destruction
means an imbalance of active MPs over TIMP and in several model
systems we have evidence to suggest the validity of this hypothesis.
 As yet we do not know precisely the mechanisms that control TIMP
production but in pathological situations TIMP levels may simply be
overcome by the presence of proteinases from inflammatory cells.
 FUTURE TRENDS

The new information that is now available concerning the mechanisms involved
in connective tissue destruction suggest that several avenues of research might
be profitably explored.

 Firstly, further experiments should be aimed at firmly establishing the

importance of cytokines such as IL 1 as mediators of the induction of MPs
by cells, as well as their mode of action and the role of second messengers.
Analysis of the regulatory mechanisms of cytokine production and action
and the role of natural inhibitors is required.
 Secondly, to obtain a fuller understanding of the control of expression of
MPs and TIMP at all levels from their genes to their extracellular activity,
the development of compounds that could alter the balance between the
synthesis of TIMP and MPs (such as corticosteroid derivatives) in a
localized environment would be particularly important.
 Thirdly, experiments are required to test whether TIMP, or fragments
derived from it or synthetic peptides, could be used to control tissue
destruction in in vivo model systems. Such experiments may also help to
elucidate the precise contribution of MPs to resorption in each particular
situation.
 Fourthly, further investigations should be made of the mechanisms that
lead to the recruitment of polymorphonuclear leucocytes in situations
 where there may be synergism between MPs and other proteinases. The
efficacy of specific inhibitors in model systems should help to delineate the
role of serine and cysteine proteinases in connective tissue turnover.
 Further research in these areas could lead to new therapies that might be
useful not only in arthritis and related diseases but also in controlling
tumour invasiveness.
 CONCLUSION:
 Most diseases in higher animal involve collagen containing connective
tissues directly/indirectly.
 Collagens are a group of 28 closely related types that have numerous
functions all over body.
 Mechanism that regulate collagen synthesis have a direct bearing on
periodontal structures in which connective tissues specially collagen
undergo dynamic changes during periodontitis and drug induced gingival
hyperplasia.
 A balanced synthesis, regulation and degradation of collagen ensures
healthy periodontal health.
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Pathology for Dental Students, 4th Edition 2012. Jaypee Publication, New Delhi; p-
133-134.
2. Ahuja T, Dhakray V., Mittal M., Khanna P.,Yadav B., Jain M. Role Of Collagen In The
Periodontal Ligament - A Review. The Internet Journal of Microbiology. 2012 Volume
10 Number 1.
3. Yue B. Biology of the Extracellular Matrix: An Overview. J Glaucoma. 2014: S20–
S23.
4. Kim S. Midwood, Leyla Valenick Williams, Jean E. Schwarzbauer. Tissue repair and
the dynamics of the extracellular matrix. The International Journal of Biochemistry &
Cell Biology 36 (2004) 1031–1037.
5. Murphy G. & Reynolds J. Current views of collagen degradation: Progress towards
Understanding the Resorption of Connective Tissues. BioEssays Vol. 2, NO. 2. 56-60.
6. Ricard S.B. The Collagen Family. Cold Spring Harb Perspect Biol 2011;3:a004978.