BIOSYNTHESIS OF COLLAGENOUS PROTEIN

SHEETHALAN.M.S.R

CONTENTSINTRODUCTION

STRUCTURE OF COLLAGEN

TYPES OF COLLAGEN

TISSUE DISTRIBUTION OF COLLAGEN

DISTRIBUTION OF COLLAGEN TYPES IN PERIODONTIUM

BIOSYNTHESIS OF COLLAGEN

REGULATION OF COLLAGEN BIO SYNTHESIS

DEGRADATION & REMODELLING

SYSTEMIC DISEASES ASSOCIATED WITH COLLAGEN ALTERATION

ALTERATION OF COLLAGEN IN PERIODONTAL DISEASE

USES OF EXOGENOUS COLLAGEN

CONCLUSION

INTRODUCTION

Collagen is protein structures distributed throughout the animal kingdom.

The word collagen is derived from Greek roots Kolla (glue) and gene.

In French the word collagene designates glue producing constituents because collagenous tissues were used as sources of glue and gelatin.

Collagen constitutes about one third of the total protein in the body.

Collagen, in the form of elongated fibrils, is mostly found in fibrous tissues such as tendon, ligament and skin, and is also abundant in cornea, cartilage, bone, blood vessels, the gut, and intervertebral disc.

INTRODUCTION Collagen constitutes one to two percent of muscle

tissue, and accounts for 6% of the weight of muscles.

Collagen forms the major structural component it also alters the cell shape, differentiation and the activities of extracellular matrix forming an important group of multifunctional connective tissue protein.

Collagen is an important component of tissues like periodontal ligament and tendon where mechanical forces need to be transmitted without loss.

STRUCTURE OF COLLAGEN

• The structure of triple helix was first described in detail by Ramachandran and Kartha (1955).

• The whole molecule is approximately 300 nm long and 1.5 nm in diameter.

• Collagen consist of triple helical structure formed by three polypeptide chains called chain.

• The chains are left handed helices that wrap around each other to form right handed, rope like triple helical rod.

The three left-handed helices are twisted together into a right-handed coiled coil, forming a triple helix or super helix.

STRUCTURE OF COLLAGEN

The triple helix may be a continuous stretch, or interrupted by non collagenous segments.(Ramachandran&Ramakrishnan,Piez 1976)

Glycine occupies every third position within the repeated amino acid sequence in the triple helical domain

 Sequence often follows the pattern Gly-Pro-X or Gly-X-Hyp.

Glycine is essential because any larger amino acid cannot fit in the center of triple helix

Hydroxyproline and Hydroxylysine are

unique amino acids . The collagen molecule is stabilized by a

number of lysine derived intra and inter molecular cross links.

TYPES OF COLLAGEN 19 different types of collagen have been

described by Prockkop and Kivirrikko 1995 ; van der Rest and Garrone 1991)

It ranges from type I to type XIX. They are divided into three groups depending on

their ability to form fibrils.

1.Fibril forming collagens.

2.FACIT collagens.

3.Non fibrillar collagens

Fibril forming collagens:

-Easily recognised forms of collagen. They form banded fibrils. Type I, II, III, V, and XI collagens belong to this

group. The triple helical structure of these molecules

contains uninterrupted stretch of 338 to 343 Gly-X-Y triplets in each alpha chain.

The molecular weight of these collagens are of 95,000 kda.

-The molecule mesaures 15x 3000 A’ (Ramachandran and Rama krishnan 1976;Piez 1976)

FACIT collagens:

- They are fibril associated collagens with interrupted triple helices

- In these proteins the collagenous domains are interrupted by non collagenous sequences and are found associated with the surface of fibril forming collagens.

- It contains type IX, XII, XIV and XVI.- The type IX, XII and XIV collagens are

unique in containing glycosaminoglycan components covalently linked to protein.

Non fibrillar collagens

-All other types forms non fibrillar collagens that do not form fibrils.

-Network forming collagens –Types IV, VIII and X.

-Beaded –Type-VI

-Anchoring fibrils-Type-VII

-Cuticle collagens-in invertebrates.

-These collagens form a sheets or protein membranes that encloses tissues and organs.

 

In addition to the above collagen groups at least ten noncollagenous proteins incorporating short ,triple helical collagen domains have been described

This group of collagen domain containing non matrix proteins includes the lung surfactant protein , acetyl choline esterases , and mannose binding protein .

These collagen are not considered true collagen as they do not form part of the extracellular matrix

TISSUE DISRIBUTION OF COLLAGEN Type I

- in most tissues including bone, tendon, ligament, skin etc.,

-Accounts for 65% to 95% of the total collagen.

-Provides tensile strength

Type II, IX, X, XI

-mostly associated with cartilage and vitreous humour.

-Type IX is associated with type II and helps the cartilage to function at low friction.

TISSUE DISRIBUTION OF COLLAGEN

Type III

- mostly in fetal and immature tissues.

-Always associated with type I and found in many tissues.

-More fibrillar and extensible than type I and traditionally called as reticular fibers.

-Second major species and the proportion ranges from 5 to 30% in adult tissues.

TISSUE DISRIBUTION OF COLLAGEN Type IV

- in basement membrane forming chicken wire network.

-It has interrupted non collagenous sequences at the ends and also in the middle of the structure allowing it to be susceptible to all proteolytic enzymes.

-They are stable to the action of collagenases.

-They also bind to angiogenic and osteoblastic factors presenting them in immobilized status to initiate and sustain angiogenesis and osteogenesis.

TISSUE DISRIBUTION OF COLLAGEN Type V- Found in most tissues associated with type I.

Type VI - Forms beaded microfibrils and found in most tissues.

Type VII- Anchoring fibrils that attaches the anchoring plaque with the basement membrane thus maintaining the integrity between dermis and epidermis.

Type VIII - In the descemet’s membrane and endothelium.

Type XII to XVI - In many tissues.

TISSUE DISRIBUTION OF COLLAGEN Type XVII- In hemi desmosomes of the skin.

Type XIX - In rhabdo myosarcoma cells.

BIOSYNTHESIS OF COLLAGENSynthesis of pro chains , their assembly to pro

collagen and their conversion into collagen fibers involves several well coordinated biosynthetic reactions occurring in the nucleus, cytoplasm and the extracellular space.

The triple helical collagen molecule is not accessible to the modification enzymes. Therefore the modifications can occur only on the nascent α chains.

Due to these reasons, the collagen molecule is first synthesized as a large precursor(pro-α chain) containing extra amino acids at both N and C terminal ends

Gene expression:

-Collagen genes are large and range in size from 5kb to 130 kb.

-More than 30 genes has been described for the different collagen types.

Fibril Forming collagen genes: Contains 42 exons for the major triple helical

region.

The exons are composed of 54 bp and start with an intact codon for glycine

They are separated by introns that are 80 to 2000 nucleotides long .

Non Fibrillar Collagen gene:

-Organization of gene varies .

eg Type IV collagen gene the exons do not follow the 54 bp rule.

Type IX collagen gene the size of the exon ranges from 21 to 400 bp

Type X collagen gene the region that encode the tripple helical domain consists of a single 2136 bp exon.

Collagen gene

↓

mRNA

↓

Pro α chains

↓

Collagen

↓

Collagen Fibers

In nucleus:

Collagen mRNAs are transcribed as precursors and undergo the usual processing of

- Capping,

- Polyadenylation and

- Splicing.

TRANSCRIPTION

The process of transcription involves the generation of a single stranded RNA using a DNA template.

It involves the three steps

1 Initiation

2 Elongation

3 Termination

Occurs in both prokaryotes and eukaryotes

Prokaryotes possess only one type of RNApolymerase

Three classes of RNA are transcribed:

1.mRNA(messenger RNA)

2.rRNA(ribosomal RNA)

3.tRNA (transfer RNA) and

other small RNA species

Eukaryotes possess three RNA polymerases:

RNA polymerase I,II,III RNA polymerase I transcribes rRNA.

RNA polymerase II transcribes mRNA and

tRNA.

RNA polymerase III transcribes other small RNAs.

TRANSCRIPTION:

Starts with the binding of a molecular complex of RNA polymerase and other accessory factors to the DNA segment.

The binding locally separates DNA strands and exposes the template sequence to be transcribed to RNA polymerase

The transcription is initiated by the binding of σ subunit of RNA polymerase, it is released after the polymerization of about 10 ribonucleotides.

Then the core polymerase( made up of α,β,ω) subunits take over and completes the transcription.

Initiation

CAPPING:

After the synthesis of 30 nucleotides the RNA transcript is modified at its 5’ end by capping.

5’-end of the mRNA is capped 5’ to 5’ with a guanine nucleotide by the enzyme guanyl transferase.

Capping appears to be necessary for recognition of mRNA by the nuclear transport apparatus.

Capping contributes mRNA stability by protecting the molecule from degradation by nucleases that attack RNA at 5’ end.

The cap also plays an important role in positioning mRNA on ribosome for initiation of translation

TERMINATION:

The RNA-polymerase stops moving on the DNA template. The RNA transcript falls off from the transcription complex.

Newly made transcripts become immediately associated with proteins to from heterogenous nuclear ribonuclear protein particle.

Processing of transcription products :

Transcription products undergo modifications like removal of introns ,addition of bases or chemical modifications of certain nucleotides

POLYADENYLATION:

-50-250 adenine nucleotides are added to 3’ end of mRNA.

-Polyadenylation generates poly(A) tail and is carried by the enzyme poly(A) polymerase in a two step reaction (Whale and Keller 1996).

-Stabilizes the mRNA, and plays an important role in transcription termination

SPLICING:

- The process of removal of introns ( non coding genes) is known as splicing.

- Introns are the nucleotide sequences in mRNA which do not code for protein.

- Exons of mRNA posses genetic code and are responsible for protein synthesis

Introns start with the sequence GU at their 5’junction with their preceeding exons and end with AG at the 3’end site.

Splicing requires the formation of a splicesome complex where the actual splicing takes place

Splicesome is formed by the association of small nuclear ribonuclear protein particles with several protein splicing factors with the splice site in the precussor RNA.

Splicing offers a mechanism by which several isoforms of a single protein can be produced by one mRNA

Transport of the mRNA to the cytoplasm:

After nuclear event, mRNA forms complex with proteins and then is transported to the cytoplasm as mRNPs through nuclear pores.

After reaching their destination, the mRNPs disassociate releasing their protein components.

The final location of mRNA may be perinuclear, near plasma membrane, or in any other geographical location within the cytoplasm

Translation:

Conversion of a messenger RNA sequence into the amino acid sequence of a polypeptide (i.e., protein synthesis).

Translation occurs in different steps resulting in the

formation of pre pro-α chains.

1. Activation of AA

2. Initiation

3. Elongation

4. Termination

ACTIVATION OF AA Polypeptide synthesis takes place in ribosomes located

either free in the cytoplasm or bound to membranes in rough endoplasmic reticulum(RER).

The ribosomes are compact ribonuclear protein particles consisting large and small subunits of 60s and 40s in eukaryotes.

Each ribosomal sub unit is made up of many proteins associated with rRNA.

A ribosome attaches to the mRNA molecule at the 5” end and moves along in the direction of 3” direction.

In this route it translates triplet codons into corresponding amino acids.

Translation begins at an AUG intiation site and peptide synthesis proceeds from the amino acid to carboxyl end.

GTP is an important co factor necessary for translation reactions

Several protein factors play a role in the translation and they are classified based on their functions they are classified as initiation, elongation, termination or release factors

The amino acids for polypeptide synthesis are presented as aminoacyl-tRNAs.

Each tRNAs contain a three nucleotide anti codon sequence complementary to the corresponding codon for the particular amino acid it specifies.

There are 64 possible codons and one amino acid may be specified by one or more codons

ELONGATION:

Factors needed for the peptide chain elongation are EF1A, EF1B and EF2.

These protien factors form a complex with GTP and aminoacyl-tRNA.

A peptide bond forms between the first methionine, which is present at the P site and the aminoacyl –tRNA at the A site.

Formation of the peptide bond is catalyzed by the enzyme peptidyl transferase.

Once a peptide bond is formed the ribosome moves to the next codon, transfering the pepitdyl tRNA to the P site and releasing the deacylated tRNA.

This sequence is repeated as the ribosomes move along the mRNA ín the 5’-to-3’direction

TERMINATION:

Termination of the peptide elongation occurs when the ribosomes recognize the termination codons UAG,UAA or UGA .

Termination involves releasing the completed polypeptide chain from the tRNA and the free tRNA from the ribosomes.

Many ribosomes work on same mRNA molecule simultaneously and these aggregates are called POLY RIBOSOMES or POLYSOMES.

This process requires proteins known as release factors.

In bacteria about 15 aminoacids are added per second at 37⁰c .

In eukaryotic cells it is at the rate of 2 aminoacids per second

Translation has a very low error rate of 1 in 10⁴ amino acids.

Initiation factor eIF2 is one of the key regulators of overall eukaryotic protein synthesis

POSTTRANSLATIONAL PROCESSING AND PROTEIN TARGETING:TARGETING OR SORTING:

Placement of newly synthesized proteins in target cell organelles is achieved either by synthesis at the required location as is seen in mitochondria and chloroplasts or by translocation if proteins are synthesized elsewhere.

The movement of proteins from the site of synthesis to a different destination is referred to as target or sorting.

Proteins targeted to nucleus are instructed to move this site by a 7 to 9 basic amino acid sequence.

Ser-Lys-Leu triplet sequence at the carboxyl terminal of proteins such as catalase places the protein in the peroxisomes.

A sequence of aprox 25 amino acids provides the signal for the polypeptide to traverse through membranes in the chloroplast and mitochondria.

Once the protein passes through the membrane this sequence is cleaved off.

Proteins destined to be retained within the membrane contains this sequence followed by another stretch of uncharged amino acids.

Movement of polypepetides across membranes of organelles and RER is known as translocation.

In eukaryotes the secretory protein are synthesized at the RER.

The amino terminus of these proteins contains a signal sequence consisting of 15 to 30 hydrophobic and one or more positively charged amino acids.

When ribosomes initiate protein synthesis this signal sequence binds to a signal recognition particle (SRP).

SRP bound ribosomes attaches a receptor on the ER membrane.

Signal sequence attached to the polypeptide enters the ER membrane

SRP then disassociates and the nascent polypeptide enters into the ER lumen

Protein Folding:

Folding of nascent polypeptides into three dimensional conformations proceeds from the amino to the carboxyl ends.

This process is assisted by accessory proteins and di sulfide linkages.

The formation of disulfide linkages is catalyzed by the enzyme protein di sulfide isomerase.

Chaperones: Set of proteins that bind with nascent polypeptides translocated into the ER lumen, facilitate their transport and resist their degradation.

They aid in folding of proteins and also prevent misfolding of proteins under normal conditions and during heat shock

C L E V A G E O F S IG N A L S E Q U E N C E:

The mRNAs are translocated to the ribosomes in the cytoplasm that is attached to RER.

Signal sequences are cleaved during polypeptide elongation as nascent pro alpha chain are being transported into rough endoplasmic reticulum.

As translation procedes , certain proline and lysil residue at Y position are oxidised to HYP & HYL by enzyme prolyl 4 hydroxylase.

The cofactors needed for these reactions are vit-C, oxygen, iron and alpha keto glutarate.

Prolyl hydroxylation is essential for the thermal stability of collagen.

The melting temperature of fully hydroxylated collagen is 39°C and that of under hydroxylated collagen is 25°C.

Lysyl hydroxylation is important because glycosylation takes place in these residues.

Glycosylation It takes place in the Hydroxy lysine

(Hyl) and Asparagine (Asn) residues with oligosaccharides like glucose, galactose in Hyl and mannose in Asn respectively.

This glycosylation shields the collagen molecules from the action of other proteolytic enzymes.

As soon as synthesis of pro alpha chain is completed , 2 pro alpha 1 ( I ) chain and one pro alpha 2 (I), in type 1 or 3 pro alpha 1, in type II AND III associate at the C- terminal ends and the association is stabilised by inter change di sulfide bond formation catalysed by enzyme ( PROTEIN DISULFIDE ISOMERASE)

Association of chains takes place near the C- terminal ends and proceeds to N- terminal in the form of a zipper.

Pro collagen molecule thus assembled is then translocated into the golgi complex where additional glycosylation, phosphorylation and sulfation takes place depending on the type of collagen.

These pro collagen molecules are packaged into vesicles, which fuse with cell membrane and release their content into the extracellular space.

IN EXTRA CELLULAR PHASE:

First the Propeptide sequence at N-C terminal ends are removed by N-C procollagen peptidases, also known as N-C proteinases.

The N-propeptidases cleaves the proline glutamine bond in pro alpha I and pro alpha III and alanine glutamine bond pro alpha 2(I).

Propeptide are removed from type I, II, III procollagen.

Fibrillar collagen formed in this manner aggregates spontaneously as ordered fibril due to presence of charged hydrophobic region on the surface.

Aggregation occurs in parallel overlapping lateral array such that adjacent molecule are staggered by approx ¼ th of the molecule.

This leads to quarter stagger arrangement which permit interaction with side chain of neighbouring molecule which gives tensile strength to the fibres .

Aggregation is nonenzymatic but regulated by presence of other types of collagen.

Removal of the propeptide extension is necessary for ordered fibril formation.

If retained at N-terminus, it leads to poor apposition of collagen molecule – prevents normal fibril growth and fibril formed are thinner and disorganised.

Defects is known as DERMATOSPARAXIS – an inherited disease seen in cattle and sheep.

STABILISATION OF COLLAGEN FIBRILLS:

The collagen fibrillar arrays are stabilized by covalent cross links .

This crosslinking requires enzyme lysyl oxidase. The enzyme converts lysine and hydroxy lysine

residues to allysine and hydroxyallysine which then condenses with norleucine and hydroxy norleucine forming divalent cross links.

They are subsequently converted into more complex cross links, merodesmosines and hydroxy pyridiniums.

Cross linked collagen molecules then combine with other extracellular matrix to form a three dimensional scaffolding.

Ultimate organisation of collagen fibers depends upon the specific functional demand of each tissues and presence of minor collagens and proteoglycans like decorin.

REGULATION OF COLLAGEN BIOSYNTHESIS:

As collagen is the main structural component of the connective tissue the quantity and proportion of collagen within each tissue need to be precisely regulated to maintain tissue integrity.

The most important step at which regulation of collagen production occurs is at the gene transcription level. By the action of promoters and enhancers elements.

The magnitude of collagen synthesis is dependent upon the levels of mRNA for its α chains .

REGULATION OF COLLAGEN BIOSYNTHESIS:

Those that upregulate the synthesis,

- PDGF, TGF-, FGF, IGF.

Those that down regulates the synthesis are,

- Cytokines like IL-1, IFN , TNF-

- Hormones like glucocorticoids

- Others like PGE 2

TGF- elevates the level of mRNA by increasing there stability.

Collagen gene is downregulated by the inflammatory cytokine TNF α and by the lymphokine interferon gamma IFN-ϒ.

Growth factors and cytokines affecting collagen synthesis are secreted by platelets, macrophages, keratinocytes and fibroblast during inflammation, and their presence at the foci of inflammation and wound sites generally correlates with changes in collagen synthesis

DEGRADATION AND REMODELLING:

Degradation of collagen and other matrix elements is a key component of normal tissue remodeling.

It also contributes greatly to pathologic alterations. Matrix degradation is a major cause of connective

tissue degradation in chronic periodontitis, rheumatoid arthritis and other inflammatory diseases whereas hyperplasias and fibrosis are associated with low levels of degradation.

Collagen degradation takes place in two different ways,

Intracellular degradation

Extracellular degradation

Collagen degradation is primarily mediated by the collagenases.

Collagenases are specialised enzymes that have evolved specifically to hydrolyze collagen because their triple helical structure is resistant to most common proteinases.

The collagenases belong to a family of enzymes called matrix metalloprotienases MMP.

They have 13 member with closely related domain structure and discrete functions.

All have 21 –kd catalytic domain that contains a Zn++ binding site.

Based on the substrate specificity they have been classified as

-Collagenases,

-Gelatinases,

-Stomelysins ,

-Matrilysins.

Intracellular degradation:

fibroblast that are in intimate contact with the fibers

identify the alterations in the structure of collagen

cleave the fibrils and phagocytose of fibrils

forming phagosome then the phagolysosome

further degraded by the lysosomal enzymes especially cathepsin.

Extracellular degradation:

The altered integrity of the extracellular matrix causes increased water to enter the collagen bundles causing loosening of the bundles thus allowing the cells that secrete degrading MMPS to enter the bundles.

Collagenases : Type 1- MMP 1(fibroblast type) Type 2 – MMP 8 (neutrophil type) Type 3 – MMP 13 (found in breast cancer) Type 4 – MMP 18 ( unknown

Gelatinases:

A – MMP 2

B – MMP 9 Stromelysin:

Type 1 – MMP 3

Type 2 – MMP 10

Type 3 - MMP 11 Matrilysin: - MMP 7 Membrane type MMP’s: MMP 14, 15, 16, 17. Enamelolysins: MMP 19, 20

The collagenases normally degrades collagen types I, II, III, VI, VIII & X.

Collagenase I also called as MMPI or fibroblast type collagenase is produced by a variety of human epithelial and mesenchymal cells including keratinocytes, fibroblast and macrophages .

This enzyme can hydrolyze type I, II, III, VI, VIII & X. collagen & gelatin.

It hyrolyzes type III collagen molecules faster than it does typeI.

Collagenase -2 also called Polymorphonuclear leukocyte (PMN) type or MMP -8 is found only in the granules of polymorphonuclear lymphocytes.

-It hydrolyzes type I & III collagens.

Collagenase -3 is a recently described enzyme found in breast cancer

The action of MMP is controlled by 3 different mechanisms.

1ST MECHANISM These enzymes are present in tissue as

inactive precursors and conversion to an active form requires activation by plasmin , trypsin or other proteinases.

These are activated when the condition demand (eg During inflammation ) and they are also regulated by other proteins such as tissue plasminogen activator.

Inflammation →Tissue Plasminogen Factor→Plasmin,Trypsin(proteinases)→MMP

2nd mechanism

Through modulation of synthesis, IL-1,TNF-alpha,PGE2 increase MMP production from both keratinocyte and fibroblasts.

TGF-beta also increase MMP production in fibroblasts but decreases in keratinocytes.in macrophages MMP production is stimulated by LPS,but decreased by IFN-gamma ,IL-4,IL-10.

3rd mechanism

The activities of MMPs are neutralised by inhibitors present in the serum like alpha 2 macroglobulin the binds to MMP1 and inhibits MMP1.

BACTERIAL COLLAGENASE:

In contrast to vertebrate enzymes, bacterial collagenases degrade host collagen molecules to small peptides.

Theses enzymes are highly potent and have broad specifications and they can degrade all native collagens fibrils regardless of the collagen type.

They do not have substrate specificity as host collagenases.

Diseases Associated With Collagen Alterations:

Most tissues contains a mixture of collagen types and damage in their structure, content, or proportion can lead to functional abnormalities of these tissues.

They can be broadly classified as

1.Inherited diseases.

2.Acquired diseases.

3 Types of alteration can affect collagens and lead to connective tissue changes:

1. Defect in the structure of collagen genes.

2. Molecular defect in the processing enzymes .

3. Mechanisms affecting the expression of collagen genes due pathologies of acquired disease.

INHERITED DISEASES:

Due to point mutations, deletions or insertions in the structural genes for collagens or their post translational processing enzymes.

Severity of diseases varies depending on several factors.

For type I collagen, mutations that affect the assembly of proα1 chains can be lethal, whereas those affecting proα2 are not.

This is because the homotrimers of α1(I) formed in the absence of proα2(I) are stable at physiologic temperature whereas the α2 (I)3 formed in the absence of α1(I) is not stable and gets degraded.

Therefore large deletions, insertions or mutations near the C-terminus of pro-α1(I) which affects its assembly into collagen are often lethal.

Replacement of glycine with other aminoacids decrease the rate of collagen folding, its thermal stability and secretion therefore they are retained intracellularly longer and degraded.

Mutations associated with genes of collagen processing enzymes are usually not lethal, but they lead to functional abnormalities of respective tissues.

Almost 200 mutations have been characterized in COL1A1 and COLIA2 genes.

IN H E R IT E D D IS E A S E S D U E T O C O L L A G E N A LT E R A T IO N S

Osteogenesis Imperfecta. Ehlers Danlos Syndrome. Chondrodysplasia. Alport’s Syndrome. Epidermolysis Bullosa

Osteogenesis Imperfecta: (Brittle bone disease)

A heterogenous group of disorders associated with

- bone fragility

- dentinogenesis imperfecta

- hearing loss

- blue sclera and soft tissue dysplasia.

Classified into 4 types:

Type 1 :

C.F: Autosomal Dominant.

Characterised by blue sclera but normal teeth and near normal stature.

Pathogenesis:

Due to mutations that decrease Type I collagen production,

Substitution of Glycine by cysteine near the N -terminal end.

Affected Gene:COL1A1

Type II:

Lethal in the perinatal period and is associated new dominant mutations.

Pathogenesis:

Due to mutations that affect the procollagen assembly by the substitution of glycine near the C –terminal end

Rearrangemnet of multiple exons leading to loss of large segment of amino acids

Mutations at the C terminal propeptide of proα(1).

Affected Gene:COL1A1, COLIA2

TYPE III:

CF: Autosomal Dominant

Progressively deforming variety.

Pathogenesis:

Mutations in proα1(I) and proα2(I) that prevents its inclusion in the collagen molecule.

Affected Gene: COL1A1, COLIA2

TYPE IV:

CF: Autosomal Dominant

Dentinogenesis Imperfecta.

Pathogenesis:

Mutation in α2(I) and small deletions in α2(I) gene

Affected Gene: COL1A2, COLIA1

Ehlers- Danlos syndrome (Rubber Man Syndrome):

Hetrogenous group of connective tissue disorder charecterized by skin fragility and hyperextensibility and hypermobility of joints.

EDS types I-III

CF: Autosomal dominant hereditary disorder whose severe form exhibits this triad of symptoms:

– “Rubber skin” ( A): Large folds of skin can be lifted off the subcutaneous tissue; minor injuries produce skin tears (dermatorrhexis).

– “Rubber joints”: Joints are hypermobile and are easily dislocated due to lax ligaments.

– Childhood history of weak connective tissue:Manifestations include inguinal hernia, intestinal diverticula, and rectal prolapse.

Pathogenesis:

Abnormal transcription of the type I procollagen chains causes deranged fibrillogenesis. This in turn produces collagen fibrils whose cross section resembles a bear paw (“bear-paw” collagen)

Autosomal dominant hereditary disorder in which tissue tears like wet toilet paper,

characterized by quartet of symptoms:

– Vulnerable skin (dermatorrhexis).

– Aneurysm.

– Alveolar tearing (spontaneous pneumothorax).

– Hollow organ ruptures in the genital and intestinal.

Pathogenesis: Mutation or multiple exon deletions within COL3A1 gene

EDS TYPE VI(OCCULAR):

CF:Blindness

Pathogenesis: Lysl hydroxylation is

severely depressed and lysine is under hydroxylysed in Type I and Type III collagens.

EDS TYPE VII(A &B):

CF: Autosomal dominant hereditary disorder leading to joint dislocation (usually the hip); the skin is rarely involved.

Pathogenesis: Impairment of pro- collagen to collagen conversion .Assembly of collagen onto functional fibrils is affected due to hindrance by the retained pro-peptide domain .

Affected Gene: COLIAI,COLIA2(Mutations at the N – propepatidase cleavage site exon 6 of COLIAI or COLIA2

EDS TYPE VIIC (Dermatosparaxis): Enzyme N –protienase itself could be affected in this condition.

EDS TYPE VIII(PERIODONTAL):

CF: Autosomal dominant

Characterised by periodontal involvement, Loss of teeth.

Underlying Molecular cause is not yet identified.

EDS TYPE IX(cutis laxia or occiptal horn syndrome):

Reduced activity of lysyl oxidase due to defective intracellular distribution of copper a co factor for lysysl oxidase.

Affected Gene: ATP7A

CHONDRODYSPLASIAS:

Approximately 50 mutations that leads to this condition has been reported.

CF: Heterogenous group of disease.

Abnormal growth and development of cartilage.

Exhibit abnormalities in type II collagen- containing tissues such as growth plates ,nucleus pulpous and viterous humor.

3 major forms of this disease :

Achondrogenesis/Hypochondrogenesis

Spondyloepiphyseal dysplasia

Stickler Syndrome.

ALPORT SYNDROME:

Progressive hereditary nephritis associated sensoneuronal deafness and occular abnormalilties.

It has an X linked inheritance pattern. Characterized by splitting and thinning of the

glomerular basement membrane. Pathogenesis: Point mutations in triple helical and C-propeptide

domains. Multiple exon deletions Affected Gene: COL4A5 GENE

Dystrophic epidermolysis bullosa:

Molecular defects is seen in type VII collagen gene. In this disease the dermal epidermal integrity is affected due to abnormal or absent anchoring fibrils.

Pathogenesis:

Mutations in COL7AI gene.

Marfan’s Syndrome:

This is the most common hereditary group of connective tissue disorders. Manifestations include ocular, skeletal, cardiovascular, and dural lesions.

There is defect in organisation of collagen.

There is more amount of soluble collagen.

Symptoms : Skeletal

dolichostenomelia (long, narrow extremities);

scoliosis (abnormal spinal curvature);

pectus excavatum (deeply depressed sternum);

flat foot (pes planus) due to the medial deviation of the medial malleolus

-Aortic aneurysm : The media of the aorta is thinned and atrophic. Its elastic fibers are thin, fragmented, and pushed apart by focal deposits of proteoglycan As a result, the aorta appears weakened and is susceptible to rupture. This means that the aortic rupture often occurs at several sites over time in the form of a recurrent dissecting aneurysm Fatal hemorrhage may result.

-Aortic valve insufficiency. Mitral valve prolapse may occasionally occur.

Dural ectasia: This may occur in the lumbosacral region.

Dislocation of the lens: deficient zonule fibers may cause ectopia lentis, resulting in impaired vision.

ACQUIRED DISEASES:

Acquired diseases are much more common than inherited.

In these disorders, the gene expression is affected, not the gene structure.

The underlying cause of the disease is unknown; the diseases are caused by variety of factors and all matrix components including collagens and proteoglycan are affected.

Examples include chronic inflammatory diseases like rheumatoid arthritis, periodontal diseases, Crohn’s disease, progressive systemic sclerosis, lupus erythematosis, sjogrens syndrome etc., and also fibrosed conditions like atherosclerosis, pulmonary fibrosis etc.,

COLLAGEN VASCULAR DISEASE:

-Autoimmune diseases that involve formation of immune complex due to autoreactive antibodies and are associated with fibrinoid collagen necrosis and collagen deposits. collagen vascular diseases include:

— Systemic lupus erythematosus

— Progressive systemic sclerosis

— Dermatomyositis

— Nodular panarteritis

— Sjogren’s syndrome

— Wegener’s granulomatosis

— Pseudo-lupus erythematosus;

— Combined connective tissue disease (Sharp’s syndrome) with lupus erythematosus, scleroderma,and dermatomyositis;

— Rheumatic fever

— Rheumatoid arthritis

Systemic inflammatory autoimmune disorder primarily attacks the synovial membrane of the minor joints in successive episodes, leading to joint stiffening.

Pathogenesis:

Occurs in patients with a genetic predisposition (HLA-DRB1 0401, 0404 and0101).

Initial viral infection somehow stimulates autoreactive Th1 cells.

This generates autoantigens, This causes B cells to form autoreactive antibodies(rheumatism factors).

-anti-idiotypes (primarily IgM against IgG) ,

-antinuclear antibodies, and

-anti-collagen type II antibodies

These antibodies cross-link to form deposits on collagen fibers (fibrinoid necrosis), leading to formation of rheumatoid granulomas.

This activates the complement system which perpetuates the inflammation.

It affects the TMJ causing loss of vertical dimension. Indirectly have perio complications like dys functionally excessive occlusal forces in the molar regions and loss of bone support from extrusion of the anterior teeth.

Sjogren’s syndrome:

Systemic autoimmune disorder with symmetrical involvement of the salivary and lacrimal glands and desiccation of the conjunctiva and oropharynx.

Clinical triad of – xerostomia, keratoconjunctivitis sicca, rheumatoid arthritis.

Primary form involves an isolated keratoconjunctivitis sicca syndrome.

Secondary form involves a keratoconjunctivitis sicca syndrome in conjunction with other autoimmune disorders such as rheumatoid arthritis or systemic lupus erythematosus.

Pathogenesis: Patients with a genetic predisposition (primarily HLA-DR3) form autoreactive antibodies mainly against splicosomal proteins as well as against the excretory epithelia of the salivary and lacrimal glands.

Systemic Lupus erythematosis

is a immunologically mediated inflammatory condition characterized by production of auto antibodies and causing multi organ damage.

Pathogenesis: antinuclear antibodies (= ANA) are primarily directed against double-stranded DNA (anti-ds-DNA), nuclear ribonucleic protein.

This leads to complement activation necrotizing immune complex vasculitis (lupus arteritis), and, in the terminal stage, “onion-skin” arteriopathy.

CF: Common in women than men,

between the ages of 20 and 50. Butterfly erythema: Skin inflammation on the cheek

side of the nose and over the zygoma. Libman-Sacks endocarditis: Rough wart-like deposits

of fibrin on the margins of the mitral and tricuspid valves increase the risk of embolism.

Progressive systemic sclerosis:

Systemic immunologic disorder that begins with progressive sclerosis of the dermal connective tissue and spreads to the vascular connective tissue of internal organs.

CF: There is progressive fibrosis of skin and mucosa and limited jaw opening

Pathogenesis: The disorder is characterized by two mechanisms:

— Formation of immune complex and, presumably induced by it,

— Excessive formation of abnormal collagen and microfibrils

Scurvy

Definition: Connective-tissue disorder associated with vitamin C deficiency.

Vitamin C is a cofactor of proline hydroxylase that maintains that substance’s enzyme iron in the bivalent form.

Pathogenesis: Vitamin C deficiency causes proline hydroxylase insufficiency. This interferes with glycosylation, which impairs collagen fibrillogenesis. Increased fragility of the vessels leads to hemorraging

Diabetes:

Characterized by hyperglycemia, polyuria and polyphagia.

CF: Numerous clinical changes including chelosis, mucosal drying and cracking , burning mouth and tongue ,diminished salivary flow.

In periodontium there is enlarged gingiva, sessile or pedunculated gingival polyps, polypoid gingival proliferations, abscess formation, periodontitis and loosened teeth.

Altered Collagen Metabolism:

Increased collagenase activity and decreased collagen synthesis is found in individuals with diabetes.

Chronic hyperglycemia affects the synthesis, maturation and maintenance of collagen and extracellular matrix.

In hyperglycemic state elevated levels of blood glucose lead to pathological biochemical processes such as glycation of protein-like collagens or lipids and non-enzymatic oxidative destruction resulting in accumulated glycation end products (AGEs).

Collagen is cross linked by these AGE formation, making it less soluble and less likely to be normally repaired or replaced.

Advanced glycation end-products can directly affect normal protein function, or indirectly act by reacting with receptors on the cell membrane of a variety of cells.

Cellular migration through cross linked collagen is impeded and tissue integrity is impaired as a result of damaged collagen in tissues for a longer period.

Increased expression oh MMP 8 &9 is seen in diabetic patients As a result the collagen in the tissues of patient with poorly

controlled diabetes is aged and more susceptible to breakdown.

Distribution of collagen types in the periodontium

Periodontium maintains the structural and functional integrity because of the remodeling capacity of the connective tissue component.

The major connective tissue component is collagen and it’s tissue distribution is as follows,

Gingiva:

Collagen are the most abundant biochemical component of gingival connective tissue accounting for more than 60% of the total tissue protein.

They are arranged in two different forms :

type I collagen - arranged as large dense bundles of thick fibers is the major species.

type III collagen - Those that are arranged in a loose pattern of short thin fibers mixed with a fine reticular network.

type V collagen - accounts for < 1 % of the total collagen.

type VI collagen - is seen in the micro fibrils throughout the lamina propria.

type IV collagen - is present in the basement membrane in lamina lucida along with laminin. It is also present in the internal basal lamina that serves as an interface through which junctional epithelium is attached to the root surface.

The basal lamina of the epithelium is invested into the underlying connective tissue through anchorage fibrils containing type VII collagen. The attachment of epithelial cells to the basement membranes and tooth surface is mediated via hemi desmosomes that contain type XVII collagen.

Periodontal ligament:

Majority of the collagen is type I, followed by type III and type V collagen.

Type VI collagen - micro fibrils

Type IV - basement membrane to which anchoring

fibrils are attached that contains type VII collagen.

Type XII collagen, is involved in the three- dimensional organisation of the extracellular matrix. This collagen is restricted to the mature tissues.

Type XIV collagen is associated with the major collagen fibrils but not the micro fibrils.

The rate of collagen turn over in the periodontal ligament is greater than any tissue of the body, being twice than that of gingiva, five times that of alveolar bone and fifteen times that of the skin.

Cementum:

Predominantly contains type I & type III collagen. The sharpey’s fibers do not have type V & type VI collagens.

Alveolar bone:

Predominantly has type I & type III collagen. The collagen fibers attached to bone,has

- larger diameter

- small in number

- less matured

- rapid turn over than the collagen attached to cementum.

ALTERATION OF COLLAGEN IN GINGIVITIS AND PERIODONTITIS

Periodontal disease is characterized by destruction of gingival and connective tissues.

In gingivitis tissue destruction begins within the perivascular extracellular matrix where most of the collagen within the foci of inflammation is degraded.

Quantitative and Qualitative changes occur in gingival collagen in patient with the above disease.

In gingiva collagen becomes more soluble(indicates new and active synthesis).

Type I & II collagen are destroyed at the foci of inflammation.

The ratio of collagen types are altered. Type V collagen increases and its amount may exceed

that of Type III. A new collagen species, Type I trimer can be detected in

inflamed gingiva

Gingival Overgrowth:

Charecterised by increased gingival mass associated with fibroepithelial changes and an accumlation of connective tissue matrix .

Type I/III ratio becomes different with loss of type I and elevated levels of type III collagen.

Two mechanisms are involved .

1 .Reduction in the level or activities of matrix degrading enzymes in these lesions.

2. Induction of collagen production may occur by direct action of drugs on collagen synthesis mechanisms.

-by directly affecting the gene transcription rate

3.Indirectly through cytokines such as TGFβ

USES OF EXOGENOUS COLLAGEN

Periodontal regeneration: The goal of periodontal therapy is to regenerate the

periodontal tissues affected by diseases to their original architectural form and function. Thus we need regeneration of cementum, periodontal ligament and alveolar bone over the diseased root surface.  

Earlier single layered collagen were used,but now they have been replaced by bi-layered collagen membranes.

These collagen are derived mostly from achilles tendon of bovine origin and also from rat’s tail.

Uses of these collagen are around infrabony defects, grade II furcation involvement, peri implant defects, over exposed implant surface, even for root coverage procedures.

Root conditioning:

Mostly citric acid is used. collagen based putative cell binding PEPGEN P-15

peptide is used to enhance fibroblast attachment and cell migration of periodontal cells to root surfaces

As a haemostatic:

Haemorrhage from donor site in free gingival graft results in operative and post operative complications. Collagen has been used for the arrest of bleeding.

Microfibrillar Collagen Haemostat (MHC) is used to control surgical bleeding both from oozing raw surface and also discrete finite bleeding from transected or penetrated vessel.

Disadvantages – migration from the application site, adherence to wet instruments, surgical gloves, mucosal tissues.

Sutures:

Surgical gut sutures:

Derived from submucosa of sheep intestine / serosa of beef cattle intestine. It is 99% pure collagen, resorbable being degraded by proteolytic enzymes.

Collagen sutures:

Made by extruding homogenized tendon achilles of beef cattle ,almost 100% pure collagen, stiffer than surgical gut,used in eye surgery since it’s tissue reaction is less than surgical gut.

References

BIOLOGY OF PERIODONTAL CONNECTIVE TISSUE- BY P . MARK BARTOLD & A .SAMPAT NARAYANAN

ORAL CELLS AN TISSUE BIOLOGY BY GARRANT TEXT BOOK OF ORAL MEDICINE – BURKIT ORAL AND MAXILLOFACIAL PATHOLOGY- NEVILLE

& ALLEN TEXT BOOK OF ORAL PATHOLOGY –SHAFERS

THANK YOU