Metabolism of hard tissues

The extracellular matrix

© Department of Medical and Clinical Biochemistry
UPJŠ in Košice, Faculty of Medicine
Metabolism of hard tissues
Outline

• Basic characteristics of extracellular matrix (ECM)

• Proteins of ECM
• Chemical composition of bones and teeth
• Bone remodeling, mineralisation
• Role of osteoclasts and osteoblasts in bone remodeling
• Regulation of bone remodeling
• Bone diseases
• Cartilage
The extracellular matrix
most mammalian cells located in tissues where
surrounded by a complex extracellular matrix
(ECM)

often referred to as “connective tissue”

the ECM contains 3 major classes of

biomolecules:
(1)the structural proteins
collagen, elastin, and fibrillin;
(2) certain specialized proteins
fibronectin, and laminin;
(3) proteoglycans and glycosaminoglycans
The extracellular matrix
the ECM involved in many normal and
pathologic processes

Functions of ECM:
- protects the organs
- provides elasticity where required (eg. in
blood vessels, lungs, skin)
- in development
- in inflammatory states
- in the spread of cancer cells
The extracellular matrix
the ECM involved in many normal and
pathologic processes

changes occur in the ECM during the aging

process

2 specialized forms of ECM

bone and cartilage
Metabolism of hard tissue
Collagen
the major protein of connective tissues
constitutes approximately 25% of the protein
of mammals
at least 28 distinct types of collagen in
human tissues
a number of proteins not classified as
collagens have collagen-like domains in
their structures
these proteins sometimes referred to as
“noncollagen collagens”
Metabolism of hard tissue
Collagen
Metabolism of hard tissue
Collagen
Functions of Collagen
- to give support to organs
- to provide alignment of cells this in turn helps
proliferation and differentiation of cells
- in blood, if collagen is exposed plateles adhere and
thrombus formation initiated

Structure of Collagen
all collagen types with a triple helical structure
in some collagens - the entire molecule triple helical
in others - the triple helix may involve only a fraction
of the structure
Metabolism of hard tissue
Collagen
Mature collagen type I
1000 amino acids
the entire molecule triple helical,
each polypeptide chain twisted into a left-handed helix
3 -chains wound into a right-handed superhelix
Metabolism of hard tissue
Collagen
Mature collagen type I
glycine at every 3rd position
repeating structure (Gly-X-Y)n an absolute requirement for the triple
helix formation
X and Y any other amino acids

on X position usually Pro

on Y position usually HyPro, HyLys
formed within posttranslational hydroxylation
Metabolism of hard tissue
Collagen
Mature collagen type I
posttranslational hydroxylation of peptide-bound Pro-
catalyzed by the enzyme prolyl hydroxylase
cofactors ascorbic acid (vitamin C) and Fe2+
Metabolism of hard tissue
Collagen
Mature collagen type I
Lys in the Y position, may also be posttranslationally modified
hyLys formed through the action of lysyl hydroxylase
an enzyme with similar cofactors
Metabolism of hard tissue
Collagen
Mature collagen type I
triple helix by lateral association
assembled into fibrils

fibrils, in turn, associate into thicker fibers

the main fibril forming collagens of types I

and II in skin and bone and in cartilage
Metabolism of hard tissue
Collagen
collagen synthesized on ribosomes as a precursor preprocollagen
contains a signal sequence - directs the polypeptide chain into the
lumen of ER
as it enters ER, the leader sequence enzymatically removed - pro--
chains
Metabolism of hard tissue
Collagen
then occurs hydroxylation of proline and lysine residues

some of selected hydroxylysines further glycosylated

galactose or glucose through an O-glycosidic linkage
Metabolism of hard tissue
Collagen
common saccharides added
glucose and galactose
(glucosyl-galactose)

enzymes: glucosyl transferase

galactosyl transferase

the extent of glycosylation different in

various sites

in tendons only 6 saccharide units

in the lens capsule 110 units
Metabolism of hard tissue
Collagen
after hydroxylation and
glycosylation

3 pro--chains assemble

intra- and inter- chain

disulfide bonds formation

triple helix formation

procollagen formed
Metabolism of hard tissue
Collagen
movement of procollagen from ER through Golgi apparatus into ECM
procollagen cleaved by specific peptidases, about:
150 AAs in N-terminus
300 AAs in C-terminus cleaved off (extension peptides)
Metabolism of hard tissue
Collagen
self-assembly into fibrils
individual tropocollagen molecules spontaneously
associate - a “quarter staggered” alignment

cross-linking
form through the action of lysyl oxidase oxidatively
deaminates – formation of reactive aldehydes
Metabolism of hard tissue
Collagen
Metabolism of hard tissue
Synthesis and processing
Metabolism of hard tissue
Degradation of collagen
• normal collagens highly stable molecules (half-live several years)
• however, connective tissue dynamic and constantly remodeled
• collagenases – enzymes that degrade collagen (large family of matrix
metalloproteinases, MMPs)
• for type I collagen, the cleavage site specific generates 3/4 and 1/4
length fragments
Metabolism of hard tissue
Collagen
several collagen types do not form fibrils in tissues
characterized by interruptions of the triple helix with stretches of
protein lacking Gly-X-Y repeat sequences

classification of collagens according to the structures they form

FACIT, fibril-associated collagen with interrupted triple helices
Metabolism of hard tissue
Abnormalities in the synthesis
of collagen

about 30 genes encode collagen

at least 8 enzyme-catalyzed
posttranslational steps
not surprising that a number of diseases
due to mutations in collagen genes
or in genes encoding some of the
enzymes
Metabolism of hard tissue
Abnormalities in the synthesis
of collagen
Ehlers-Danlos syndrome

principal clinical features

hyperextensibility of the skin,
abnormal tissue fragility, and increased
joint mobility
genetic defects,
improper synthesis and assembly of
collagens type I, III and V
at least 10 types
Metabolism of hard tissue
Abnormalities in the synthesis
of collagen
Alport Syndrome
a number of genetic disorders affecting
type IV collagen

basal membrane of kidney abnormal

glomerular apparatus

hematuria, hearing loss

eventually progress to kidney failure

Metabolism of hard tissue
Abnormalities in the synthesis
of collagen
Epidermolysis bullosa
type VII collagen abnormal
skin blisters and breaks

Scurvy
affects the structure of collagen
due to a deficiency of ascorbic acid
major sign bleeding gums
Metabolism of hard tissue
Elastin
a connective tissue protein
responsible for properties of
extensibility and elastic recoil in tissues
not as widespread as collagen
present in large amounts in lung, large
arterial blood vessels, and some elastic
ligaments
smaller quantities also found in skin,
ear cartilage, and several other tissues
only one genetic type of elastin
Metabolism of hard tissue
Elastin
synthesized as a soluble monomer of 70
kDa tropoelastin
some of the prolines hydroxylated to
HyPro by prolyl hydroxylase
hyLys and glycosylated hyLys not present
tropoelastin not synthesized in a pro-
form
elastin does not contain
repeat Gly-X-Y sequences,
triple helical structure,
or carbohydrate moieties
Metabolism of hard tissue
Elastin
after secretion, certain Lys residues
oxidatively deaminated by lysyl oxidase
the major cross-links formed in elastin
through desmosines
result from the condensation
of 3 of these lysine-derived aldehydes
with an unmodified lysine
once cross-linked in its mature,
extracellular form, elastin highly
insoluble and extremely stable
Metabolism of hard tissue
Elastin
The major differences between collagen and elastin
Metabolism of hard tissue
Elastin disorders
Williams-Beuren syndrome
the gene for elastin in chromosome 7
abnormalities in connective tissues

Copper deficiency
Cu deficiency blocks the formation of
aldehydes
some Lys residues oxidized by Cu-
containing lysyl oxidase
Metabolism of hard tissue
Fibrillin
a large glycoprotein (about 350 kDa)
a structural component of microfibrils
10-to 12 nm fibers found in many tissues
appear to provide a scaffold for deposition of elastin
Metabolism of hard tissue
Fibrillin
Marfan syndrome
mutations in the gene (on
chromosome 15) for fibrillin

affects:
the eyes
(eg, causing dislocation of the lens, known as
ectopia lentis)

the skeletal system

most patients are tall and exhibit long digits
(arachnodactyly)

the cardiovascular system

weakness of the aortic media Abraham Lincoln Niccolo Paganini
Metabolism of hard tissue
Fibronectin
a major glycoprotein of ECM
also found in a soluble form in plasma
consists of two identical subunits
joined by two disulfide bridges near
C- terminals
contains at least seven functional
domains
functions of these domains include:
binding of heparin and fibrin, collagen,
DNA, and cell surfaces
Metabolism of hard tissue
Fibronectin
binds to cells via a transmembrane
receptor protein - integrin
Arg-Gly-Asp (RGD) sequence that
binds to the receptors
the sequence shared by a number of
other proteins present in the ECM
bonded to integrins

a cell interactions with major proteins of the ECM

a and b indicate α and β polypeptide chains of integrins
Metabolism of hard tissue
Fibronectin
the fibronectin receptor interacts
indirectly with actin microfilaments

interaction through known attachment

proteins:
talin, vinculin,α-actinin and paxillin

the interaction of fibronectin with its

receptor provides one route whereby
the outside of the cell can
communicate with the inside
Metabolism of hard tissue
Laminin
a major protein component of renal
glomerular and other basal laminas

consists of 3 distinct elongated

polypeptide chains linked together to
form an elongated cruciform shape

binding sites for type IV collagen,

heparin, and integrins on cell surfaces

an important role in glomerular filtration

Metabolism of hard tissue
Bones - functions

1. Support: provides framework that supports

and anchors all soft organs
2. Protection: skull and vertebrae surround soft
tissue of the nervous system
3. Movement: skeletal muscles use the bones
as levers to move the body
4. Storage: minerals and fat stored in the interior
of the bones
5. blood cell formation: hematopoesis occurs
within the cavities of the bones
Metabolism of hard tissue
Bones
Chemical composition of bones:
consists of cells, osteoid and bone
matrix

bone contains both organic and

inorganic material

Organic:
mainly protein - type I collagen

Inorganic component:
hydroxyapatite
Metabolism of hard tissue
Bones
Chemical composition of bones:
Cells:
• osteoclasts (large cells which resorb matrix)
• osteoblasts (forms bone matrix)
• osteocytes (mature cells)
• osteogenic cell
Metabolism of hard tissue
Bones
Chemical composition of bones:
Osteoid:
(the nonmineralized organic matrix)

makes up 1/3 of the matrix

includes proteogylcans, glycoproteins

and collagen

these components contribute to the

flexibility and tensile strength of bone
Metabolism of hard tissue
Bones
Chemical composition of teeth:

enamel – dentine – cementum

enamel and dentine

different composition

cementum and dentine

very similar in composition
Metabolism of hard tissue
Bones
Organic composition of teeth
1 collagen
2 elastin
3 proteoglycans
chondroitin sulphate,
glucuronic acid
N-acetyl galactoseamine
4 pyruvate, lactate, citrate
5 acylglycerols and cholesterol
6 enzymes of: CAC, glycolysis, proteolysis, ALP
7 non-collagenous proteins – osteocalcin, sialoprotein – nucleation centres
Metabolism of hard tissue
Bones
Organic composition of teeth
Organic constituents of enamel
(approx. % of dry weight)
- insoluble protein 0.3-0.4
- collagen traces
- soluble protein 0.05
- lipid 0.6
- citrate 0.1
Total 1.1
Metabolism of hard tissue
Bones
Organic composition of teeth
Organic constituents of dentine
(approx. % of dry weight)
- collagen 18
- citrate 0.9
- lipid 0.33
- non-collagenous matrix 1.6
Total 20.83
non-collagenous matrix (in %):
- GAGs (mostly chondroitin sulphate) 12
- glycoproteins 46
- peptides 4.2
- serum proteins 2.6
Metabolism of hard tissue
Bones
Inorganic components of bones and teeth:
calcium phosphate in the form of
hydroxyapatite (65% by mass)
Ca5(PO4)3(OH)

10% calcium carbonate, other minerals

(fluoride, potassium, magnesium)

tiny crystals of Ca-salts deposited in and

around the collagen fibers of the ECM

the crystals exceptionally hard and resist

compression
Metabolism of hard tissue
Bones
Inorganic components of bones and teeth:
apatites calcium phosphates salts
difficulties with their stoichometry
Molecular Type Ca/P ratio Name
Ca(H2PO4)2 H2O 0.50 Monohydrate Calcium Phosphate, (MCPH)
Ca(H2PO4)2 0.50 Monocalcium Phosphate, (MCP)
Ca(HPO4) H2O 1.00 Dicalcium Phosphate Dihydrate, (DCPD)
Ca8H2(PO4)6 H2O 1.33 Octacalcium Phosphate, (OCP)
1.40-1.50 Amorphous Calcium Phosphate, (ACP)
a- and b-Ca3(PO4)2 1.50 Tricalcium Phosphate, (TCP)
Ca5(PO4)3(OH) 1.67 Hydroxyapatite, (HAP)
Metabolism of hard tissue
Bones
Inorganic components of bones and teeth:
hydroxyapatite
the thermodynamically the most stable
calcium phosphate salt
because of properties considerable interest
in many areas
the formula Ca5(PO4)3(OH), but usually
written Ca10(PO4)6(OH)2
the OH- ion can be replaced
by F-, Cl- or CO32-
Metabolism of hard tissue
Bones
Organic components of bones and teeth:
type I collagen the major protein
comprising 90% to 95% of the organic
material
type V collagen also present in small
amounts
a number of noncollagen proteins
some of them relatively specific to bone
Metabolism of hard tissue
Bone remodeling
bone - a dynamic structure
undergoes continuing cycles of remodeling
resorption followed by deposition of new bone tissue
coupled processes of resorption by osteoclasts and formation by
osteoblasts
Metabolism of hard tissue
Bone remodeling
Osteoclasts
multinucleated cells derived from
pluripotent hematopoietic stem cells

possess an apical membrane domain,

exhibiting a ruffled border that plays a
key role in bone resorption

in the ruffled border

present the resorption area - the
microenvironment of
low pH and lysosomal enzymes
Metabolism of hard tissue
Bone remodeling
Osteoclasts
a special proton translocating ATPase

carbonic anhydrase - protons

lowered local pH increases the

solubility of hydroxyapatite and
breakdown into Ca2+, H3PO4

starting of demineralization

lysosomal acid proteases such as

cathepsins also released
Metabolism of hard tissue
Bone remodeling
Osteoblasts

mononuclear cells derived

from pluripotent mesenchymal
precursors

synthesize most of the proteins

found in bone

responsible for the deposition of

the new bone matrix (osteoid) and
its subsequent mineralization
Metabolism of hard tissue
Bone remodeling
Osteoblasts

control mineralization by
regulating the passage of calcium
and phosphate ions across
membranes

the mechanisms of mineralization

not fully understood
Metabolism of hard tissue
Bone remodeling
Mineralisation
a process ensuring the formation of functional hard (mineralized) tissues

a unique way of storing of bone mineral into bone mass

represents the final stage of ossification

the mineral crystal must exceed the solubility product KS

of ions Ca2+ and PO43-

for Ca3(PO4)2 = of the order of 10-26

Metabolism of hard tissue
Bone remodeling - Mineralisation

Basic conditions of mineralisation:

− sufficient energy supply – a necessary conditions in the case of energy
source block (glycolysis) mineralisation and overall osiffication halted
− local changes of pH – very important not the pH of the cell (although the
pH of osteoblasts is 8.5 - significantly higher than pH of conventional cells),
but the pH of the bone cerebrospinal fluid
− increase in concentration of calcium and phosphates in specific place
− formation of nucleation centres
− presence of regulatory factors (hormones and vitamines)
Metabolism of hard tissue
Bone remodeling
Mineralisation
explained by at least 3 theories:

1. homogenous nucleation – booster mechanism

due to action of the enzymes the concentration of the calcium and
phosphate ions increases, this leads to their precipitation

2. heterogenous nucleation – seeding or epitactic mechanism

a presence of a seeding or nucleating substance acts as a mould
(or) template on which the crystals are deposited
possible seeding substances: collagen, chondroitin sulfate, lipid
substances, phosphoproteins
Metabolism of hard tissue
Bone remodeling
Mineralisation
explained by at least 3 theories:

3. matrix vesicle theory

mineralization due to the presence of vesicles containing apatite
crystals near each cartilage cell which aggregate and form a matrix
which is mineralized
vesicles secreted by osteoblasts
vesicles with many transporters in the membrane
Metabolism of hard tissue
Bone remodeling
Mineralisation
matrix vesicle theory
the influx of phosphate ions into the matrix
vesicle mediated by several proteins such as:
TNAP (tissue non-specific alkaline
phosphatase)
ENPP1 (ectonucloetide pyrophosphatase),
Pit1 (also known as NaPi – sodium
phosphate transporter)

membranes of matrix vesicles with high

concentration of different phospholipids with
high affinity to Ca2+
Metabolism of hard tissue
Bone remodeling - Mineralisation
Matrix vesicle theory
• by the catalytic activity of ENPP1 pyrophosphate (PPi) generated
(extracellular ATPs used as a substrate)
• the resultant PPi prevents crystal overgrowth
Metabolism of hard tissue
Bone remodeling - Mineralisation

Matrix vesicle theory

• TNAP hydrolyzes PPi into
phosphate ion monomers
• monomers then transported into
the matrix vesicle through Pit1
• accumulation of Ca2+ and PO43−
inside matrix vesicles then
induces crystalline nucleation
Metabolism of hard tissue
Bone remodeling
Mineralisation
matrix vesicle theory
Metabolism of hard tissue
Bone remodeling
Mineralisation
Other theories of mineralisation

- decrease of water content in hard tissue during development,

thickening of Ca2+ and phosphates

- collagen theory - ability of collagen fibrils to bind to each other by

cordination of metal ions (Ca2+) and phosphates

- mitochondrial theory – the content of calcium in mitochondria much

higher than in cytosol
Metabolism of hard tissue
Bone remodeling
Mineralisation
Activation and inhibition factors of mineralisation

activators
- chelation group containing proteins
osteocalcin
binds Ca specifically to one residue
γ-carboxyglutamate (Gla)
Metabolism of hard tissue
Bone remodeling
Mineralisation
Activation and inhibition factors of mineralisation

activators
- chelation group containing proteins
osteocalcin
binds Ca specifically to one residue
γ-carboxyglutamate (Gla)
- phosphoserine groups of phosphoproteins

inhibitors
- diphosphate and proteoglycans - prevent formation of nucleation centres
Metabolism of hard tissue
Bone remodeling
Mineralisation
Activation and inhibition factors of mineralisation
alkaline phosphatase
- metaloprotein
- active in the form of dimer
- hydrolysis of monoesters of H3PO4
- important its phosphatase activity – ensuring of the necessary phosphate
concentration
- significant also diphosphatase activity – removal of disphosphate –
inhibitor of mineralisation
Metabolism of hard tissue
Bone remodeling
Bone remodeling cycle

the bone resorption and the bone

formation run in a cycle:

1. osteoclast resorption

2. matrix formation

3. mineralization

4. resting phase
Metabolism of hard tissue
Regulation of bone remodeling
RANK – RANKL – OPG system
RANK receptor activator of nuclear factor-κB
membrane protein
on the surface of osteoclasts involved in their
activation

RANKL receptor activator of nuclear factor- κ B ligand

produced by osteoblasts
the primary mediator of osteoclasts

OPG osteoprotegerin
produced by osteoblasts
inhibits binding of RANKL to RANK
Metabolism of hard tissue
Regulation of bone remodeling
RANK – RANKL – OPG system
activated RANK activates TRAF
(TNF receptor-associated cytoplasmic factors)

TRAF bind to cytosolic domain of RANK

signal spreads to cascade of kinases

ends in induction of genes coding proteins that

directly control bone resorption
Metabolism of hard tissue
Regulation of bone remodeling
RANK – RANKL – OPG system
activated RANK activates TRAF
(TNF receptor-associated cytoplasmic factors)

TRAF bind to cytosolic domain of RANK

signal spreads to cascade of kinases

ends in induction of genes coding proteins that

directly control bone resorption
Metabolism of hard tissue
Bone diseases
Osteopetrosis
marble bone disease
characterized by increased bone
density
a rare condition by inability to resorb
bone
due to mutations in the gene encoding
carbonic anhydrase II (CA II)
normal bone resorption does not
occur, and osteopetrosis results
Metabolism of hard tissue
Bone diseases
Osteoporosis
generalized progressive reduction
in bone tissue mass per unit
volume causing skeletal weakness
the ratio of mineral to organic
elements unchanged in the
remaining normal bone
among other factors, estrogens
appear to be intimately involved in
the causation of osteoporosis
affect the OPG production
Metabolism of hard tissue
Bone diseases
Paget’s disease

disorder of bone remodeling

involves abnormal bone

destruction and regrowth

results in deformity of the affected

bones

characterized by areas of
accelerated bone turnover
Metabolism of hard tissue
Cartilage
an avascular tissue

obtains most of its nutrients from

synovial fluid

exhibits slow but continuous

turnover

various proteases (eg, collagenases

and stromelysin) can degrade
schematic representation of the molecular
collagen and the other proteins found organization in the cartilage matrix
in cartilage
Metabolism of hard tissue
Cartilage
type II collagen the principal protein

a number of other minor

types of collagen also present

elastic cartilage contains elastin

fibroelastic cartilage contains type I

collagen

also number of proteoglycans

present
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