The extracellular matrix of connective tissueThe extracellular matrix of connective tissue
Collagen and elastinCollagen and elastin
Proteoglycans, fibronectin, laminin
Biochemical markers of bone resorption and remodelationBiochemical markers of bone resorption and remodelation
Biochemistry IIBiochemistry II
Lecture 13 2008 (J.S.)
Connective tissues (soft connective tissue, ligaments, cartilage, bone)Connective tissues (soft connective tissue, ligaments, cartilage, bone)
are of mesenchymal origin.
The proportion of cells is relatively small:The proportion of cells is relatively small:
fibroblasts and fibrocytes (chondroblasts and chondrocytes, osteoblasts and
osteocytes, too) produce the major components of extracellular matrix,
histiocytes (fixed and free macrophages) scavenge foreign particles and tissuehistiocytes (fixed and free macrophages) scavenge foreign particles and tissue
detritus,
mast cells (mastocytes) releasing heparin and histamine,
adipose cells (adipocytes),adipose cells (adipocytes),
leukocytes, namely lymphocytes, plasmocytes, and monocytes (circulating
macrophages, leaving vessels through diapedesis).macrophages, leaving vessels through diapedesis).
The extracellular matrix includes
a fibrillar component – fibrils of collagen and reticulin,a fibrillar component – fibrils of collagen and reticulin,
in some connective tissue types elastin fibres, and
an interfibrillar component (the amorphous ground substance) –an interfibrillar component (the amorphous ground substance) –
proteoglycans and a glycosaminoglycan hyaluronic acid,
fibronectin, laminin and other cell adhesion glycoproteins,
and, in bones, voluminous insoluble mineral component.
2
and, in bones, voluminous insoluble mineral component.
Collagen fibresCollagen fibres
are included in all connective tissue types. They consist of bundles or
networks of thin collagen microfibrils.networks of thin collagen microfibrils.
Collagen
is not a single protein but a major class of glycoproteins in the intracellular matrix of connective
tissue. In mammals, collagen represents up to 25 % of all body proteins mass. Nineteen
collagen types (collagens I – XIX) are known till now. They differ from each other in their
occurrence, in part in the structure and function, too.
All collagen types include (at least in part) the characteristic triplestranded
helix (triple helix), which occurs in other proteins rather
rarely.
Only five collagen types are typical fibrillar proteins, the molecules of which form long
fibrous structures. In the other collagen types, parts of polypeptide chains are arranged intofibrous structures. In the other collagen types, parts of polypeptide chains are arranged into
one or more globular domains.
In the following text, most attention is paid to the collagen type I,In the following text, most attention is paid to the collagen type I,
the most prevailing collagen type in soft connective tissue and
bones.
3
bones.
Some structural divergences in collagen types I, II, III, and IV
Collagen I is the most common
type (skin, bones, tendons, dentin),
resisting to tensile strength. Slightlyresisting to tensile strength. Slightly
glycosylated (< 1 % saccharides),
no cysteinyl residues.
Collagen II is the major type present
in the hyaline cartilage of joints.
High degree of glycosylation,High degree of glycosylation,
no cysteinyl residues.
Collagen III (skin, aorta, uterus)
is an elastic type in the form ofis an elastic type in the form of
thin reticuline fibrils. Very low
glycosylation, cysteinyl residues areglycosylation, cysteinyl residues are
present, small number of disulfide bridges.
Collagen IV is the typical type of basement membranes (among others renal
glomeruli, capsule of the eye lens) forming the non-fibrillar network that stabilizesglomeruli, capsule of the eye lens) forming the non-fibrillar network that stabilizes
a thin membrane. Its flexible triple helices include some non-helical segments and at
their C-ends there are globular domains. Saccharidic component about 15 %,
4
their C-ends there are globular domains. Saccharidic component about 15 %,
cysteinyl residues and disulfide bridges are present.
The structure of collagen fibrils
electron optical picture
of soft connective tissue
microfibrils – either a felty tangle or
arranged bundles of protofibrils
interacting with proteoglycans
1 µm
0.3 - 3 µm
interacting with proteoglycans
protofibrils are formed through 10 - 30 nm
0.3 - 3 µm
protofibrils are formed through
aggregation of tropocollagen
units
tropocollagen unit 300 nm x 1.5 nm
10 - 30 nm
tropocollagen unit 300 nm x 1.5 nm
(triple helix, in type I collagen
two chains α1I and one chain α2I) 1.5 nm
5
Triple-stranded helices of tropocollagen
Polypeptide chains of fibrillar collagen types (named αααα-chains)
include the multirepeatedinclude the multirepeated
sequences Gly-X-Y.
X and Y can be residues of any amino acids, but mostly prevail
in position X proline, 3-hydroxyproline, Glu, His, Leu, Phe,in position X proline, 3-hydroxyproline, Glu, His, Leu, Phe,
in position Y 4-hydroxyproline, Thr, Lys, Arg.
In α-chains, each third amino acid residue is residue of glycine,In α-chains, each third amino acid residue is residue of glycine,
approximately one quarter of residues are residues of
proline or hydroxyproline.proline or hydroxyproline.
The secondary structure of α-chains is, due to the high
incidence of proline or hydroxyproline residues, a structure called.incidence of proline or hydroxyproline residues, a structure called.
the steep left-handed helix with 3.3 amino acid residues per
turn. Those chains cannot turn in the way that would enableturn. Those chains cannot turn in the way that would enable
stabilization by means of hydrogen bonds between the groups
within the particular chain (as it is usual in α-helices).
6
The secondary structure of α-chains is stabilized by
formation of trimers, three helical α-chains are woundformation of trimers, three helical α-chains are wound
together to form right-handed triple helices that are
maintained by interchain hydrogen bonds between themaintained by interchain hydrogen bonds between the
NH-groups of glycine residues, and the carbonyl groups
of residues X in an adjacent chain. These long trimersof residues X in an adjacent chain. These long trimers
are called tropocollagen units (or molecules)..
At the both N- and C-ends of α-chains, short sequences occur, which areAt the both N- and C-ends of α-chains, short sequences occur, which are
lacking in any regular structure; these segments are called
N-terminal and C-terminal telopeptides.
Two or even all three chains of tropocollagen are of the same type. E.g., the type I collagen
consists of two chains α1 I, the third chain is distinct ( α2 I), type II collagen in the cartilage
include three chains of the type α1 I).
Within the helical regions, there are no interchain disulfide bridges.
Covalent interchain crosslinks are formed in the course of
7
collagen "ripening".
The synthesis of collagen in fibroblasts, chondroblasts, and osteoblasts:
INTRACELLULAR:
The synthesis of separate procollagen chains (pro-α1, pro-α2, etc.)
Cotranslational hydroxylation of prolyl and lysyl residues and
glycosylation of hydroxylysyls (Hyl) within the endoplasmic reticulum
The synthesis of separate procollagen chains (pro-α1, pro-α2, etc.)
Formation of procollagen from three chains (triple-helical middle parts).
Secretion of procollagen by exocytosis.
EXTRACELLULAR:
Removal of N- and C-terminal propeptides – tropocollagen is formed
Aggregation of tropocollagen units into protofibrils
Interactions of protofibrils with proteoglycans – microfibrils are formed
Collagen ripening – oxidation of lysine side chains to allysine,
formation of aldimines and aldols, then also of hydroxypyridinium and
Interactions of protofibrils with proteoglycans – microfibrils are formed
other types of intermolecular crosslinks.
Degradation of collagen is initiated specifically by collagenases
8
Degradation of collagen is initiated specifically by collagenases
(Zn2+-metalloenzymes), then by various non-specific proteinases.
Ribosomal synthesis of separate procollagen chains (pro-α , pro-α ,
Details of the collagen biosynthesis:
Ribosomal synthesis of separate procollagen chains (pro-α1, pro-α2,
etc.), the chains enter the endoplasmic reticulum.
Cotranslational hydroxylation of prolyl and lysyl residues andCotranslational hydroxylation of prolyl and lysyl residues and
glycosylation of hydroxylysyl (Hyl) residues:
Both hydroxylation of Pro and Lys and O-glycosylation of hydroxylysine in the middle partsBoth hydroxylation of Pro and Lys and O-glycosylation of hydroxylysine in the middle parts
(as well as N-glycosylation of C-terminal sequences) of procollagen chains is the prerequisite
for the formation of disulfide interchain bonds within the N-terminal "extension" sequences of
nascent procollagen chains, and even for the formation of disulfide bonds in their C-terminalnascent procollagen chains, and even for the formation of disulfide bonds in their C-terminal
parts. The resulting stabilization of globular structure of the terminal sequences supports
winding of the chain middle parts and formation of the triple helix.
Three pro-α-chains
(hydroxylated Pro
Disulfide bridges
between chains in
N-terminal "extension“
"Extension“ C-terminal
parts of procollagen
(hydroxylated Pro
and glycosylated Hyl)N-terminal "extension“
parts of procollagen
9
Hydroxylation of proline and lysine residuesHydroxylation of proline and lysine residues
is catalyzed by the monooxygenase (hydroxylating) system, in which
2-oxoglutarate acts as an unusual reductant (it is oxidatively decarboxylated to
succinate in the reaction). Fe2+ ions and L-ascorbate are essential cofactors:succinate in the reaction). Fe2+ ions and L-ascorbate are essential cofactors:
(ascorbate, Fe2+)
monooxygenase
proline 2-oxoglutarate 4-hydroxyproline succinate
5-Hydroxylation of lysyl residues is analogous to the hydroxylation of proline.
proline 2-oxoglutarate 4-hydroxyproline succinate
Glycosylation of 5-hydroxylysine residues
In type I tropocollagen, only a few glucosyl-galactosyl or galactosyl groups occurIn type I tropocollagen, only a few glucosyl-galactosyl or galactosyl groups occur
attached through O-glycoside bond to hydroxylysine side chain. The glycosylation
is catalyzed by two specific UDP-glycosyl:hydroxylysyl-glycosyltransferases.
10
Winding of the procollagen middle parts results in the triple helix
Procollagen is converted to tropocollagen
Specific procollagen peptidases catalyze the removal of globular (andSpecific procollagen peptidases catalyze the removal of globular (and
stabilized by disulfide bonds) N-terminal and C-terminal propeptides
by hydrolysis within the non-helical segments. In clinical biochemistry,
the abbreviations PINP and PICP are used for those propeptides.the abbreviations PINP and PICP are used for those propeptides.
11
procollagen I N-terminal propeptides tropocollagen procollagen I C-terminal propeptides
Aggregation of tropocollagen units – formation of protofibrilsAggregation of tropocollagen units – formation of protofibrils
After processing and close to the surface of fibrocytes,
tropocollagen units associate spontaneously to form protofibrils,tropocollagen units associate spontaneously to form protofibrils,
parallel bundles, the diameter of which corresponds to about 5 – 10
units. Tropocollagen units in protofibrils are shifted by ¼ of theirunits. Tropocollagen units in protofibrils are shifted by ¼ of their
length, so that the regularity of gaps and overlaps generates the
banding pattern in the fibrils. .
N-ends C-ends
67 nm
12
Protofibrils of collagen interact with
glycosaminoglycan chains of
Proteoglycan
Collagen protofibrilsglycosaminoglycan chains of
proteoglycans:
Collagen protofibrils
Chondroitin or
keratan sulfates
Bundles or felty tangles of protofibrils
Core protein
keratan sulfates
Bundles or felty tangles of protofibrils
of fibrillar collagen types compose
thick microfibrils.thick microfibrils.
Hyaluronic acid
13
Further stabilization of collagen (maturing, ripening)
– formation of covalent crosslinks– formation of covalent crosslinks
In crosslink formation, the decisive role belongs to the side chains of lysine
or non-glycosylated hydroxylysine residues.or non-glycosylated hydroxylysine residues.
The initial reaction – the only one enzymatic reaction involved in crosslink
formation – is the oxidative deamination of lysine or hydroxylysine side
chains in the non-helical ends of fibril-forming tropocollagens, catalyzed bychains in the non-helical ends of fibril-forming tropocollagens, catalyzed by
a specific extracellular aminooxidase lysyl oxidase.
Lysyl oxidase is copper-containing enzyme, coenzyme a quinone TOPA quinone:Lysyl oxidase is copper-containing enzyme, coenzyme a quinone TOPA quinone:
Oxidative deamination of ε-amino group gives
a reactive aldehyde group – the reactivea reactive aldehyde group – the reactive
residue of amino acid called allysine
substitutes the residue of lysine in polypeptide
TOPA quinone
NH NH
substitutes the residue of lysine in polypeptide
tropocollagen chain.
allysine (2-aminoadipaldehydate)
NH
CH-CH2-CH2-CH2-CH2-NH2 →→→→ CH-CH2-CH2-CH2-CH=O
OC OC
14
allysine (2-aminoadipaldehydate)
Interchain covalent crosslinks
originate in non-catalyzed reactions between the side chains of allysineoriginate in non-catalyzed reactions between the side chains of allysine
and lysine residues.
Simple covalent bridges join only two adjacent polypeptide chains,Simple covalent bridges join only two adjacent polypeptide chains,
two types of those crosslinks are possible:
– Aldimine type, when the aldehyde group of allysine reacts with the
εε-amino group of lysine. The product is an aldimine (Schiff base):
–CH2-NH2 + O=CH-CH2– –CH2-N=CH-CH2– (+ 2H → –CH2-NH-CH2-CH2–)
The reaction is rapid, bur the product unstable. It is stabilized very slowly by
hydrogenation, the crosslink is then called lysinonorleucine bridge.
– Aldol type, when two aldehyde groups of allysine react with each other– Aldol type, when two aldehyde groups of allysine react with each other
(aldol condensation). One aldehyde group remains free in the
bridge, so that it can take part in another reaction.bridge, so that it can take part in another reaction.
(→ –C=CH-CH2– + H2O)
CH=O
–CH2-CH=O + O=CH-CH2– –CH-CH(OH)-CH2–
CH=O
The resulting aldol is unstable, but it can be stabilized by elimination of
water (a double bond is formed) to the dehydroallysinealdehyde bridge..
CH=OCH=O
15
water (a double bond is formed) to the dehydroallysinealdehyde bridge..
Aldimine bridges can react with the aldehyde group of another allysine
side chain. Then the heterocyclic pyridinium ring is closed that bindsside chain. Then the heterocyclic pyridinium ring is closed that binds
three polypeptide chains together through stable covalent bonds –
the hydroxypyridinium type of crosslink is formed..the hydroxypyridinium type of crosslink is formed..
In each tropocollagen unit, only four of all lysine residues (two near the N-ends
and other two near the C-ends of the chains) can take part in formation of those
hydroxypyridinum crosslinks.hydroxypyridinum crosslinks.
Therefore, hydroxypyridinium crosslinks join N-ends of chains with Cends
of adjacent tropocollagen units.ends of adjacent tropocollagen units.
The hydroxypyridine ring is a very stable
structure. It can be determined even afterNH-CH-CO structure. It can be determined even after
hydrolysis of all peptide bonds in collagen
fragments, e.g., excreted into the urine, as
deoxypyridinoline or pyridinoline.
NH
HO
NH-CH-CO
CH2
CH2-CH2 CH
CO deoxypyridinoline or pyridinoline.
The only difference between those two
structures is the presence or absence of an
alcoholic group on the carbon 5 (highlighted
HO
N +
2 2
CO
alcoholic group on the carbon 5 (highlighted
in the formula). This hydroxyl is present,
when hydroxylysine was one of the
NH-CH-CO
CH2
CH2-CH2-CH2
16
when hydroxylysine was one of the
reactants.
NH-CH-CO
Elastic fibres
are characteristic components of elastic connective tissue in the arterialare characteristic components of elastic connective tissue in the arterial
walls, pulmonary alveoli, skin, ligamentum nuchae, and elastic cartilage.
The structure and formation of elastic fibresThe structure and formation of elastic fibres
The elastic deformability of elastin derives from the presence of elastic fibres,
which consist of spatial network (located in the fibre axis) formed by crosslinking of
a soluble protein tropoelastin into insoluble "mature“ elastin.a soluble protein tropoelastin into insoluble "mature“ elastin.
Elastin network in elastic fibres is surrounded by a microfibrillar sheath that consists
of glycoprotein fibrilin and fibromodulin. During the formation of elastic fibres,of glycoprotein fibrilin and fibromodulin. During the formation of elastic fibres,
those two proteins are arranged in the form of oxytalan fibrils, which serve as
a mould that is then filled by tropoelastin, and in which tropoelastin is crosslinked to
mature elastin.mature elastin.
Elastin
is an unusual protein. Nonpolar amino acids prevail distinctly. In theis an unusual protein. Nonpolar amino acids prevail distinctly. In the
tropoelastin chains (about 750 AA residues -.33 % glycine, 10 % proline
with a small portion of hydroxyproline, and over 40 % valine, alanine, and
other hydrophobic amino acids). Hydroxylysine is absent, no saccharidic
component. The characteristic feature of elastin is the alternation of large
nonpolar segments with segments including lysine, important for
17
nonpolar segments with segments including lysine, important for
crosslinking of the chains.
Elastin is not a typical fibrillar protein, even when it is the major
component of elastic fibres and determines their elastic deformability.
The chains of soluble tropoelastin (taken sometimes as monomers or
subunits of elastin) don't have a regular secondary structure, theirsubunits of elastin) don't have a regular secondary structure, their
conformation is usually declared as disarranged.
The tropoelastin chains are joined by means of numerous covalentThe tropoelastin chains are joined by means of numerous covalent
crosslinks during the process of maturation. This tropoelastin
crosslinking produces insoluble elastin, an elastic spatial network is
formed. Hydrophobic interactions between numerous nonpolar amino
acid residues (namely valine) support the elasticity.
Scheme of the elastic network of elastin
stretching
relaxing
18
Covalent crosslinks in elastin
are formed in a similar way as in collagen – in non-catalyzed reactions between theare formed in a similar way as in collagen – in non-catalyzed reactions between the
side chains of lysine and allysine residues (allysine is produced by oxidative
deamination catalyzed by lysyl oxidase).deamination catalyzed by lysyl oxidase).
Two chains can be bound through simple aldol or aldimine bridge,
acyclic merodesmosine crosslink joins three side chains (the reaction of aldol with
the lysine side chain).the lysine side chain).
Four side chains of allysine and lysine (usually of only two tropoelastin chains) form
a tetrafunctional crosslink (the reaction of merodesmosine with allysine) with the
stable pyridinium structure called either desmosine or isodesmosine:stable pyridinium structure called either desmosine or isodesmosine:
CH 2-CH 2CH 2-CH 2
CH2-CH2-CH2
CH2-CH2 CH2-CH2
N CH 2-CH 2-CH 2
CH 2
CH 2-CH 2-CH 2
+
N
CH2
CH -CH -CH
+
CH 2-CH 2-CH 2
CH2-CH2-CH2
19
desmosine isodesmosine
Synthesis, ripening, and degradation of elastin
The synthesis of elastin occurs only in the last phase of foetal development, it isThe synthesis of elastin occurs only in the last phase of foetal development, it is
completed practically soon after the childbirth. The later synthesis of considerable
amounts of elastin is not supposed.
INTRACELLULAR synthesis in fibrocytes
and leiomyocytes of blood vessel walls PROELASTIN
peptide fragments?
posttranslational processing
soluble TROPOELASTIN
peptide fragments?
posttranslational processing
(incl. limited proline hydroxylation)
SECRETION OF TROPOELASTIN into the
EXTRACELLULAR matrix incorporation into the fibrilin mould,
spatial arrangement (coacervation),
allysine is formed (lysyloxidase),partial degradation
of tropoelastin chains
allysine is formed (lysyloxidase),
non-catalyzed formation of desmosines
(crosslinking), interactions with proteoglycans
insoluble ELASTIN
specific elastases antielastase factors
(a very long biological half-life)
peptide fragments
elastases
supporters of breakdown
ozone, (NO)x, tobacco smoke,
in vessels – intercalation of LDL,
bile and fatty acids
antielastase factors
α1-antitrypsin,
α2-macroglobulin,
HDL in blood vessels
20
peptide fragments
(some of them are potent immunogens)
bile and fatty acids
Elastin exhibits a very long biological half-life (the half-life of elastin in the wall ofElastin exhibits a very long biological half-life (the half-life of elastin in the wall of
aorta is referred to be about 40 years). After the childbirth, the synthesis of elastin is
distinctly reduced, if it is possible at all.
The loss of tissue elasticityThe loss of tissue elasticity
(namely of skin, large vessels, lung) in aging organisms is indisputable. Much
attention is paid to the elastin degradation.
Wrinkles are the sign of low skin elasticity that decreases by 2 - 5 % per decade
(extrapolated to neonatal period). Wrinkles represent rather a cosmetic problem,
though it is extremely interesting from the commercial point of view to those whothough it is extremely interesting from the commercial point of view to those who
produce cosmetics and feign the knowledge how to remove wrinkles.
The loss of vessel wall elasticity in ageing doesn't quite depend on the lowThe loss of vessel wall elasticity in ageing doesn't quite depend on the low
elastin content, but rather on elastin modification due to partial degradation caused
by activity of elastase (released mostly by polymorphonuclear leukocytes),
supported by the interactions of elastin fibres with LDL or bile acids.supported by the interactions of elastin fibres with LDL or bile acids.
The loss of lung tissue elasticity is associated directly with the degradation of
elastin in the walls of alveoli. Elastase is released from circulating neutrophils but,elastin in the walls of alveoli. Elastase is released from circulating neutrophils but,
under normal conditions, elastase is nearly completely inhibited by α1-antitrypsin.
Inherited deficit of α1-antitrypsin or factors that diminish its effect (smoking, ozone in
the ground smog) distinctly increase the risk of pulmonary emphysema.
21
the ground smog) distinctly increase the risk of pulmonary emphysema.
Proteoglycans
If not thinking of dense collagen connective tissue and bone, proteoglycansIf not thinking of dense collagen connective tissue and bone, proteoglycans
represent the most voluminous component of amorphous ground substance in
connective tissue, which fill in the space among fibres and cells.connective tissue, which fill in the space among fibres and cells.
In proteoglycans, numerous chains of heteropolysaccharides –
glycosaminoglycans (GAG) bind through glycosidic bonds the
core protein.core protein.
Glycosaminoglycans
The common feature of various glycosaminoglycans (e.g. dermatan
sulfate, keratan sulfate, hyaluronic acid) is the regular alternating of
units N-acetylated amino sugars and glycuronic acids. Both types ofunits N-acetylated amino sugars and glycuronic acids. Both types of
units are oft sulfated (except hyaluronic acid):.
22Segment of the hyaluronic acid molecule →3) GlcNAc (β1–4) GlcA (β1–3) GlcNAc (β1–4) GlcA(β1→
Hyaluronate is the only glycosaminoglycan that occurs in the ground substanceHyaluronate is the only glycosaminoglycan that occurs in the ground substance
as a heteropolysaccharide without any covalent bond to proteins. Other GAGs
are attached to proteins through glycosidic bonds forming proteoglycans.
ProteoglycansProteoglycans
Core protein of proteoglycans is a relatively large protein, Mr about 200 000. In
spite of that size, it represents only about 5 – 15 % mass of the proteoglycan.spite of that size, it represents only about 5 – 15 % mass of the proteoglycan.
At the N-end of the chain, there is a globular domain, through which proteoglycans can bind
non-covalently (with the assistance of two other glycoproteins, link proteins) hyaluronic acid
forming so complicated aggregates.
GAG chains
forming so complicated aggregates.
Glykosaminoglycans are linked to the core protein mostly
by their sequence Gal-Gal-Xyl. Nearly all those bonds areby their sequence Gal-Gal-Xyl. Nearly all those bonds are
O-glycosidic bonds to serine residues. The chains include
10 –100 monosaccharide units.
N-end of the
The number of GAG chains in monomeric proteoglycans
called agrecans is very approximately 100. The agrecan
structure resembles a bottle-brush. N-end of the
core protein
structure resembles a bottle-brush.
Proteoglycans are highly hydrated, and numerous
carboxylate and sulfate groups bind due to negative electric
charges large amounts of hydrated cations.
Monomeric proteoglycan
23
charges large amounts of hydrated cations.
Monomeric proteoglycan
(agrecan)
A large number of simple monomeric proteoglycans (agrecans) bind their globular
domains of core proteins non-covalently to a long chain of hyaluronic acid,domains of core proteins non-covalently to a long chain of hyaluronic acid,
each of these interactions is supported by two molecules of link glycoproteins.
Huge aggregates are formed in this way namely in hyaline cartilages. TheyHuge aggregates are formed in this way namely in hyaline cartilages. They
contribute to the resistance of a cartilage to mechanical pressure and to its
elasticity. The solidity of a cartilage depends on the number and arrangement of
collagen fibrils.collagen fibrils.
C-end of the
core protein ofcore protein of
proteoglycan
Electron optical picture of proteoglycanshyaluronate
link
glycoprotein
24
Electron optical picture of proteoglycans
in extracellular matrix of an cartilage
hyaluronate
Fibronectin
is a glycoprotein of the extracellular matrix, released by fibrocytes, endothelialis a glycoprotein of the extracellular matrix, released by fibrocytes, endothelial
cells, and other cell types. The liver cells secrete fibronectin into the blood
plasma (concentration 150 - 700 mg / l). It mediates interactions betweenplasma (concentration 150 - 700 mg / l). It mediates interactions between
collagen, proteoglycans and cells. Its name descents from Latin "fibra
nectere", metaphorically speaking a cellular adhesive.
Two polypeptide chains (230 kDa) of fibronectin are joined by disulfide bondsTwo polypeptide chains (230 kDa) of fibronectin are joined by disulfide bonds
and include binding domains composed of repeated sequences.
Deficit of fibronectin on the surface of tumour cells can support their release
and formation of metastases.
+
Binding domains for
and formation of metastases.
protofibril of collagen
NH3
+
NH3
+
fibrin
heparin
collagen
integrin
fibronectin
NH3
collagen
integrin
integrins
of cellular membranes actin filament
25-ooc COO-
LamininLaminin
Laminins are members of the family of non-collagen glycoproteins in basement
membranes. They are large heterotrimers (Mr 950 000). Three chains are joinedmembranes. They are large heterotrimers (Mr 950 000). Three chains are joined
through disulfide bridges and they are arranged into a cross-like form with four arms.
Similarly to fibronectin, laminins include several binding sites, e.g. for integrins of
cellular membranes, heparin, and heparan sulfate.cellular membranes, heparin, and heparan sulfate.
Laminin aggregates reversibly into a flat polymeric network at high concentrations of
Ca2+ ions. Those polymers bind through another protein (either nidogen or entactin)
type IV collagen and core proteins of proteoglycans. These interactions are thetype IV collagen and core proteins of proteoglycans. These interactions are the
cause of anchoring epithelial cells onto components of basement membranes.
Binding sites for
integrins orclustering
heparin
integrins or
nidogen / entactin
(interaction with collagen
or cellular surface)
clustering
of laminin
26
heparin
The boneThe bone
The bone is a tissue exhibiting very high metabolic activity, though it
doesn't seem so at first glance.doesn't seem so at first glance.
The unremitting bone remodelation, both osteoresorption and bone
formation, continues after the growth of bones is finished.formation, continues after the growth of bones is finished.
The bulkiness and consistency of bones depends on the balance
between resorption and formation.
Composition of bones
Water 25 % in compact bone (12 % cementum, 10 % dentin,Water 25 % in compact bone (12 % cementum, 10 % dentin,
1 % tooth enamel)
Organic components 30 %
Mineral components 40 %
In dry bone tissue organic components 40 – 45 %In dry bone tissue organic components 40 – 45 %
mineral components 60 – 55 %
27
Extracellular matrix:
organic components - collagen type Iorganic components - collagen type I
proteoglycans
sialoprotein
osteocalcinosteocalcin
citrate
mineral components - hydroxylapatite 3Ca3(PO4)2.Ca(OH)2mineral components - hydroxylapatite 3Ca3(PO4)2.Ca(OH)2
with octacalcium phosphate Ca8(HPO4)8.5H2O
and amorphous calcium phosphate Ca3(PO4)2
on the whole 85 %on the whole 85 %
CaCO3 10 %
CaF2 0.3 %
CaCl 0.2 %CaCl2 0.2 %
Mg3(PO4)2 1 %
alkaline salts 2 %
Bone cells: osteoclasts (modified macrophages, bone resorption),
osteoblasts (a type of fibroblasts, bone formation), and
osteocytes (transformed aged osteoblasts) affect the activity of
previous cell types by paracrine secretion of interleukins, tumour
necrosis factor (TNF), osteoprotegerin, prostaglandins and other
28
necrosis factor (TNF), osteoprotegerin, prostaglandins and other
various growth factors.
Bone cells
Osteoblasts occur on the surfaces of growingOsteoblasts occur on the surfaces of growing
or remodelated bones, less frequently inside
adult bones. They synthesize and insert osteoidadult bones. They synthesize and insert osteoid
(extracellular matrix), deposit the bone
minerals, and consecutively settle and
transform into osteocytes. The surface oftransform into osteocytes. The surface of
osteoblasts binds molecules of alkaline
phosphate that support mineralization of the
matrix.matrix.
Osteoclasts occur at sites of active bone
resorption, in resorption pits. They include
many lysosomes exhibit high activity of acidmany lysosomes exhibit high activity of acid
phosphatase. Osteoclasts liquidate fragments
of collagen and other organic components
through phagocytosis.through phagocytosis.
Osteocytes are prevailing cellular elements of
mature bone. They are dispersed in lacunasmature bone. They are dispersed in lacunas
and form a cellular net by means of contacts
between their projections. Life span of
osteocytes is estimated to about 25 years,
29
osteocytes is estimated to about 25 years,
extinct cells initiated bone resorption.
Control of bone remodelation
Bone resorption
parathyrin
calcitriol
calcitonin
stimulated by
inhibited by calcitonin
estrogens
inhibited by
(parathyrin)stimulated by
Bone formation
(parathyrin)
calcitriol
androgens
estrogens
stimulated by
inhibited by
estrogens
glucocorticoids
30
Biochemical markers of bone metabolism
are usefull in clinical biochemistry in tests of osteoporosis and other
Biomarkers of bone formation
are usefull in clinical biochemistry in tests of osteoporosis and other
metabolic osteopathies.
Biomarkers of bone formation
Catalytic concentration of the bone isoenzyme of alkaline phosphatase
(ALP) in serum – the isoenzyme is a marker of osteoblast activity,(ALP) in serum – the isoenzyme is a marker of osteoblast activity,
of which it is an ectoenzyme. Total activity of ALP can serve only as a
very rough estimate due to prevalence of other isoenzymes.
Concentration of osteocalcin (bone Gla protein, BGP) in serum –Concentration of osteocalcin (bone Gla protein, BGP) in serum –
osteocalcin is a modulator of bone remodelation secreted by
osteoblasts, its production is induced by calcitriol. Osteocalcin is theosteoblasts, its production is induced by calcitriol. Osteocalcin is the
main non-collagen protein in the extracellular matrix of bones.
Concentration of N-terminal or C-terminal propeptides of procollagen I
in serum.in serum.
procollagen I N-terminal tropocollagen procollagen I C-terminal
31
procollagen I N-terminal tropocollagen procollagen I C-terminal
propeptide (PINP) propeptide (PICP)
Biomarkers of bone resorption
Catalytic concentration of the bone isoenzyme of acid phosphatase
(ACP) in serum. This isoenzyme is one of the six ACP isoenzymes (called
also tartrate-resistant ACP) considered a marker of osteoclast activity.also tartrate-resistant ACP) considered a marker of osteoclast activity.
Concentration of C-terminal telopeptide of collagen I (ICTP, or of
C-terminal octapeptide, CTx) in serum or urine. C-terminal telopeptidesC-terminal octapeptide, CTx) in serum or urine. C-terminal telopeptides
are terminal non-helical sequences of tropocollagen I, without crosslinks.
Excretion of N-terminal telopeptide of collagen I (INTP, NTx) into the
urine – N-terminal non-helical sequences of tropocollagen I, which may
include some crosslinks, too..
Determination of deoxypyridinoline and pyridinoline in the urine, andDetermination of deoxypyridinoline and pyridinoline in the urine, and
determination of galactosyl hydroxylysine in the urine.
galactosyl-hydroxylysine collagen I C-terminalgalactosyl-hydroxylysine
(after alkaline hydrolysis)collagen I N-terminal
telopeptide (INTP)
collagen I C-terminal
telopeptides (ICTP)
Excretion of hydroxyproline (free or total) in the urine was in common use for years,
32
Excretion of hydroxyproline (free or total) in the urine was in common use for years,
but currently it is taken as an obsolete test of bone resorption.