collagen synthesis methods in leather processing

collagen synthesis methods

1. 1
HOW IS COLLAGEN SYNTHESIZED?

2. 2
How is collagen made?
STEP 1: Synthesis of -chains of pre-procollagen
on ribosomes and endoplasmic reticulum

3. 3
How is collagen made?
STEP 2: Hydroxylation of proline residues to obtain
hydroxyproline (an aminoacid unique to collagen).
a reaction that substitutes a hydroxyl group, OH, for a
hydrogen atom, H, in the proline
the hydroxylation reaction secures the chains in the triple
helix of collagen
hydroxylation is catalyzed by the enzyme prolyl-4-
hydroxylase
Vitamin C is essential for enzyme action, scurvy!
OHOH
OH
Alpha-chain

4. 4
How is collagen made?

STEP 3: Hydroxylation of lysine
residues to obtain hydroxylysine
hydroxylysine is needed to permit the
cross-linking of the triple helices into the
fibers

the enzyme peptidyl proline hydroxylase
is essential
OHOH
OH
Alpha-chain
OH
OH

5. 5
How is collagen made?

STEP 4: Glycosylation of some
hydroxlysine residues
glucose and galactose are added by
enzymes galactosyl transferase and
glycosyl transferase
may affect fibril size
OHOH
OH
Alpha-chain
OH
OH
Glu
Gal

6. 6
How is collagen made?

STEP 5: Assembly of the three alpha chains
to from procollagen
formation of disulphide bonds between parts of
the polypeptide chains known as registration
peptides at the C-terminal
three chains associate, align and the triple helix
forms in a zipper-fashion giving procollagen
C-Terminal
N-Terminal
s
s
s
s
s
s
s
s
s
s
s
s

7. 7
How is collagen made?
STEP 5 (cont):
C-Terminal
N-Terminal
s
s
s
s
s
s
s
s
s
s
s
s
PROCOLLAGEN

8. 8
How is collagen made?
STEP 6: Secretion of procollagen molecules by
exocytosis into the extra cellular space
STEP 7: Cleavage of registration peptides in the
extra cellular space, by procollagen peptidases.
The resulting molecule is collagen (finally!)

9. 9
How is collagen made?
STEP 8: Self-assembly or polymerization of
collagen molecules form collagen fibrils.
STEP 9: Cross-linkage between adjacent collagen
molecules that stabilizes the fibrils.
= collagen molecule

10. 10
How is collagen made?
STEP 9 (cont):
Types of crosslinks based on the number of
molecules involved

bivalent (reducible): linking the N or C terminal
(i.e. telopeptides) of one molecule to the helical
region on another
trivalent (stable or mature crosslink): linking the N
or C terminals of two molecules to the helical
region of the third

11. 11
How is collagen made?
STEP 9 (cont):
•Lysyl oxidase deamines
(removes NH
2) the lysine or
hydroxylysine residue on the
telopeptide of collagen
molecule 1 (CM1)  aldehyde
forms
•Aldehyde links with the lysine
or hydroxylysine residue on the
helical region of another
collagen molecule (CM2) to
form bivalent crosslink (see the
next slide)
-chain CM1
CM2

12. 12
How is collagen made?
STEP 9 (cont): Bivalent crosslinks
CM1+CM2
Ketoamine crosslink
OR
CM1+CM2
Aldimine crosslink

13. 13
How is collagen made?
STEP 9 (cont): A third telopeptide can be added to
the bivalent xlink to obtain stable trivalent xlink
CM1+CM2+CM3
Pyridinoline (lysylpyridinoline if helical
residue is lysyl; hydroxypyridinoline if
helical residue is hydroxylysl
OR
CM1+CM2+CM3
Pyrrole crosslink

14. 14
How is collagen made?
STEP 9 (cont): How it looks in 3D…
BIVALENT

15. 15
GLYCOSAMINOGLYCANS AND
PROTEOGLYCANS

16. 16
Polysaccharides: Terminology
monosaccharide:
simple sugar (a single monomer)
cannot be decomposed by
hydrolysis (i.e. by addition of
water)
colorless
crystalline
sweet taste
have the same general formula,
C
nH
2nO
n

17. 17
Polysaccharides: Terminology
Mannose
C
6
H
12
O
6
)                     

Glucose
C
6
H
12
O
6
difference?

18. 18
Polysaccharides: Terminology
polysaccharide (a.k.a.
glycan):
consists of more than two
monosaccharides linked
together by glycosidic
bonds
glycosidic linkage forms
via a dehydration reaction
e.g. starch:
polysaccharide that
consists entirely of
glucose monomers
DISACCHARIDE

19. 19
Polysaccharides: Terminology
heteropolysaccharide: consists of more
than one type of monosaccharide.
glycosaminoglycan (GAG):
heteropolysaccharide consisting of a
characteristic repeating disaccharide unit
Disaccharide unit contains an N-acetylated
hexosamine (a 6-carbon sugar with an added
amino group, lobster shell)
~1 nm

20. 20
Polysaccharides: Terminology
glycoprotein: Conjugated proteins
containing one or more covalently linked
carbohydrate residues (not strictly, but
collagen can be considered as a
glycoprotein, remember glycosylation)
proteoglycan: A type of glycoprotein with
a very high carbohydrate content.

Proteoglycan subunits consist of a core
protein to which glycosaminoglycans are
covalently linked

21. 21
Glycosaminoglycans and
Proteoglycans
Regulate diffusion and flow of
macromolecules through connective
tissues
Provide resilience
negative charge  cannot move closer
in compression
negative charge  holds water 
osmotic swelling pressure
viscosity  stiff at high loading rates
(car suspension)

22. 22
Glycosaminoglycans
most abundant polysaccharides in the
body are the glycosaminoglycans (GAGs)
long unbranched polysaccharides
containing a repeating disaccharide unit
highly negatively charged, with extended
conformation that imparts high viscosity
to the solution

23. 23
Glycosaminoglycans
located primarily on the surface of cells
or in the extracellular matrix (ECM)
their rigidity provides structural integrity
to cells and provides passageways
between cells, allowing for cell migration
low compressibility, ideal for a lubricating
fluid in the joints

24. 24
Glycosaminoglycans
GAG polymers are very large (with
molecular weights of 100,000 -
10,000,000, compared to collagen
250,000)
can displace a large volume of water
 excellent lubricators and shock
absorbers

25. 25
Glycosaminoglycans
The specific GAGs of physiological significance:
Keratansulfates:
composed of galactose+ GlcNAc-6-sulfate
linkage is (1, 4)
Heparin and Heparansulfates:
composed of D-glucuronate-2-sulfate (or
iduronate-2-sulfate) + N-sulfo-D-
glucosamine-6-sulfate
linkage is (1, 4)
(heparanshave less sulfate than heparins)
Chondroitin4-and 6-sulfates :
composed of D-glucuronate+ GalNAc-4-or
6-sulfate
linkage is (1, 3)
(the figure contains GalNAc4-sulfate)
Dermatansulfates:
composed of L-iduronate(many are sulfated)
+ GalNAc-4-sulfate
linkages is (1, 3)
Hyaluronates:
composed of D-glucuronate+ GlcNAc
linkage is (1, 3)
Keratansulfates:
composed of galactose+ GlcNAc-6-sulfate
linkage is (1, 4)
Heparin and Heparansulfates:
composed of D-glucuronate-2-sulfate (or
iduronate-2-sulfate) + N-sulfo-D-
glucosamine-6-sulfate
linkage is (1, 4)
(heparanshave less sulfate than heparins)
Chondroitin4-and 6-sulfates :
composed of D-glucuronate+ GalNAc-4-or
6-sulfate
linkage is (1, 3)
(the figure contains GalNAc4-sulfate)
Dermatansulfates:
composed of L-iduronate(many are sulfated)
+ GalNAc-4-sulfate
linkages is (1, 3)
Hyaluronates:
composed of D-glucuronate+ GlcNAc
linkage is (1, 3)
Keratansulfates:
composed of galactose+ GlcNAc-6-sulfate
linkage is (1, 4)
Heparin and Heparansulfates:
composed of D-glucuronate-2-sulfate (or
iduronate-2-sulfate) + N-sulfo-D-
glucosamine-6-sulfate
linkage is (1, 4)
(heparanshave less sulfate than heparins)
Chondroitin4-and 6-sulfates :
composed of D-glucuronate+ GalNAc-4-or
6-sulfate
linkage is (1, 3)
(the figure contains GalNAc4-sulfate)
Dermatansulfates:
composed of L-iduronate(many are sulfated)
+ GalNAc-4-sulfate
linkages is (1, 3)
Hyaluronates:
composed of D-glucuronate+ GlcNAc
linkage is (1, 3)
Keratansulfates:
composed of galactose+ GlcNAc-6-sulfate
linkage is (1, 4)
Heparin and Heparansulfates:
composed of D-glucuronate-2-sulfate (or
iduronate-2-sulfate) + N-sulfo-D-
glucosamine-6-sulfate
linkage is (1, 4)
(heparanshave less sulfate than heparins)
Chondroitin4-and 6-sulfates :
composed of D-glucuronate+ GalNAc-4-or
6-sulfate
linkage is (1, 3)
(the figure contains GalNAc4-sulfate)
Dermatansulfates:
composed of L-iduronate(many are sulfated)
+ GalNAc-4-sulfate
linkages is (1, 3)
Hyaluronates:
composed of D-glucuronate+ GlcNAc
linkage is (1, 3)

26. 26
Glycosaminoglycans
GAG Localization
Hyaluronate
synovial fluid, vitreous humor, ECM
of loose connective tissue
Chondroitin
sulfate
cartilage, bone, heart valves
Heparan sulfate
basement membranes,
components of cell surfaces
Heparin
mast cells lining the arteries of the
lungs, liver and skin
Dermatan sulfateskin, blood vessels, heart valves
Keratan sulfate
cornea, bone, cartilage aggregated
with chondroitin sulfates

27. 27
Glycosaminoglycans
Hyaluronic Acid:
retains water
regulates water flow
largest GAG (500-several thousand
disaccharides) (how long is it?)
extended structure in solution; thus,
renders viscosity to the solution
present in all connective tissues
10%-20% of all GAGs in skin and aorta
1% of all GAGs in cartilage

28. 28
Glycosaminoglycans
Chondroitin Sulphate:
20-60 disaccharide units
abundant in cartilage and
intervertebral disc (10% of wet weight)
Dermatan Sulphate:
absent in cartilage
identified in meniscus, tendon, skin
and joint capsule

29. 29
Glycosaminoglycans
Keratan Sulphate:
5%-20% of GAGs in hyaline
cartilage
30%-40% of GAGs in nucleus
pulposus (depends on age)

30. 30
Proteoglycans
proteoglycan = protein + GAG
a few to a several hundred
glycosaminoglycan chains covalently
attached to a protein core
akin to a bottle brush!
the majority of GAGs in the body are
linked to core proteins, forming
proteoglycans (also called
mucopolysaccharides)
cartilage proteoglycans:
average molecular weight 2,500,000
~250 nm long, ~40 nm wide

31. 31
Proteoglycans
linkage of GAGs to the
protein core via a specific
trisaccharide unit (two
galactose + a xylose)
trisaccharide linker is
coupled to the protein core
through an O-glycosidic
bond to a serine residue in
the protein.
protein cores of
proteoglycans are rich in
serine, which allows
multiple GAG attachments.
core protein
GAG
extensions

32. 32
Proteoglycans
Proteoglycans form large
aggregates
proteglycans attached to a
hyaluronate backbone
can be as long as 4000 nm and a
diameter of 500 nm
Function of aggregation:
increased water retention
increased stiffness
regulate collagen fibril deposition
Aggregated proteoglycans
aggrecan
versican

33. 33
Proteoglycans
Aggrecan monomer:
a protein backbone of 210-250 kDa
both chondroitin sulphate and keratan sulphate chains
attached to backbone
chondroitin sulphate chains (100 - 150 per monomer),
being located in the C terminal 90%
the keratan sulphate (30 - 60 per monomer) is
preferentially located towards the N terminal
N-terminal
C-terminus
CS: Chondroitin sulphate, G: globular domain, LP: linking protein

34. 34
Proteoglycans
Aggrecan aggregate:
up to 100 aggrecan monomers interact with
hyaluronic acid
an aggregate of very high molecular weight
formed