CSB327 Lecture 2 Notes – Molecular organization and classification of collagens (September
1 – Lecture topics
Type I collagen will be used as the prototype for fibrillar collagens.
Type I collagen is the dominant collagen in the skin and bone.
3 – Supramolecular Assemblies of Collagens based on rotary shadowing electron microscopy
This is an overview. Different collagens are classified by the superstructures that they
form. You can see structural diversity between collagens. Type IV collagen is the
signature collagen of basement membrane, which forms a network. FACIT collagens
interact with Type I collagen and promote proper assembly of fibrils.
4 – Classification of Collagens
I want to emphasize the nomenclature. The chain composition of Type I collagen is
1,1,2. This means that Type I collagen is a blend of α1 and α2 chain. This means that you
have a heterogeneous polymer. The molecular weight shows you that they are the same
length in the fibril analysis. Type I is almost ubiquitous, but more abundant in bone and
so on. α1(I)2α2(I) heterotrimer means you have two α1-chains of Type I collagen and
one α2-chain of Type I collagen. There are two transcriptional units that are responsible
for the formation of Type I collagen molecule. The type of collagen is in brackets.
Type II collagen is the signature collagen of cartilage. It is a homotrimer, which means
there is only one gene that codes for Type II collagen. There are three α1-chains of Type
Type III collagen is α1(II3) homotrimer.
5 – Fibrillar collagens
Type V collagen is a nucleator.
6 – Genetic Organization of Type I Collagen
You do not need to memorize this. There is N-propeptide and C-propeptide for Type I
collagen. Exon duplication created tandems of Gly-X-Y. Type I collagen is made of two
α1-chains and one α2-chains. The N-terminal globular domain and the C-terminal
domain must be cleaved in the extracellular space for collagen fibrils to form. If you
cleave them prematurely, then it can self-polymerize inside the cell. It is fundamental
that the molecule does not pre-assemble into a polymer inside the cell, so there are
globular structures at the ends. By this stage, the signal peptide is not attached
anymore. You are shooting a collagen molecule with intact C- and N- termini into the ER.
Telopeptides are important. 7 – Fibroblast cell imaged in culture medium
The major cell that make connective tissue are fibroblast cells. Fibroblast cells are non-
epithelial cells that do not have the polarized character of epithelial cells. They are a
major source of ECM molecule in our hearts. The cardio fibroblast cells make collagen
and ECM functional. Fibroblast cells refer to a cell type that makes a major amount of
ECM. It can be adhesive and they are morphologically heterogeneous.
8 – Overview of fibrillar collagen synthesis and assembly
α-chain hydroxylation triple helix make its way out of secretory pathway
through Golgi into extracellular space collagen molecule gets cleaved form
staggered and repeated arrangement fibrils fibres
Pathologies of collagen molecules refer to collagen molecules after secretion. Defects
here have lead to consequences of poor bone and poor skin formation. A mutation here
has dramatic effects at this end. It is an epistatic event.
9 – Collagen Biosynthesis Pathway
You would see vesicles like the ER, Golgi network, and collagen fibrils. Unless otherwise
mentioned, I am going to talk about collagen fibrils most of the time. You can see
distinct collagen fibrils. The orientation is in a weave-like fashion. The collagen fibrils are
uniformly sized. The fibrils are not all contacting each other. The fibrils are not fused.
10 – Tissue specific three dimensional (3D) arrays of type I collagen
You tend to see the fibrils, assembled into fibers, in a specific orientation. The cornea
has to be transparent and precisely organized. One of the major biochemical properties
of collagen is tensile strength. Collagen fibre is tougher than steel. Bones are formed in
concentric rings of ECM of mineralization. Our skin is weave-like. The three-dimensional
organization reflects the direction of the force. Approximately 3 kg of our body weight is
11 – Post-translational modifications of collagens in the endoplasmic reticulum
Formation of protein aggregates (monomeric complexes) happens in the ER.
12 – Isomerization of proline residues in the ER: a rate limiting step
The Pro residue is in the cis conformation and needs to be changed into the trans
conformation for it to form the polyproline left-handed helix as an α-chain that will be
transformed into a right-handed triple helical structure. Without this step, you have a
right-handed helix that doesn’t properly assemble into a collagen molecule. Pro
residues can bend the chain. This is a rate limiting step, as synthesis of α-chains occurs
in the ER, PPI will convert it into an isoform that is capable of assembling into a helix.
Without PPI, you cannot make collagen. What is the biological significance of PPI? It is a
rate limiting step. 13 – ER Processing of Fibrillar Collagens
The next major event that occurs is the formation of hydroxyl groups that are added to
the chain as synthesis occurs. Note that the signal peptides are already cleaved off. As
you make an α-chain, it becomes hydroxyl. X can be Pro but it is rare. You cannot
assemble a collagen molecule from Pro-α-chains. You cannot assemble a collagen
molecule until the C-terminal region has been made. If you miss hydroxylation, the helix
is unstable at our body temperature. Hydroxylation is to stabilize the triple helix.
14 – Hydroxylation of proline and lysine residues
Hydroxylation is critical. Prolyl-3-hydroxylase and Prolyl-4-hydroxylase is responsible for
the hydroxylation of Pro residues. Prolyl-4-hydroxylase is the predominant one. As
primates, we cannot make vitamin C, but it is co-factor that is required by these
enzymes. I will focus on the vitamin C aspect.
15 – Hydroxylation of Collagen Proline Residues in the ER
Pro-986 is conserved in evolution and defects in hydroxylation cause mutations also. 4-
hyroxyproline is the predominant form. Hydrogen bonds from Gly and HO-Pro residues
are responsible for the stability of the helix.
17 – Scurvy
Scurvy is due to a lack of vitamin C. This was a big problem for sailors. You get vitamin C
from fresh fruits. The vasculature of the basement membrane is associated with Type IV
collage and needs to be hydroxlated. If you do not do anything about this, then you will
19 – Curing Scurvy
They used to think it was the bad air once you crossed the warm climates that caused
scurvy. Limes stopped scurvy.
20 – Denaturation of collagen containing a normal content of H0-proline and abnormal collagen
containing no HO-proline
HO-Pro residues are important for the stability of the triple helix. This is the melting
curve of a triple helix. Normal collagen is stable beyond our body temperature.
Resistance to denaturation is around 40°C. Without HO-proline H-bonds, the triple helix
is stable above 20°C and you lose about 50% helix content. This emphasizes the
importance of hydroxylation. The most fundamental function of hydroxylation is to
maintain stability of the triple helix. Is it only HO-proline residues that are important?
No, Gly residues are also important. What are the temperature requirements?
What does it mean it takes a couple months for you to start showing signs of scurvy?
This means that the matrix, collagens, are stable for the most part, but they keep
turning over. You are remodelling the entire matrix. You start to fall apart because in
matrix remodelling, you are not making proper collagens, and it all falls apart. What does it emphasize about matrix turnover with respect to scurvy? That it isn’t a
permanent state. You make collagens but it is not permanent.
21 – Inhibition of collagen secretion with α,α′-dipyridyl
You can look at the impact of hydroxylation by using α,α’-dipyridyl, which blocks
hydroxylation but has no impact on protein synthesis. They used radioisotopes so that
you can follow the synthesis of protein in tissues. They chose C-proline is an abundant
amino acid in collagens. Hydroxylation leads to decreased triple helix stability and
therefore collagen secretion.
ER quality control is an important event for protecting our cells if one of your genes for
matrix molecules is defective, which you inherited from one of the parents. I don’t want
you to remember the molecular structure of this compound. I want you to remember its
use. You don’t have protein synthesis affect. It is hydroxylation.
22 – Glycosylation
There are glucose and galatose residues added onto the triple helix. The role of those
residues is not totally understood, but recently it is found that turnover of matrix
molecules may be promoted by the sugar residues that are coating the collagen. How do
you internalize a collagen fibril? Macrophages seem to be able to go around a particle
that is much larger than the plasma membrane and internalize the particle. Fibrils are
out of range for any macrophage. You end up with some cleavage with
metalloproteinases which are specific for the ECM that can break down matrix. As a
result, you can internalize the partially broken down matrix to go to the lysosomes and
finish the job. You don’t totally break down the matrix. This glycosylation seems to be
important for that.
23 – Disulfide bond formation and molecular chaperones
There is a whole bunch of molecular chaperones. We talked about the prolyl cis-trans
isomerase as a chaperone in the ER that helps orient the proline residues in the trans
state to allow the chain to form triple helix. We talked about hydroxylases which will
stabilize the helix. There are BiP chaperones. You have a PDI, which is a key one because
before you align them together, you have to make sure that they are in register so when
you form the triple helix, they are all in one place. The formation of disulfide bridges in
the C-terminal end help register the α-chains in space so that they will form a proper
helix. This is an important enzyme that I want you to remember.
What does a PDI do? It helps form disulfide bonds. If it misses a disulfide bond, then it
can correct it. It has the ability to proofread.
You start from the C-terminal end, but you need the cooperation of several key events
in the ER that ensures that this occurs properly. 24 – Extracellular processing of Type I collagen
We are looking at the pro-collagen molecule. The pro-domains are targeted by two
specific enzymes. There is an N-peptidase which is a member of the ADAMTS family.
Another major subclass of MMP is called the ADAMTS family. A disintegrin differentiates
ADAMTS from MMP. Integrins are responsible for interacting with ECM molecules.
Disintegrins has this sequence that likes to bind to integrin and sterically interferes with
its interaction with matrix molecules.
These two enzymes, ADAMTS-2 and ADAMTS-3, cleave the N-terminal. BMP1 is not
bone morphogenic protein. BMP1 is an MMP. BMP1 removes the pro-sequences at the
plasma membrane surface. They are not intracellular enzymes, otherwise if they were
added in the ER, you will remove the ends at the membrane.
You must remove the C-terminal pro-peptide for any fibril to form. There are examples
where the flexibility of the collagen fibril is enhanced by not completely cleaving the N-
terminal pro-peptide. No polymerization can occur if you don’t cleave the C-terminal
25 – Tropocollagen
I drew this plasma membrane to emphasize an extracellular compartment. This would
be inside the cell. This would be the plasma membrane. This would be a tropocollagen.
When the N- and C- pro-peptides are removed, it is called a tropocollagen molecule.
Removing the sequences will change the solubility of the tropocollagen molecule such
that it can self-assemble into fibrils.
The telopeptides are the non-helical component of the tropocollagen molecule. They
are special because if molecules are going to be assembling together and they create a