what-when-how
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interactions with molecular chaperones take
place to prevent premature triple helix formation.
Some lysine residues are hydroxylated by lysyl
hydroxylase.
25,26
4.
After completion of the synthesis of the carboxy-
terminal propeptide, this part of the molecule folds,
forms intra-chain disulfide bonds,
27
and interacts
either directly or indirectly with the lipid bilayer of
the rER.
28
5.
Chain selection and association occurs through the
carboxy-terminal propeptides attached to the rER
membrane and is limited by diffusion.
6.
A nucleus for triple helix formation is formed by the
C-propeptide that staggers the chains in the right
order.
27
Inter-chain disulfide bonds are formed in the
C-propeptide.
7.
Hydroxylation of proline residues and some lysine
residues continues and triple helix formation
proceeds from the carboxy-terminal end towards the
amino-terminal end. The fast propagation of triple
helix formation is interrupted by the occurrence of
cis peptide bonds involving proline residues. These
peptide bonds need to be isomerized into trans to
allow triple helix formation to continue. This step
is catalyzed by peptidyl-prolyl cis-trans isomerases
(PPIases).
29
8.
After completion of the folding of the major helix,
the amino-terminal propeptides associate and form
the small triple helix within this domain. Interaction
with chaperones prevents premature fibril formation
within the rER. Further modifications can occur
during the transport through the Golgi stack by
cisternal maturation. The long-standing problem
of how a 300 nm long procollagen molecule is
transported to the Golgi in 50 nm transport vesicles
has been investigated.
30,31
Most recently it was
shown that TANGO1
/
Mia3 plays a crucial role
during this process.
32-34
9.
The amino- and carboxy-terminal propeptides are
cleaved by ADAMTS2 and BMP-1, respectively.
35,36
Oxidation of lysine residues is catalyzed by
lysyloxidase and inter-chain intermolecular crosslinks
are formed between two or three molecules.
37,38
N- and O-glycosylation. Other obligatory steps are pro-
teolytic cleavage of the N- and C-propeptides, oxidation
of lysyl residues and inter-chain crosslinking. As a result
collagen biosynthesis is probably one of the most compli-
cated processes in our body. To make it even more com-
plicated many enzymes involved in this process exist
as molecular complexes. A well-known example is the
tetrameric prolyl 4-hydroxylase (P4H)
/
protein disulfide
isomerase (PDI) complex. Prolyl 4-hydroxylase accom-
plishes the most frequent modification of proline into
4(R)-hydroxyproline in the Yaa position of the GlyXaaYaa
collagen sequence (
Figure 6.2
). Prolyl 4-hydroxylase has
been originally purified in the tetramer form by affin-
ity chromatography.
45
Later studies have identified the
β subunit of the α
2
β
2
tetramer as protein disulfide isom-
erase (PDI).
46,47
Prolyl 4-hydroxylation is absolutely
required for the assembly and stability of collagen triple
helix.
48
The absence of the α subunit of the P4H1 enzyme
leads to early embryonic death in mice due to insufficient
mechanical stability of the basement membrane.
49
A NEW PARADIGM IN OI
OI has been around for many years. A 3000-year-old
mummy was recently diagnosed with OI.
50
In the 20th
century the disease was studied extensively and con-
tinuously. The first classification of the OI types based
on clinical features was introduced in 1978 by Sillence.
51
Since then the original classification has been revised
multiple times.
2,52,53
Autosomal dominant forms of OI are the most abun-
dant.
53
However, recessive forms account for about 10%
of cases. The new paradigm in OI was introduced by the
discovery of the genes involved in collagen folding and
post-translational modifications that are responsible for
the recessive forms of brittle bone disease.
We extracted rER proteins from chick embryos and
analyzed proteins, which eluted from a gelatin Sepharose
column and were subsequently subjected to velocity sed-
imentation on a sucrose gradient (
Figure 6.3
).
18
These are
soluble rER proteins that have direct or indirect affinity
to denatured collagen and therefore are likely involved
in procollagen biosynthesis. There is a good reason for
reproducing this figure in our chapter: during the last
six years virtually every protein indicated on this gel
was associated with severe recessive forms of OI. The
first protein in this cohort was cartilage associated pro-
tein (CRTAP), which is one of the three components of
the prolyl 3-hydroxylase 1 (P3H1)
/
CRTAP
/
cyclophilin
B (CypB) complex responsible for 3-hydroxylation of
type I collagen. Discovery of the prolyl 3-hydroxylase
1 complex introduced a revolutionary new vision of a
previously well-studied condition, autosomal dominant
OI with
COL1A1
and
COL1A2
gene mutations. In this
Thus numerous molecules assisting procollagen syn-
thesis are present in the rER.
39-42
These helper molecules
can be roughly separated into two classes serving the
function of post-translational modification enzymes and
molecular chaperones. Molecular chaperones include
general chaperones, protein disulfide isomerase, disul-
fide reductase, disulfide oxidase, peptidyl-prolyl cis-
trans isomerase, and molecules involved in transport,
translocation, degradation and disaggregation. Post-
translational modifications are proline 4-hydroxylation,
proline 3-hydroxylation, lysine hydroxylation,
43,44
and
