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In Depth Tutorials and Information
P3H1
FKBP65
P4H
HSP47
PDI
CRTA P
KD30
Cyp B
FKBP13
KD10
25
27
29
7
13
15
23
Fr #
5
9
17
19
21
11
S value
8.1
7.2
6.4
5.5
4.7
3.8
3.0
2.1
FIGURE 6.3
Proteins (derived from chick rER extracts) eluted from the gelatin Sepharose column were labeled with [
14
C]-formaldehyde and
subjected to velocity sedimentation on a sucrose gradient (50 mM Tris, pH 7.5, containing 150 mM NaCl and 5-20% sucrose). The sucrose gradi-
ent was fractionated and the samples were analyzed on SDS-polyacrylamide gels. Proteins were then detected and quantified by fluorography.
The sample was run on a 5-20% sucrose gradient at 4°C in PBS. All the known proteins are labeled on the left-hand side and consecutive frac-
tions of the sucrose gradient were loaded onto the gel. Sedimentation is from right to left. The most distant sedimenting peak is the tetramer of
4-proline hydroxylase (P4H and PDI) centered at fraction 10 (with an
s
-value of 8.1S). P3H1 sediments to fraction 12
/
13. In these fractions, we
also find the much smaller cyclophilin B (CYPB) and CRTAP. If these proteins were sedimenting alone they would be found in much higher
fractions. Indeed, free Cyp B is found centered in fraction 22
/
23. Also, the free PDI (not part of the tetramer of P4H) sediments to fraction 18
/
19.
These data show that P3H1 is complexed with cyclophilin B and CRTAP. If this complex is in a 1:1:1 ratio of proteins, the expected molecular
weight would be around 155 kDa.
collagen folding.
59
We suggest that the complex binds at
the junction of unfolded and folded collagen triple helix
since it shows affinity for both denatured and folded col-
lagen. Once the triple helix is folded, the P3H1
/
CRTAP
/
CypB complex might be replaced by another chaperone
Hsp47 or FKBP65 to stabilize the triple helix and prevent
premature fibril formation (see
Figure 6.1
).
chapter we aim to summarize what is known about the
role of each protein in OI and discuss possible mecha-
nisms involved in disease development.
DISCOVERY OF THE P3H1
/
CRTAP
/
CYPB
COMPLEX
While 4-hydroxyproline (4-Hyp) occurs at almost
every Yaa position proline in collagens the appear-
ance of the 3-hydroxyproline (3-Hyp) is a rare event.
However, 3-Hyp is found in almost all types of collagens
in a -Gly-3Hyp-4Hyp-Gly- sequence.
54,55
Modification
of proline to 3-hydroxyproline is accomplished by pro-
lyl 3-hydroxylases (P3Hs), enzymes, distinct from prolyl
4-hydroxylase.
56,57
The first partial purification of the prolyl 3-hydroxylase
was reported in 1979.
58
An activity of the enzyme eluted
at 160 kDa on a molecular sieve column was demon-
strated. In 2004, we identified the prolyl 3-hydroxylase
family of enzymes.
24
P3H1 is an 85 kDa protein, but it
forms a complex with two other proteins. These pro-
teins have been identified as cartilage associated protein
(CRTAP), the function of which is unknown, and peptidyl-
prolyl cis-trans isomerase cyclophilin B (CypB).
18,24
The
P3H1
/
CRTAP
/
CypB complex has a 1:1:1 stoichiometry
which results in a 155 kDa complex.
18,24
Thus, the original
appearance of the prolyl 3-hydroxylase as a 160 kDa pro-
tein was justified. The CypB within the complex exhib-
ited peptidyl-prolyl cis-trans isomerase activity and the
complex was shown to have chaperone activity during
ROLE OF THE PROLYL 3-HYDROXYLASE 1
COMPLEX IN OI
In 2006 Morello et al. demonstrated that mutations in
CRTAP cause recessive OI and that the CRTAP knockout
mice display osteochondrodysplasia characterized by
severe osteoporosis and decreased osteoid production.
18
These results gave rise to a new era in understanding
of the pathogenesis of recessive OI. It is now clear that
mutations, not only in the type I collagen chains but also
in enzymatic proteins responsible for collagen modifica-
tion as well as in chaperone proteins, can lead to OI.
After the initial CRTAP publication,
18
first reports
that alterations in
LEPRE1
(gene for P3H1)
17
as well
as in CypB
13
cause very similar phenotypes followed
quickly. The crucial role of the complex components in
bone development was recognized.
60
Up-to-date clinical
cases and mouse models involving CRTAP, P3H1 and
CypB keep accumulating (
https:
//
oi.gene.le.ac.uk
/
home.
mutations in every component of the complex and pos-
sible mechanisms leading to the disease.
