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binding of Hsp47 to its folded, native conformation.
22
In vivo
studies have shown that Hsp47 knockout mice
only survive to 10.5 days post coitum and display a lack
of proper collagen folding.
110
In the absence of Hsp47
secretion of type I and type IV collagens was delayed in
mice null cells
20,111
and these collagens accumulated in
the dilated ER. Secreted collagen formed thin, frequently
branched fibrils and was found to be susceptible to tryp-
sin
/
chymotrypsin digestion.
20,110
All accumulated data
clearly demonstrates that Hsp47 is indispensable for the
proper folding of collagen triple helix.
Increased production of Hsp47 in OI patient cells
with collagen mutations was noted more than 10 years
ago.
112
Recently, it was first demonstrated in dachs-
hunds
100
and then in humans
101
that missense mutations
in the
SERPINH1
gene (which codes for the Hsp47 pro-
tein) cause autosomal recessive OI. In both documented
cases an autosomal-recessive missense mutation resulted
in the substitution of a single amino acid in a conserved
region (c.977C>T, p.Leu326Pro in dogs and c.233T>C,
p.Leu78Pro in humans). Homology modeling of the
mutant Hsp47 suggested that Leu326Pro substitution
might alter the alpha-helical region of the protein affect-
ing the overall conformation of Hsp47.
100
No studies on
collagen modification or secretion were done in this case.
In the human mutation, the amount of Hsp47 pro-
tein was greatly reduced in cells and the protein stability
was altered.
101
Type I collagen synthesized by the human
patient cells was analyzed and found to migrate normally
on SDS polyacrylamide gel electrophoresis.
101
Based on
gel migration, the authors suggested that collagen modi-
fication is normal. However, no amino acid analysis was
performed to quantitate the extent of hydroxylysines and
sugars. Pulse-chase experiments demonstrated delayed
secretion of type I collagen compared to control. The
3-hydroxyl group at Pro986 was in place suggesting the
functional mechanism of Hsp47 to be independent of that
of the P3H1
/
CRTAP
/
CypB complex. The authors specu-
lated that the OI causing mutation accelerated transit of
type I procollagen from the ER to the Golgi, but slightly
slowed the overall transit time from inside the cell to the
extracellular environment. They concluded that Hsp47
monitors proper triple helix formation on the boundary of
ER and Golgi and therefore acts downstream of the pro-
lyl 3-hydroxylase complex. The exact mechanism causing
OI remains unknown. Quantitative evaluation of the type
I collagen modifications would provide more information
on the effect of the delayed secretion on collagen quality.
Another hypothesis is that the 3-hydroxylation site may
represent the binding substrate for other molecules that are
important in collagen fibrillogenesis such as small leucine-
rich proteoglycans (SLRPs).
86
Asporin, biglycan and deco-
rin are likely candidates. Knockout mice deficient in two
of these proteins showed phenotypes that overlap with the
mouse phenotypes of the P3H1, CRTAP and CypB nulls.
Weak disorganized tendon collagen fibrils were observed
in both biglycan and decorin null mice.
86-89
The tendon
phenotype of the biglycan
/
ibromodulin double null
mice
90
is also similar to that of the P3H1 null mouse.
11
In
addition, biglycan-deficient mice demonstrated low bone
mass.
88
Moreover, it was shown that decorin binds near the
C-terminus of native type I collagen and close to the region
located within the cyanogen bromide peptide fragment
α1(I) CB6.
91
The main 3-hydroxylation site of type I colla-
gen at Pro986 is also located in CB6.
61
To date no studies
describing SLRPs binding to non-3-hydroxylated collagen
derived from the deficiency in any of the complex compo-
nents have been published. However, this is an attractive
hypothesis as SLRPs were shown to be involved in the lat-
eral growth of tendon fibrils.92
92
THE ROLE OF COLLAGEN CHAPERONES
IN OI
The heat shock protein 47 (Hsp47)
93
and the rough
endoplasmic reticulum-resident FK506-binding pro-
tein (FKBP65)
94
are two more proteins eluted from the
gelatin Sepharose (
Figure 6.3
). Both of them were previ-
ously described as probable collagen chaperones.
71,95-99
Recently it has been demonstrated that mutations in each
of these proteins cause severe recessive OI.
4,8,100-103
Hsp47
Hsp47 was first identified in chick embryos as a 47 kDa
collagen-binding protein resident in the ER.
104
Most mem-
bers of the heat shock protein family function as molecular
chaperones.
105
Misfolded proteins aggregate and do not
get transported from the endoplasmic reticulum to their
final destinations. Instead such aggregates are stored in
the ER causing “ER stress.” Chaperones are normally acti-
vated in response to ER stress.
106
Hsp47 is unique in the
way that its expression is dramatically enhanced by heat
shock
104
but not by ER stress. The molecular function of
Hsp47 has been extensively studied.
107
The expression
pattern of Hsp47 closely follows that of collagen.
98
It has
been well characterized as a collagen-specific chaperone.
95
Unlike other chaperones, Hsp47 preferentially binds to the
folded collagen triple helix. This was first suggested by
in vitro
studies with collagen-like peptides.
108,109
Based on
the measured binding constants it was argued that fold-
ing of the triple helix requires stabilization by preferential
FKBP65
FKBP65, the 65 kDa FK506-binding protein, belongs to
the family of immunophilins
113
and resides in the ER.
114
Similar to the CypB, FKBP65 is a peptidylprolyl cis-trans
isomerase.
115
However, the enzymatic activity of FKBP65
