what-when-how
In Depth Tutorials and Information
Covering the Gap between Genotype and
Phenotype
To approach development of new therapies for
recessive OI treatment we first need to understand the
mechanisms which link the mutations to the clinical
phenotypes. Therefore, it is extremely important to per-
form biochemical studies in each case. However, it is not
always possible to analyze human cases in substantial
detail. Therefore mouse models are of great importance.
During the past few years mouse models with knock-
outs in CRTAP, P3H1 and CypB have been developed
and investigated.
11,15,18
The CRTAP, P3H1 and CypB null
mice demonstrated similar phenotypes. Comparison
of the three mouse phenotypes combined with data
obtained from the patient's samples provide the basis for
understanding the underlying molecular mechanisms.
about a compound heterozygous mutation in P3H1
which resulted in the removal of the ER retention sig-
nal sequence (KDEL) in which Pro986 of the α1 chain of
type I collagen was still 85% 3-hydroxylated.
3
However,
in this case, the truncated form of P3H1 presumably
still has functional enzymatic activity.
Similarly to the mouse models, patients with muta-
tions in CypB tend to have slightly more 3-Hyp at Pro986
compared to those with mutations in CRTAP and P3H1.
13
However, one report presented a case of a human CypB
mutation where Pro986 of type I collagen was fully
3-hydroxylated.
63
Two proteins in the complex, P3H1 and CRTAP, have
been suggested to mutually stabilize each other.
64
In con-
trast, CypB can be stable on its own
65
and can perform
its isomerase activity in the absence of both partner pro-
teins.
66,67
Moreover, CypB participates in several other ER
multiprotein complexes
68-70
where neither CRTAP nor
P3H1 have been identified. A recent study by our group
showed that mutation in CypB alters its interaction with
the P-domain of calreticulin and with lysyl hydroxylase
1 (LH1).
71
Also CypB was implicated in performing mul-
tiple diverse functions unrelated to collagens such as
immunosuppression,
72
chemotaxis,
73
hepatitis C virus
replication
74
and prolactin signaling.
75
Combining all
existing knowledge about CypB, the range of expected
phenotypes resulting from mutations in this protein
may be far beyond OI; however, the major phenotype
observed in CypB null mice is a bone phenotype.
Summarizing all cases it is clear that the ability of the
complex to accomplish 3-hydroxylation is substantially
affected regardless of the mutated gene (P3H1, CRTAP
or CypB).
What Happens to Type I Collagen when the
P3H1 Complex is not Working Properly?
One of the important questions that needs to be
answered in order to understand the mechanism underly-
ing the OI phenotype is: Is the observed phenotype due to
the lack of the 3-hydroxyl group or due to the absence of
chaperone activity of the P3H1
/
CRTAP
/
CypB complex or
both? Currently there is no short and certain answer to this
question. Existing data allow speculations either way and
this has been discussed in the literature.
2
Some researchers
believe that the higher residual 3-hydroxylation of Pro986
in the α1 chain of type I collagen in individuals with CypB
mutations plus the involvement of CypB in different func-
tions indicates that the role of the chaperone activity is
more important.
13
Others, however, suggest a possible
direct function of the 3-hydroxyl group in association with
collagen triple helices, which makes lack of the 3-Hyp the
dominant factor.
61
To consider this question in more detail
we need to understand the consequences of the chaperone
activity of the complex and the function of the 3-hydroxyl
group in type I collagen. Here we will first discuss the
consequences for type I collagen in the case of complex
malfunction.
THE EXTENT OF TYPE I COLLAGEN
MODIFICATION WHEN COMPLEX
C
OMPONENTS ARE ALTERE
D
In most cases with alterations in the complex com-
ponents (with the exception of one
63
), type I collagen
chains, when run on an SDS polyacrylamide gel, dem-
onstrated characteristic shifts towards higher molecular
weight compared to the wild-type controls.
3,11,15,18
These
shifts are consistent with an overmodification of the col-
lagen chains and have been observed for a number of
cases in dominant forms of OI as well.
76-78
The Amount of 3-Hydroxyproline in Type I
Collagen is Generally Greatly Reduced when
Components of the Complex are Missing or
Mutated
The only fully 3-hydroxylated site in the α1 chain
of type I collagen, Pro986, has been shown to lack the
3-hydroxyl group in the CRTAP and P3H1 knock-
out mice.
11,18
Residual 3-Hyp was detected at Pro986
in the CypB null mouse.
15
In patients the amount of
3-Hyp at this site is usually decreased but is not nec-
essarily absent.
10,12,62
There is one very recent report
Type I Collagen is Overmodified in Cases of
P3H1 Mutations
Detailed amino acid analysis using base hydrolysis
confirmed that P3H1 null collagen (extracted from several
tissues of the P3H1 knockout mice) was overmodified
with galactosyl- and glucosylgalactosyl-hydroxylysine
