Generalized Connective Tissue Disease in Crtap-/- Mouse
Dustin Baldridge1, Jennifer Lennington1, MaryAnn Weis2, Erica P. Homan1, Ming-Ming Jiang1, Elda
Munivez1, Douglas R. Keene3, William R. Hogue4, Shawna Pyott5, Peter H. Byers5,6, Deborah Krakow7,
Daniel H. Cohn7, David R. Eyre2, Brendan Lee1,8*, Roy Morello1¤
1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America, 2Department of Orthopaedics and Sports
Medicine, University of Washington, Seattle, Washington, United States of America, 3Shriners Hospitals for Children, Portland, Oregon, United States of America, 4Center
for Orthopaedic Research, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America, 5Department of Pathology, University of
Washington, Seattle, Washington, United States of America, 6Department of Medicine, University of Washington, Seattle, Washington, United States of America, 7Medical
Genetics Institute, Cedars-Sinai Medical Center, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America,
8Howard Hughes Medical Institute, Houston, Texas, United States of America
Mutations in CRTAP (coding for cartilage-associated protein), LEPRE1 (coding for prolyl 3-hydroxylase 1 [P3H1]) or PPIB
(coding for Cyclophilin B [CYPB]) cause recessive forms of osteogenesis imperfecta and loss or decrease of type I collagen
prolyl 3-hydroxylation. A comprehensive analysis of the phenotype of the Crtap-/- mice revealed multiple abnormalities of
connective tissue, including in the lungs, kidneys, and skin, consistent with systemic dysregulation of collagen homeostasis
within the extracellular matrix. Both Crtap-/- lung and kidney glomeruli showed increased cellular proliferation.
Histologically, the lungs showed increased alveolar spacing, while the kidneys showed evidence of segmental
glomerulosclerosis, with abnormal collagen deposition. The Crtap-/- skin had decreased mechanical integrity. In addition
to the expected loss of proline 986 3-hydroxylation in a1(I) and a1(II) chains, there was also loss of 3Hyp at proline 986 in
a2(V) chains. In contrast, at two of the known 3Hyp sites in a1(IV) chains from Crtap-/- kidneys there were normal levels of 3-
hydroxylation. On a cellular level, loss of CRTAP in human OI fibroblasts led to a secondary loss of P3H1, and vice versa.
These data suggest that both CRTAP and P3H1 are required to maintain a stable complex that 3-hydroxylates canonical
proline sites within clade A (types I, II, and V) collagen chains. Loss of this activity leads to a multi-systemic connective tissue
disease that affects bone, cartilage, lung, kidney, and skin.
Citation: Baldridge D, Lennington J, Weis M, Homan EP, Jiang M-M, et al. (2010) Generalized Connective Tissue Disease in Crtap-/- Mouse. PLoS ONE 5(5): e10560.
Editor: Christoph Winkler, National University of Singapore, Singapore
Received December 13, 2009; Accepted April 15, 2010; Published May 11, 2010
Copyright: ? 2010 Baldridge et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH grants AR051459 (RM), HD022657 (BL, DHC, DK, DRE), DE1771 (BL), AR36794 (DRE), the Baylor College of Medicine
IDDRC HD024064 (BL), the Osteogenesis Imperfecta Foundation (RM, SP, PHB), the Rolanette and Berdon Lawrence Bone Disease Program of Texas (BL, RM), and
the Freudmann Fund at the University of Washington. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤ Current address: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America
The Crtap gene encodes cartilage-associated protein (CRTAP), a
resident protein of the rough endoplasmic reticulum (rER) that
can form a trimeric complex with prolyl 3-hydroxylase 1 (P3H1,
also known as Leprecan1 and encoded by LEPRE1) and
Cyclophilin B (CYPB, encoded by PPIB) . The complex is
responsible for the 3-hydroxylation of specific prolyl residues
(Pro986) in the proa1 chains of both type I and II procollagen .
This enzymatic modification is catalyzed by the Fe++and 2-
oxoglutarate-dependent dioxygenase domain which is present at
the C-terminal portion of P3H1 . Although CRTAP is not
enzymatically active, it is required in vivo for proper collagen prolyl
3-hydroxylation to take place . Cyclophilin B, the other
member of the complex, has peptidyl-prolyl cis-trans isomerase
(PPIase) activity and is thought to facilitate the molecular winding
of the collagen triple helix. Importantly, recent studies have
demonstrated that the CRTAP/P3H1/CYPB complex also has
chaperone activity in the rER . The trimeric complex was
shown to be active in two independent chaperone assays, to have
PPIase activity and, like HSP47, to interact with folded triple-
helical collagen, perhaps to inhibit intracellular collagen fibril
Mice lacking both copies of the Crtap gene have a severe
osteochondrodysplasia with rhizomelia and osteoporosis. At the
tissue level, Crtap-/- mice have normal numbers of osteoblasts that
deposit very little osteoid. Collagen fibrillogenesis is affected in that
there is an increased diameter of collagen fibrils in the skin .
The phenotype of the Crtap-/- mice led to the identification of
CRTAP mutations in patients with recessively inherited forms of
osteogenesis imperfecta (OI). The severity of this form of OI
disease varies based upon the nature of the CRTAP mutations
[1,4]. Subsequently, mutations in the LEPRE1 gene, that encodes
prolyl 3-hydroxylase 1, the second component of the rER-resident
complex, were identified in patients who had no mutations in type
I collagen genes or CRTAP . Several reports followed describing
additional novel CRTAP, LEPRE1, and now also PPIB mutations
in patients with severe recessive forms of OI from different parts of
PLoS ONE | www.plosone.org1 May 2010 | Volume 5 | Issue 5 | e10560
the world [6,7,8,9,10]. The majority of CRTAP or LEPRE1
reported mutations are null alleles associated with severe
phenotypes. There are only a handful of patients, most with
missense mutations, who survive childhood.
OI, whether dominant or recessive, is a generalized connective
tissue disorder which can present with a variety of signs that
include early osteoporosis, dentinogenesis imperfecta, hearing loss,
blue sclerae, scoliosis, ligament and skin laxity, and growth
deficiency [11,12]. All affected tissues express high levels of type I
collagen. Some features of OI, such as abnormal pulmonary
function, have been explained as a consequence of multiple rib
fractures and/or orthopedic complications of the spine (scoliosis,
kyphosis and vertebral compressions) that lead to poor pulmonary
ventilation and cause a progressive decrease in cardio-respiratory
fitness . However, the involvement of extra-skeletal tissues in
the OI disease process could also be explained by nonstructural
functions played by type I collagen in these organs. Alternatively,
especially in cases of recessive forms of OI with mutations in
members of the prolyl 3-hydroxylation complex, other collagen
types may not be properly processed. Basement membrane
collagens are more heavily modified by prolyl 3-hydroxylation
and decreased hydroxylation could in theory lead to a multi-
systemic phenotype. To explore these hypotheses, we conducted a
thorough histological evaluation of extra-skeletal tissues in Crtap-/-
mice. We have identified abnormalities affecting the lung, kidney,
and skin to provide a more complete understanding of the
pathophysiology of recessive OI that should guide a rational
clinical assessment of these patients and may identify alternative
Crtap expression in extra-skeletal tissues
Based on Northern blot analysis, we showed that Crtap is
expressed in most murine tissues  and characterized the
distribution of the protein in all components of the skeleton .
Here we extended these studies to non-skeletal tissues including
those that do not express high levels of fibrillar collagens. We
identified Crtap expression in postnatal lung, kidney and skin from
wildtype mice. CRTAP was found throughout the lung paren-
chyma principally in all pneumocytes (Figure 1B–C). In the
metanephric kidney, CRTAP was found in both visceral
(podocytes) and parietal epithelial cells of the glomerulus. In
addition, throughout the kidney, including the pelvis, there was
interstitial staining which appears to localize to peritubular
capillaries (Figure 1E–F, H–I). In skin there was generalized
staining of the dermal fibroblasts and within blood vessels
(Figure 1K–L). The localization of the CRTAP protein in
multiple tissues extends the previous mRNA expression studies,
identifies the cells in which the protein is produced and,
unexpectedly, showed that CRTAP is present in tissues not
particularly rich in types I or type II collagen. This last finding, in
combination with the previously described broad expression
patterns of P3H1 and CYPB [2,15,16], suggests that CRTAP,
within the described ternary complex, may play a role in the prolyl
3-hydroxylation of other types of collagens. At the same time, we
cannot exclude novel functions of CRTAP which could be
unrelated to collagen prolyl 3-hydroxylation in these tissues.
Tissue alterations in Crtap-/- mice
To identify the effects of loss of the protein, we evaluated target
tissues in Crtap-/- mice at neonatal (P10) and adult time points (5–
9 month-old). In Crtap-/- lungs, there was a diffuse increase in
alveolar airway space often accompanied by a thinning of the
alveolar walls (Figure 2A–C). This was already visible at P10 and
became more obvious in the adult lung, as assessed by an
increased Mean Linear Intercept (MLI) (Figure 2G). In the
kidney we saw no differences at P10 (data not shown). However, in
the adult Crtap-/- kidney we found that some glomeruli had
abnormal PAS and picrosirius red staining compared with WT
controls (Figure 2H–K). The picrosirius red, specific for collagen,
revealed areas of intense staining within some glomeruli, consistent
with a focal glomerulosclerosis that were never observed in WT
kidney sections. These alterations were correlated with mesangial
cell hyperplasia and with collagen fibril deposition as seen by
transmission EM (Figure 2L–M and inset). The skin of Crtap-/-
mice was notably lax, a property noted when handling the animals
(data not shown). The skin was thin, the layers were disorganized,
and collagen fibrils abnormal (Figure 3A–D). The decreased
thickness of the skin of adult mice reflected a dramatic decrease in
dermal thickness rather than in the adipose layer (Figure 3 E-F-).
The mechanical properties of the skin were also examined by
using a load-to-failure technique. Consistent with the gross laxity
and the decreased thickness, the Crtap-/- skin tolerated signifi-
cantly less load and was less stiff than the wildtype skin (Figure 3
G–H). It is known that defects in extracellular matrix proteins such
as type I collagen are correlated with changes in the proliferation
and survival of surrounding cells . Therefore, to determine if
there is an altered cellular phenotype which might result from
and/or contribute to the observed histological alterations, we
studied cellular proliferation and apoptosis in the affected tissues at
P10. While there was no statistical difference in the number of
BrdU positive cells observed in the skin (data not shown), the lungs
of Crtap-/- mice showed a statistically significant increase of BrdU
positive cells compared to WT controls (N=5 mice, p,0.003)
(Figure 4A–C). In P10 Crtap-/- kidney, there were similar
numbers of BrdU positive cells in the tubular compartment;
however, the number of BrdU positive cells within the glomerular
compartment was increased in the Crtap-/- mice compared to WT
controls (N=5 mice, p,0.04) (Figure 4D–F). There was no
difference in apoptosis between the tissues from the two genotypes
(data not shown). These data show that the altered histological
phenotype in Crtap-/- lung and kidney was correlated with
evidence of increased cellular proliferation, which may be a result
of disrupted extracellular matrix regulation. Alternatively, the
abnormal increase in cell proliferation in these tissues could be
indicative of defective terminal differentiation and synthesis of
extracellular matrix proteins such as type I collagen.
Collagen prolyl 3-hydroxylation in Crtap-/- tissues
One of the consequences of loss of Crtap is the near complete
absence of prolyl 3-hydroxylation at proline 986 in a1(I) and a1(II)
chains of type I and II collagens . While this proline is 95% 3-
hydroxylated in a1(I) and a1(II) chains from WT mice, it is ,1%
3-hydroxylated in Crtap-/- mice. The homologous proline at P986
in the a2(V) chain of type V collagen from bone and skin was 98–
100% 3-hydroxylated in the WT, but ,1% from bone and skin in
Crtap-/- mice (Figure 5A–B), indicating that type V procollagen is
a substrate for the CRTAP complex. Of note, the a1(I) chain from
the kidney also lacked 3-Hyp at P986 (results not shown).
Type IV collagen is known to have a high level of prolyl 3-
hydroxylation (10–15% of residues) in normal tissues. At two sites
in the proa1(IV) chain, known to be highly 3-hydroxylated in
bovine tissues , the hydroxylation of the target prolyl residues
in type IV collagen from kidney in WT and Crtap-/- mice were
similar (Figure 5C). These findings indicate that prolyl 3-
hydroxylation of some collagens does not depend on the presence
of CRTAP or is carried out by an independent system.
Crtap Extraskeletal Phenotype
PLoS ONE | www.plosone.org2 May 2010 | Volume 5 | Issue 5 | e10560
Coordinated loss of both CRTAP and P3H1 proteins when
either gene is mutant in human primary fibroblasts
Because P3H1 enzymatic activity requires the presence of
CRTAP in both mice and humans , and a similar clinical
phenotype is seen in patients with mutations in either CRTAP or
LEPRE1 [4,5,6,7,8,9], we searched for a common mechanism of
disease caused by mutations in the two human genes. CRTAP
(Figure S1A) and P3H1 (Figure S1C) are both localized to the
perinuclear region of the cell in normal primary human fibroblasts
consistent with localization in the rough endoplasmic reticulum, as
previously reported .
CRTAP protein was not detectable in cultured human
fibroblasts derived from a patient with a homozygous frameshift
mutation in CRTAP, c.24_31del  that leads to nonsense
mediated decay of the mRNA (Figure S1E). P3H1 protein was
also lost (Figure S1G), without changes in P3H1 mRNA level
(data not shown). The expected loss of P3H1 protein was seen in
fibroblasts from a patient with a homozygous frameshift mutation
in LEPRE1, c.232delC, which results in loss of P3H1 mRNA 
(Figure S1K) and was accompanied by loss of the CRTAP
protein (Figure S1I); no effects were noted on CRTAP mRNA
level (data not shown). These findings indicate that CRTAP and
P3H1 are each essential components of the prolyl 3-hydroxylation
complex, and that they interact for mutual stabilization.
Our recent studies and those from other groups highlighted the
important role that CRTAP plays in bone mass homeostasis and
recessive forms of OI [1,4,6,9]. Although the usual clinical features
that bring individuals with OI to clinical attention are fractures or
other evidence of bone fragility, OI has many features of a multi-
Figure 1. Immunofluorescence staining of CRTAP protein in wild-type mouse tissues. (A) Lung H&E stained section. (B) CRTAP protein
expression in all pneumocytes. (C) Merge of CRTAP with DAPI. (D) Kidney medulla H&E stained section. (E) CRTAP protein seems to localize to
peritubular capillaries. (F) Merge of CRTAP with DAPI. (G) H&E stained section of kidney glomeruli. (H) CRTAP protein is in visceral and parietal
epithelial cells of the glomerulus. (I) Merge of CRTAP with DAPI. (J) Skin H&E stained section, including hair follicle. (K) CRTAP protein is seen as
intense foci in fibroblasts distributed throughout the dermis. Epidermal and follicular staining is consistent with background staining seen in Crtap-/-
skin stained with anti-CRTAP antibody (data not shown). (L) Merge of CRTAP with DAPI. All pictures are at 406magnification, and scale bars are 50
micrometers. Specificity of staining was confirmed in all cases by using the same CRTAP antibody onto Crtap-/- tissue sections (data not shown).
Crtap Extraskeletal Phenotype
PLoS ONE | www.plosone.org3 May 2010 | Volume 5 | Issue 5 | e10560
systemic disorder, and there is widespread expression of the major
genes involved that express the two chains of type I procollagen.
While bone, cartilage, tendon, and skin are the classical tissues
affected in connective tissue disease, other organs, including lung
and kidney, have a significant stromal component containing
collagen fibrils and hence may also be affected. To gain additional
insight into the pathophysiology of OI, we exploited the mouse
model of inactivation of Crtap, a component of the prolyl 3-
hydroxylation system important for type I procollagen processing.
We found that CRTAP is produced in many extra-skeletal tissues
that do not express very high levels of type I collagen, such as the
lung and kidney. Most of these tissues analyzed from the Crtap-/-
mice had mild alterations and an increase in cell proliferation,
even with virtually complete lack of 3-hydroxylation of Pro 986 in
chains of type I and II collagens, as previously described. The
prolyl residue at the same position of the triple helical domain in
the proa2(I) chain of type V collagen (encoded by COL5A2, a
member of the same evolutionary clade as the type I and type II
collagen genes) was similarly not hydroxylated. These data
demonstrate both that CRTAP functions in tissues other than
the skeleton and targets additional substrates that contribute to the
Type V collagen is a heterotrimeric, minor component of all
type I collagen fibrils that contains several 3-hydroproline residues
and helps regulate fibrillogenesis and fibril diameter of type I
collagen [19,20]. It is possible that the lack of 3-hydroxylation of
this proline in type V collagen could contribute to the increased
diameter of type I collagen fibrils observed in Crtap-/- mice .
Moreover, type V collagen is required to initiate collagen fibril
assembly , and alteration of type V collagen could help
mediate the striking decrease in osteoid volume in Crtap-/- mice
. Finally, mutations in the COL5A1 and COL5A2 genes that
encode the chains of type V collagen cause Ehlers-Danlos
syndrome types I and II [22,23], which are characterized, among
other defects, by skin laxity, a feature of the Crtap-/- mice. Skin
laxity has also been observed in some patients with autosomal
dominant OI forms resulting from type I procollagen gene
mutations, and is a common finding in several connective tissue
disorders . Thus, the overlapping OI/Ehlers-Danlos features
observed in Crtap-/- mice and in some patients with recessive
Figure 2. Lung and kidney abnormalities in Crtap-/- mice. Mutant lung H&E stained sections at P10, at 106 magnification (A) and 406
magnification (B), and in the adult at 406 magnification (C). Wildtype lung H&E stained sections at P10, at 106 magnification (D) and 406
magnification (E), and in the adult at 406 magnification (F). (G) Quantification of alveolar airway space by Mean Linear Intercept (MLI) shows
increased airway space in Crtap-/- mouse lungs. Mutant adult kidney stained sections shows that some glomeruli have abnormal staining by PAS (H)
and picrosirius red (I) at 406magnification, suggestive of focal glomerulosclerosis. These changes were never observed in corresponding wildtype
sections (J–K). Transmission EM images of Crtap-/- and WT glomeruli (L–M, scale bar =2 mm) showing mesangial hyperplasia and abnormal collagen
fibril deposition in the mutant (arrows and inset, scale bar in inset is 0.5 mm).
Crtap Extraskeletal Phenotype
PLoS ONE | www.plosone.org4 May 2010 | Volume 5 | Issue 5 | e10560
forms of OI could be explained by the extended substrate for
CRTAP and effects on prolyl 3-hydroxylation in collagens in
addition to types I and II. Because prolyl 3-hydroxylation of chains
of type IV collagen was normal, it is likely to be a substrate for a
different hydroxylase (at least three are known in both mice and
humans) and does not appear to depend on the presence of
CRTAP. The morphological changes in the kidney therefore may
instead be caused by abnormal collagen I and/or V expression or
modification, although this remains to be proven. Additional
functions of CRTAP cannot be excluded, unrelated to prolyl 3-
Figure 3. Skin defects in Crtap-/- mice. (A) Adult Crtap-/- skin H&E section at 20X shows thinning of the dermis relative to wildtype (C). (B) Adult
Crtap-/- skin picrosirius red section at 20X shows disorganization of tissue layers and ECM in contrast to wildtype (D). Quantification of adult total skin
thickness (E), measured from epidermis to bottom of adipose layer, and adult dermis thickness (F) demonstrates thinning of Crtap-/- skin. Significant
decrease in measurements of skin peak load (G) and stiffness (H) in Crtap-/- skin compared to wild-type controls.
Crtap Extraskeletal Phenotype
PLoS ONE | www.plosone.org5 May 2010 | Volume 5 | Issue 5 | e10560
hydroxylation, which may contribute to the tissue defects
Our results have important clinical implications for patients
with recessive OI caused by mutations in CRTAP or LEPRE1. The
absence of both encoded proteins in patient fibroblasts when either
the CRTAP or LEPRE1 gene has a null mutation, most likely
explains the overlapping skeletal phenotype seen in individuals
with recessive OI that results from mutations in these genes
[1,4,5,6,7,8,9]. It remains to be seen if these patients also have
overlapping non-skeletal phenotypes, as it is unknown if CRTAP
and P3H1 have functions that are dependent on one another in
other tissues such as the lung and kidney. Reciprocal CRTAP/
P3H1 co-stabilization has now been described by others and
shown to be independent of CYPB expression [10,25].
The phenotypes described in the Crtap-/- mouse lung, kidney,
and skin may present as subclinical phenotypes in the human
Figure 4. Crtap-/- mice have increased cell proliferation in lung and kidney. BrdU staining of Crtap-/- (A) and wildtype (B) lung at P10 and
206magnification, n=5. (C) Quantification of BrdU positive cells per field shows a significantly increased number of dividing cells in Crtap-/- lung
compared to wildtype. BrdU staining of Crtap-/- (D) and wildtype (E) kidney at P10 and 206magnification. (F) Quantification of BrdU positive cells
per glomerulus shows increased number of dividing cells in Crtap-/- glomeruli compared to wildtype.
Crtap Extraskeletal Phenotype
PLoS ONE | www.plosone.org6 May 2010 | Volume 5 | Issue 5 | e10560
patients or, given the marked severity of the bony phenotype in
most, may not have been studied. Regardless, our findings
emphasize the importance of thorough monitoring of OI patients
for non-skeletal consequences of their connective tissue disease.
For instance, renal abnormalities are reported in OI patients that
may not be detected without monitoring. In one series, 17 out of
47 individuals with OI had persistent hypercalciuria, correlating
with severity of the OI, with one patient having isolated
microscopic hematuria ; in a separate study 4 out of 58 OI
children were found with nephrolithiasis . It is likely that the
large majority of these OI cases are due to mutations in the type I
collagen genes, but it is unclear if the OI caused by CRTAP,
LEPRE1, or PPIB mutations may also have renal abnormalities.
Furthermore, it must be acknowledged that the hypercalciuria
may be secondary to abnormal bone mineral metabolism,
although our results suggest that a primary kidney defect should
also be considered. Regardless, because individuals with OI are
often supplemented with vitamin D and calcium, which are known
to increase calcium flux in the kidney, it is especially important for
the treating physician to be attentive to any renal complications.
Glomerulopathy has been observed in another mouse model of
osteogenesis imperfecta, the homotrimeric a1(I) collagen oim/oim
mouse , suggesting that distinct molecular abnormalities in OI
can result in a similar skeletal and extraskeletal phenotype. The
subtle nature of this abnormality is confirmed by the absence of
proteinuria by dipstick analysis in both our Crtap-/- mice (data not
shown) and in the OI patients with hypercalciuria , as well as
the normal levels of serum and urinary calcium, phosphorous, and
magnesium previously reported in the Crtap-/- mice .
The most common causes of death in individuals with severe OI
are respiratory problems, including pneumonia . After
exclusion of type II perinatal lethal OI, a pathological evaluation
of the cause of death in 82% of the more severe OI type III and
39% of milder OI type I and OI type IV was considered
respiratory . Increasing scoliosis correlates with increasing
restrictive lung disease . However, the authors of that study
note that ‘‘the presence of more severe restrictive lung disease with
relatively smaller curve magnitudes in the population with OI
indicates the possibility of intrinsic pulmonary abnormality’’ .
In addition, there are isolated reports of abnormal collagen in the
lungs of individuals with severe OI [30,31,32]. Therefore, these
data combined with our results suggest that a primary lung defect
in individuals with OI may be a consequence of abnormal collagen
synthesis, in addition to secondary consequences of skeletal
As with other connective tissue diseases, skin laxity and skin
fragility have been observed in some individuals with OI, and it
has long been known that cultured dermal fibroblasts from
individuals with OI make abnormal proteins [33,34]. Even if there
is no obvious clinical skin abnormality, there can be alterations of
the mechanical properties of the skin in OI, similar to that seen in
the Crtap-/- mice . Furthermore, recently described mice that
are null for the Ppib gene, which encodes CYPB, also demonstrate
Figure 5. Tandem mass spectra identifies missing 3Hyp in
Crtap-/- mouse at a2(V) P986. (A) Full scan spectra over the LC
elution window of the tryptic peptide containing P986 from wildtype
and Crtap-/- a2(V) from bone. (B) MS/MS spectra that identify the main
forms of the variably hydroxylated peptides. From wildtype, P986
is almost fully 3-hydroxylated and the earlier P at 978 is not
4-hydroxylated, whereas from Crtap-/- P986 is not 3-hydroxylated and
P978 is about 55% 4-hydroxylated. (The 765.62+ion in A is the version
lacking P978 4Hyp.) (C) Mass spectral analysis of a 3Hyp-containing
peptide from mouse kidney type IV collagen. Two full scan spectra are
shown over the LC elution window of the a1(IV) tryptic peptide of
sequence shown. This peptide from near the C-terminus of the main
triple-helix (residue 1440 from NCBI mouse database) reveals two sites
of 3Hyp that are heavily occupied in both wt and Crtap-/- a1(IV). MS/MS
spectra (not shown) established the identities of the main post-
translational variants for the labeled ions.
Crtap Extraskeletal Phenotype
PLoS ONE | www.plosone.org7 May 2010 | Volume 5 | Issue 5 | e10560
skin laxity as well as weakness, suggesting that loss of components
of the prolyl 3-hydroxylation complex may lead to common skin
findings . In addition, sections of the dermis of mice that are
heterozygous for a null allele of Col1a1 appear strikingly similar to
the Crtap-/- skin sections . These findings suggest that
abnormal skin, albeit subclinical in most cases, may be a part of
the phenotype of both classical OI and OI caused by mutations in
CRTAP or LEPRE1.
In summary, absence of CRTAP in the mouse results in the
pathophysiology of multiple organ systems, and CRTAP is
required at the molecular level for proper prolyl 3-hydroxyation
of several types of fibrillar collagen and for the expression of the
P3H1 protein in humans.
Materials and Methods
All research involving animals was conducted according to
relevant national and international guidelines. Trained veterinar-
ians supervised animal care according to standard conditions of
the Baylor College of Medicine (BCM) Center of Comparative
Medicine (CCM). The BCM animal program is fully accredited by
the Association for Assessment and Accreditation of Laboratory
Animal Care International and is operated in compliance with the
Guide for the Care and Use of Laboratory Animals. CCM also
operates in coordination with the Institutional Animal Care and
Use Committee whose membership and procedures comply with
Public Health Service policy.
Animal Tissue Collection and Processing
Crtap-/- mice and wildtype littermates were sacrificed, and
lungs, kidneys, testes, and skin from the upper back were dissected,
fixed, paraffin embedded, and sectioned according to standard
methods as previously described . Specifically, the skin was
placed on Whatman filter paper immediately after dissection in
order to maintain tissue integrity, and it was sectioned exactly
perpendicular to the plane of embedding. The lungs of each
mouse were inflated to a constant pressure of 25 cm with formalin
fixative and then sutured closed at the trachea. The kidneys were
cut longitudinally in order to ensure proper fixation. The Crtap-/-
mouse colony was maintained in a mixed 129Sv/ev-C57Black/6J
genetic background and housed in the Baylor College of Medicine
Histological Staining, Tissue Immunofluorescence, BrdU,
and Apoptosis Assays
Standard protocols were followed for the following stains:
Hematoxylin and Eosin, Toluidine Blue, and Picrosirius Red.
Immunofluorescence on mouse tissues was done as previously
described . A rabbit polyclonal antibody raised against
CRTAP protein was used. Crtap-/- tissues were used as a control
for background and specificity of staining. Briefly, the paraffin
sections were xylene treated, rehydrated, and heated for 20
minutes in a steamer for antigen retrieval. Subsequently they were
incubated in blocking solution (3% normal Donkey serum, 0.1%
BSA, 0.1% Triton X-100 in PBS), 1:100 dilution of CRTAP
antisera, 1:600 donkey ant-rabbit secondary antibody conjugated
to Alexa flour 594 (Invitrogen), and finally mounted with Prolong
Gold anti-fade reagent with DAPI (Invitrogen). Cell proliferation
status was assessed by BrdU incorporation using a Zymed BrdU
labeling reagent kit and following the manufacturer recommen-
dations. At 10 days post-natal growth, mice were injected with
10 mL/g of concentrated BrdU reagent two hours before tissue
collection, and imaged using an anti-BrdU antibody conjugated to
Alexa flour 488 (Invitrogen). Apoptotic cells were labeled with
green fluorescent signal using the ApopTag Plus In Situ Apoptosis
Flourescein Detection kit (Millipore). At the end of each described
procedure, images were captured using a Zeiss Axioplan 2
The mean linear intercept (MLI) method was used to obtain
quantitative analysis of the distal airway space enlargement, as
previously described . At least 10 histological fields per mouse
were captured at 20X magnification from all lobes of both lungs.
The ImageJ software grid analysis plug-in (rsb.info.nih.gov/ij) was
used to overlay a grid consisting of 10 horizontal lines and 14
vertical lines onto each 521 micrometer X 697 micrometer image.
Intersections of alveolar walls with each grid line were manually
counted, and the MLI was calculated as the total length of lines
analyzed divided by the total number of intercepts counted.
Freshly dissected tissues were fixed in 1.5% glutaraldehyde/
1.5% paraformaldehyde with 0.05% tannic acid in 0.1 M
Cacodylate buffer, pH 7.4 for 60 minutes on ice, rinsed in
0.1 M cacocylate overnight, then postfixed for 60 minutes in
cacodylate buffered 1% OsO4, rinsed, then dehydrated in a
graded ethanol series from 30–100%. The samples were washed in
propylene oxide and embedded in Spurrs epoxy. Ultrathin
sections were stained in Uranyl Acetate followed by Reynolds
lead citrate and examined using a FEI Tecnai G2 TEM.
Skin Tension Test
Samples were prepared from the dorsal skin of adult (9 month-
old) wildtype and Crtap-/- male mice (n=3). The skin was
harvested and cut into 1 cm wide by 4 cm long pieces. The long
axis of the sample coincided with the superior–inferior direction of
the mouse. The samples were clamped between two aluminum
plates at the superior and inferior ends of the sample. Tension tests
were performed on a MTS (Eden Prairie, MN) servohydraulic
materials test machine. The samples were preloaded to 0.2
Newtons and then loaded to failure in tension at a constant rate of
10 mm/min. Peak load and stiffness were recorded using
TestWorks 4 software (Eden Prairie, MN).
Preparation of collagens
Types I and V collagens were prepared from WT and Crtap-/-
bone. Bone was defatted at 4uC in 0.5 M EDTA, 0.05 M Tris-
HCl, pH 7.5. Type IV collagen was prepared from WT and
Crtap-/- kidney. The kidney was equilibrated in saline containing
protease inhibitors. Tissues were finely minced and collagens
solubilized by pepsin (1:20, w/w, pepsin/dry tissue) in 3% acetic
acid for 24 h at 4uC. Bone collagens I and V were serially
precipitated at 0.7 and 1.8 M NaCl respectively. Kidney collagens
were serially precipitated at 0.7 and 1.8 M NaCl and type IV
collagen was reprecipitated from the 1.8 M fraction at 2.5 M
NaCl after dissolving in 1 M NaCl, 0.05 M Tris-HCl, pH 7.4. For
SDS-PAGE, the method of Laemmli was used with 6% gels for
Collagen a-chains were cut from SDS-PAGE gels and subjected to
in-gel trypsin digestion. Electrospray MS was performed on the
tryptic peptides using an LCQ Deca XP ion-trap mass spectrometer
equipped with in-line liquidchromatography (LC)(ThermoFinnigan)
using a C8 capillary column (300 mm6150 mm; Grace Vydac
Crtap Extraskeletal Phenotype
PLoS ONE | www.plosone.org8 May 2010 | Volume 5 | Issue 5 | e10560
208 MS5.315) eluted at 4.5 ml min. Sequest search software
(ThermoFinnigan) was used for peptide identification using the
NCBI protein database.
Human Cell Culture and Immunofluorescence
Primary human fibroblasts were cultured in DMEM with 4 mM
L-glutamine and 4500 mg/L glucose (HyClone) plus 10% FBS,
100 units/mL penicillin, and 100 micrograms/mL streptomycin.
The cells were split into glass LAB-TEK 4-well chamber slides
(Nunc), and 24 hours later were fixed with 4% paraformaldehyde,
treated with 0.1% Triton X-100, blocked in 10% donkey serum
and 1% BSA, and then sequentially incubated with 1:250 dilution
of CRTAP antisera or P3H1 mouse MaxPab polyclonal antibody
(Abnova), 1:500 donkey anti-rabbit secondary antibody or donkey
anti-mouse secondary antibody conjugated to Alexa Flour 594
(Invitrogen), and mounted with Prolong Gold anti-fade reagent
with DAPI (Invitrogen). The cells were also co-stained with
Phalloidin conjugated to Alexa Flour 488 (Invitrogen) in order to
visualize the actin cytoskeleton and the overall cell outline.
Student’s t-test was used to identify statistically significant
differences, with p,0.05 considered significant. All bar graphs
display the mean, plus or minus the standard error of the mean.
control primary human fibroblasts and in patients with recessive
Immunofluorescence of CRTAP and P3H1 protein in
osteogenesis imperfecta due to mutations in CRTAP or LEPRE1
(which codes for P3H1). In the control fibroblasts, the CRTAP (A)
and P3H1 (C) staining patterns are each consistent with ER
localization. In CRTAP mutant cells, there is loss of staining of
both CRTAP (E) and P3H1 (G). In LEPRE1 mutant cells, there is
also loss of staining of both CRTAP (I) and P3H1 (K). Panels (B),
(D), (F), (H), (J), and (L) are merges of CRTAP or P3H1 staining
with green fluorescent labeled phalloidin and DAPI to show
cellular morphology. All images are at 20X magnification.
Found at: doi:10.1371/journal.pone.0010560.s001 (3.05 MB TIF)
The authors would like to thank Terry K. Bertin for genotyping, Yuqing
Chen for her original contribution to the generation of Crtap-/- mice, Dr.
John Hicks (Texas Children’s Hospital, Houston, TX) for evaluating the
immuno-fluorescent staining of tissues, and Dr. Larry J. Suva (University of
Arkansas for Medical Sciences, Little Rock, AR) for facilitating the skin
Conceived and designed the experiments: DB JL SP PHB DE BL RM.
Performed the experiments: DB JL MW EPH MMJ EM DRK WH DE
RM. Analyzed the data: DB JL MW EPH DRK WH DK DC DE BL RM.
Contributed reagents/materials/analysis tools: DK DC. Wrote the paper:
DB MW PHB DE BL RM.
1. Morello R, Bertin TK, Chen Y, Hicks J, Tonachini L, et al. (2006) CRTAP is
required for prolyl 3- hydroxylation and mutations cause recessive osteogenesis
imperfecta. Cell 127: 291–304.
2. Vranka JA, Sakai LY, Bachinger HP (2004) Prolyl 3-hydroxylase 1: Enzyme
characterization and identification of a novel family of enzymes. J Biol Chem.
3. Ishikawa Y, Wirz J, Vranka JA, Nagata K, Bachinger HP (2009) Biochemical
characterization of the prolyl 3-hydroxylase 1.cartilage-associated protein.
cyclophilin B complex. J Biol Chem 284: 17641–17647.
4. Barnes AM, Chang W, Morello R, Cabral WA, Weis M, et al. (2006) Deficiency
of cartilage-associated protein in recessive lethal osteogenesis imperfecta.
N Engl J Med 355: 2757–2764.
5. Cabral WA, Chang W, Barnes AM, Weis M, Scott MA, et al. (2007) Prolyl 3-
hydroxylase 1 deficiency causes a recessive metabolic bone disorder resembling
lethal/severe osteogenesis imperfecta. Nat Genet 39: 359–365.
6. Baldridge D, Schwarze U, Morello R, Lennington J, Bertin TK, et al. (2008)
CRTAP and LEPRE1 mutations in recessive osteogenesis imperfecta. Hum
Mutat 29: 1435–1442.
7. Bodian DL, Chan TF, Poon A, Schwarze U, Yang K, et al. (2009) Mutation and
polymorphism spectrum in osteogenesis imperfecta type II: implications for
genotype-phenotype relationships. Hum Mol Genet 18: 463–471.
8. Willaert A, Malfait F, Symoens S, Gevaert K, Kayserili H, et al. (2009) Recessive
osteogenesis imperfecta caused by LEPRE1 mutations: clinical documentation
and identification of the splice form responsible for prolyl 3-hydroxylation. J Med
Genet 46: 233–241.
9. Van Dijk FS, Nesbitt IM, Nikkels PG, Dalton A, Bongers EM, et al. (2009)
CRTAP mutations in lethal and severe osteogenesis imperfecta: the importance
of combining biochemical and molecular genetic analysis. Eur J Hum Genet.
10. van Dijk FS, Nesbitt IM, Zwikstra EH, Nikkels PG, Piersma SR, et al. (2009)
PPIB mutations cause severe osteogenesis imperfecta. Am J Hum Genet 85:
11. Cheung MS, Glorieux FH (2008) Osteogenesis Imperfecta: update on
presentation and management. Rev Endocr Metab Disord 9: 153–160.
12. Burnei G, Vlad C, Georgescu I, Gavriliu TS, Dan D (2008) Osteogenesis
imperfecta: diagnosis and treatment. J Am Acad Orthop Surg 16: 356–366.
13. McAllion SJ, Paterson CR (1996) Causes of death in osteogenesis imperfecta.
J Clin Pathol 49: 627–630.
14. Morello R, Tonachini L, Monticone M, Viggiano L, Rocchi M, et al. (1999)
cDNA cloning, characterization and chromosome mapping of Crtap encoding
the mouse cartilage associated protein. Matrix Biol 18: 319–324.
15. Tiainen P, Pasanen A, Sormunen R, Myllyharju J (2008) Characterization of
recombinant human prolyl 3-hydroxylase isoenzyme 2, an enzyme modifying
the basement membrane collagen IV. J Biol Chem 283: 19432–19439.
16. Gothel SF, Marahiel MA (1999) Peptidyl-prolyl cis-trans isomerases, a
superfamily of ubiquitous folding catalysts. Cell Mol Life Sci 55: 423–436.
17. Hynes RO (2009) The extracellular matrix: not just pretty fibrils. Science 326:
18. Schuppan D, Glanville RW, Timpl R (1982) Covalent structure of mouse type-
IV collagen. Isolation, order and partial amino-acid sequence of cyanogen-
bromide and tryptic peptides of pepsin fragment P1 from the alpha 1(IV) chain.
Eur J Biochem 123: 505–512.
19. Birk DE, Fitch JM, Babiarz JP, Doane KJ, Linsenmayer TF (1990) Collagen
fibrillogenesis in vitro: interaction of types I and V collagen regulates fibril
diameter. J Cell Sci 95 (Pt 4): 649–657.
20. Kypreos KE, Birk D, Trinkaus-Randall V, Hartmann DJ, Sonenshein GE
(2000) Type V collagen regulates the assembly of collagen fibrils in cultures of
bovine vascular smooth muscle cells. J Cell Biochem 80: 146–155.
21. Wenstrup RJ, Florer JB, Brunskill EW, Bell SM, Chervoneva I, et al. (2004)
Type V collagen controls the initiation of collagen fibril assembly. J Biol Chem
22. Wenstrup RJ, Langland GT, Willing MC, D’Souza VN, Cole WG (1996) A
splice-junction mutation in the region of COL5A1 that codes for the carboxyl
propeptide of pro alpha 1(V) chains results in the gravis form of the Ehlers-
Danlos syndrome (type I). Hum Mol Genet 5: 1733–1736.
23. Burrows NP, Nicholls AC, Richards AJ, Luccarini C, Harrison JB, et al. (1998)
A point mutation in an intronic branch site results in aberrant splicing of
COL5A1 and in Ehlers-Danlos syndrome type II in two British families.
Am J Hum Genet 63: 390–398.
24. Hakim AJ, Sahota A (2006) Joint hypermobility and skin elasticity: the
hereditary disorders of connective tissue. Clin Dermatol 24: 521–533.
25. Chang W, Barnes AM, Cabral WA, Bodurtha JN, Marini JC (2009) Prolyl 3-
Hydroxylase 1 and CRTAP are Mutually Stabilizing in the Endoplasmic
Reticulum Collagen Prolyl 3-Hydroxylation Complex. Hum Mol Genet.
26. Chines A, Boniface A, McAlister W, Whyte M (1995) Hypercalciuria in
osteogenesis imperfecta: a follow-up study to assess renal effects. Bone 16:
27. Vetter U, Maierhofer B, Muller M, Lang D, Teller WM, et al. (1989)
Osteogenesis imperfecta in childhood: cardiac and renal manifestations.
Eur J Pediatr 149: 184–187.
28. Phillips CL, Pfeiffer BJ, Luger AM, Franklin CL (2002) Novel collagen
glomerulopathy in a homotrimeric type I collagen mouse (oim). Kidney Int 62:
29. Widmann RF, Bitan FD, Laplaza FJ, Burke SW, DiMaio MF, et al. (1999)
Spinal deformity, pulmonary compromise, and quality of life in osteogenesis
imperfecta. Spine (Phila Pa 1976) 24: 1673–1678.
Crtap Extraskeletal Phenotype
PLoS ONE | www.plosone.org9 May 2010 | Volume 5 | Issue 5 | e10560
30. Falvo KA, Bullough PG (1973) Osteogenesis imperfecta: a histometric analysis. Download full-text
J Bone Joint Surg Am 55: 275–286.
31. Shapiro JR, Burn VE, Chipman SD, Jacobs JB, Schloo B, et al. (1989)
Pulmonary hypoplasia and osteogenesis imperfecta type II with defective
synthesis of alpha I(1) procollagen. Bone 10: 165–171.
32. Thibeault DW, Pettett G, Mabry SM, Rezaiekhaligh MM (1995) Osteogenesis
imperfecta Type IIA and pulmonary hypoplasia with normal alveolar
development. Pediatr Pulmonol 20: 301–306.
33. Penttinen RP, Lichtenstein JR, Martin GR, McKusick VA (1975) Abnormal
collagen metabolism in cultured cells in osteogenesis imperfecta. Proc Natl Acad
Sci U S A 72: 586–589.
34. Holbrook KA, Byers PH (1982) Structural abnormalities in the dermal collagen
and elastic matrix from the skin of patients with inherited connective tissue
disorders. J Invest Dermatol 79 Suppl 1: 7s–16s.
35. Hansen B, Jemec GB (2002) The mechanical properties of skin in osteogenesis
imperfecta. Arch Dermatol 138: 909–911.
36. Choi JW, Sutor SL, Lindquist L, Evans GL, Madden BJ, et al. (2009) Severe
osteogenesis imperfecta in cyclophilin B-deficient mice. PLoS Genet 5:
37. Bonadio J, Saunders TL, Tsai E, Goldstein SA, Morris-Wiman J, et al. (1990)
Transgenic mouse model of the mild dominant form of osteogenesis imperfecta.
Proc Natl Acad Sci U S A 87: 7145–7149.
38. Morello R, Bertin TK, Schlaubitz S, Shaw CA, Kakuru S, et al. (2008) Brachy-
syndactyly caused by loss of Sfrp2 function. J Cell Physiol 217: 127–137.
39. McComb JG, Ranganathan M, Liu XH, Pilewski JM, Ray P, et al. (2008)
CX3CL1 up-regulation is associated with recruitment of CX3CR1+ mononu-
clear phagocytes and T lymphocytes in the lungs during cigarette smoke-induced
emphysema. Am J Pathol 173: 949–961.
Crtap Extraskeletal Phenotype
PLoS ONE | www.plosone.org10 May 2010 | Volume 5 | Issue 5 | e10560