COLLAGEN XI CHAIN MISASSEMBLY IN CARTILAGE OF THE CHONDRODYSPLASIA (cho) MOUSE
+*Fernandes R J; *Weis, M A; *Scott, M A; **Seegmiller, R E; *Eyre, D R
+*University of Washington, Seattle, WA
[email protected]
respectively by in-gel trypsin digestion and microbore LC/mass
INTRODUCTION
spectrometry with database searching (Table 1). In the cho/+
Type XI collagen, although a minor structural protein of cartilage, is
heterozygote cartilage, both α1(XI) and α1(V) chains were identified.
essential for normal embryonic skeletal development and the cohesive
Western blotting using the antibody to α2(XI) N-telopeptide clearly
properties of cartilage. This collagen accounts for 3% to 10% of the
showed that this chain was cross-linked to the α1(V) collagen chain in
collagenous protein of adult and fetal cartilage (1). Mutations in the gene
cho/cho cartilage (Figure 2) suggesting a polymer of the hybrid type
Col11a1 cause chondrodysplasia and mild osteoarthritis in mice and
V/XI collagen molecules had formed.
humans (2). In fetal cartilage, type XI collagen consists of molecules
These results suggest that in the absence of α1(XI) chains (as in the
containing three genetically distinct chains, α1(XI), α2(XI) and α3(XI)
cho/cho mouse), α1(V) collagen chains can be incorporated into stable
in a 1:1:1 ratio. However, from mature articular cartilage, the purified
(pepsin resistant) type V/IX hybrid molecules. The 1:1 ratio of
type XI collagen fraction includes a significant proportion of the α1(V)
α1(V): α2(XI) chains suggests a molecule consisting of of α1(V),
chain (3), suggesting the existence of type V/ XI hybrid molecules in the
α2(XI), α3(XI) chains, but other combinations are possible. Since
tissue. Type XI collagen molecules are cross-linked by lysyl oxidaseα1(V), α3(V), α1(XI) and α2(XI) belong to the same clade of collagen
mediated bonds and it is becoming clear that type XI collagen serves as
gene products, substitution of α1(V) for α1(XI) might be expected.
the template and regulator of the type II collagen fibrillar network (4,5).
Although type V/XI hybrid molecules can form and polymerize in the
The neonatal lethal chondrodysplasia (cho) mouse carries a deletion
cho/cho mouse rib cartilage, these molecules are probably not being
of a cytidine residue 570 nucleotides downstream of the translation
exported or functioning to form a normal matrix. Thick collagen fibrils
initiation codon in Col11a1 resulting in a reading frame shift and the
are observed in cho/cho cartilage matrix by electron microscopy (8).
introduction of a premature stop codon (6). The cho/cho mouse develops
This suggests a crucial role for the α1(XI) chain in early skeletal
a severe chondrodysplasia and dies at birth. The cho/+ heterozygote
development, possibly in maintaining the thin collagen fibril diameters
survives, and develops early onset osteoarthritis (7). The α1(XI) chain is
normally seen in fetal cartilage. As normal cartilage matures, the α1(V)
not translated into protein in the homozygous mouse (cho/cho) cartilage
chain becomes a more prominent component (3) and interestingly,
and it has been hypothesized that type XI collagen molecules cannot
thicker collagen fibril diameters are observed in the matrix.
assemble (6). We have used the cho mouse to address this hypothesis
This study emphasizes the complexity of the collagen network of
and the possibility that alternative type V/ XI hybrid molecules can form
cartilage. Exactly how type V/XI isoforms can regulate the formation of
in the absence of α1(XI) chains.
distinctive collagen networks in fetal and mature cartilage will be a
Here we report on the consequences of this mutation on type XI
challenge to understand.
collagen chain assembly in the cartilage of chondrodysplasia mice.
METHODS
Tissue acquisition and genotype determination. Neonatal fetuses were
obtained from matings of heterozygous mice. The genotype of the
fetuses was determined by PCR amplification of a region of the Col11a1
gene followed by restriction enzyme analysis.
Collagen extraction. Rib plates from control (+/+) mice and from mice
homozygous and heterozygous for the cho mutation were minced and
Figure 1. SDS-PAGE of collagen extracted from the rib cartilage of +/+
extracted with 4 M GuHCl. Collagen in the residue was solubilized by
and cho/cho mice. Arrowhead points to a band migrating in the region of
pepsin digestion. Pepsin digests were fractionated into collagen types II,
α1(XI) chains in the cho/cho cartilage.
and IX by precipitation at 0.8M and 2.2M NaCl, respectively.
Electrophoresis and Western blotting. Pepsin solubilized collagen chains
Band Collagen
Sequence
Mass
were resolved by Laemmli SDS-PAGE and stained with Coomassie
chain
(M+H)
blue. Collagen chains transferred to PVDF membrane were probed with
1
α1(V)
GPAGAAGPIGIP*GR
1207.37
polyclonal antibody (pAb) 5892 which specifically recognizes α2(XI)
GDP*GPSGPP*GIP*GDDGER
1725.71
N-telopeptide stubs that are normally cross-linked to the triple helical
2
α2(XI)
P*GATGQAGPP*GPVGPP*GLP*GLR
2015.22
region of α1(XI) chain (2). Thus this antibody detects the α1(XI) chain
α1(ΙΙ)
3
VGPP*GANGNP*GPAGPP*GPAGK
1814.94
or any other collagen chain cross-linked to the α2(XI) N-telopeptide.
GEAGAQGPMGPSGPAGAR
1568.70
Pepsin solubilized rat chondrosarcoma (RCS-LTC) type II and XI
Table 1. Tryptic peptides identified by in-gel trypsin digestion and mass
collagen were used as standards.
spectrometry. Identities of bands 1, 2, and 3 from Figure 1 are shown.
Mass Spectrometry. A ThermoFinnnigan LCQ Deca XP mass
spectrometer with electrospray ionization source and in-line C8 RPHPLC was used. After Coomassie Blue staining on SDS-PAGE,
individual protein bands were digested in-gel by trypsin. The resulting
peptides were subjected to microbore column liquid chromatography
(µLC) interfaced directly to a tandem mass spectrometer equipped with
a micro-electrospray ionization source. For protein identification,
Figure 2. Western blot of type XI collagen extracted from homozygous
peptide fragments were compared with the FBSC non-redundant protein
(cho/cho), wild-type (+/+) and heterozygous (cho/+) mouse rib cartilage.
database using SEQUEST, an automated database search algorithm
In cho/cho cartilage, the pAb 5892 recognized the α1(V) chain
designed for use with tandem mass spectrometry data.
indicating that the α2(XI) N-telopeptide is cross-linked to it.
RESULTS AND DISCUSSION
As shown in Figure 1, SDS-PAGE resolved pepsin extracted α1(XI)
REFERENCES
and α2(XI) chains extracted from +/+ cartilage. The α3(XI) chain is the
1. Eyre, DR. (1991) Sem. Arth. Rheum. 21:2-; 2. Spranger, J. (1998)
product of the col2a1 gene and does not easily resolve from the α1(II)
Ped. Radiol. 28:745-; 3. Wu, JJ and Eyre, DR (1991) Trans. Orthop.
chain. An unexpected banding pattern was observed in the cho/cho
Res. Soc. 16:27; 4. Wu, JJ and Eyre, DR (1995) JBC 270: 18865; 5.
cartilage. Two bands in a 1:1 ratio were observed migrating slower than
Blaschke, UK, et al., (2000) JBC 275:10370; 6. Li, Y. et al., (1995) 80:
the α1(II) collagen chains. Band 1 (Figure 1, open arrowhead) migrating
423-; 7. Rodriguez, RR et al., (2004) OA and Cart 12:314; 8. Seegmiller,
slightly slower than the α1(XI) collagen band (compare cho/cho and
RE et al., (1971) JCB 48:580-.
We acknowledge NIH grants AR52896(RJF) and AR37318(DRE)
+/+ lanes in Figure 1), and band 2 migrating in the region of the α2(XI)
AFFILIATED INSTITUTION FOR CO-AUTHOR
chain. Using purified type XI collagen, the identities of these bands were
** Brigham Young University, Provo, UT
firmly established as mouse α1(V) and α2(XI) collagen chains
53rd Annual Meeting of the Orthopaedic Research Society
Paper No: 0175