2002 - University of Washington Bone and Joint Sources

Figure 2: Electrophoresis of molecular sieve fractionated type II collagen from fetal bovine epiphyseal
cartilage on SDS-5% PAGE. Collagen samples were run under non-reducing and reducing conditions.
Arrows show the type II collagen fraction that appears to be disulfide bonded. The reduced protein
was blotted to PVDF membrane and identified by N-terminal sequence analysis.
To define precisely the sites of
intermolecular cross-linking, a mAb
2B4 affinity column was used to purify
cross-linked CB peptides that
contained α1(IX)NC1 from a CB digest
of human type II collagen. A
methionine in this domain ensures its
release as a short peptide. Peptides
eluted from the affinity column were
further purified by RP-HPLC then
SDS-PAGE. Western blot analysis was
performed with mAb 2B4.
Amino-terminal sequence analysis
of the purified peptide identified it as
α1(IX)NC1 cross-linked to a helical site
in α1(II) CB9,7, which most likely is the
known 930 hydroxylysine cross-linking
residue. The discovery of this domain
of type IX collagen linked to the type II
collagen triple helix was not predicted
from earlier methods of finding as
cross-linking sites in the collagen IX
molecule (Figure 1). It was known that
type IX collagen is extensively crosslinked
in cartilage matrix through lysyl
oxidase-mediated bonds to both N-
and C- telopeptides of type II collagen
and between the type IX collagen
molecules themselves through the
α3(IX)NC1 domain. The finding of
α1(IX)NC1 links to type II collagen,
taken with other properties of fibril
associated collagen molecules, suggest
that the initial docking and crosslinking
of type IX molecules on type II
fibrils occurs through the COL1/NC1
domain interacting with the hole region
of the quarter-staggered assembly
(Figure 3).
RECOMMENDED READING
Mendler M, Eich-Bender S G, Vaughan
L, Winterhalter KH, Bruckner P. (1989)
Cartilage contains mixed fibrils of
collagen types II, IX, and XI. J. Cell Biol.
108, 191-197.
Figure 3: Collagen IX / II cross-linking interactions: working model.
Wu JJ, Woods P E, Eyre DR. (1992)
Identification of cross-linking sites in
bovine cartilage type IX collagen reveals
an antiparallel type II-type IX
molecular relationship and type IX to
type IX bonding. J. Biol. Chem. 267,
23007-23014.
Diab M, Wu JJ, Eyre DR. (1996)
Collagen type IX from human cartilage:
a structural profile of intermolecular
cross-linking sites. Biochem. J. 314, 327-
332.
Bönnemann CG, Cox GF, Shapiro F, Wu
JJ, Feener CA, Thompson T, Anthony
DC, Eyre DR, Darras B, Kunkel LM.
(2000) A mutation in the a3 chain of
type IX collagen causes autosomal
dominant multiple epiphyseal dysplasia
with mild myopathy. Proc. Natl. Acad.
Sci. USA 97, 1212-1217.
van der Rest M, Mayne R. (1989) Type
IX collagen. In: Structure and Function
of Collagen Types (Mayne R. and
Burgeson RE, eds.), pp. 195-221,
Academic Press.
Atley LM, Shao P, Ochs V, Shaffer K,
Eyre DR. (1998) Matrix
metalloproteinase-mediated release of
immunoreactive telopeptides from
cartilage type II collagen. Trans.
Orthop. Res. Soc., 23, 850.
44 2002 ORTHOPAEDIC RESEARCH REPORT