Download functional differences underlying matrilin

FUNCTIONAL DIFFERENCES UNDERLYING MATRILIN-3 MUTATIONS IN SPONDYLO-EPI-METAPHYSEAL
DYSPLASIA AND IN HAND OSTEOARTHRITIS
Luo, J; Zhang, Y; Wang, Z; Wei, L; Chen, Q
Department of Orthopaedics, Brown Medical School/Rhode Island Hospital, Providence, RI
INTRODUCTION: Matrilins are extracellular matrix molecules
involved in the development and homeostasis of cartilage and bone.
Although all four members of matrilins are expressed in the skeletal
system, matrilin-3 (Matn3) is particularly interesting because mutations
in Matn3 cause a variety of skeletal diseases. Matrilin-3 is composed of
a single N-terminal vWFA domain followed by four EGF repeats and a
coiled-coil domain, whereas the other matrilins are each composed of
two vWFA domains separated by EGF repeats. Mutations in the vWFA
domain of Matn3 resulted in multiple epiphyseal dysplasia (MED),
characterized by delayed and irregular ossification of the epiphyses and
early-onset of osteoarthritis. Recently, mutations in Matn3 have been
shown to lead to two more diseases: spondylo-epi-metaphyseal dysplasia
(SMED), a chondrodystrophy occurring during skeletal development in
childhood, and hand osteoarthritis (OA), a much milder disease
occurring in adults during aging. Interestingly, both of these point
mutations occur in the first EGF repeat of Matn3, but not in the vWFA
domain where the MED mutations reside. Intriguingly these two point
mutations occur next to each other: C304S point mutation is associated
the SMED, and T303M point mutation is related to hand OA. Our study
is trying to answer how mutations in the neighboring amino acid
residues in the same domain lead to two very different diseases?
METHODS: A cDNA transcript encoding full length mouse matrilin-3
was cloned by RT-PCR from total RNA isolated from the rib cartilage of
C57 newborn mice. The full-length transcript was cloned into an
expression vector pcDNA3.1/v-5-His. Point mutations mimicking
SMED, hand OA, and both were generated by overlapping PCR.
Normal and mutated Matn3 cDNA were transfected into COS-1 cell line
or MCT chondrocyte cell line using Lipofectamin 2000 (Invitrogen),
respectively. Culture medium and cell lysates were collected each day
after transfection until the fifth day for Western blot analysis. The cell
plate from the fifth day incubation was processed for
immunofluorenscence using an antibody against the recombinant
matrilin-3.
RESULTS: To understand the mechanisms underlying mutations
associated with SMED or hand OA, we cloned a full-length mouse
matrilin-3 cDNA and generated three cDNAs containing the point
mutation of SMED (CS), hand OA (TM), or both mutations,
respectively, in the corresponding mouse sequence. These mutant
cDNAs along with the wildtype Matn3 (WT) were transfected into MCT
chondrocyte cell lines. As shown by western blot analysis (Fig. 1, left
panel), wildtype and hand OA-related mutant matrilin-3 was detected in
medium one day after transfection, while Matn3 harboring SMED
mutation could not be detected in medium until three days after
transfection. The secretion of the Matn3 mutant harboring both SMED
and hand OA mutations was also delayed. Thus, there was a significant
delay of the secretion of matrilin-3 protein harboring SMED mutation,
but not that harboring hand OA mutation. Furthermore, this delay was
observed in both COS cells, which does not synthesize endogenous
Matn3, and MCT chondrocytes that synthesize endogenous Matn3 (Fig.
1, right panel). To determine whether mutated matrilin-3 is trapped
within the cell, we performed immunocytochemistry of transfected cells
using an antibody against the V5 tag of the recombinant matrilin-3.
Immunocytochemical analysis showed that MCT cells transfected with
Matn3 cDNA harboring either SMED mutation or both SMED and hand
OA mutations contain a greatly expanded cytoplasm with numerous
matrilin-3 positive vesicles (Fig. 2, A and C). The expanded cytoplasm
with numerous matrilin-3 positive vesicles was observed in MCT
chondrocytes under both proliferation and hypertrophic culture
conditions. In contrast, cells transfected with a Matn3 cDNA harboring
hand OA mutation exhibit a normal sized cytoplasm containing normal
number of vesicles, similar to the wildtype Matn3 transfected cells (Fig.
2, B and D).
diseases including MED, SMED and hand OA, but the molecular
mechanisms are unknown. We have shown previously that MEDrelated point mutation in the vWFA domain of matrilin-3 causes a
significant delay of matrilin secretion and an altered chondrocyte
cytoplasm including expanded ER and numerous secretion vesicles. The
site of MED-related mutation is located in β-fold region in the center of
vWFA domain. This suggests that the secretion defect of MED Matn3
may be due to abnormal protein folding. In contrast, SMED and hand
OA-related point mutation sites are located in the first EGF domain.
Our results showed that the SMED-related mutation led a significant
delay of matrilin secretion and an altered chondrocyte cytoplasm
including expanded ER and numerous vesicles. These phenotypes are
very similar to that from the MED-related point mutation in the vWFA
domain. This suggests a common mechanism between MED and SMED
pathogenesis. SMED-related mutation is a point mutation from cysteine
to serine. Because cysteine is very important for folding and
conformational structure of the protein, this mutation may affect the
folding of the protein, thereby interfering with secretion of the protein.
Therefore, even though the MED mutations occur in the vWFA domain
and the SMED mutation occurs in the first EGF domain, they may share
similar molecular mechanism that results in similar phenotypes. On the
other hand, the hand OA-related point mutation does not result in
delayed secretion of Matn-3, even though the mutated threonine is
located next to the cysteine residue in the same EGF domain related to
SEMD. This suggests that the pathological mechanism of hand OA may
be distinct from those of MED and SEMD. We are in the process of
analyzing the mechanism underlying Matn3 related OA.
Figure 1. Western blot analysis of medium and cell lysates collected
from cells transfected with matrilin-3 cDNA encoding wildtype (WT),
Hand OA (TM), SMED (CS), or both mutations (B).
Figure 2. Immunofluorenscence analysis of MCT chondrocytes
expressing wildtype Matn3 (A), Hand OA Matn3 (B), SMED Matn3
(C), or Matn3 containing both mutations (D).
DISCUSSION: Point mutations have been identified within the vWFA
and the first EGF domain of matrilin-3. They cause different skeletal
52nd Annual Meeting of the Orthopaedic Research Society
Paper No: 0008