Identification of a complex between fibronectin and aggrecan G3 domain in synovial
fluid of patients with painful meniscal pathology
Gaetano J. Scuderib, Naruewan Woolfa, Kaitlyn Denta, S. Raymond Golishb, Jason M. Cuellarb,1,
Vanessa G. Cuellarb,1, David C. Yeomansb, Eugene J. Carrageeb, Martin S. Angstb,
Robert Bowsera, Lewis S. Hannaa,⁎
aResearch and Development, Cytonics Corporation, 555 Heritage Drive, Suite 115, Jupiter, FL 33458, USA
bStanford University, Stanford, CA, USA
a b s t r a c ta r t i c l ei n f o
Received 10 February 2010
Received in revised form 6 April 2010
Accepted 22 April 2010
Available online 11 May 2010
Objectives: We previously described a panel of four cytokines biomarkers in knee synovial fluid for acute
knee pain associated with meniscal pathology. The cytokine biomarkers included interferon gamma (IFN-γ),
interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein-1 beta
(MIP-1β). Validation studies using other immunologic techniques confirmed the presence of IL-6, MCP-1 and
MIP-1β, but not IFN-γ. Therefore we sought the identity of the IFN-γ signal in synovial fluid.
Methods: Knee synovial fluid was collected from patients with an acute, painful meniscal injury, as well
as asymptomatic volunteers. A combination of high-pressure chromatography, mass spectrometry and
immunological techniques were used to enrich and identify the protein components representing the IFN-γ
Results: A protein complex of fibronectin and the aggrecan G3 domain was identified in the synovial
fluid of patients with a meniscal tear and pain that was absent in asymptomatic controls. This protein
complex correlated to the IFN-γ signal. A novel enzyme-linked immunosorbent assay (ELISA) was developed
to specifically identify the complex in synovial fluid.
Conclusions: We have identified a protein complex of fibronectin and aggrecan G3 domain that is a
candidate biomarker for pain associated with meniscal injury.
© 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Degenerative joint disease and joint injury are associated with
increased turnover of articular cartilage proteins, inflammation and
alterations to other joint tissue proteins [1,2]. Degenerative joint
disease in the knee is often idiopathic, however it has also been
strongly associated with prior injury such as meniscal damage. The
inflammatory milieu induced by such injury may therefore lay the
groundwork for future degeneration and osteoarthritis. The profile of
inflammatory proteins within synovial fluid after acute knee injury
may represent diagnostic or prognostic biomarkers for the degener-
ative joint disease or osteoarthritis that may ensue.
Expression and fragmentation of the extracellular matrix protein
fibronectin has been shown to occur in the synovial fluid of arthritic
patients and joint injury [3,4]. Fibronectin fragment induced knee
injury in an animal model results in further cartilage damage and loss
of proteoglycans . Fibronectin also induces microglial activation
and stimulation of cytokine production and activation of matrix
metalloproteases . It is well knownthat inflammatorycytokines are
associated with fibronectin and its fragments in the pathophysiology
of degenerative joint disease .
Aggrecan, a high molecular weight proteoglycan present in
articular cartilage, undergoes extensive degradation and turnover
during normal cartilage metabolism, aging and joint diseases .
Aggrecanases are activated during cartilage degradation and diseases
. Recently, itwasdemonstratedthatpatterns ofaggrecanfragments
differ between acute injury and chronic degeneration relative to
healthy controls . Therefore both fibronectin and aggrecan exhibit
increased fragmentation in degenerative joint conditions and after
articular cartilage damage. It is possible that fibronectin, aggrecan and
their fragments interact in the synovial fluid to facilitate signaling
cascades that augment joint and cartilage degeneration.
Clinical Biochemistry 43 (2010) 808–814
Abbreviations: IFN-γ, interferon gamma; IL-6, interleukin 6; MCP-1, monocyte
chemotactic protein 1; MIP-1β, macrophage inflammatory protein-1 beta; ELISA,
enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; HPLC, high
performance liquid chromatography; SEC, size exclusion chromatography; AEC, anion
exchange chromatography; TMB, tetramethylbenzidine; LC-MS/MS, liquid chromatog-
raphy based mass spectrometry; FN1, fibronectin; ACAN, aggrecan.
⁎ Corresponding author.
E-mail address: Lewis.email@example.com (L.S. Hanna).
1Current address: New York University Hospital for Joint Diseases, New York City,
0009-9120/$ – see front matter © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/clinbiochem
We recently described a panel of four cytokines biomarkers for
acute knee pain associated with meniscal pathology , and one
cytokine in the epidural space of spinal disc disease . The cytokine
biomarker panel was identified from synovial fluid using multiplex
inflammatory cytokine profiling and included interferon gamma
(IFN-γ), interleukin 6 (IL-6), monocyte chemotactic protein 1
(MCP-1), and macrophage inflammatory protein-1 beta (MIP-1β).
Validation studies using other immunologic techniques confirmed the
presence of IL-6, MCP-1 and MIP-1β, but not IFN-γ.
To further determine the identity of the IFN-γ signal in the
multiplex inflammatory cytokine panel, we used a combination of
column chromatography and mass spectrometry to enrich and
determine the amino acid sequence identity of the IFN-γ signal.
Additional immunoassays were used to confirm the sequence
identity. Our study identified a protein complex from knee synovial
fluid containing fibronectin and aggrecan. In addition, this protein
complex was present in a painful knee with meniscal pathology but
absent from asymptomatic healthy volunteers.
Materials and methods
Subjects and synovial fluid collection
Our prospective study included 15 adult patients without
rheumatoid arthritis who were diagnosed with painful intra-articular
derangement of the knee by history, physical examination and
magnetic resonance imaging (MRI) who elected for arthroscopic
debridement following a failure of non-operative pain management.
Inclusion criteria were an age of 18 years or older, knee pain of recent
onset (less than 6 months), and physical examination findings and
MRI results consistent with intra-articular pathology. Exclusion
criteria were an age of less than 18 years, a recent history (within
3 months) of an intra-articular injection of a corticosteroid, and a past
or current history of autoimmune disease (such as rheumatoid
arthritis). The mean±standard deviation age was 77.2±5.2 years,
and there were 7 males and 8 females in the study group.
Institutional Review Board (IRB) approval was obtained for the
study, and knee synovial fluid was collected upon informed patient
consent by needle aspiration. Synovial fluid was placed in polypro-
pylene tubes containing a protease inhibitor (10 mM AEBSF, Sigma
Aldrich, St. Louis, MO) and stored at −80 °C. Prior to use, the synovial
fluid was treated with 5 mg/mL Hyaluronidase and clarified by
centrifugation at 5000 g.
Multiplex cytokine assay
Multiplex immunoassay was performed using Bio-Rad's Bio-Plex
200 with Bio-Plex human cytokine 4, 17, and 27 multiplex panels. The
assay was performed as recommended by the manufacturer.
Antibodies and chemicals
Horseradish peroxidase (HRP) labeled anti-fibronectin antibody
was obtained from US Biological, Swampscott, MA; anti-aggrecan G3
domain antibody was obtained from Santa Cruz Biotechnology, Santa
Cruz, CA; human fibronectin was obtained from BD Biosciences, San
Jose, CA; all other chemicals were obtained from Sigma Aldrich.
HPLC and protein purification
High performance liquid chromatography (HPLC) assays and
purification were performed on a Bio-Rad BioLogic DuoFlow HPLC
system. Size exclusion chromatography (SEC) was performed using
two Bio-Rad SEC400-5 columns in series. SEC separation was
performed in isocratic mode using 50 mM Tris/HCl, 100 mM NaCl
pH 7.0 buffer.
Anion exchange chromatography (AEC) was performed using a
Bio-Rad UNO Q1 column. Buffer A: 50 mM Tris/HCL pH 7.0; Buffer B:
50 mM Tris/HCl, 1.0 M NaCl pH 7.0. The protein was loaded in 90%
buffer A/10% buffer B and eluted with a linear gradient from 90%
buffer A/10% buffer B to 70% buffer A/30% buffer B in 20 min.
Ammonuim bicarbonate (1 M) was added to solution sample to
buffer pH to 8. Cysteine residues were reduced and alkylated with
2.5 mM TCEP and 20 mM iodoacetamide. Sample was digested using
2 ng of trypsin at 37 °C for 3 h. Digestion mixtures was loaded onto a
precolumn (360 mm od×100 mm id fused silica, Polymicro Technol-
ogies, Phoenix, AZ) packed with 3 cm irregular C18 (5–15 mm non-
spherical, YMC, Inc., Wilmington, NC) and washed with 0.1 M HOAc
for 5 min before switching in-line with the resolving column (7 cm
spherical C18, 360 mm×100 mm). Once the columns are in-line, the
peptides are gradient eluted with a gradient of 0–100%A in 30 min
where A is 0.1 M HOAc in nanopure H20 and B is 0.1M HOAc in 80%
MeCN. All samples were analyzed using a Thermo Electron LTQ or
LTQ-Orbitrap (San Jose, CA). Electrospray was accomplished using an
Advion Triversa Nanomate (Advion Biosystems, Ithica, NY) with a
voltage of 1.7 kV and a flow rate of 300 nL/min. The mass
spectrometer was operated using data dependent scanning with the
top 5 most abundant ions in each spectrum being selected for
sequential MS/MS experiments. All MS/MS spectra were searched
with Sequest using appropriate human database (ipi database v3.32).
Database search results are tabulated and visually inspected using
Scaffold (Proteome Software, Portland, OR).
Anti-aggrecan G3 domain antibody was immobilized on Affi-Gel
according to the manufacturer's (Bio-Rad) recommended procedure
to obtain an affinity chromatography for the purification of aggrecan
G3 related molecules. The eluted peak from the AEC column
containing the Bio-plex IFN-γ signal was further enriched on the
aggrecan G3 affinity column. The column was equilibrated with
10 mM phosphate, 150 mM NaCl buffer pH 7.8. The AEC fractions
were pooled and concentrated using spin filter with 30 kDa molecular
weight cutoff. The column was then washed with the same buffer and
eluted with 15 mM phosphate buffer pH 3.0. The eluted fraction was
assayed for aggrecan and fibronectin proteins by slot blot analysis.
Gel electrophoresis and Western blotting
Gel electrophoresis was performed using Tris/HCl 18% or 4–20%
polyacrylamide gradient gels (Bio-Rad). The samples were treated
with SDS sample buffer at room temperature and loaded on the gel
immediately. The gel was run at constant voltage of 200 V for 1 h
using 20 mM Tris base, 192 mM Glycine, 0.1% SDS, pH 8.3 as running
buffer. The gel was stained with silver stain (Bio-Rad Silver Stain Kit)
according to the manufacturer's recommended procedure.
For Western analysis, proteins were transferred to nitrocellulose
membrane. The transfer was performed at constant voltage of 100 V
for 40 min using Towbin buffer. The membranes from Western or slot
blot analysis was blocked with 20 mM Tris, 0.5 M NaCl, 1% casein pH
7.4 overnight and developed using anti-aggrecan G3, anti-fibronectin
or anti-interferon-γ antibodies.
Enzyme-linked immunosorbent assay (ELISA) plates were coated
with anti-aggrecan G3 domain antibody in PBS/tween 20/thimerosal
and blocked with 1% BSA overnight at 4 °C. Samples were incubated at
room temperature for 1 h followed by 6 washes (using Bio-Rad's Bio-
G.J. Scuderi et al. / Clinical Biochemistry 43 (2010) 808–814
Plex Pro II wash station). HRP-labeled anti-fibronectin antibody was
then added and the plate was incubated at room temperature for 1 h
followed by 6 washes. Tetramethylbenzidine (TMB) substrate was
added and incubated at room temperature in the dark for 5 min. The
reaction was stopped with sulfuric acid and the plate was read using
Bio-Rad's Benchmark Plus microplate spectrophotometer at 450 nm.
All assays were performed in triplicate. Human fibronectin at 1 µg/mL
is used as negative control.
By cytokine profiling of synovial fluid, we previously identified
significant increase in the concentrations of IL-6, MCP-1, MIP-1β and
IFN-γ in patients with cartilage degeneration pathology . In the
current study, we measured levels of these same cytokines in the
synovial fluid from 15 patients with intra-articular pathology of the
knee undergoing arthroscopic debridement as described in Material
The knee synovial fluid from 5 of these patients in pain with
meniscal pathology that exhibited the highest levels of all 4 cytokine
biomarkers (IFN-γ, IL-6, MCP-1, and MIP1-β) was used for the
subsequent analysis. While immunoblot confirmed the presence of
IL-6, MCP-1 and MIP1-β, we could not confirm the presence of IFN-γ
by immunoblot or ELISA using multiple IFN-γ antibodies and
commercially available ELISA kits (data not shown). We therefore
sought other methodologies to enrich for the IFN-γ signal detected by
the Bio-Plex assay and determine the protein identity corresponding
to this cytokine signal.
Size exclusion chromatography performed via high performance
liquid chromatography (SEC-HPLC) analysis of the synovial fluid
displayed a complex protein elution profile (Fig. 1). Direct compar-
isons between a symptomatic and non-symptomatic subject demon-
strate the difference in the presence or absence of high molecular
weight proteins (Fig. 1). As expected, silver stained polyacrylamide
gels for SEC fractions 9–18 showed high molecular species in the early
fractions and lower molecular weight species in the later fractions
(Fig. 2). We assayed column fractions for the presence of the
4-cytokine biomarkers using the Bio-plex assay. IFN-γ signal resided
in fractions 13, 14 and 15, elution time 32–38 min (Table 1). This
elutiontimeis characteristic ofproteins400–700 kDa molecularmass.
The other three cytokines were present in fractions 17–19, a mass
range more typical of the respective cytokine proteins. The molecular
mass of the IFN-γ signal was confirmed by fractionating the clarified
synovial fluid using spin filters. The IFN-γ signal was retained on a
spin filter with a 300 kDa cutoff membrane and flowed through a spin
filter with a 1000 kDa cutoff membrane (data not shown).
Identification of fibronectin–aggrecan complex
To determine the protein identity corresponding to the IFN-γ
signal, we pooled SEC fractions 14 and 15 and digested the proteins
with trypsin for subsequent amino acid sequence analysis by liquid
chromatography based mass spectrometry (LC-MS/MS). A predom-
inant protein identified from these fractions corresponded to
fibronectin. We identified 15 unique peptides representing 320 of
2386 amino acids (13.4% coverage) of human fibronectin (Swiss-Prot
identification number P02751) sequence (Fig. 3). While other
proteins were identified in these SEC fractions, fibronectin is an
extracellular matrix protein previously implicated in cartilage joint
disease . The molecular mass of fibronectin in its dimeric form is
approximately 524 kDa in-line with the SEC fractions containing the
IFN-γ signal have approximately 400–700 kDa molecular mass. Since
human fibronectin did not cross react with INF-γ antibody labeled
beads in the Bio-Plex assay (data not shown), additional proteins may
interact with fibronectin within these SEC fractions that give rise to
the antibody cross reactivity.
To further characterize proteins within these SEC fractions, we
performed Western blot analysis with antibodies to fibronectin and
other potential binding partners representing various cartilage and
Fig. 1. SEC elution profile of a 0.1 mL injection of clarified synovial fluid. A) synovial fluid from symptomatic patient; B) synovial fluid from asymptomatic healthy volunteer. The
column was run under isocratic condition using 50 mM Tris/HCl, 100 mM NaCl, pH 7.0 buffer. Protein elution was monitored by absorption at 215 nm (y-axis). Time elution from the
column (minutes) and column fraction number are noted on the x-axis.
Fig. 2. SEC fractions 9–18 were analyzed on a Tris/HCl 4–20% polyacrylamide gradient
SDS gel and protein bands visualized by silver stain. The bands in fractions 14 and 15 are
in the molecular weight range of 400–600 kDa.
G.J. Scuderi et al. / Clinical Biochemistry 43 (2010) 808–814
gel containing SEC fractions 14 and 15 is shown in Fig. 4A. We
determined that protein bands immunoreactive for fibronectin and
aggrecan G3 co-migrated in the gel (Figs. 4B and C). A polyclonal
antibody to IFN-γ also weakly immunolabeled the same protein band
Enrichment of the fibronectin/aggrecan complex by anion exchange
chromatography and anti-aggrecan G3 affinity column chromatography
To further validate our SEC results and obtain amino acid sequence
information for fibronectin interacting proteins, we enriched for the
Bio-plex IFN-γ signal by anion exchange chromatography (AEC) of
synovial fluid from a patient suffering from a painful meniscus tear.
The AEC elution profile is shown in Fig. 5A and all fractions were
analyzed by Bio-plex cytokine assay to identify fractions containing
the IFN-γ signal. Slot blot analysis demonstrated the presence of both
fibronectin and aggrecan G3 in these same fractions (data not shown).
An ELISA was developed to identify the presence of a fibronectin/
aggrecan G3 protein complex in synovial fluid. The ELISA format was a
modification of the classical sandwich ELISA, where the capture
antibody recognized aggrecan G3 domain and the detection antibody
was HRP-labeled anti-fibronectin antibody. We used this ELISA and
determined the presence of a fibronectin–aggrecan protein complex
in the AEC fractions (Table 2). The fibronectin–aggrecan G3 complex
was present in AEC fractions 19–20 (Fig. 5A), corresponding to the
Bio-plex IFN-γ signal.
AEC fractions 19 and 20, containing the fibronectin–aggrecan
protein complex, were pooled and concentrated using a 30 kDa
molecular weight cutoff spin filter. This sample was further purified
using the aggrecan G3 affinity column as described in Materials and
methods, the elution profile is shown in Fig. 5B. We noted a large peak
of protein that failed to bind to the affinity column (fractions 2–5), a
peak of proteins that eluted off the column at pH 3.0 (fractions
IFN-γ signal in SEC fractions by Bio-plex assay.
SEC fractions obtained from symptomatic subject shown in Fig. 1 were assayed for IFN-
γ cytokine levels by the multiplex Bio-plex assay (Bio-rad). IFN-γ levels are shown for
fractions containing the peak IFN-γ signal.
Fig. 3. Tandem mass spectrometric analysis for the fibronectin peptide, GATYNIIVEALKDQQR. A) The MS spectrum of the [M+2H]+2 precursor ion that wasisolated and dissociated
for MS/MS analysis. The spectrum was acquired on a Thermo hybrid LTQ-Orbitrap instrument using the Orbitrap as the mass analyzer. The [M+2H]+2 ion was within 4 ppm of the
theoretical m/z value (m/z 909.9891). B) The amino acid sequence of the peptide, shown with the b- and y-type product ions detected by MS/MS analysis. C) The MS/MS spectrum
of the fibronectin peptide following dissociation in the LTQ ion trap. The observed b- and y-type ions are labeled in the spectrogram.
Fig. 4. SDS gel electrophoresis and immunoblot analysis of proteins contained in the
IFN-γ containing fractions. Fractions 14 and 15 were analyzed by silver stain (A), and
immunoblot for aggrecan G3 (B), fibronectin (C), and IFN-γ (D). The arrow denotes a
single band that is detected by silver stain and immunoreactive for the aggrecan G3
domain, fibronectin, and IFN-γ.
G.J. Scuderi et al. / Clinical Biochemistry 43 (2010) 808–814
18–24), and a final peak of proteins that eluted during the re-
equilibration of the column (fractions 35–38). Due to protein
aggregation and loss of protein–protein interaction that typically
occurs at low pH, we used a slot blot analysis to explore the presence
or absence of fibronectin and aggrecan G3 in these affinity column
fractions (Fig. 6). We detected both fibronectin and aggrecan in flow
through fractions 2–5 suggesting saturation of the column, no
aggrecan or fibronectin in the pH 3.0 elution fractions 18–24, but
the presence of both aggrecan and fibronectin in fractions 35–38.
Fibronectin tends to aggregate at pH 3.0 and we believe this explains
why the eluted peak containing fibronectin and aggrecan appears at
the buffer front during the re-equilibration of the column.
ELISA results for the clinical samples
Finally, we used the novel ELISA to the fibronectin–aggrecan G3
complex to measure its level in the synovial fluid of 15 symptomatic
subjects undergoing arthroscopic debridement of the knee. The
optical density of the ELISA immunoassay for the symptomatic
group ranged from 0.87 to 15.43 (mean 7.6; std.dev. 4.2).
It is well accepted that disc and joint cartilage degeneration is
associatedwith aging.However, itis notknownwhythe degeneration
is painful in some individuals and not in others. Aggrecan, the major
Fig. 5. Purification of the protein complex by anion exchange chromatography (AEC) and affinity column chromatography. A) Elution profile of the AEC and the gradient program
used in the elution. Column fraction numbers are listed above the profile; fractions 19 and 20 contained the INF-γ signal by Bio-Plex and the Fibronectin–aggrecan G3 complex by
ELISA assay. Fractions 19 and 20 containing the peak levels of the protein complex were pooled, concentrated and further enriched by aggrecan G3 affinity column chromatography.
B) Elution profile of the aggrecan G3 affinity chromatography and the gradient program used in the elution. Column fraction numbers are listed above the profile.
ELISA complex assay for the starting material and anion exchange chromatography
1 µg fibronectin, negative
10.030.04 0.04 0.0417.7
Knee synovial fluid from a patient in pain with a meniscal injury was applied to an
anion exchange column (AEC) and eluted with a linear salt gradient as described in
Materials and methods. The fibronectin/aggrecan G-3 ELISA was used to analyze the
AEC fractions for the presence of the protein complex. Fibronectin at 1 µL/mL was used
as negative control. A pool of positive patient samples was used as positive control.
Starting sample is the patient sample prior to AEC fractionation.
G.J. Scuderi et al. / Clinical Biochemistry 43 (2010) 808–814
component of cartilage, and fibronectin degradation were observed in
both groups and considered part of the cartilage generation/
degeneration cycle. We recently described a panel of four cytokines
(IFN-γ, MCP-1, IL-6 and MIP-1β) that correlated to pain in patients
with meniscal injury of the knee . In this study, we have identified
a complex of fibronectin with an aggrecan fragment that is associated
with painful cartilage degeneration or damage. The presence of this
protein complex correlated to the IFN-γ signal in synovial fluid
detected by multiplex analysis.
Since we could not confirm the presence of IFN-γ in synovial fluid
by immunoblot or ELISA specific to IFN-γ, we enriched for the Bio-
plex IFN-γ signal by column chromatography. It was evident from size
exclusion chromatography and spin filters with nominal molecular
weight cutoff that the protein(s) responsible for the IFN-γ signal had
an apparent molecular mass of 400–600 kDa, much larger than the
molecular mass of 25 kDa. LC-MS/MS of the partially purified IFN-γ
signal demonstrated the presence of fibronectin that was confirmed
by western and ELISA assays. Fibronectin alone did not show a
positive signal in the Bio-Plex IFN-γ assay. Aggrecan is a multidomain
protein where the C-terminal domain (G3) consists of an EGF, lectin
and CRP sequences . Age-related increases in aggrecan G3 have
been shown to occur in articular cartilage , and increased
aggrecan fragments have been observed after knee injury and in
osteoarthritis . The known interaction of fibronectin with lectins
suggests that a complex could be formed between fibronectin and
aggrecan G3 domain. Western blot analysis of the partially purified
IFN-γ signal by SEC-HPLC showed one band immunopositive for
antibodies to fibronectin, aggrecan G3 domain, and also cross-reacted
with a polyclonal antibody to IFN-γ, suggesting the formation of a
protein complex between these proteins that corresponds to the IFN-
A heterogeneous ELISA assay where a microplate was coated with
anti-fibronectin and anti-aggrecan G3 domain was used for detection
and the reverse where the microplate was coated with anti-aggrecan
G3 domain and detection was performed using anti-fibronectin
further confirmed the presence of a complex between fibronectin
and aggrecan G3 domain (data not shown). We also enriched the
complex using anion exchange chromatography followed by affinity
chromatography using immobilized anti-aggrecan antibody. The
purified fraction contained both fibronectin and aggrecan G3 by
Western blot analysis. The presence of fibronectin in the elution
fraction further supports the presenceof the complex in knee synovial
in patients with painful meniscal injury.
Our study showed 100% agreement between the results of the
cytokine profiling and the presence or absence of the fibronectin–
aggrecan G3 complex as determined by the developed ELISA assay.
Furthermore, analyzing more than 150 joint samples from patients
and healthy volunteers in the same age group showed 98% agreement
between the IFN-γ and the fibronectin/aggrecan G3 ELISA results.
Many questions remain regarding the protein binding site
between fibronectin and aggrecan G3, and the nature of the cross
reactivity between the protein complex and the IFN-γ antibodies on
the Bio-plex beads. Lundell et al. identified direct interaction between
the fibronectin type III (FN III) domains of tenascins and the C-type
lectin domains . Fibronectin has 14 FN III domains. Sharma et al.
demonstrated the importance of the linker region (182AIDAP)
between FN 13 and FN 14 in the interaction of fibronectin with
integrin α4β1 . The linker between FN 7, FN 8 and FN 9 also has
the same tilt angle, twist angle and buried surface area in the interface
as FN 13 and FN 14 . These data suggests a ratio of 2:1 aggrecan G3
domain:fibronectin. Further studies are underway to assemble the
complex in vitro and determine the molar ratio of the aggrecan G3
domain to fibronectin in the complex.
We suggest that the cross reactivity between the Bio-plex IFN-γ
beads and the fibronectin–aggecan complex occurs via a conforma-
tion epitope generated upon formation of the protein complex. Pure
fibronectin or aggrecan failed to exhibit a positive Bio-plex IFN-γ
signal, and direct sequence comparisons failed to identify significant
sequence identity between IFN-γ and either aggrecan G3 or
fibronectin (data not shown).
This study is unique in its observation of full-length fibronectin
involved in the complex formation whereas studies to-date indicate
that fibronectin fragments and not the full-length fibronectin
contribute to cartridge degradation [19,20]. Full-length fibronectin
at levels of 15–80 ng/mL was detected in 60% of the synovial fluid
samples obtained from our healthy control subjects. Fibronectin
fragments were not detected in the synovial fluid of any healthy
control subject by Western blot or column chromatography. At this
time, we do not know if the presence of fibronectin in the synovial
fluid of healthy volunteers is a prelude to cartilage degeneration. One
potential mechanism involves the early generation of aggrecan
fragments that may associate with full-length fibronectin. Further
studies are necessary to explore the relationship between the
fibronectin–aggrecan G3 complex identified in this study and the
continued fragmentation of fibronectin, aggrecan and other structural
proteins during cartilage degenerative disorders.
In conclusion, we have identified a protein complex between
fibronectin and aggrecan G3 domain present in the synovial fluid of
patients with meniscal degeneration/injury and pain, and this complex
mayplaya roleinthe developmentof futuredegenerative jointdisease.
We would like to thank Dr. Jennifer Busby, Kristie Rose and Valerie
Cavett at Scripps Florida, Jupiter FL, for performing the LC/MS/MS
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
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