Identication of a complex between broneuid of patients with painful meniscal path

a,Ca

FL 3

Received in revised form 6 April 2010Accepted 22 April 2010Available online 11 May 2010

ative joint disease or osteoarthritis that may ensue.

Clinical Biochemistry 43 (2010) 808814

Contents lists available at ScienceDirect

Clinical Bioc

j ourna l homepage: www.e lsevDegenerative joint disease and joint injury are associated withincreased turnover of articular cartilage proteins, inammation andalterations to other joint tissue proteins [1,2]. Degenerative jointdisease in the knee is often idiopathic, however it has also beenstrongly associated with prior injury such as meniscal damage. Theinammatory milieu induced by such injury may therefore lay thegroundwork for future degeneration and osteoarthritis. The prole ofinammatory proteins within synovial uid after acute knee injury

Expression and fragmentation of the extracellular matrix proteinbronectin has been shown to occur in the synovial uid of arthriticpatients and joint injury [3,4]. Fibronectin fragment induced kneeinjury in an animal model results in further cartilage damage and lossof proteoglycans [5]. Fibronectin also induces microglial activationand stimulation of cytokine production and activation of matrixmetalloproteases [6]. It is well known that inammatory cytokines areassociated with bronectin and its fragments in the pathophysiologyof degenerative joint disease [7].

Aggrecan, a high molecular weight proteoglycan present inarticular cartilage, undergoes extensive degradation and turnoverduring normal cartilage metabolism, aging and joint diseases [8].Abbreviations: IFN-, interferon gamma; IL-6, intchemotactic protein 1; MIP-1, macrophage inammenzyme-linked immunosorbent assay; HRP, horseraperformance liquid chromatography; SEC, size exclusioexchange chromatography; TMB, tetramethylbenzidine;raphy based mass spectrometry; FN1, bronectin; ACAN Corresponding author.

E-mail address: Lewis.hanna@cytonics.com (L.S. Han1 Current address: New York University Hospital for

NY, USA.

0009-9120/$ see front matter 2010 The Canadiandoi:10.1016/j.clinbiochem.2010.04.069may represent diagnostic or prognostic biomarkers for the degener-IntroductionKeywords:FibronectinAggrecanBiomarkerCartilageSynovial uidPain

as asymptomatic volunteers. A combination of high-pressure chromatography, mass spectrometry andimmunological techniques were used to enrich and identify the protein components representing the IFN-signal.

Results: A protein complex of bronectin and the aggrecan G3 domain was identied in the synovialuid of patients with a meniscal tear and pain that was absent in asymptomatic controls. This proteincomplex correlated to the IFN- signal. A novel enzyme-linked immunosorbent assay (ELISA) was developedto specically identify the complex in synovial uid.

Conclusions: We have identied a protein complex of bronectin and aggrecan G3 domain that is acandidate biomarker for pain associated with meniscal injury.

2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.knee pain associated with meniscal pathology. The cytokine biomarkers included interferon gamma (IFN-),interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and macrophage inammatory protein-1 beta(MIP-1). Validation studies using other immunologic techniques conrmed the presence of IL-6, MCP-1 andMIP-1, but not IFN-. Therefore we sought the identity of the IFN- signal in synovial uid.

Methods: Knee synovial uid was collected from patients with an acute, painful meniscal injury, as wellArticle history:Received 10 February 2010Objectives:We previously described a panel of four cytokines biomarkers in knee synovial uid for acuteGaetano J. Scuderi b, Naruewan Woolf a, Kaitlyn DentVanessa G. Cuellar b,1, David C. Yeomans b, Eugene J.Robert Bowser a, Lewis S. Hanna a,a Research and Development, Cytonics Corporation, 555 Heritage Drive, Suite 115, Jupiter,b Stanford University, Stanford, CA, USA

a b s t r a c ta r t i c l e i n f oerleukin 6; MCP-1, monocyteatory protein-1 beta; ELISA,dish peroxidase; HPLC, highn chromatography; AEC, anionLC-MS/MS, liquid chromatog-, aggrecan.

na).Joint Diseases, New York City,

Society of Clinical Chemists. Publishctin and aggrecan G3 domain in synovialology

S. Raymond Golish b, Jason M. Cuellar b,1,rragee b, Martin S. Angst b,

3458, USA

hemistry

i e r.com/ locate /c l inb iochemAggrecanases are activated during cartilage degradation and diseases[9]. Recently, it was demonstrated that patterns of aggrecan fragmentsdiffer between acute injury and chronic degeneration relative tohealthy controls [10]. Therefore both bronectin and aggrecan exhibitincreased fragmentation in degenerative joint conditions and afterarticular cartilage damage. It is possible that bronectin, aggrecan andtheir fragments interact in the synovial uid to facilitate signalingcascades that augment joint and cartilage degeneration.

ed by Elsevier Inc. All rights reserved.

809G.J. Scuderi et al. / Clinical Biochemistry 43 (2010) 808814We recently described a panel of four cytokines biomarkers foracute knee pain associated with meniscal pathology [11], and onecytokine in the epidural space of spinal disc disease [12]. The cytokinebiomarker panel was identied from synovial uid using multiplexinammatory cytokine proling and included interferon gamma(IFN-), interleukin 6 (IL-6), monocyte chemotactic protein 1(MCP-1), and macrophage inammatory protein-1 beta (MIP-1).Validation studies using other immunologic techniques conrmed thepresence of IL-6, MCP-1 and MIP-1, but not IFN-.

To further determine the identity of the IFN- signal in themultiplex inammatory cytokine panel, we used a combination ofcolumn chromatography and mass spectrometry to enrich anddetermine the amino acid sequence identity of the IFN- signal.Additional immunoassays were used to conrm the sequenceidentity. Our study identied a protein complex from knee synovialuid containing bronectin and aggrecan. In addition, this proteincomplex was present in a painful knee with meniscal pathology butabsent from asymptomatic healthy volunteers.

Materials and methods

Subjects and synovial uid collection

Our prospective study included 15 adult patients withoutrheumatoid arthritis who were diagnosed with painful intra-articularderangement of the knee by history, physical examination andmagnetic resonance imaging (MRI) who elected for arthroscopicdebridement following a failure of non-operative pain management.Inclusion criteria were an age of 18 years or older, knee pain of recentonset (less than 6 months), and physical examination ndings andMRI results consistent with intra-articular pathology. Exclusioncriteria were an age of less than 18 years, a recent history (within3 months) of an intra-articular injection of a corticosteroid, and a pastor current history of autoimmune disease (such as rheumatoidarthritis). The meanstandard deviation age was 77.25.2 years,and there were 7 males and 8 females in the study group.

Institutional Review Board (IRB) approval was obtained for thestudy, and knee synovial uid was collected upon informed patientconsent by needle aspiration. Synovial uid was placed in polypro-pylene tubes containing a protease inhibitor (10 mM AEBSF, SigmaAldrich, St. Louis, MO) and stored at80 C. Prior to use, the synovialuid was treated with 5 mg/mL Hyaluronidase and claried bycentrifugation at 5000 g.

Multiplex cytokine assay

Multiplex immunoassay was performed using Bio-Rad's Bio-Plex200 with Bio-Plex human cytokine 4, 17, and 27 multiplex panels. Theassay was performed as recommended by the manufacturer.

Antibodies and chemicals

Horseradish peroxidase (HRP) labeled anti-bronectin antibodywas obtained from US Biological, Swampscott, MA; anti-aggrecan G3domain antibody was obtained from Santa Cruz Biotechnology, SantaCruz, CA; human bronectin was obtained from BD Biosciences, SanJose, CA; all other chemicals were obtained from Sigma Aldrich.

HPLC and protein purication

High performance liquid chromatography (HPLC) assays andpurication were performed on a Bio-Rad BioLogic DuoFlow HPLCsystem. Size exclusion chromatography (SEC) was performed usingtwo Bio-Rad SEC400-5 columns in series. SEC separation wasperformed in isocratic mode using 50 mM Tris/HCl, 100 mM NaCl

pH 7.0 buffer.Anion exchange chromatography (AEC) was performed using aBio-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.

Mass spectrometry

Ammonuim bicarbonate (1 M) was added to solution sample tobuffer pH to 8. Cysteine residues were reduced and alkylated with2.5 mM TCEP and 20 mM iodoacetamide. Sample was digested using2 ng of trypsin at 37 C for 3 h. Digestion mixtures was loaded onto aprecolumn (360 mm od100 mm id fused silica, Polymicro Technol-ogies, Phoenix, AZ) packed with 3 cm irregular C18 (515 mm non-spherical, YMC, Inc., Wilmington, NC) and washed with 0.1 M HOAcfor 5 min before switching in-line with the resolving column (7 cmspherical C18, 360 mm100 mm). Once the columns are in-line, thepeptides are gradient eluted with a gradient of 0100%A in 30 minwhere 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 orLTQ-Orbitrap (San Jose, CA). Electrospray was accomplished using anAdvion Triversa Nanomate (Advion Biosystems, Ithica, NY) with avoltage of 1.7 kV and a ow rate of 300 nL/min. The massspectrometer was operated using data dependent scanning with thetop 5 most abundant ions in each spectrum being selected forsequential MS/MS experiments. All MS/MS spectra were searchedwith Sequest using appropriate human database (ipi database v3.32).Database search results are tabulated and visually inspected usingScaffold (Proteome Software, Portland, OR).

Afnity chromatography

Anti-aggrecan G3 domain antibody was immobilized on Af-Gelaccording to the manufacturer's (Bio-Rad) recommended procedureto obtain an afnity chromatography for the purication of aggrecanG3 related molecules. The eluted peak from the AEC columncontaining the Bio-plex IFN- signal was further enriched on theaggrecan G3 afnity column. The column was equilibrated with10 mM phosphate, 150 mM NaCl buffer pH 7.8. The AEC fractionswere pooled and concentrated using spin lter with 30 kDamolecularweight cutoff. The column was then washed with the same buffer andeluted with 15 mM phosphate buffer pH 3.0. The eluted fraction wasassayed for aggrecan and bronectin proteins by slot blot analysis.

Gel electrophoresis and Western blotting

Gel electrophoresis was performed using Tris/HCl 18% or 420%polyacrylamide gradient gels (Bio-Rad). The samples were treatedwith SDS sample buffer at room temperature and loaded on the gelimmediately. The gel was run at constant voltage of 200 V for 1 husing 20 mM Tris base, 192 mM Glycine, 0.1% SDS, pH 8.3 as runningbuffer. 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 nitrocellulosemembrane. The transfer was performed at constant voltage of 100 Vfor 40 min using Towbin buffer. The membranes fromWestern or slotblot analysis was blocked with 20 mM Tris, 0.5 M NaCl, 1% casein pH7.4 overnight and developed using anti-aggrecan G3, anti-bronectinor anti-interferon- antibodies.

ELISA assay

Enzyme-linked immunosorbent assay (ELISA) plates were coatedwith anti-aggrecan G3 domain antibody in PBS/tween 20/thimerosaland 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-

Plex Pro II wash station). HRP-labeled anti-bronectin antibody wasthen added and the plate was incubated at room temperature for 1 hfollowed by 6 washes. Tetramethylbenzidine (TMB) substrate wasadded and incubated at room temperature in the dark for 5 min. Thereaction was stopped with sulfuric acid and the plate was read usingBio-Rad's Benchmark Plus microplate spectrophotometer at 450 nm.All assays were performed in triplicate. Human bronectin at 1 g/mLis used as negative control.

Results

By cytokine proling of synovial uid, we previously identiedsignicant increase in the concentrations of IL-6, MCP-1, MIP-1 andIFN- in patients with cartilage degeneration pathology [11]. In thecurrent study, we measured levels of these same cytokines in thesynovial uid from 15 patients with intra-articular pathology of theknee undergoing arthroscopic debridement as described in Materialand methods.

The knee synovial uid from 5 of these patients in pain withmeniscal pathology that exhibited the highest levels of all 4 cytokinebiomarkers (IFN-, IL-6, MCP-1, and MIP1-) was used for the

spin lter with a 300 kDa cutoff membrane and owed through a spinlter with a 1000 kDa cutoff membrane (data not shown).

id from symptomatic patient; B) synovial uid from asymptomatic healthy volunteer. Theffer. Protein elution was monitored by absorption at 215 nm (y-axis). Time elution from the

Fig. 2. SEC fractions 918 were analyzed on a Tris/HCl 420% polyacrylamide gradientSDS gel and protein bands visualized by silver stain. The bands in fractions 14 and 15 arein the molecular weight range of 400600 kDa.

810 G.J. Scuderi et al. / Clinical Biochemistry 43 (2010) 808814subsequent analysis. While immunoblot conrmed the presence ofIL-6, MCP-1 and MIP1-, we could not conrm the presence of IFN-by immunoblot or ELISA using multiple IFN- antibodies andcommercially available ELISA kits (data not shown). We thereforesought other methodologies to enrich for the IFN- signal detected bythe Bio-Plex assay and determine the protein identity correspondingto this cytokine signal.

Size exclusion chromatography performed via high performanceliquid chromatography (SEC-HPLC) analysis of the synovial uiddisplayed a complex protein elution prole (Fig. 1). Direct compar-isons between a symptomatic and non-symptomatic subject demon-strate the difference in the presence or absence of high molecularweight proteins (Fig. 1). As expected, silver stained polyacrylamidegels for SEC fractions 918 showed high molecular species in the earlyfractions and lower molecular weight species in the later fractions(Fig. 2). We assayed column fractions for the presence of the4-cytokine biomarkers using the Bio-plex assay. IFN- signal residedin fractions 13, 14 and 15, elution time 3238 min (Table 1). Thiselution time is characteristic of proteins 400700 kDamolecularmass.The other three cytokines were present in fractions 1719, a massrange more typical of the respective cytokine proteins. The molecularmass of the IFN- signal was conrmed by fractionating the clariedsynovial uid using spin lters. The IFN- signal was retained on a

Fig. 1. SEC elution prole of a 0.1 mL injection of claried synovial uid. A) synovial ucolumnwas run under isocratic condition using 50 mM Tris/HCl, 100 mMNaCl, pH 7.0 bu

column (minutes) and column fraction number are noted on the x-axis.Identication of bronectinaggrecan complex

To determine the protein identity corresponding to the IFN-signal, we pooled SEC fractions 14 and 15 and digested the proteinswith trypsin for subsequent amino acid sequence analysis by liquidchromatography based mass spectrometry (LC-MS/MS). A predom-inant protein identied from these fractions corresponded tobronectin. We identied 15 unique peptides representing 320 of2386 amino acids (13.4% coverage) of human bronectin (Swiss-Protidentication number P02751) sequence (Fig. 3). While otherproteins were identied in these SEC fractions, bronectin is anextracellular matrix protein previously implicated in cartilage jointdisease [7]. The molecular mass of bronectin in its dimeric form isapproximately 524 kDa in-line with the SEC fractions containing theIFN- signal have approximately 400700 kDa molecular mass. Sincehuman bronectin did not cross react with INF- antibody labeledbeads in the Bio-Plex assay (data not shown), additional proteins mayinteract with bronectin within these SEC fractions that give rise tothe antibody cross reactivity.

To further characterize proteins within these SEC fractions, weperformed Western blot analysis with antibodies to bronectin andother potential binding partners representing various cartilage and

synovialuid proteins. A representative silver stain SDS-polyacrylamidegel containing SEC fractions 14 and 15 is shown in Fig. 4A. Wedetermined that protein bands immunoreactive for bronectin andaggrecan G3 co-migrated in the gel (Figs. 4B and C). A polyclonalantibody to IFN- also weakly immunolabeled the same protein band(Fig. 4D).

Enrichment of the bronectin/aggrecan complex by anion exchangechromatography and anti-aggrecan G3 afnity column chromatography

To further validate our SEC results and obtain amino acid sequenceinformation for bronectin interacting proteins, we enriched for theBio-plex IFN- signal by anion exchange chromatography (AEC) ofsynovial uid from a patient suffering from a painful meniscus tear.The AEC elution prole is shown in Fig. 5A and all fractions wereanalyzed by Bio-plex cytokine assay to identify fractions containingthe IFN- signal. Slot blot analysis demonstrated the presence of bothbronectin and aggrecan G3 in these same fractions (data not shown).

An ELISA was developed to identify the presence of a bronectin/aggrecan G3 protein complex in synovial uid. The ELISA format was amodication of the classical sandwich ELISA, where the captureantibody recognized aggrecan G3 domain and the detection antibodywas HRP-labeled anti-bronectin antibody. We used this ELISA and

determined the presence of a bronectinaggrecan protein complexin the AEC fractions (Table 2). The bronectinaggrecan G3 complexwas present in AEC fractions 1920 (Fig. 5A), corresponding to theBio-plex IFN- signal.

AEC fractions 19 and 20, containing the bronectinaggrecanprotein complex, were pooled and concentrated using a 30 kDamolecular weight cutoff spin lter. This sample was further puriedusing the aggrecan G3 afnity column as described in Materials andmethods, the elution prole is shown in Fig. 5B. We noted a large peakof protein that failed to bind to the afnity column (fractions 25), apeak of proteins that eluted off the column at pH 3.0 (fractions

Table 1IFN- signal in SEC fractions by Bio-plex assay.

Fraction 8 9 10 11 12 13 14 15 16 17 18 19IFN- 0.3 0 0 0 0 12.4 58.7 8.4 1.7 2 1.4 0

SEC fractions obtained from symptomatic subject shown in Fig. 1 were assayed for IFN- cytokine levels by the multiplex Bio-plex assay (Bio-rad). IFN- levels are shown forfractions containing the peak IFN- signal.

Fig. 4. SDS gel electrophoresis and immunoblot analysis of proteins contained in theIFN- containing fractions. Fractions 14 and 15 were analyzed by silver stain (A), andimmunoblot for aggrecan G3 (B), bronectin (C), and IFN- (D). The arrow denotes asingle band that is detected by silver stain and immunoreactive for the aggrecan G3domain, bronectin, and IFN-.

811G.J. Scuderi et al. / Clinical Biochemistry 43 (2010) 808814Fig. 3. Tandemmass spectrometric analysis for the bronectin peptide, GATYNIIVEALKDQQR.for MS/MS analysis. The spectrumwas acquired on a Thermo hybrid LTQ-Orbitrap instrumentheoreticalm/z value (m/z 909.9891). B) The amino acid sequence of the peptide, shown wiof the bronectin peptide following dissociation in the LTQ ion trap. The observed b- and yA) TheMS spectrum of the [M+2H]+2 precursor ion that was isolated and dissociatedt using the Orbitrap as the mass analyzer. The [M+2H]+2 ion was within 4 ppm of theth the b- and y-type product ions detected by MS/MS analysis. C) The MS/MS spectrum-type ions are labeled in the spectrogram.

Fig. 5. Purication of the protein complex by anion exchange chromatography (AEC) and afused in the elution. Column fraction numbers are listed above the prole; fractions 19 and 2ELISA assay. Fractions 19 and 20 containing the peak levels of the protein complex were poolB) Elution prole of the aggrecan G3 afnity chromatography and the gradient program us

Table 2ELISA complex assay for the starting material and anion exchange chromatographyfractions.

Sample Dilution Absorption units(450 nm)

Averageabsorption

CV%

1 g bronectin, negativecontrol

1 0.03 0.04 0.04 0.04 17.7

Positive control 10 2.69 2.45 2.70 2.61 5.4Starting sample 10 1.47 1.53 1.53 1.51 2.1fx. 10 1 0.04 0.04 0.04 0.04 1.6fx. 11 1 0.04 0.04 0.04 0.04 1.7fx. 12 1 0.04 0.04 0.05 0.04 13.2fx. 13 1 0.04 0.04 0.04 0.04 2.4fx. 14 1 0.04 0.04 0.04 0.04 6.6fx. 15 1 0.05 0.04 0.05 0.04 10.7fx. 16 1 0.05 0.04 0.04 0.04 13.8fx. 17 1 0.06 0.04 0.05 0.05 13.5fx. 18 1 0.13 0.12 0.10 0.12 14.4fx. 19 1 0.32 0.20 0.22 0.25 26.7fx. 20 1 0.22 0.41 0.31 0.31 30.3fx. 21 1 0.07 0.07 0.11 0.08 30.8fx. 27 2 0.04 0.04 0.04 0.04 0.0fx. 30 4 0.04 0.04 0.05 0.04 7.3

Knee synovial uid from a patient in pain with a meniscal injury was applied to ananion exchange column (AEC) and eluted with a linear salt gradient as described inMaterials and methods. The bronectin/aggrecan G-3 ELISA was used to analyze theAEC fractions for the presence of the protein complex. Fibronectin at 1 L/mL was usedas negative control. A pool of positive patient samples was used as positive control.Starting sample is the patient sample prior to AEC fractionation.

812 G.J. Scuderi et al. / Clinical Biochemistry 43 (2010) 8088141824), and a nal peak of proteins that eluted during the re-equilibration of the column (fractions 3538). Due to proteinaggregation and loss of proteinprotein interaction that typicallyoccurs at low pH, we used a slot blot analysis to explore the presenceor absence of bronectin and aggrecan G3 in these afnity columnfractions (Fig. 6). We detected both bronectin and aggrecan in owthrough fractions 25 suggesting saturation of the column, noaggrecan or bronectin in the pH 3.0 elution fractions 1824, butthe presence of both aggrecan and bronectin in fractions 3538.Fibronectin tends to aggregate at pH 3.0 and we believe this explainswhy the eluted peak containing bronectin and aggrecan appears atthe buffer front during the re-equilibration of the column.

ELISA results for the clinical samples

Finally, we used the novel ELISA to the bronectinaggrecan G3complex to measure its level in the synovial uid of 15 symptomaticsubjects undergoing arthroscopic debridement of the knee. Theoptical density of the ELISA immunoassay for the symptomaticgroup ranged from 0.87 to 15.43 (mean 7.6; std.dev. 4.2).

Discussion

It is well accepted that disc and joint cartilage degeneration isassociatedwith aging. However, it is not knownwhy the degenerationis painful in some individuals and not in others. Aggrecan, the major

nity column chromatography. A) Elution prole of the AEC and the gradient program0 contained the INF- signal by Bio-Plex and the Fibronectinaggrecan G3 complex byed, concentrated and further enriched by aggrecan G3 afnity column chromatography.ed in the elution. Column fraction numbers are listed above the prole.

813G.J. Scuderi et al. / Clinical Biochemistry 43 (2010) 808814component of cartilage, and bronectin degradation were observed inboth groups and considered part of the cartilage generation/degeneration cycle. We recently described a panel of four cytokines(IFN-, MCP-1, IL-6 and MIP-1) that correlated to pain in patientswith meniscal injury of the knee [11]. In this study, we have identieda complex of bronectin with an aggrecan fragment that is associatedwith painful cartilage degeneration or damage. The presence of thisprotein complex correlated to the IFN- signal in synovial uiddetected by multiplex analysis.

Since we could not conrm the presence of IFN- in synovial uidby immunoblot or ELISA specic to IFN-, we enriched for the Bio-plex IFN- signal by column chromatography. It was evident from sizeexclusion chromatography and spin lters with nominal molecularweight cutoff that the protein(s) responsible for the IFN- signal hadan apparent molecular mass of 400600 kDa, much larger than the

Fig. 6. Slot blot of aggrecan G3 afnity chromatography elution fractions from Fig. 5B.A) Slot blot for bronectin. B) Slot blot for aggrecan G3. Fractions 25 represent columnow through, both aggrecan G3 and bronectin are observed; fractions 1824represent protein peaks eluted at pH 3.0, no bronectin or aggrecan were observedin this fraction; fractions 3538 represent proteins eluted at the buffer front during re-equilibration of the column, this peak contained both aggrecan G3 and bronectin.molecular mass of 25 kDa. LC-MS/MS of the partially puried IFN-signal demonstrated the presence of bronectin that was conrmedby western and ELISA assays. Fibronectin alone did not show apositive signal in the Bio-Plex IFN- assay. Aggrecan is a multidomainprotein where the C-terminal domain (G3) consists of an EGF, lectinand CRP sequences [13]. Age-related increases in aggrecan G3 havebeen shown to occur in articular cartilage [14], and increasedaggrecan fragments have been observed after knee injury and inosteoarthritis [15]. The known interaction of bronectin with lectinssuggests that a complex could be formed between bronectin andaggrecan G3 domain. Western blot analysis of the partially puriedIFN- signal by SEC-HPLC showed one band immunopositive forantibodies to bronectin, aggrecan G3 domain, and also cross-reactedwith a polyclonal antibody to IFN-, suggesting the formation of aprotein complex between these proteins that corresponds to the IFN- signal.

A heterogeneous ELISA assay where a microplate was coated withanti-bronectin and anti-aggrecan G3 domain was used for detectionand the reverse where the microplate was coated with anti-aggrecanG3 domain and detection was performed using anti-bronectinfurther conrmed the presence of a complex between bronectinand aggrecan G3 domain (data not shown). We also enriched thecomplex using anion exchange chromatography followed by afnitychromatography using immobilized anti-aggrecan antibody. Thepuried fraction contained both bronectin and aggrecan G3 byWestern blot analysis. The presence of bronectin in the elutionfraction further supports the presence of the complex in knee synovialin patients with painful meniscal injury.

Our study showed 100% agreement between the results of thecytokine proling and the presence or absence of the bronectinaggrecan G3 complex as determined by the developed ELISA assay.Furthermore, analyzing more than 150 joint samples from patientsand healthy volunteers in the same age group showed 98% agreementbetween the IFN- and the bronectin/aggrecan G3 ELISA results.

Many questions remain regarding the protein binding sitebetween bronectin and aggrecan G3, and the nature of the crossreactivity between the protein complex and the IFN- antibodies onthe Bio-plex beads. Lundell et al. identied direct interaction betweenthe bronectin type III (FN III) domains of tenascins and the C-typelectin domains [16]. 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 bronectin withintegrin 41 [17]. The linker between FN 7, FN 8 and FN 9 also hasthe same tilt angle, twist angle and buried surface area in the interfaceas FN 13 and FN 14 [18]. These data suggests a ratio of 2:1 aggrecan G3domain:bronectin. Further studies are underway to assemble thecomplex in vitro and determine the molar ratio of the aggrecan G3domain to bronectin in the complex.

We suggest that the cross reactivity between the Bio-plex IFN-beads and the bronectinaggecan complex occurs via a conforma-tion epitope generated upon formation of the protein complex. Purebronectin or aggrecan failed to exhibit a positive Bio-plex IFN-signal, and direct sequence comparisons failed to identify signicantsequence identity between IFN- and either aggrecan G3 orbronectin (data not shown).

This study is unique in its observation of full-length bronectininvolved in the complex formation whereas studies to-date indicatethat bronectin fragments and not the full-length bronectincontribute to cartridge degradation [19,20]. Full-length bronectinat levels of 1580 ng/mL was detected in 60% of the synovial uidsamples obtained from our healthy control subjects. Fibronectinfragments were not detected in the synovial uid of any healthycontrol subject by Western blot or column chromatography. At thistime, we do not know if the presence of bronectin in the synovialuid of healthy volunteers is a prelude to cartilage degeneration. Onepotential mechanism involves the early generation of aggrecanfragments that may associate with full-length bronectin. Furtherstudies are necessary to explore the relationship between thebronectinaggrecan G3 complex identied in this study and thecontinued fragmentation of bronectin, aggrecan and other structuralproteins during cartilage degenerative disorders.

In conclusion, we have identied a protein complex betweenbronectin and aggrecan G3 domain present in the synovial uid ofpatients withmeniscal degeneration/injury and pain, and this complexmay play a role in the development of future degenerative joint disease.

Acknowledgment

Wewould like to thank Dr. Jennifer Busby, Kristie Rose and ValerieCavett at Scripps Florida, Jupiter FL, for performing the LC/MS/MSexperiments.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.clinbiochem.2010.04.069.

References

[1] Goldring MB, Goldring SR. Osteoarthritis. J Cell Physiol 2007;213:62634.[2] Lohmander LS, Dahlberg L, Eyre D, Lark M, Thonar EJMA, Ryd L. Longitudinal and

cross-sectional variability in markers of joint metabolism in patients with knee

pain and articular cartilage abnormalities. Osteoarthritis Cartilage 1998;6:35161.

[3] Homandberg GA. Cartilage damage by matrix degradation products bronectinfragments. Clin Orthop 2001;391:S1007.

[4] Przybysz M, Borysewicz K, Szechinski J, Katnik-Prastowska I. Synovial bronectinfragmentation and domain expressions in relation to rheumatoid arthritisprogression. Rheumatology 2007;46:10715.

[5] Williams JM, Zhang J, Kang H, Ummadi V, Hoandberg GA. The effects of hyaluronicacid on bronectin fragment mediated cartilage chondrolysis in skeletally maturerabbits. Osteoarthritis Cartilage 2003;11:449.

[6] Milner R, Crocker SJ, Hung S, Wang X, Frausto RF, del Zoppo GJ. Fibronectin- andvitronectin-induced microglial activation and matrix metalloproteinase-9 expres-sion is mediated by integrins 51 and v5. J Immunol 2007;178:815867.

[7] Homandberg GA, Davis G, Maniglia C, Shrikhande A. Cartilage chondrolysis bybronectin fragments causes cleavage of aggrecan at the same site as found inosteoarthritic cartilage. Osteoarthritis Cartilage 1997;5:4503.

[8] Ilic MZ, Handley CJ, Robinson HC, Mok MT. Mechanism of catabolism of aggrecanby articular cartilage. Arch Biochem Biophys 1992;294:11522.

[9] Nagase H, Kashiwagi M. Aggrecanases and cartilage matrix degradation. ArthritisRes Ther 2003;5:94103.

[10] Struglics A, Larsson S, Hansson M, Lohmander LS. Western blot quantication ofaggrecan fragments in human synovial uid indicates differences in fragmentpatterns between joint diseases. Osteoarthritis Cartilage 2009;17:497506.

[11] Cuellar JM, Scuderi GJ, Cuellar VG, Golish SR, Yeomans DC. Diagnostic utility ofcytokine biomarkers in the evaluation of acute knee pain. J Bone Joint Surg Am2009;91:231320.

[12] Scuderi GJ, Cuellar JM, Cuellar VG, Yeomans DC, Carragee EJ, Angst MS. Epiduralinterferon gamma-immunoreactivity. Spine 2009;34:23117.

[13] Luo W, Guo C, Zheng J, Chen T-L, Wang PY, Vertel BM, et al. Aggrecan from start tonish. J Bone Miner Metab 2000;18:516.

[14] Dudhia J, Davidson CM, Wells TM, Vynios DH, Hardingham TE, Bayliss MT. Age-related changes in the content of the C-terminal region of aggrecan in humanarticular cartilage. Biochem J 1996;313:93340.

[15] Lohmander LS, Ionescu M, Jugessur H, Poole AR. Changes in joint cartilageaggrecan after knee injury and in osteoarthritis. Arthritis Rheum 1999;42:53444.

[16] Lundell A, Olin AI, Morgelin M, al-Karadaghi S, Aspberg A, Logan DT. Structuralbasis for interactions between tenascins and lectican C-type lectin domains:evidence for a crosslinking role for tenascins. Structure 2004;12:1495506.

[17] Sharma A, Askari JA, Humphries MJ, Jones EY, Stuart DI. Crystal structure of aheparin- and integrin-binding segment of human bronectin. EMBO J 1999;18:146879.

[18] Leahy DJ, Aukhil I, Erickson HP. 2.0A crystal structure of a four-domain segmentof human bronectin encompassing the RGD loop and synergy region. Cell1996;84:15564.

[19] Yasuda T. Cartilage destruction by matrix degradation products. Mod Rheumatol2006;16:197205.

[20] Ding L, Guo D, Homandberg GA. The cartilage chondrolytic mechanism ofbronectin fragments involves MAP kinases: comparison of three fragments andnative bronectin. Osteoarthritis Cartilage 2008;16:125362.

814 G.J. Scuderi et al. / Clinical Biochemistry 43 (2010) 808814

Identification of a complex between fibronectin and aggrecan G3 domain in synovial fluid of pat.....IntroductionMaterials and methodsSubjects and synovial fluid collectionMultiplex cytokine assayAntibodies and chemicalsHPLC and protein purificationMass spectrometryAffinity chromatographyGel electrophoresis and Western blottingELISA assay

ResultsIdentification of fibronectinaggrecan complexEnrichment of the fibronectin/aggrecan complex by anion exchange chromatography and anti-aggrec.....ELISA results for the clinical samples

DiscussionAcknowledgmentSupplementary dataReferences