Proteoglycans in the Human Sclera - iovs.arvojournals.org human sclera to provide background...

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Proteoglycans in the Human Sclera Evidence for the Presence of Aggrecan Jody A. Rada, Virginia R. Achen, Cheryll A. Perry, and Petra W. Fox Purpose. The proteoglycans synthesized and accumulated within the adult human sclera (aged 50 to 80 years) were identified by their size, glycosaminoglycan side chains, and core proteins in an effort to characterize the proteoglycan content of the human sclera. Methods. Sclerae, unlabeled, or radiolabeled in organ culture with 35 SO 4 or 3 H-proline, were extracted in 4M guanidine-HCl and separated by Sepharose CL-2B and Superose 6 forced- pressure liquid chromatography. Peak fractions, identified by glycosaminoglycan content or radioactivity, were pooled and subjected to G-50 chromatography or sodium dodecyl sulfate- polyacrylamide gel electrophoresis before and after digestion with specific glycosidases. Scleral proteoglycan core proteins were identified in Western blot analysis using specific antisera to decorin, biglycan, and aggrecan. Reverse transcription-polymerase chain reaction analyses were carried out on human scleral fibroblast RNA to confirm the transcription of one scleral proteoglycan. Proteoglycans were localized on sections of scleral tissue using specific antisera. Results. After chromatography on CL-2B, scleral proteoglycans could be resolved into three major peaks, PG-1, PG-2, and PG-3. The largest scleral proteoglycan, PG-1, contained chondroi- tin sulfate and keratan sulfate glycosaminoglycan side chains. Results of Western blot analyses indicated that the core protein of PG-1 is the aggrecan core protein, migrating at «350 kDa. Reverse transcription-polymerase chain reaction analyses confirmed that human scleral fibroblasts transcribe aggrecan in vitro and in vivo. PG-2 and PG-3 were identified as biglycan and decorin in Western blot analyses using antibiglycan and antidecorin antibodies, respec- tively. Immunostaining results indicated that aggrecan, biglycan, and decorin are distributed throughout the thickness of the human sclera. Conclusions. The adult human sclera contains three major proteoglycans; aggrecan, biglycan, and decorin. It is likely that these proteoglycans contribute to the structural properties of the sclera and that the ratios of these proteoglycans will change with age, specific region, and condition of the sclera. Invest Ophthalmol Vis Sci. 1997;38:l740-1751. A he tough outer coat of the eye, the sclera, is a connec- tive tissue that provides the structural framework that de- fines the shape and, therefore, the focal length of the eye. At the same time, the sclera must prevent distortion of the light-sensitive components of the visual pathway when the eyeball is moved by the extraocular muscles, From the Department of Anatomy and Cell Biology, University of North Dakota School of Medicine, Grand Forks. Supported by National Institutes of Health grant EYO9391 (JAR) and funds from the Howard Hughes Medical Institute Undergraduate Research Program Grant HHHMI 71195-539401 (PWF). Submitted for publication September 3, 1996; revised March 26, 1997; accepted March 27, 1997. Proprietary interest categoiy: N. Reprint requests: Jody A. Rada, Department of Anatomy and Cell Biology, University of North Dakota School of Medicine, Box 9037, Grand Forks, ND 58202. and it must provide sufficient elasticity to resist any fluc- tuations in intraocular pressure. The requirements of scle- ral strength, elasticity, and resiliency are largely met by a connective tissue, consisting of scleral fibroblasts embed- ded in a matrix of interwoven collagen fibrils of varying diameters, in close association with proteoglycans. Proteo- glycans serve several biologic functions that include regu- lation of hydration in vivo, maintenance of structural in- tegrity, growth regulation, matrix organization, and cell adhesion; they also bind certain growth factors. 1 The principal proteoglycans of connective tissue have been characterized through complementary DNA clon- ing of the core proteins. Decorin (also referred to as PG II) and biglycan (also referred to as PG I) are two, closely related, small proteoglycans with apparent molecular 1740 Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9 Copyright © Association for Research in Vision and Ophthalmology Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933425/ on 09/05/2018

Transcript of Proteoglycans in the Human Sclera - iovs.arvojournals.org human sclera to provide background...

Proteoglycans in the Human ScleraEvidence for the Presence of Aggrecan

Jody A. Rada, Virginia R. Achen, Cheryll A. Perry, and Petra W. Fox

Purpose. The proteoglycans synthesized and accumulated within the adult human sclera (aged50 to 80 years) were identified by their size, glycosaminoglycan side chains, and core proteinsin an effort to characterize the proteoglycan content of the human sclera.

Methods. Sclerae, unlabeled, or radiolabeled in organ culture with 35SO4 or 3H-proline, wereextracted in 4M guanidine-HCl and separated by Sepharose CL-2B and Superose 6 forced-pressure liquid chromatography. Peak fractions, identified by glycosaminoglycan content orradioactivity, were pooled and subjected to G-50 chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after digestion with specific glycosidases. Scleralproteoglycan core proteins were identified in Western blot analysis using specific antisera todecorin, biglycan, and aggrecan. Reverse transcription-polymerase chain reaction analyseswere carried out on human scleral fibroblast RNA to confirm the transcription of one scleralproteoglycan. Proteoglycans were localized on sections of scleral tissue using specific antisera.

Results. After chromatography on CL-2B, scleral proteoglycans could be resolved into threemajor peaks, PG-1, PG-2, and PG-3. The largest scleral proteoglycan, PG-1, contained chondroi-tin sulfate and keratan sulfate glycosaminoglycan side chains. Results of Western blot analysesindicated that the core protein of PG-1 is the aggrecan core protein, migrating at «350kDa. Reverse transcription-polymerase chain reaction analyses confirmed that human scleralfibroblasts transcribe aggrecan in vitro and in vivo. PG-2 and PG-3 were identified as biglycanand decorin in Western blot analyses using antibiglycan and antidecorin antibodies, respec-tively. Immunostaining results indicated that aggrecan, biglycan, and decorin are distributedthroughout the thickness of the human sclera.

Conclusions. The adult human sclera contains three major proteoglycans; aggrecan, biglycan,and decorin. It is likely that these proteoglycans contribute to the structural properties of thesclera and that the ratios of these proteoglycans will change with age, specific region, andcondition of the sclera. Invest Ophthalmol Vis Sci. 1997;38:l740-1751.

A he tough outer coat of the eye, the sclera, is a connec-tive tissue that provides the structural framework that de-fines the shape and, therefore, the focal length of theeye. At the same time, the sclera must prevent distortionof the light-sensitive components of the visual pathwaywhen the eyeball is moved by the extraocular muscles,

From the Department of Anatomy and Cell Biology, University of North DakotaSchool of Medicine, Grand Forks.Supported by National Institutes of Health grant EYO9391 (JAR) and funds fromthe Howard Hughes Medical Institute Undergraduate Research Program GrantHHHMI 71195-539401 (PWF).Submitted for publication September 3, 1996; revised March 26, 1997; acceptedMarch 27, 1997.Proprietary interest categoiy: N.Reprint requests: Jody A. Rada, Department of Anatomy and Cell Biology,University of North Dakota School of Medicine, Box 9037, Grand Forks, ND58202.

and it must provide sufficient elasticity to resist any fluc-tuations in intraocular pressure. The requirements of scle-ral strength, elasticity, and resiliency are largely met by aconnective tissue, consisting of scleral fibroblasts embed-ded in a matrix of interwoven collagen fibrils of varyingdiameters, in close association with proteoglycans. Proteo-glycans serve several biologic functions that include regu-lation of hydration in vivo, maintenance of structural in-tegrity, growth regulation, matrix organization, and celladhesion; they also bind certain growth factors.1

The principal proteoglycans of connective tissue havebeen characterized through complementary DNA clon-ing of the core proteins. Decorin (also referred to as PGII) and biglycan (also referred to as PG I) are two, closelyrelated, small proteoglycans with apparent molecular

1740Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9Copyright © Association for Research in Vision and Ophthalmology

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Human Scleral Proteoglycans 1741

masses of 120 and 200 kDa, respectively, and proteincores of «45 kDa.2 Decorin contains one chondroitin-dermatan sulfate glycosaminoglycan side chain, whereastwo chondroitin-dermatan sulfate side chains may beattached to the biglycan core protein. Additionally, twosimilar, though distinct, large proteoglycans have beenidentified in many connective tissues. One form is thelarge chondroitin sulfate proteoglycan present in carti-lage, called aggrecan3'4; and the other form, called versi-can, is a proteoglycan identified in cultured human fi-broblasts.5 Although these two proteoglycans have largecore proteins of similar size (>350 kDa as determinedby sodium dodecyl sulfate-polyacrylamide gel electro-phoresis [SDS-PAGE]), versican contains 15 to 17 chon-droitin sulfate glycosaminoglycan side chains, whereas ag-grecan contains more than 100 chondroitin sulfate chainsand more than 30 keratan sulfate chains.

Decorin and biglycan core proteins and mRNA havebeen identified on sections of fetal human sclera6; andat the ultrastructural level, proteoglycans have beendemonstrated in the human sclera as cuprolinic-blue-positive, electron-dense filaments localized around andradiating from the d band of collagen fibrils.7 However,although there have been several studies on the proteo-glycan content of the bovine sclera,8"11 there is littledocumentation on the composition of proteoglycans inthe human sclera. The bovine sclera has been shown tocontain two types of dermatan sulfate proteoglycans: alarge proteoglycan, in which the core protein is >100kDa, and a group of small dermatan sulfate proteogly-cans whose core proteins have an apparent molecularweight of «46 kDa.8'9 The human sclera contains hyalur-onan, chondroitin sulfate, dermatan sulfate, and he-paran sulfate.1213 The presence of keratan sulfate hasbeen reported by some14 but not by others.12 With theexception of hyaluronan, it is likely that these glycosami-noglycans exist in a proteoglycan form; however, withoutcharacterizing the core proteins of these proteoglycans,the identity of the scleral proteoglycans cannot be ascer-tained. The purpose of the current study is to identifyand characterize the proteoglycans present within thehuman sclera to provide background information forfuture studies on disorders in which scleral anatomy andmechanical properties are altered.

In this article, we document the presence of threeproteoglycans in human sclerae aged 50 to 80 years andprovide the first evidence of the presence of aggrecan,the cartilage proteoglycan, in the human sclera.

MATERIALS AND METHODS

Extraction of Proteoglycans

Sclerae from human donor eyes, aged 50 to 80 years,were received within two days of death from the Medi-

cal Eye Bank of Western Pennsylvania and the Na-tional Disease Research Interchange. Human tissuewas handled according to the tenets of the Declarationof Helsinki, and the research was approved by theUniversity of North Dakota's institutional reviewboard. On receipt, sclerae were immediately frozen at—80°C or placed in organ culture for radiolabeling.Two batches of 10 previously frozen eyes were usedto extract scleral proteoglycans for molecular sievechromatography of nonradiolabeled material. For bio-synthesis experiments, both sclerae from each of sixdonors were radiolabeled and chromatographed sepa-rately as described later. To extract proteoglycans, ad-herent muscle, fat, lamina cribrosa, and optic nervehead were removed from each sclera; and the entiresclera (anterior, equatorial, and posterior regions) wasminced with a razor blade into pieces less than 2 mm3.The minced tissue was extracted in 4 M guanidine-HC1, containing 0.01 M sodium acetate, 0.01 M so-dium EDTA, 0.005 M benzamidine-HCl, and 0.1 Me-amino-n-caproic acid at 4°C overnight,15 followed bya reextraction in the same solvent for 2 to 4 hours at4°C. The two extracts were combined for each tissueand dialyzed exhaustively in distilled water or in 0.01M Na2SO4-, dialyzed again in distilled water for radio-labeled sclerae, and lyophilized.

Organ Culture and Radiolabeling

Sclerae used for proteoglycan biosynthesis experi-ments were cleaned of adherent adnexia, cut into but-tons «all mm in diameter with the aid of a surgicaltrephine, and radiolabeled in organ culture with 0.5ml of Dulbecco's Modified Eagle Medium (DMEM)containing 15% fetal bovine serum and either 35SO4

(250 /LtCi/ml) or 3H-proline (100 /iCi/m\) for 24hours, to label proteoglycans or total protein, respec-tively. After radiolabeling, the scleral punches wereminced and extracted with 4 M guanidine-HCl asdescribed above. The radiolabeled scleral extractswere dialyzed exhaustively in 0.01 M Na2SO4', dialyzedagain in distilled water, and lyophilized.

Chromatography

Lyophilized scleral extracts that were not radiola-beled, or that were radiolabeled with 35SO4, were re-constituted into column buffer (4 M guanidine-HCl,containing 0.02 M Tris, pH 6.8, and 0.1% CHAPS)and applied to a Sepharose CL-2B column (100 X 1.6cm). An aliquot from each fraction was measured forglycosaminoglycan content using the dimethylmethy-lene blue assay16 or for the presence of radioactivityby liquid scintillation counting, and tubes containingthe peak fractions were pooled, dialyzed, and lyophi-lized.

Lyophilized extracts from sclerae that were radio-

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labeled with 3H-proline were reconstituted in 6 M ureacontaining 0.05 M Tris, pH 6.8; 0.1% CHAPS; and 0.15M NaCl and applied to Diethylaminoethyl (DEAE)-Sepharose step columns (3.7 X 1.5 cm) equilibratedin the same solvent. The unbound, or glycoprotein,fraction was washed through; and the bound, or pro-teoglycan, material was eluted with 6 M urea con-taining 1.15 M NaCl. The proteoglycan fraction wasdialyzed against water, lyophilized, and applied to aSepharose CL-2B column, as described. An aliquotfrom each fraction was counted for radioactivity todetect the presence of DEAE-purified, 3H-proline-la-beled proteoglycans; and tubes containing the peakfractions were pooled, dialyzed, and lyophilized.

To characterize one pool of 35SO4-labeled proteo-glycans identified by Sepharose CL-2B chromatogra-phy (PG-1), the types of glycosaminoglycans detectedwere identified by their sensitivity to chondroitinaseABC (c'ase ABC) and endo-/?-galactosidase (endo-/?-gal) using molecular sieve chromatography on Sepha-dex G-50 to separate the resistant glycosaminoglycansfrom those which were degraded by the enzyme treat-ment.17"19 Briefly, 35SO4-labeled proteoglycans ob-tained from fraction PG-1 after chromatography onSepharose CL-2B were digested with c'ase ABC orendo-/?-galactosidase (endo-/3-gal; Seikagaku America,Ijamsville, MD) in 0.1 M Tris, pH 7.4, containing 500mM phenylmethylsulfonyl fluoride, 100 raM N-ethyl-maleimide, 100 mM EDTA, and 36 mM pepstatin Aovernight at 37°C. Digestion mixtures were applied toa G-50 Sephadex column (50 X 1 cm) in phosphate-buffered saline (PBS) containing 0.02% sodium azide.An aliquot from each fraction was counted for radioac-tivity, and the chromatographic profile was used tocalculate percentages of enzyme-resistant, 35SO4-la-beled glycosaminoglycans (eluting in the void vol-ume) and the percentage of enzyme-sensitive, 35SO4-labeled glycosaminoglycans (eluting in the includedvolume).

35SO4-labeled proteoglycans in fractions PG-2 andPG-3 were further separated by Forced Pressure Liq-uid Chromatography (FPLC) on Superose 6 (HR 10/30; Pharmacia, Piacataway, NJ), which was equili-brated and eluted with 4 M guanidine-HCl con-taining 0.02 M Tris HC1, pH 6.8. Aliquots of eachfraction were measured for radioactivity, and tubescontaining peak fractions were pooled, dialyzed, andlyophilized.

Electrophoretic Techniques

Proteoglycans in fractions PG-1, PG-2, and PG-3 werecharacterized by Western blot analyses, using antiseragenerated against human aggrecan (generously pro-vided by Dr. Robin Poole, Joint Diseases Laboratory,McGill University, Montreal, Canada), a peptide of

human biglycan20 (generously supplied by Dr. PeterRoughley, Joint Diseases Laboratory, McGill Univer-sity, Montreal, Canada), and antisera against a syn-thetic peptide containing the exon 5 sequence of hu-man decorin21 (generdusly supplied by Dr. DavidMcQuillan, Center for Extracellular Matrix Biology,Texas A & M University, Houston). Additionally, amonoclonal antibody to the human large proteogly-can, versican22 (#2-B-l; Seikagaku America) was em-ployed in Western blot analysis. Fractions of PG-1, PG-2, and PG-3 proteoglycans were digested with c'aseABC, endo-/?-gal, keratanase II (K'ase II; SeikagakuAmerica), or both, as described above; and digestedand undigested samples were electrophoresed on 5%or 10% sodium dodecyl sulfate-polyacrylamide gels.Proteoglycans were transferred to nitrocellulose, re-acted with antibodies to the proteoglycan core pro-teins, and detected with the Western Star chemilumi-nescent substrate (Tropix, Bedford, MA). Westernblot analyses were repeated two to three times for eachproteoglycan pool.

Cell Culture

Human sclerae were cleaned of adhering muscle, fat,retina, conjunctiva and vitreous and minced intopieces less than 2 mm2. Scleral pieces were then placedinto 60-mm culture dishes and were covered with ster-ile coverslips to hold the sclerae in place. Fibroblastswere grown from the explanted donor sclerae in Dul-becco's Modified Eagle Medium (DMEM) with 15%fetal bovine serum. After 2 to 4 weeks of culture, thecoverslips and scleral explants were removed, and cul-tures were allowed to become confluent. For RNAisolation, scleral fibroblasts were passaged into ten100-mm dishes and grown to confluency.

Reverse Transcription -Polymerase ChainReaction Analyses

To confirm that scleral fibroblasts synthesize ag-grecan, total RNA was isolated from human scleralfibroblasts grown in culture, as well as from donorhuman sclerae by guanidinium thiocyanate-phenol-chloroform extraction.23 Briefly, to isolate RNA fromcultured cells, the growth medium was removed, andthe cell layers were extracted in denaturing solution(50 ml of solution D [4 M guanidinium thiocyanate;25 mM sodium citrate, pH 7; 0.5% sarcosyl; 0.1 M 2-mercaptoethanol]; 5 ml of 2 M NaAc, pH 4; and 50ml phenol). The cells were disrupted in denaturingsolution by scraping, and the extract was removed andplaced into sterile test tubes. Chloroform (10 ml/105ml denaturing solution) was added to each homoge-nate, shaken vigorously for 15 seconds, and cooled onice for 15 minutes. After centrifugation (30 minutesat 10,000g), the aqueous phase was removed and

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Human Scleral Proteoglycans 1743

mixed with an equal volume of chloroform and centri-fuged again. The aqueous layer was removed, andRNA was precipitated with an equal volume of isopro-panol at — 20°C for 90 minutes. After centrifugation(10,000g for 15 minutes), the supernatant was dis-carded, and the pellet was reextracted in 5 ml of dena-turing solution, followed by chloroform extractionand isopropanol precipitation of RNA, as described.The RNA was quantitated and its purity assessed byspectrophotometry at 260nm and 280 nm. A smallportion of each preparation 10% was evaluated on adenaturing 10% agarose gel after staining with ethid-ium bromide. To extract RNA from intact humansclerae, liquid nitrogen was used to snap-freeze freshlyobtained human sclerae, which were stored at —80°Cuntil RNA extraction. For RNA extraction, frozensclerae (^5 gm) were pulverized in a cryogenic millunder liquid nitrogen (Spex, Metuchen, NJ), and thefrozen powder was transferred to a beaker containing105 ml of denaturing solution and homogenized witha Virtis rotor-stator assembly (Vitris, Gardiner, NY).Chloroform (10 ml/105 ml denaturing solution) wasadded to each homogenate, and the extraction wascarried out as described above.

Two PCR primers, which flanked a region (1244to 1634 bp) of the published sequence of the humanaggrecan gene4 that was the most different from thesequence of human versican,24 were selected for R T -PCR. The forward primer (5'-GCAGCGTGATCCTTA-CCGTAAAG-3') corresponded to regions 1244 to1266 of the human aggrecan sequence, whereas thebackward primer (5'-TCACACTGCTCATAGCCT-GCTTC-3') corresponded to regions 1634 to 1612 ofthe published sequence. Analyses of human scleralfibroblasts and scleral tissue were carried out by RT-PCR on 1 /zg and 0.25 fig of total RNA, respectively,using an RT-PCR kit (GeneAMP, Perkin Elmer, Nor-walk, CT), after the standard protocol, which involvedincubating RNA at 42°C in the presence of reversetranscriptase and the backward primer for 15 minutesto generate complementary DNA, followed by PCR for35 cycles with forward and backward primers in thepresence of AmpliTaq (Perkin Elmer) at 61 °C. Theamplified products were purified from the gel, usingthe Qiaex gel extraction kit (Qiagen, Chatsworth, CA),and their sequences were confirmed by nucleic acidsequencing using, the dideoxy termination-chainmethod (Sequenase version 2, United States Biochem-ical, Cleveland, OH).

Immunohistochemistry

Human sclerae were embedded and frozen in OCTcompound (Miles, Elkhart, IN), and sectioned usinga cryostat. Frozen sections were mounted on gelatin-coated slides; and in some cases, sections were di-

gested with c'ase ABC (0.06 U in 50 fi\ working buffer[0.1 M Tris, pH 7.4, containing 500 mM phenylmethyl-sulfonyl fluoride; 100 mM N-ethylmaleimide; 100 mMEDTA; and 36 mM pepstatin A]) at 37°C for 1 hourbefore immunostaining. Immunostaining was carriedout on frozen sections using the TrueBlue immunode-tection kit (KPL, Gaithersburg, MD) and the standardprotocol. Briefly, endogenous peroxidase activity wasblocked using the KPL blocking solution. Sectionswere then rinsed in PBS, blocked with 10% normalgoat serum, and incubated with primary antibodiesdiluted in PBS. Primary antibodies used were a Fab'fragment specific to the human aggrecan core protein(generously provided by Dr. Robin Poole, McGill Uni-versity), antihuman biglycan antibodies,20 and an anti-sera against a synthetic peptide containing the exon5 sequence of human decorin.21 All antisera were di-luted 1:100 in PBS, which corresponded to immuno-globulin G concentrations of 21.6 ng/fil, 8.8 ng/fi\,and 31.8 ng/fi\ for antiaggrecan, antibiglycan andantidecorin antisera, respectively. Normal rabbit se-rum (diluted 1:100 in PBS for an IgG concentrationof 20 ng/fil) was substituted for the primary antiserafor negative control samples. Sections were thenwashed in PBS and incubated with goat antirabbit IgGconjugated to horseradish peroxidase (Sigma, St.Louis, MO) diluted 1:500 in PBS. Sections werewashed in PBS, reacted with the TrueBlue peroxidasesubstrate, and counterstained with KPL contrast red.Sections were then dehydrated, dried, and mountedin Permount (Fisher, Pittsburgh, PA).

RESULTS

Isolation of Scleral Proteoglycans

The elution positions of scleral proteoglycans couldbe identified after chromatography on Sepharose CL-2B by measuring the concentration of glycosaminogly-cans in each fraction (Fig. 1A) or by measuring theamount of radioactivity in fractions from sclerae thathad been previously radiolabeled with 35SO4 (Fig. IB),or 3H-proline after purification of 3H-labeled proteo-glycans by DEAE chromatography (Fig. 1C). In allthree chromatographic profiles, three peaks wereidentified: PG-1, a small peak representing the largestscleral proteoglycan and accounting for «6% of newlysynthesized scleral proteoglycans; PG-2, a slightlyfaster-migrating shoulder to the third and largestpeak, PG-3; and PG-3, which represents the smallestscleral proteoglycan. The data presented in Figure 1Arepresent the results of two separate experiments us-ing 10 pooled sclerae, the results of Figure IB arerepresentative of five different chromatographic pro-files of scleral proteoglycans, whereas Figure 1C repre-

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1744 Investigative Ophthalmology 8c Visual Science, August 1997, Vol. 38, No. 9

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sents the results of one experiment. Fractions indi-cated by the bars were combined for further analysis.

Characterization of PG-2 and PG-335SO4-labeled PG-2 and PG-3 fractions from SepharoseCL-2B were chromatographed on a Superose 6 FPLCcolumn to separate further the proteoglycans in thesefractions (Fig. 2). Based on the FPLC profiles, it wasestimated that PG-2 accounted for «20% of 35SO4-labeled scleral proteoglycans, and PG-3 represented«74% of the total 35SO4-labeled proteoglycans. ThePG-2 and PG-3 fractions separated by Superose 6 chro-matography were pooled separately and investigatedby Western blot analyses using antibodies specific tothe core proteins of biglycan and decorin (Fig. 3).Equal protein amounts of PG-2 and PG-3 (40 ng) wereseparated by sodium dodecyl sulfate-polyacrylamideelectrophoresis followed by electrotransfer to nitrocel-lulose. Antibiglycan antisera detected the 45-kDa bigly-can core protein in c'ase ABC digests of PG-2 (Enz +,Fig. 3A). No bands could be detected in the undi-gested PG-2 samples because the proteoglycan formof biglycan is unreactive with this antisera in Westernblot analyses.20 A faint band migrating at 45 kDa wasalso apparent in the c'ase ABC-digested PG-3 fraction,indicating that some biglycan was present in PG-3 (Fig.3A). Densitometric analyses of this 45-kDa band indi-cate that of the total amount of biglycan present in thehuman sclera, «89% is present in PG-2, and «11%is present in PG-3 proteoglycan fractions (data notshown).

An identical Western blot specimen was incubated

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Human Scleral Proteoglycans 1745

PG-2 PG-3Enz

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FIGURE 3. Western blot analyses of PG-2 and PG-3. PG-2 and PG-3, which had been purifiedby Sepharose CL-2B and Superose 6 forced-pressure liquid chromatography, were digestedwith chondroitinase Enz +, and reacted with (A) antibodies to the core protein of biglycanor (B) antibodies to the core protein of decorin. Note that antibiglycan antibodies reactwith a core protein of 45 kDa in chondroitinase ABC-treated PG-2 samples and weakly inchondroitinase ABC-digested PG-3 samples. Antideconn antibodies react with a core proteinmigrating as a doublet at 43 and 47 kDa in chondroitinase ABC-digested PG-3 samples (B).PG-2 = biglycan; PG-3 = decorin; Enz = enzyme alone.

with antibodies against a synthetic peptide containingthe exon 5 sequence of human decorin21 (Fig. 3B).Case ABC digestion of PG-3 produced a doublet ofcore proteins at 43 and 47 kDa that reacted stronglywith the antidecorin antiserum (Fig. 3B). Two lighter-staining bands migrating at ^32 kDa and 25 kDa alsoappeared in the c'ase ABC PG-3 sample, which mayrepresent fragments of the decorin core protein.25 Nobands were detected in the PG-2 samples.

Characterization of PG-1

The glycosaminoglycan composition of 35SCvlabeledPG-1 was evaluated by chromatography on SephadexG-50 before and after digestion with endo-/?-gal andc'ase ABC (Fig. 4). Undigested PG-1 eluted as a singlepeak in the void volume of Sephadex G-50, After diges-tion with endo-/?-gal, the void-volume peak was re-duced, and a small peak appeared in the includedvolume, representing degraded keratan sulfate (Fig.4A). The endo-/?-gal-resistant peak was then digestedwith c'ase ABC, resulting in the complete digestion ofthe remaining 35SO4-labeled glycosaminoglycans andin the appearance of a large included peak, represent-ing degraded chondroitin sulfate (Fig. 4B). From mea-surements of the area under each peak after G-50chromatography, it was determined that PG-1 containschondroitin sulfate (75%) and keratan sulfate (25%)glycosaminoglycan side chains, and it was suggestedthat PG-1 represents aggrecan and that the cartilagerepresents proteoglycan. To date, aggrecan, the carti-lage proteoglycan, is the only large proteoglycan con-taining chondroitin sulfate and keratan sulfate glycos-aminoglycan side chains, indicating that PG-1 proba-bly represents aggrecan.

To confirm that the PG-1 proteoglycan containsthe aggrecan core protein, aliquots of PG-1 were di-gested with c'ase ABC and endo-/?-gal, k'ase II, orboth, and were subjected to Western blot analyses us-ing antibodies specific to die human aggrecan coreprotein (Fig. 5). No bands could be detected in undi-gested PG-1 samples, or in. samples digested widi c'aseABC alone. After digestion widi c'ase ABC and endo-/?-gal, a band migrating at «350 kDa could be seen,which represented the aggrecan core protein. Afterdigestion with c'ase ABC, endo-/?-gal, and K'ase II, amuch more intense band appeared at 350 kDa, indi-cating that a large population of keratan sulfate re-mained undigested after endo-/3-gal digestion and re-quired additional treatment with k'ase II to releasethe core protein. The 280-kDa band in the c'ase ABC-en do-/3-gal-K'ase II-digested sample was also presentin the k'ase II enzyme preparation (compare with en-zyme-alone [marked ENZ] lane). The smear in dieundigested PG-1 lane probably represents an artifactof the chemiluminescent detection. When die sameblot was stripped and reprobed with a monoclonalantibody to human versican, no reaction productswere seen, indicating that versican is not present inPG-1 (data not shown).

Reverse Transcription -Polymerase ChainReaction Analyses

Reverse transcription-polymerase chain reactionanalyses were carried out to verify aggrecan transcrip-tion by human scleral fibroblasts. Total RNA was ex-tracted from cultures of human scleral fibroblasts andfrom human scleral tissue and was determined to beof high purity and integrity on the basis of findings

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1746 Investigative Ophthalmology & Visual Science, August 1997, Vol. 38} No. 9

300

250 -

•5 200 -

150 -

Undigested

Endo-p-Gal

20 30

Tube Numbers

140

120 H

100

80 -

60 -

40 -

20 -

—•- Case ABC digestion of Endo -p- Galresistant fraction

B

%o 20 30

Tube Numbers

40 50

FIGURE 4. Sephadex G-50 chromatography of 35SO4-labeledPG-1 before and after digestion with endo-/3-galactosidaseand chondroitinase ABC. (A) Treatment of PG-1 with endo-/?-galactosidase resulted in the appearance of a small peakin the included volume of Sephadex G-50. (B) Treatment ofendo-/3-galactosidase-resistant material with chondroidnaseABC resulted in complete digestion of remaining PG-1. PG-1 = aggrecan.

in spectrophotometry at 260 and 280 nm (260:280ratio > 1.7), and by the appearance of the 28S and18S rRNA bands after electrophoresis on a denaturinggel and ethidium bromide staining (data not shown).An RT-PCR primer set was selected that would am-plify a 391-bp region that included a region of theinterglobular domain and the G2 domain of the hu-man aggrecan gene, between base numbers 1244 and1634.4 As can be seen in Figure 6, when upstream anddownstream primers were included in the reaction, a391-bp product was visible in RNA samples from thecultured human scleral fibroblasts (lane 2) as well asfrom RNA extracted directly from human sclerae(lane 3). No bands were seen when reverse tran-

scriptase or either the upstream or downstreamprimer was omitted from the reaction (data notshown). The identity of the 391-bp product from scle-ral fibroblasts and tissue was confirmed to be the am-plified region of the human aggrecan gene by directsequencing of the RT-PCR products using the for-ward primer. An 89-bp region of the 391-bp RT-PCRproduct amplified from RNA isolated from humanscleral fibroblasts (lane 2) was sequenced and shownto be identical to that of the published sequence ofhuman aggrecan, whereas a 100-bp region of the RT—PCR product amplified from RNA extracted directlyfrom human sclerae (Fig. 6, lane 3) was shown toalign to the sequence of human aggrecan, with fourmismatches (data not shown). The identity of the«90-bp band, seen in lanes 2 and 3, is unknown.

No aggrecan RT-PCR products were seen whenhuman cornea] fibroblast RNA was tested using thesame RT-PCR primers, indicating that human cor-neal fibroblasts do not express the aggrecan gene(data not shown).

Immunohistochemistry

Specific antisera to the aggrecan, biglycan, and de-corin core proteins were used to localize these anti-gens on sections of human scleral stroma (Fig. 7).Immunostaining with an Fab' fragment specific to the

Case ABCEndo-f3-Gal

K'ase II

ENZ

200 k D -

116 k D -

FIGURE 5. Western blot analysis of PG-1. PG-1, purified bySepharose CL-2B chromatography was digested with chon-droitinase ABC, endo-/3-galactosidase, and keratanase II, fol-lowed by reaction with antibodies to the core protein ofaggrecan. A band migrating at «350 kDa is apparent insamples digested with chondroitinase ABC and endo-/?-galand is greatly enhanced after digestion with chondroitinaseABC, endo-/?-galactosidase, and keratanase II. PG-1 = ag-grecan.

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Human Scleral Proteoglycans

1 2

FIGURE 6. Identification of aggrecan mRNA in cultured hu-man scleral fibroblasts and human scleral tissue by reversetranscription-polymerase chain reaction. Upstream anddownstream primers were selected that would amplify a 391-bp region of human aggrecan mRNA. When upstream anddownstream primers were used, a 391-bp product is visiblein RNA isolated from cultured scleral fibroblasts (lane 2)and in RNA extracted directly from human sclera (lane 3).Lane 1: 0x molecular weight markers.

core protein of aggrecan suggested that aggrecan isdistributed throughout the thickness of the sclera(Fig. 7A). Before immunostaining with antibiglycanantisera, frozen sections were digested with c'ase ABCto expose the antigenic sites on the core protein.20

Immunostaining of c'ase ABC-digested, frozen sec-tions with antibiglycan indicated that biglycan is alsodistributed throughout the thickness of the adult hu-man sclera (Fig. 7B) similar to the immunostainingpattern obtained using antidecorin antisera (Fig. 7C),Minimal staining was detected on frozen sectionswhen nonimmune rabbit IgG was substituted for theprimary antibody (Fig. 7D).

DISCUSSION

Previously reported data on the glycosaminoglycancomposition of the human sclera indicate that thesclera contains chondroitin sulfate (28% to 48%), der-matan sulfate (29% to 49%), heparan sulfate (2% to

1747

12%), and hyaluronan (19% to 33%).Vi The amountof each glycosaminoglycan present varies within differ-ent regions of the sclera and with the age of thesclera.1213 The results presented in this study demon-strate the presence of three major proteoglycans inthe adult human sclera, identified as PG-1, PG-2, andPG-3, based on their elution from Sepharose CL-2B.The smallest proteoglycan, PG-3, represented »74%of the newly synthesized sulfated glycosaminoglycansin the sclera and was shown to contain chondroitin-dermatan sulfate side chains and a core protein thatmigrated as a doublet on sodium dodecyl sulfate-polyacrylamide gels at 43 and 47 kDa. The slightlylarger, less abundant proteoglycan (PG-2) accountedfor «20% of the newly synthesized sulfated glycosami-noglycans and consists of chondroitin-dermatan sul-fate side chains attached to a 45-kDa core protein.Western blot analyses with specific antisera confirmedthat PG-2 and PG-3 were biglycan and decorin, respec-tively. Our immunostaining results indicate that at thelight microscopic level, biglycan and decorin colocal-ize and are distributed throughout the thickness ofthe adult human sclera in close association with thecollagen fibrils. These results are somewhat differentfrom those of Bianco et al,6 in which they report (with-out micrographs) that biglycan core protein andmRNA are limited to the inner portion of the devel-oping human sclera (at 14 to 17 weeks of gestationalage), whereas decorin is distributed throughout thethickness of the fetal sclera. To our knowledge, thecurrent study is the only report on the distribution ofthese core proteins in the adult human sclera andsuggests that the distribution of biglycan in the adultsclera is very different from tiiat in die sclera of thehuman fetus.

In general, decorin localizes in all classic connec-tive tissue matrixes rich in collagen type I or II, includ-ing dermal matrix, tendon, bone, articular cartilage,cornea, sclera, interstitium of kidney, heart, and lungin which it remains bound at the surface of maturecollagen fibrils.2'6'26 In vitro, decorin binds to collagenstype I and II, in which it delays the lateral assemblyof collagen molecules, resulting in thinner diametercollagen fibrils.27'28 Biglycan has also been found inmost of these tissues; but in fetal tissues, it has a muchmore restricted distribution, in that it is localized inthe pericellular matrixes and cell surfaces of certaincells.6 In adult tissues, biglycan has been observed tocolocalize with collagen I and III in atheroscleroticand restenotic lesions of human coronary arteries.29

It has been observed in hypertrophic and burn scarsof the human dermis30 and has been identified in thehuman gingivae, in which it appears to form fine,filamentous structures on extracellular matrix fibers.91

Although biglycan does not appear to show a specific

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1748 Investigative Ophthalmology & Visual Science, August 1997, Vol. 38, No. 9

I

FIGURE 7. Immunolocalization of aggrecan, biglycan, and decorin on frozen sections of theposterior human sclera. Sections were treated with an Fab' subunit or specific immunoglobu-lin G antibodies, followed by peroxidase-labeled goat antirabbit immunoglobulin G andTrueBlue color development. All sections were counterstained with contrast red. Blue stain-ing indicates the presence of the antigen. (A) After staining of posterior human sclera withan Fab' subunit directed against the aggrecan core protein, aggrecan can be seen scatteredthroughout the thickness of the sclera. (B) Frozen sections were digested with chondroitinaseABC before staining with antisera against a peptide of the human biglycan core protein.Immunostaining indicates that biglycan is present throughout the thickness of the humansclera. (C) Immunostaining with antisera against a peptide of the human decorin coreprotein indicates that decorin is distributed throughout the thickness of the human sclera,similar to the distribution of biglycan. (D) Control sections of sclera, initially treated withnonimmune rabbit immunoglobulin G, followed by staining with peroxidase-labeled goatantirabbit immunoglobulin G and TrueBlue color development. O = outer sclera; I - innersclera, adjacent to choroid; All bars = 150 [im.

effect on collagen fibril formation in vitro,32 biglycanbinds to purified collagen type I fibrils33 and is associ-ated with collagen fibrils in the human dermis33 andarticular cartilage.34 The results of the current studyindicate that in die adult sclera, biglycan is not locatedin a pericellular distribution, as has been observed infetal tissues,6 but is distributed throughout the scleralstroma, in close association with collagen fibrils, simi-lar to the distribution observed in dermis.

The third and largest proteoglycan extracted fromthe human sclera and separated on Sepharose CL-2B(PG-1) was identified as aggrecan, on the basis of itssize, glycosaminoglycan side chains, core protein size,

and immunoreactivity. The presence of aggrecanmRNA in scleral tissue and in human scleral fibro-blasts confirmed that human scleral fibroblasts tran-scribe the aggrecan gene. Our results indicate that inthe human sclera, aggrecan carries chondroitin sulfateand keratan sulfate side chains based on the presenceof c'ase ABC, endo-/3-gal- and k'ase II-sensitive glycos-aminoglycans. The finding that treatment with k'aseII and endo-/3-gal significantly increases the amount ofaggrecan core protein visible in Western blot analysesabove the amount seen with endo-/3-gal treatmentalone, suggests that a relatively large population ofkeratan sulfate in scleral aggrecan contains 6-O sul-

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Human Scleral Proteoglycans 1749

fated galactose in the galactosidic linkage region. Thepresence of keratan sulfate in the human sclera hasbeen examined in several studies with conflicting re-sults. Borcherding et al14 concluded that keratan sul-fate was present in the sclera (4 to 5.6 fjM/gm defatteddry weight) on the basis of levels of hexosamine,whereas Brown et al,12 reported only negligible levelsof keratan sulfate within the human sclera, using spe-cific enzymatic digestion and measuring the undi-gested glycosaminoglycans with dimethylmethyleneblue. The results of our chromatographic studies showthat PG-1 represents «6% of the total scleral proteo-glycans, and that 25% of the 35SO4-labeled glycosami-noglycans in PG-1 are keratan sulfate moieties thatare sensitive to endo-/3-gal. We therefore estimate thatkeratan sulfate accounts for at least 1.5% of the total35SO4-labeled glycosaminoglycans synthesized by theaged human sclera. The amount of keratan sulfate inthe human sclera may be higher than 1.5%, inasmuchas Western blot results indicate that there is a popula-tion of keratan sulfate on aggrecan that is resistant toendo-/?-gal. However, given the limited sensitivity ofthe dimethylmethylene blue assay, and the relativelylow amount of keratan sulfate in the human sclera, itis conceivable that keratan sulfate would have beenundetected in the previous study.

The presence of aggrecan in the human sclerawas surprising, because aggrecan typically has beenrestricted to cartilage. However, large proteoglycanshave been isolated from the bovine sclera8"10'35 andhave been shown to contain specific epitopes relatedto the hyaluronate-binding region of cartilage proteo-glycan.9 Furthermore, electron microscopic analysisindicated that the large proteoglycans from bovinesclera have the same general domain organization andestimated size as that of the large cartilage proteogly-

can.11,36

Recently, aggrecan accumulation has been re-ported in compressed regions of bovine and humantendons.37 Furthermore, compressive loading of bo-vine tendons in vitro resulted in increased synthesisof large proteoglycans.38 The mechanisms by whichcompression leads to aggrecan synthesis and accumu-lation are unknown; however, previous results haveshown that hydrostatic pressure,39 fluid loss, pH de-creases,40'41 and changes in cytoskeletal actin organiza-tion42 can regulate proteoglycan biosynthesis. Theo-retical mechanical analysis of the human sclera indi-cates that under the normal intraocular pressure of15 mm Hg, the sclera of an emmetropic eye is sub-jected to an average tensile stress of »112.5 mm Hg/mm2, and this stress can increase significantly as thesclera thins or the radius of curvature increases.43 Al-though intraocular pressure is much lower than thecompressive forces employed in the tendon studies

(«7000 mm Hg) it is possible that aggrecan accumu-lates in the sclera in response to the tensile stressesimposed during an extended period. In the sclera,aggrecan probably plays an important role, as it doesin cartilage,44 in providing the sclera with compressivestiffness and the ability to resist the tensile stressesresulting from changes in intraocular pressure.

The presence of aggrecan in the human scleraalso may be related to the association of scleritis withvarious rheumatic diseases. Scleritis is a major ophthal-mologic complication accompanying rheumatoid ar-thritis and polychondritis,45"47 and immunity to ag-grecan has been demonstrated in patients with relaps-ing polychondritis48 and rheumatoid arthritis.49

Therefore, an autoimmune response to aggrecan maynot only be manifested in the joints, but also mayinclude such extraarticular areas as the sclera in whichaggrecan is also located.

In summary, we report that the adult humansclera contains three major proteoglycans; aggrecan,biglycan, and decorin. It is probable that the relativesynthesis and accumulation of these proteoglycans willchange with age, specific region, and condition of thesclera. These studies are currently in progress and willbe the subject of a later report.

Key Words

aggrecan, biglycan, decorin, proteoglycans, sclera

Acknowledgments

The authors thank Drs. Robin Poole, Peter Roughley, andDavid McQuillan for generously supplying the antisera usedin this study, Dr. John Hassell for his help with the Superose6 FPLC chromatography, and Karla J. Glick for her skilledhelp with photographic work.

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