Int. J. Biochem.Vol. 24, No. 10, pp. 1573-1583, 1992 Printedin Great Britain.All rightsreserved

Copyright0

ISOLATION AND CHARACTERIZATION GINGIVAL PROTEOGLYCANS VERSICAN P.

BRATT , 1,4 M

M. ANDERSON,~ B. MANSSON-RAHEMTULLA,’

and F.

0020-71IX192 $5.00 + 0.00 1992 PergamonPressLtd

OF BOVINE AND DECORIN

J. W. STEVENS,’

C. Znou2

RAHEMTULLA~*

‘Departments of Community and Public Heaith Dentistry and 20ral Biology, University of Alabama School of Dentistry, University Station, Birmingham, AL 35294, U.S.A. [Tel. (2(X5)934-1693;Fnx (205)934-25291, 3Department of Medicine and Veterans Administration Medical Center, University of Alabama at Birmingham, UAB Station, Birmingham, AL 35294, U.S.A. and 4Department of Cariology, University of LJme& UmeH, Sweden

(Received 3 March 1992) Abstract-l.

We have isolated, chemically and immunologically characterized versican and decorin from bovine gingiva. 2. Versican was of large molecular weight and the molecular size of the core protein was estimated to be greater than 200 kDa. 3. The glycosaminoglycan chains were susceptible to chondroitinase ABC and N-linked oligosaccharides were present on the protein core of the molecule. 4. Immunologica studies provided evidence that a hyaluronic acid binding region was present in the core protein of versican. 5. The overall structure was similar to that of versican isolated from bovine sclera. 6. Decorin had a molecular weight of 102 kDa and its glycosaminoglycan chain was completely digested by specific glycosidases. 7. The partially deglycosyiated core protein had a molecular weight of 55 kDa and N-linked oligosaccharides were present on the molecule.

INTRODUCTION

The extracellular matrix of gingival tissue consists of complex matrix proteins, i.e. collagen, noncollagenous glycoproteins and proteoglycans (Bartold, 1987; Narayanan and Page, 1983; Rahemtulla, 1992). Proteoglycans are a diverse group of complex, often multidomain proteins to which glycosaminoglycan chains are covalently attached. A significant degree of structural diversity is found among the proteoglycan population dependent upon the size of the core proteins, nature and number of glycosaminoglycan chains, and presence of N- and O-linked oligosaccharides (Hascall and Hascall, 1981; Heinegdrd and Paulsson, 1984). The prevailing proteoglycans of soft connective tissues are a large molecular weight proteoglycan, versican, and a small molecular weight proteoglycan, decorin. While both of these proteoglycans have been isolated from aorta (McMurtrey et al., 1979; Salisbury and Wagner, 1981), sclera (C6ster and Fransson, 1981) and tendon (Vogel and Heinegbrd, 1985), only decorin has been isolated and characterized from skin, (Damle et al., 1979), periodontal ligaments (Pearson and Gibson, 1982), human uterus

*To whom correspondence addressed.

and reprint requests should be

(Uldbjerg ef al., 1983a) and human uterine cervix (Uldbjerg et al., 1983b). Another proteoglycan, known as biglycan, originally isolated from cartilage (Rosenberg et al., 1985) and bone (Fisher et aI., 1989), is also present in soft connective tissues. Its mRNA has been detected in skin, periodontal ligament and several other soft connective tissues, but was not detected in fibroblasts derived from gingival tissue (Fisher et al., 1989). Fibromodulin, the latest addition to the proteoglycan family, was recently isolated from cartilage and chemically characterized (HeinegHrd et al., 1986). It has been cloned and sequenced and is present in a wide variety of connective tissues (Oldberg et al., 1989). The presence of glycosaminoglycans and proteoglycans in gingival tissues was established several years ago. Initially, histochemical techniques were employed to demonstrate the presence of these molecules in gingiva (Melcher, 1967; Quintarelli, 1960; Stahl et al., 1958; Thonard and Scherp, 1962). Several early biochemical studies have demonstrated the presence of hyaluronic acid, chondroitin sulfate, dermatan sulfate and heparan sulfate in human, porcine and bovine gingival tissues (Bartold et al., 1981,1982; Ciancio and Mather, 1971; Hiramatsu et al., 1978; Sakamoto et al., 1978). Although these studies reported varying amounts of the different glycosaminoglycans in the tissues, all studies established that

1573

P. BItArT et al.

1574

dermatan sulfate is the major glycosaminoglycan of gingiva. Several studies have indicated that the sulfated glycosaminoglycans exist as protein-bound ~lysac~harides in the gingival tissue (Bartold er ai., 1983; Dziawiatkowski et al., 1977; Embery et at., 1979; Wiebkin et al., 1979). investigations by Pearson and Pringle (1986) have revealed that 2 types of proteoglycans are present in bovine gingiva. The small molecular weight proteoglycan was identified as proteode~atan sulfate, while the large moiecular weight proteoglycan was only partially characterized. No heparan sulfate proteoglycans were detected in this preparation (Pearson and Pringle, 1986). The specific physiological roles of proteoglycans in gingival tissues are not known, however, several functions have been ascribed to them. It has been postulated that proteoglycans of gingiva form a front which acts as a defense mechanism during the initiation of periodontal disease. The properties of these molecules include molecular sieving which regulates the penetration and flow of potential irritants including toxins and hydrolytic enzymes produced by the plaque bacteria (Bartold et al., 1983). Because soft connective tissue proteoglycans (decorin) interact with collagen (reviewed in Scott, 1986) and influence fibrillogenesis (Vogel and Heinegbrd, 1985; Vogel et al., 1984), they are postulated to play a role in maintaining the structural integrity of gingival tissue, The long-term objective of our research program is to arrive at a deeper understanding of the structure, function, metabolism and regulation of proteoglycans in normal and diseased periodontal tissue. In order to reach this goal, we require chemical amounts of proteoglycans for the production of probes for immunologica and molecular biological studies. Large quantities of proteoglycans are also required to facilitate exploration of possible biological roles for these molecules. In the present study we describe a rapid and reproducible method for the isolation of bovine gingival proteoglycans. This study also describes chemical and immunolo~cal properties of the principal proteoglycans of bovine gingival tissue. MATERIALS

AND METHODS

Tissue collection

Buccal and lingual gingiva from bovine mandibtes was collected at the local abattoir. An incision was made along the mucogingival junction and the gingiva was removed using a sharp elevator. The tissue was cut into small pieces, immediately submerged in liquid nitrogen, and later powdered by passing through a Wiley Mill while still frozen. Extraction of proteoglycans

One hundred g of finely powdered gingiva was stirred over night at 4°C in 1 liter of 4 mol/l guanidinium chloride (Sigma Chemical Co., St Louis, MO.) in 50 mmol/l sodium acetate buffer, pH 5.6. To avoid artifactual degradation of the connective tissue macromolecules, the extracting solution contained several protease inhibitors, i.e. 10 mmol/l EDTA, 10 mmot/l 6-aminocaproic acid, 5 mmol/l benza-

midine, 1 mmol/l sodium iodoacetate. I mg/l soybean trypsin inhibitor and 10 mmol/l phenylmethylsulfonyl fluoride, all obtained from Sigma Chemical Co. The mixture was centrifuged at 18,OOOgin a SorvalI RC-53 Refrigerated Superspeed Centrifuge using a GSA rotor (DuPont, Wilmington, Del.) for 30min at 4°C. The supernatants were pooled and the pellets were resuspended in 1 1 of buffered guanidinium chloride solution with protease inhibitors and re-extracted. The pooled extract was concentrated by ultraftlitration through a YM-IO membrane filter (Amicon, Danvers, Mass.), and part of the guanidinium chloride was replaced by adding 50 mmol/l sodium acetate, pH 5.5, containing 7 mol/l urea (Sigma) to the extract which was dialyzed and concentrated by ultrafiltration. The concentrated extract was dialyzed overnight against 4 liters of the urea buffer at 4°C.

After dialysis, the extract was applied to a 10 x 2Scm DEAE Sephacel column (Pharmacia LKB Biotechnology, Piscataway, N.J.), which was equilibrated in 50mmol/l sodium acetate buffer, pH 5.5, containing 7 mol/l urea. After application of the sample, the column was washed with 1liter of the same buffer and the bound material was eluted with a linear salt gradient of O-1 mol/l NaCl in 7 mol/l urea buffer at a flow rate of 0.25 ml/min. Eighty 5 ml fractions were collected, analyzed for hexuronic acid by the carbazole-borate method (Bitter and Muir, 1962) and protein by the Bradford method (Bradford, 1976) as modified by Read and Northcote (1981). The conductivity of the fractions was measured with a Radiometer CDM 83 Conductivity meter (London Co., Cleveland, Ohio). The main peak of hexuranic acid containing material eluting at 0.5-0.6 M NaCl was pooled, extensively dialyzed against deionized water, and lyophilized. The lyophilized material was dissolved in 50 mmol/l Tris-HCl, pH 7.4, containing 4 mol/l guanidinium chloride at a concentration of 5 mgjml, and 1ml aliquots were chromatographed on a Superose 6 molecular sieve FPLC column (Pharmacia LKB Biotechnology) using a Waters HPLC (Waters, Milford, Mass.) delivery system. The flow rate was 0.5 ml/min and thirty 1.Oml fractions were collected. The recovery of the proteoglycans from the column was monitored at 280 nm and the individual fractions were analyzed for hexuronic acid by the carbazole-borate method (Bitter and Muir, 1962). The proteoglycan fractions from repeated chromatographic runs were pooled, dialyzed extensively against deionized water, and lyophilized. These fractions were rechromatographed as described above to obtain fractions of higher purity. The individual fractions containing the highly purified proteoglycans were pooled, dialyzed extensively against deionized water and lyophilized. Enzymaiic digestion by chondroitinases

Purified preparations of proteoglycans were dissolved in 50 mmol/l Tris-HCl, pH 7.4, and incubated with 0.04 units chondroitinase ABC (ICN Biomedical, Costa Mesa, Calif.) per mg of proteogiycans. The enzyme-proteoglycan mixture was incubated at 37°C for 16 hr after which the reaction mixture was applied to a column (44 x 1cm) of Sephadex G-50 (Pharmacia LKB Biotechnology) which was eluted with 200 mmol/l ammonium hydrogen carbonate, pH 8.15, at a flow rate of lOml/hr. One ml fractions were collected and analyzed either for hexuronic acid by the carbazole reaction (Bitter and Muir, 1962) or absorbance measured at 232 nm.

Proteoglycans of gingivai tissues In experiments where the core proteins of the isolated proteoglycans were to be analyzed, 50 pg of the sample was digested with chondroitinase ABC as described above and aliquots of the digest applied to ~lya~~lamide gels and electrophoresed as described below. Removal of N-linked oligosaccharides from the protein cores of proteoglycans was accomplished using N-glycanase @enzyme Co., Boston, Mass.) as described by Plummer er al. (1984). Fifty pg of the protein core was dissolved in 25 ~1 0.5% SDS containing 0.1 mol/l 2-mecaptoethanol (Aldrich, Milwaukee, Wis.) and boiled for 3 min. To the sample mixture was added 22~1 of 48Ommol~l sodium phosphate buffer, pH 8.6, 12.5~1 of 0.30mmol/l EDTA, 12.5 ~1 of 7.5% NP-40 and 3 ~1 of N-glycanase to achieve a final concentration of the enzyme of 10 units/ml and the reaction mixture was incubated at 37°C for 16 hr. The reaction was stopped by boiling the mixture, electrophoresis buffer was added, and aliquots of the enzyme-treated samples were eiectrophoresed as described below. Amino acid analysis

Samples of the proteoglycans were hydrolyzed with 6.0 mol/l HCl for 20 hr at 110°C. Following hydrolysis the samples were dried using methanol/water/t~ethylamine (Ald~ch) in a ratio of 2:2: 1 and then derivatized using methanol/water~t~ethylamine/phenyl isothiocyanate (Pierce, Rockford, Ill.) in proportions of 97: 1: 1: 1. This material was chromatographed on a Pica Tag column using Waters Pica Tag HPLC system as recommended by the supplier and the recovery from the column was monitored at 254nm. ~[ectrophoret~~ techniques

The reagents used for sodium dodecyl sulfatepolyacrylamide electrophoresis (SDS-PAGE) were obtained from Fisher Scientific (Norcross, Ga.). SDS-PAGE was performed essentially as described by Laemmli (1970) and modified by Butler et al. (1981) with either 3-15 or S-IS% polyac~lamide gels el~trophores~ in a minigel apparatus (Bio-Rad, Richmond, Calif.) for 35 min at 200 V. Gels were stained first with Coomassie Brilliant Blue R (Sigma), destained in a mixture of 10% each of methanol and acetic acid and restained in 0.5% Alcian Blue (Sigma) which was dissolved in 7% acetic acid and subsequently destained in 7% acetic acid. Molecular weight markers (Pharmacia LKB Biot~hnology) included phosphorylase b (~,~), bovine serum albumin (67,000), ovalbumin (43,000). carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100) and a-lactalbumin (14,400). Immunological techniques

Decorin and versican from bovine sclera were isolated and purified by the procedure described by Ciister and Fransson (1981) and cartilage proteoglycan fraction Dl was prepared from bovine nasal cartilage by the method described by Heineggrd and Sommarin (1987). For the preparation of antiserum against small molecular weight proteoglycan of bovine gingiva, 300 pg of the sample was subjected to 5-15% SDS-PAGE. After staining the gel with CuCl, (Lee er a!., 19871,the major band migrating just above the 90 kDa molecular weight standard was excised from the gel, destained by incubation in several changes of 250mmol/l EDTA/250mmol/l Tris-HCl, pH 9.0, and washed with deionized water. The destained gel pieces containing decorin were mixed and emulsified in Freund’s complete adjuvant (Gibco, Grand Island, N.Y.) and injected

1575

subcutaneously into several sites on the back of a female New Zealand white rabbit. Subsequent injections of polyacrylamide gel containing bovine gingival decorin in Freund’s incomplete adjuvant (G&co) were made at 14 day intervals. Blood was collected prior to the first injection and 7 days after each injection. The blood was allowed to clot for 30min at room temperature, and overnight at 4”C, the serum was decanted from the blood clot and trace red blood cells present in the decanted serum were removed by centrifugation at 12,000 g using a Sorval SS-34 rotor. Several other antibodies which are available in our laboratory have been used in the present study. Polyclonal antibodies against bovine sclera decorin, monoclonal antibody 5-D-5 against bovine sclera versican, and monoclonal antibody l-C-6 against a specific region within the hyaluronic acid binding region (Gl) of bovine nasal cartilage proteoglycan were used. The polyclonal antibodies against sclera decorin are directed to epitopes present on the core protein of the molecule (manuscript in preparation). The murine monoclonal antibody 5-D-5 recognizes a segment on the protein core close to the second globular domain (G2) of bovine sclera versican (Rahemtulla et al,, 1988; manuscript in preparation). Murine monoclonal antibody l-C-6 recognizes epitopes common to many hyaluronic acid binding domains of proteoglycans, specifically, two epitopes located on the first tandem repeat loops of the Gl and G2 domains (Hejan et al., 1987; Stevens, 1987; Stevens et al., 1984). Enzyme linked immunosorbent assay @LISA) and competitive ELISA were used to study the ability of the antibodies to interact with the antigen and test substances. Proteoglycans to be tested were dissolved at a concentration of 1 fig/ml in 50mmol/l sodium acetate buffer pH 5.6, containing 4mol/l guanidinium chloride. An aliquot of 2OOpl/well of this solution was added to the 96 wells of Immulon 4 assay plates (DynaTech, Chantilli, Va) and incubated overnight at room temperature in a humidified chamber. The plates were washed 3 times with PBS-Tween (20 mmol/l sodium phosphate, pH 7.4, containing 0.15 mol/l NaCl and 0.05% Tween 20) and blocked with 1% BSA in PBS-Tween for 90 min at 37°C. Serial dilutions (200 ~1) of antiserum in PBS-Tween containing the polyclonal antibodies or ascites containing the monoclonal antibodies were added to the individual wells and the mixture allowed to incubate at 37°C for 90min. The plates were extensively washed with PBS-Tween. Secondary reagent (200 #I), goat anti-rabbit or goat anti-mouse IgG conjugated to alkaline phosphatase (Southern Biotechnology, Birmingham, Ala) at I :2000 dilutions in PBS-Tween was added to the wells, and the mixture was incubated at 37°C for 1 hr. The substrate for alkaline phosphatase (Sigma lwR) was prepared at a concentration of 1 mgjml dissolved in 97mmol/l diethanolamine, pH 9.8, containing 0.49mmol/l MgCI, and 0.02% NaN, and 200 ~1 aliquots of this solution were added to the wells. The color development from the hydrolysis of p-nitrophenylphosphate was monitored at 405 nm with a Titertek Multiscan ELISA plate reader (Flow Laboratories, McLean, Va). In competitive ELISA, general procedures were the same as described above, but the antigen or test substances in concentrations ranging from 0.0156 to 10 pg/ml, were mixed with an appropriate dilution of the antiserum or the ascites and incubated overnight at 4°C. The plates were washed and blocked and the antigen/test substance-antibody mixture was added to the wells. After addition of the secondary

antibody, alkaline phosphate substrate was added and the developed color was measured at 405 nm. To prepare reduced and alkylated antigens for ELISA, fraction Dl from bovine nasal cartilage, bovine gingival versican and decorin were dissolved at a concentration 1 mg/ml in 0.5 mol/l Tris-HCl, pH 8.5, containing 6 mol/l guanidinium chloride. To the individual solutions dithiothreitol (Miles, Elkhart, Ind.) was added to a final concentration of 10 mmol/l and the mixture incubated at 37°C for 2 hr, after which iodoacetamide (Sigma) was added to a final concentration of 20 mmol/l and the mixture incubated further for 2 hr in the dark at 4°C. The mixture was dialyzed

against 10mmol/l Tris-HCl, pH 8.5, containing 2mol/l guanidinium chloride for 4 hr and then twice against 100 mmol/l ammonium hydrogen and carbonate lyophilized. These substances were tested in competitive ELISA as described above. RESULTS

Extraction and purification of proteoglycans

Noncollagenous proteins and proteoglycans were extracted from gingival tissue with a buffered solution of 4 mol/l of guanidinium chloride solution containing several protease inhjbitors. Concentration by ultrafiltration of the resulting extract yielded a thick viscous liquid which was difficult to handle. We routinely diluted the extract with 50mmol/l sodium acetate buffer, pH 5.6, containing 7 mol/l urea, to facilitate the application of the sample on the ion exchange column. After sample appli~tion, the column was extensively washed with the starting acetate/urea buffer. In this step, most of the proteins which do not bind to the ion-exchange column were eluted. Under the low pH conditions used for ionexchange chromatography only the highly charged glycoproteins and proteoglycans bound to the ion exchange-gel matrix. When no detectable proteins were present in the wash fractions, the bound material was eluted with a salt gradient of cl.0 mol/l of sodium chloride in the acetate/urea buffer. As shown in Fig. 1, a major portion of the bound material, containing protein and hexuronic acid, was eluted from the column in the early part of the gradient. Hyaluronic acid is weakly bound to the ion-exchange matrix and thus, part of the hexuronic acid positive material present in this pool is most likely composed of hyaluronic acid. This fraction has not been analyzed further in the present study. The major hexuronic acid positive peak which eluted at a salt concentration of 0..5-0.6mo1/1 (Fig. 1) was pooled, extensively dialyzed and lyophilized. When analyzed by SDS-PAGE, it was found that this material consisted of 2 fractions, one with an apparent molecular weight of 102 kDa while the other did not enter the gel (results not shown). The proteoglycan preparation obtained from ion-exchange chromatography was dissolved in 50mmol/l Tris-HCl, pH 7.4, containing 4mol/l guanidinium chloride and chromatographed on a molecular sieve column, Superose 6, which was eluted with the same buffer. The chromatographi~ profile is

7 t su)

0.9

6

2

0.8

e

0.7

s

z

5

0.6

g

8 1 g-

4 3

0.5 0.4

I5 *

0.3

; =x

3

2

0.2

1

0.1

0

0 0

10

20

30

Fraction

40

50

60

70

80

number

Fig. 1. Ion-exchange chromatography of guanidinium chloride extract obtained from bovine gingiva. The extract was dialyzed against acetate/urea buffer and applied to the ion-exchange column. The column was washed extensively with the starting buffer and the bound proteins and proteoglycans were eluted with a salt gradient of 0-l mol/l of NaCl. Individual fractions were analyzed for protein (-), hexuronic acid (0) and conductivity (0). The high absorbance values at 595 nm shown for fractions l&28 was obtained by sampling a smaiier aliquot of the fraction and the result multiplied by an appropriate dilution factor.

depicted in Fig. 2(A). The void volume fraction which consisted of high molecular weight proteoglycans, indicated by the presence of both protein and hexuranic acid, was pooled for further purification. The second peak, which eluted in the included volume, contained predominantly low molecular weight proteoglycans. These fractions from the 2 peaks were pooled separately and further purified as described below. A third peak which gave an absorbance at 280 nm but did not contain any hexuronic acid was most likely urea which had not been dialyzed from the pooled fractions. Upon rechromatography of the individual pooled peaks using the same column and conditions as previously described, the void volume peak eluted from the column as a symmetrical peak when analyzed at 280 nm for protein, while the profile of hexuronic acid containing material displayed a shoulder on the descending part of the peak (Fig. 2B). This void voiume peak, designated Pool I, (excluding the shoulder fractions) was pooled for further analysis. The major hexuronie acid containing peak, when rechromatographed on the Superose 6 column using the same conditions, again eluted at an included volume on the column. However, a small amount of the 280 nm absorbing material eluted at the void volume of the column. This fraction was excluded from the pooled material and not further analyzed. The major hexuronic acid containing peak, designated Pool II, was pooled for additional analysis (Fig. 2C). Characterization of proteoglycans

The amino acid composition of the isolated proteoglycans from bovine gingiva, Pool I and II, resembled that of proteoglycans from other sources. Both the

Proteoglycans of gingival tissues Table

0.80

1577

I. Amino

1.00

(A)

acid composition bovine tissues

of versican

from

Residues/1000

0.60 0.80

sc1era*

060

Aspartic acid Threonine

0.40

96 58

Serine 0 20

(6)

l

.

0.30

1.

i 0.20

-

O.?O

l. ‘8. .

‘; E

..IL

. -. .. _.

g

6 D b :: a

‘.r*&.+,-d_ 0 06

0.03

5

10

Fraction

15

20

25

number

Fig. 2. Molecular sieve chromatography of partially purified proteoglycans. (A) Proteoglycans obtained from ionexchange chromatography were applied to a column of Superose 6 which was eluted with 50 mol/l of Tris-HCl, pH 7.5, containing 4 mol/l guanidinium chloride. The eluant was monitored at 280 nm (-) and by hexuronic acid (0). Partially purified versican, which eluted at the void volume of column and decorin were individually pooled. (B) Versican from the previous purification step was rechromatographed on the Superose 6 column under identical conditions. The eluant was monitored at 280 nm (-) and by hexuronic acid (0). Versican eluted at the void volume of the column was pooled as described in the Results section for further analysis. (C) Decorin obtained from the Superose 6 column was rechromatographed on the same column and under identical conditions. The eluant was monitored at 280 nm (-) and by hexuronic acid (a). Decorin eluted at the included volume of the column was pooled as described in the Results section for further analysis. samples were rich in aspartic acid, glutamic acid, glycine, serine, alanine and leucine (Tables 1 and 2). The amino acid composition of Pool I differed from that of Pool II in that it contained more threonine, serine, glutamic acid and alanine, with less aspartic acid, isoleucine and lysine. The overall composition of Pool I was similar to that of a high molecular weight proteoglycan isolated from bovine sclera (Cbster and Fransson, 1981). However, it differed significantly from that of the large molecular weight

92

Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine

138 83

110 65 8 62 41 88 16 43 19 45 37

Gingivat

Gingivaf

86 50 128 135 41 176 67 ND 41 ND 33 71 28 30 15 49 48

85 80 87 137 79 105 95 ND 58 15 38 73 16 36 20 38 38

*From CBster and Fransson (198 1); tfrom Pearson and Pringle (1986); and fpresent study. ND, not determined.

proteoglycan isolated from bovine gingiva (Table 1) by Pearson and Pringle (1986). The amino acid composition (Table 2) of the small molecular weight proteoglycan isolated for this study shows resemblance to that of bovine sclera (Cdster and Fransson, 1981) and bovine gingiva previously reported (Pearson and Pringle, 1986). The purified preparations of the proteoglycans Pool I and Pool II were examined for their susceptibility to specific glycosidases by enzymatic digestion with chondroitinase ABC as described in the Materials and Methods section. The digestion products were analyzed by gel chromatography on a column of Sephadex G-50. Both the pools were completely digested by this enzyme as indicated by the presence of hexuronic acid positive fraction near or at the total volume of the column (results not shown). Based on the results of the amino acid analysis and the nature of the glycosaminoglycan side chains, we

Table 2. Amino

acid composition

of decorin

from bovine tissues

Residues/l000 Sclera* Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine

123 49 68 122 74 84 54 9 59 7 55 115 15 34 25 76 32

Gingivat 117 47 71 112 72 81 56 ND 59 9 56 123 28 33 27 72 36

*From Ciister and Fransson (1981); tfrom Pearson (1986); and ipresent study. ND, not determined.

Gin&a1 II8 46 63 112 83 105 50 23 65 II 57 121 I3 33 24 71 29 and Pringle

1578

P. BRAT? et al.

conclude that both the pools belong to the proteoglycan family. The purified preparations of native and deglycosylated proteoglycans were analyzed by SDS-PAGE. We found that intact large molecular weight proteoglycan did not enter the 3-15% gradient gel (Fig. 4A; lane 2); while the chondroitinase ABC digested material entered the resolving gel with an apparent molecular weight greater than 200 kDa (Fig. 4A; lane 3). Two distinct bands were visible in this preparation. After deglycosylation with chondroitinase ABC and N-glycanase, the resulting core protein migrated further into the gel as a single band indicating that N-linked oligosaccharides were present, and that the differences in the 2 bands seen after chondroitinase digestion were due to different glycosylation patterns on the core protein (Fig. 4A; lane 4). The electrophoretic mobility of the purified small molecular weight proteoglycan displayed an apparent molecular weight of 102 kDa (Fig. 4B; lane 2). When this molecule was digested with chondroitinase ABC, the molecular weight was reduced to 55 kDa (Fig. 4B; lane 3). A further reduction of the molecular weight to 45 kDa was observed after deglycosylation with N-glycanase (Fig. 4B; lane 4).

from bovine sclera and large molecular weight proteoglycan from bovine gingiva by competitive ELISA. As shown in Fig. 3(A), the antigen (small molecular weight proteoglycan) effectively inhibited binding of the antibodies to the bound antigen confirming the specificity of the antibodies. The crossreactivity of the antibodies to decorin isolated from bovine sclera was remarkably similar to that of the small molecular weight proteoglycan from gingiva (Fig. 3A). The antiserum could detect the small molecular weight proteoglycan at a concentration of 40 ng/ml and above at a dilution of 1: 1000. Using the

0 90

Immunological properties

Antibodies to bovine gingival decorin were detected in rabbit serum after 4 injections of antigen and a maximum titer was observed after the eighth injection. Titer curves revealed that a 1: 1000 dilution of the antiserum was a suitable working dilution and within 1 hr of incubation with the substrate an absorbance of 1 unit at 405 nm could be obtained. These antibodies were tested for their reactivity to the antigen and cross-reactivity to the decorin isolated

60

60

Fig. 3. Competitive inhibition ELISA for decorin and versican. (A) The plates were coated with 1 pg/ml of bovine gingival decorin. Bovine gingival decorin (0) and bovine sclera decorin (A) were used as inhibitors in concentrations indicated in the figure. Polyclonal antibodies raised against bovine gingival decorin at a final dilution of 1: 1000 was used in the experiment. (B) Plates were coated with 1 pgg/ml of bovine sclera decorin. Bovine gingival decorin (0) and bovine sclera decorin (A) were used as inhibitors in concentrations indicated in the figure. Polyclonal antibodies against bovine sclera decorin were used at a final concentration of 1:lOOO. (C) Plates were coated with 1 pg/ml of bovine gingival versican and bovine gingival versican (0) and bovine sclera versican (A) were used as inhibitors in concentrations shown in the figure. Monoclonal antibody S-D-5 at a concentration of I:6000 was used. (D) Plates were coated with 1 pg/ml of reduced and alkylated fraction Dl of bovine nasal cartilage proteoglycans. The monoclonal antibody l-C-6, diluted to 1: 1000, was used. Dl of cartilage proteoglycan (0) bovine gingival versican (0) and bovine gingival decorin (a) at concentrations shown in the figure were used as inhibitors. All fractions were reduced and alkylated.

30

0 90

ID)

60

30

0 0 01

Antigen

added

Fig. 3

tpg/mL)

Proteoglycans of gingival tissues

(A

Fig. 4. Polyacrylamide gel electorphoresis of proteoglycans obtained from bovine gingiva. (A) 3315% SDS-PAGE of intact and deglycosylated versican. Lane 1: Pharmacia low molecular weight standards from top to bottom of the gel; phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100) and a-lactalbumin (14,400). Lane 2: 15 pg intact versican. Lane 3: 20 pg of versican digested with chondroitinase ABC. Lane 4: 10 pg of versican digested first with chondroitinase ABC and then with N-glycanase. (B) 515% SDS-PAGE. Lane 1: Pharmacia low molecular weight standards (same as shown in A). Lane 2: 15 fig intact decorin. Lane 3: 2Opg of decorin digested with chondroitinase ABC. Lane 4: 10 pg of decorin digested with chondroitinase ABC and then with TV-glycanase.

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competitive ELISA we have also tested the crossreactivity of the large molecular weight proteoglycan from gingiva to the antibodies and found that at concentrations of 5 pg/ml and higher, it displayed l&12% inhibition (results not shown). However, this low value could be due to non-specific binding. The immunological relationship between the small molecular weight proteoglycan from gingiva and sclera decorin was also determined using antibodies raised against bovine sclera decorin in a competitive ELISA. Results shown in Fig. 3(B), revealed that decorin from both sources inhibited the binding of the test substances in a similar manner, indicating that the 2 molecules were immunologically identical. We have also tested the ability of a monoclonal antibody against bovine sclera versican to cross-react with the large molecular weight proteoglycan from bovine gingiva. This monoclonal antibody specifically recognizes epitope(s) present on the protein core which are located close to the second globular domain, G2, on the protein core of the molecule. The results depicted in Fig. 3(C) demonstrated that this epitope was present on the protein core of the large molecular weight proteoglycan from bovine gingiva and the results further imply that these 2 molecules are immunologically related. In order to determine if hyaluronic acid binding region, Gl, is present in the large molecular weight proteoglycan from bovine gingiva, we used a monoclonal antibody directed against a very specific region within the Gl segment. Fraction Dl from bovine nasal cartilage proteoglycan which served as a control substance gave a gradual inhibition in competitve ELISA (Fig. 3D). When the reduced and carboxymethylated large molecular weight proteoglycan from bovine gingiva was used as a competing test substance, a gradual inhibition was noted for concentrations up to 1 pg/ml and at higher concentrations a negative inhibition was observed. These results implied that, to a certain extent, this epitope was present in the large molecular weight proteoglycan from bovine gingiva. This antibody did not crossreact with the small molecular weight proteoglycan from bovine gingiva (Fig. 3D). DISCUSSION

The isolation method developed by us has allowed us to rapidly and reproducibly obtain chemical amounts of proteoglycans from bovine gingival tissues. We have isolated and identified 2 proteoglycans which differ in molecular size, chemical composition and immunological properties. The products are of high purity as judged by electrophoresis, amino acid analysis and cross-reactivity to specific antibodies. Based on the amino acid and glycosaminoglycan composition and the cross-reactivity to specific antibodies of the large molecular weight proteoglycan, we conclude that this fraction is versican. Its amino

acid composition is very similar to that of versican isolated from bovine sclera (Caster and Fransson, 1981), but differs significantly from the composition of versican from bovine gingiva reported by Pearson and Pringle (1986). These differences can be ascribed to partial purification of the molecule achieved by these authors. In our study, the glycosaminoglycan chains of versican were susceptible to chondroitinase ABC indicating that both chondroitin and dermatan sulfate chains may be present. Such hybrid glycosaminoglycans have previously been reported to be present in gingival proteoglycans (Pearson and Pringle, 1986) and to be found among proteoglycans isolated from bovine sclera (Ciister and Fransson, 1981). Available methods have not allowed us to more accurately determine the molecular weight of the core protein of versican, however, based on its electrophoretic migration on SDSPAGE we suggest a value of 200 kDa. The molecular weight of the core protein of versican from other sources has been reported to be in the range of 290-400 kDa (Johansson et al., 1985; Krusius et al., 1987) and the calculated value obtained from sequence data is 263 kDa (Zimmerman and Ruoslahti, 1989). Therefore our suggested value of greater than 200 kDa is a good estimate for the protein core of the gingival versican. After deglycosylation with chondroitinase ABC and subsequent electrophoresis of the material by SDS-PAGE, 2 closely migrating bands were visible (indicated by arrows in Fig. 4A; lane 3). These results imply that it is likely that the core proteins have different glycosylation patterns. Upon N-glycanase digestion, which specifically removes N-linked oligosaccharides, the migration pattern of the resulting digestion product was altered and only 1 diffuse band was visible (Fig. 4A; lane 4). These findings establish that N-linked oligosaccharides are present on the core protein of versican, an observation consistent with sequence studies by Zimmerman and Rouslahti (1989) which indicated that there are 15520 N-glycosylation sites present in the versican from fibroblasts. The presence of hyaluronic acid binding region in bovine gingival versican was demonstrated by immunological methods. Evidence for the presence of this region was obtained using monoclonal antibody l-C-6, which is specific to an epitope present in the hyaluronic acid binding region of cartilage proteoglycan (Hejan et al., 1987; Stevens, 1987; Stevens et al., 1984). It is evident from our results that although there is cross-reactivity of the antibody to versican from bovine gingiva, its hyaluronic acid binding region may not be immunologically identical to that of cartilage proteoglycan. This tentative conclusion is based on the results demonstrating that versican at a concentration higher than 1 pg/ml gave a negative inhibition (Fig. 3D). The hyaluronic acid binding region, Gl, is also present in versican from other soft connective tissues such as bovine sclera, bovine aorta and bovine tendon as shown by Morgelin et al. (1989)

Proteoglycans of gingival tissues using

rotary-shadowing

electron

microscopy.

The

l-C-6 antibody has also been used to localize a hyaluronic acid binding region epitope in an aggregating proteoglycan from brain tissue (Ripellino et al., 1989). Although the core proteins of versican from soft connective tissues share many common features, minor differences are observed in these molecules isolated from different tissues (Miirgelin et al., 1989; Zimmerman and Rouslahti, 1989). A close immunological relationship between versican from bovine gingiva and bovine sclera was observed in our study. The monoclonal antibody used in this study is specific to an epitope on versican and will bind to versican from aorta, tendon and sclera but does not crossreact to cartilage proteoglycan (Rahemtulla et al., 1988; manuscript in preparation). Based on the results from our studies, we conclude that versican from bovine gingiva structurally resembles versican from other soft connective tissues. The second proteoglycan pool comprises of a small molecular weight proteoglycan, with an amino acid composition similar to that of decorin from sclera (Table 2). The glycosaminoglycan chains of this molecule are susceptible to chondroitinase ABC digestion. It has an apparent molecular weight of 102 kDa and the protein core obtained after removal of the glycosaminoglycan chains electrophoreses as a broad band with an apparent molecular weight of 55 kDa. A decrease in core glycoprotein size to 45 kDa was apparent after N-glycanase digestion, providing evidence that the core protein is N-glycosylated. Our results are in good agreement with those published earlier for decorin from other tissues (Caster and Fransson, 1981; Damle et al., 1979; McMurtrey et al., 1979; Oegema et al., 1979; Pearson and Gibson, 1982; Salisbury and Wagner, 1981; Uldbjerg et al., 1983a,b). Decorin isolated from several other connective tissues exhibits polydisperse properties with relative molecular weights ranging from 80 to 140 kDa, and a molecular weight of 45-55 kDa has been reported for the partially deglycosylated protein core of decorin. The polyclonal antibodies which were raised in rabbits against decorin isolated from bovine gingiva cross-reacted with decorin isolated from bovine sclera. A similar cross-reactivity was observed with antibodies raised against decorin from sclera when gingival decorin was used. Indeed, results of the inhibition studies (Figs 3A and B) displayed overlapping curves suggesting that the molecules are immunologically alike. The anti-decorin antibodies have been used in immunodetection of intact and partially deglycosylated decorin and further studies to elucidate the nature of the epitopes to which these antibodies bind are in progress and will be published elsewhere. We have not detected biglycan or fibromodulin in bovine gingival tissue. An absence of biglycan in gingival fibroblasts has been reported earlier (Fisher

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have noted the absence of biglycan in periodontal tissue cells (Larjava et al., 1992). In human cartilage, biglycan expression is high in the fetal tissue and low in the adult tissue (Melching and Roughley, 1989). Therefore, it is reasonable to assume that a low undetectable amount of biglycan may be present in bovine gingiva since we have used tissue from adult animals. The initial step in the isolation procedure described here is based on the anionic properties of the proteoglycans. While the proteoglycan molecules bind strongly to the ion-exchange column, fibromodulin, which lacks the glycosaminoglycan chain(s), is presumably eluted earlier from the column and would have escaped our detection if present in our preparations. Earlier studies (referred to in the Introduction) have demonstrated the presence of heparan sulfate in gingival tissues. We have not detected heparan sulfate proteoglycans in our preparation and absence of this glycosaminoglycan could be due to the analytical techniques used. Earlier studies have employed histochemical techniques and the biochemical analyses have been of preliminary nature. In support of this finding, it is noteworthy that early biochemical work performed on proteoglycans of gingiva have not detected heparan sulfate in bovine gingiva (Dziawiatkowski et al., 1977; Pearson and Pringle, 1986). In summary, we have developed a rapid and reproducible method with which large amounts of proteoglycans can be isolated from bovine gingiva. Our results have confirmed previously published results (Pearson and Pringle, 1986) as well as added new information regarding the biochemical and immunological properties of versican. Acknowledgements-The authors acknowledge the technical help of Aruna Das and the efforts of Anglea Steele and Cheryl S. Kiser in preparation of the manuscript. We also wish to thank the administration of John Morel1 & Co. for allowing us to collect bovine gingival tissues at their slaughter house in Montgomery, Alabama. This investigation was supported by grants from NIH/NIDR BRSG RR 05300; SlS-DE099275; R03 DE09134; ROI DE08466; Summer Research Fellowship grant T35 HL07473 to M.M.A.; support from Sweden America Foundation and the Swedish Medical Research Council, Grant No. K88-24P-07319-04B to P.B.; and Veterans Administration Merit Review Award to J.W.S. REFERENCES

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Isolation and characterization of bovine gingival proteoglycans versican and decorin.

1. We have isolated, chemically and immunologically characterized versican and decorin from bovine gingiva. 2. Versican was of large molecular weight ...
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