Bovine Pericardial Proteoglycan: Biochemical, Immunochemical and Ultrastructural Studies

Matrix Vol. 911989, pp. 301- 310

Bovine Pericardial Proteoglycan: Biochemical, Immunochemical and Ultrastructural Studies DAN SIMIONESCU 1, RENATO v. IOZZ02 and NICHOLAS A. KEFALlDES 3 Connective Tissue Research Institute, Department of Medicine, and Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Summary The major proteoglycan in bovine parietal pericardium is a low molecular weight dermatansulfate proteoglycan. It possesses structural and immunologic characteristics similar to those of the small proteoglycan found in tendon. We demonstrate that digestion of purified pericardial proteoglycan with low levels of Vs protease results in the liberation of the glycosaminoglycan chain and of a 40 kDa resistant fragment. A similar 40 kDa fragment can be obtained by Vs protease digestion of the proteoglycan deglycosylated by chondroitinase ABC. Although the protein core size of the pericardial proteoglycan is similar to that of tendon PG II, the size of the glycosaminoglycan chain liberated from the former is smaller. The pericardial proteoglycan and its V8 protease products reacted with an anti-PG II antiserum by immunoblotting. The anti-PG-II antibody localized in the pericardial tissue by the immunoperoxidase technique. The presence of intra chain disulfide bonds in the structure of pericardial proteoglycan core protein and Vs resistant fragment was demonstrated by their decreased electrophoretic mobility after disulfide reduction. Digestion of pericardial proteoglycan with Cathepsin C resulted in a rapid liberation of the glycosaminoglycan chain from the core protein, indicating that its attachment site was very close to the amino terminus. Ultrastructural examination of pericardial tissue utilizing Cuprolinic Blue revealed a periodic association of the proteoglycan with the die band on the collagen fibrils. Electron microscopic immunohistochemical studies confirmed the perifibrillar association of pericardial proteoglycan. The present data demonstrate that, although the pericardial proteoglycan possesses some unique structural features, it shares structural and immunological characteristics to place it in the category of the small PG II family. Key words: bovine parietal pericardium, derma tan-sulfate, proteoglycan.

Introduction The pericardium is a fibro-serous membrane in which the heart and the commencement of the great vessels are con1 Permanent address: Cardiovascular Surgery Research Department, The Medical Research Center of the Academy of Medical Sciences, P. O. Box 118 Tirgu-Mures 4300, Romania. 2 Current address: Department of Pathology ,Jefferson Medical

© 1989 by Gustav Fischer Verlag, Stuttgart

College, Thomas Jefferson University, Philadelphia, PA 19107, USA. 3 To whom reprint requests should be sent: Connective Tissue Research Institute. 3624 Market Street, Philadelphia, PA 19104, USA. 4 The abbreviations used are: PG, proteoglycan; DS, dermatan sulfate; Vs protease, extracellular protease isolated from Staph. Aureus strain Vg; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; BME, ~-mercaptoethanol; GAG, glycosaminoglycan; ELISA, enzyme-linked immunosorbent assay.

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tained. The inner, serous pericardium consists of a visceral mesothelial layer that covers the heart muscle and a facing parietal mesothelium that lines the inner surface of the fibrous layer. A small amount of fluid fills the cavity between the two mesothelia (Leak et al., 1987; Crofts and Trowbridge, 1988). The outer, fibrous sac is a dense connective tissue layer composed of densely packed collagen fibrils, elastic fibers, fibroblasts, adipocytes, blood vessels and nerve fibers (Ishihara et al. , 1981). The pericardium performs in vivo mostly mechanical functions; as a reaction to acute injury (e. g., inflammation) the pericardium develops fibrous adhesions which may cause impairment of cardiac function. Only recently an animal model has been developed to study this reaction (Leak et al., 1987). Chemically treated bovine parietal (fibrous) pericardium has been used as a biomaterial in cardiovascular surgery for more than a decade, especially in the form of pericardial trileaflet bioprosthetic heart valves (Ionescu and Tandon, 1979; Schoen et al., 1986). After 10years of follow-up pericardial valves perform satisfactorily, although complications such as calcification and mechanical failure of the biological tissue limit their durability. Although the pericardial morphology and mechanical properties are well documented, knowledge of the biochemical nature of pericardial connective tissue components is incomplete. Type I collagen is the predominant (90%) extracellular protein (Schoen et aI., 1986) and only recently a low molecular weight dermatan-sulfate proteoglycan (DS-PG)3 has been demonstrated in bovine parietal pericardium (Simionescu et al., 1988). Similar small PG's have been previously described in tendon (Vogel and Fisher, 1986), bone (Day et al., 1987), skin (Pearson et al., 1983) and cartilage (Rosenberg et al., 1985). The terminology PG I and PG II for the two classes of small proteoglycans was first suggested by Rosenberg et al. (1985). The common features of these small PG's are: Mr of about 120 kDa, 1-2 glycosaminoglycan chains attached to a 42-45 kDa core protein and lack of binding to hyaluronic acid. Differences between the members of the small PG family have been accounted for by the nature and size of the GAG chains, glycosylation and secondary structure of the core protein and physiological roles. In the present study, we are reporting on the structural, immunological and ultrastructural features of pericardial PG.

Experimental Procedures Materials Pericardial PG was extracted from bovine parietal (fibrous) pericardium and purified as previously described (Simionescu et al., 1988). Tendon PG II and rabbit antiserum were kindly provided by Dr. Kathryn Vogel (University of New Mexico, Albu-

querque, NM). Chondroitinase ABC and Cathepsin C (Dipeptidylaminopeptidase I) were obtained from Sigma Chemical Co., St. Louis, MO. Vs protease from Staph. aureus strain Vs (E.e. number 3-4-21-19) was from ICN Biochemicals, Cleveland, OH. SDS-PAGE on 5-15% linear gradient gels was performed with a Hoefer 600 apparatus using chemicals and molecular weight markers from Bio-Rad; electroblotting onto Immobilon-P (Millipore Corp.) transfer membranes was done in a Bio-Rad Trans-Blot Cell and immunostaining was performed with a Vectastain ABC Kit from Vector Laboratories, Inc., Burlingame, CA. Sepharose CL-6B, columns and accessories were obtained from Pharmacia-LKB Industries, Inc., Piscataway, NJ. A1cian Blue was obtained from Eastman Kodak Co., Rochester, NY and Cuprolinic blue from BDH.

Analytical Procedures SDS-PAGE

SDS-PAGE was performed on linear 5-15% gradient gels with a 3% stacking gel as described by Laemmli (1970) with slight modifications (Hames, 1981). Gels were stained overnight with 0.1 % Coomassie Blue R in methanol!acetic acid/water - 5:2:5 (v/v), destained in 15% methanol!7.5% acetic acid until the background was very low, photographed and stained overnight in 0.5% A1cian Blue (in 7% acetic acid) destained in 7% acetic acid and rephotographed (Vogel and Fisher, 1986). Enzymatic Digestions Chondroitinase ABC Digestion To a solution of 300!Ag PG in 100 !AI 50 mM phosphate buffer pH 8, 20 !AI of chondroitinase ABC (0.5 U/ml) in the same buffer were added and samples were incubated at 37 °C for 60 min. Samples containing 25 !Ag PG were mixed with SDS-PAGE sample buffer (62.5 mM Tris-HCl, 4 mM urea, 10% glycerol, 2% SDS, 0.025% bromphenol blue, pH6.8) and were boiled for 5 min in the presence of 2% BME, before being applied to the gel.

V8Protease Digestion 90!Ag PG were incubated at 37°C with 0.1 !Ag (0.0055 U) Vs in 35 !AI of 50 mM phosphate buffer pH 8; for timecourse studies, samples containing 25 !Ag PG were retrieved at 10, 30 and 60 min, and processed as described above. When Vs followed chondroitinase ABC digestion, samples were digested with chondroitinase ABC 60 min then mixed with 0.05 mU Vg protease/!Ag PG in the same buffer and incubated at 37°C; for time-course studies, samples were retrieved at 10, 30 and 60 min and processed as described above.

Pericardial Proteoglycan

Cathepsin C Digestion Cathepsin C digestion was performed by mixing 64!ig pericardia I PG dissolved in 50!i1 buffer-activator, as described by Callahan et al. (1972), with 0.5 U enzyme and incubating at 37°C. At designated intervals, samples containing 25!ig PG were retrieved and processed for SDSPAGE (see above). To assess the role of disulfide reduction upon mobility in SDS-PAGE of enzymatically digested PG, samples of pericardial PG were digested for 60 min at 37°C (as above) with chondroitinase ABC and V8 (separately). Each sample was mixed with sample buffer and divided in two aliquots; one was boiled for 5 min with 2% (w/v) BME while the other was boiled without BME. Equivalent amounts of each preparation (approximately 25!ig PG) were applied to the gel; reduced and unreduced lanes were separated by at least 3 empty lanes.

Immunoblotting and Immunohistochemistry Samples of pericardial and tendon PG were digested separately as described above with either chondroitinase ABC alone for 60 min, or 60 min with chondroitinase followed by 60 min with V8. Aliquots containing 12-15 !ig PG were applied to a 5-15% SDS-PAGE. Electroblotting onto immobilon PDVF membranes was performed as described initially by Towbin et al. (1979) for nitrocellulose paper at 4°C, for 4-5 hat 60-65 V (0.3 A). Blotted membranes were washed with phosphate buffered saline containing 0.01 % Tween 20 and incubated overnight at 4°C with diluted rabbit anti-tendon PG II antiserum. After washing, bound IgG was detected using the Vectastain ABC Kit (4-chloro-1-naphthol and HzO z were used as substrate). Frozen sections (5 !lm) cut from pericardial tissue were incubated with various dilutions of anti-tendon PG II antiserum and visualized by immunoperoxidase as described before (lozzo, 1984).

Analysis of Glycosaminoglycans To liberate the GAG chain, pericardial PG was treated with alkaline borohydride (0.05 M NaOHll M NaBH 4, 4°C) for 48 h (Carlson, 1968). The solution was neutralized with glacial acetic acid and dialyzed against 0.2 M NaC!. An aliquot was applied to a Sepharose CL 6B column (1.5 X 93 em) which was equilibrated with 0.2 M NaCI. Elution was carried out at 9 ml/h at room temperature and fractions were analyzed for hexuronic acids by the meta hydroxydiphenyl method using D-glucuronolactone as standard (Blumenkrantz and Asboe-Hansen, 1973). The void volume and total volume of the column were determined using Blue Dextran and Phenol Red, respectively. Do was calculated for the GAG peak and the molecular mass was estimated by the method of Wasteson (1971).

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Electron Microscopic Histochemistry Fragments of bovine pericardium were fixed for 18 hat 4°C in 2.5% glutaraldehyde, 25 mM sodium acetate, pH5.7, 0.05% Cuprolinic Blue in the presence of either 0.1 M or 0.3 M MgCl 2 according to the critical electrolyte concentration method (Scott and Orford, 1981; Scott and Haigh, 1985 ). Control specimens were fixed for 18 h in the absence of the dye. After fixation samples were rinsed, stained en bloc with dilute sodium tungstate, dehydrated in graded ethanol solutions and embedded in EPON. Ultrathin sections were observed after uranyl acetate staining in a Hitachi 600C electron microscope (at 50kV). In order to estimate quantitatively the average collagen fibril diameter, the width of 100 collagen fibers was measured from randomly selected pictures that were in focus (magnifications at X 60000-90000). No longitudinal or cross sectional areas were measured. Cuprolinic blue-stained PGs appeared as linear structures of variable lengths and orientation; to estimate the actual filament length 100 fully extended, collagen-associated filaments were measured and the mean observed values were corrected as described previously (Iozzo, 1984).

Results Enzymatic Digestions Pericardial PG appeared on a 5 -15% gradient SDSPAGE gel as a Coomassie Blue-Alcian Blue positive broad band of a Mr of about 115 k (Fig. 1, lanes 1 and 5). Exposure of pericardial PG to V8 protease (Fig. 1, lanes2-4) yielded a Coomassie Blue stainable 40 kDa protein and an Alcian Blue stainable polydisperse band; conversion of the pericardial PG to these products was complete after about 60 min. The position of the Alcian Blue stained band (open arrowheads, Fig. 1) was identical to that of alkaline borohydride-liberated GAG chains from pericardial PG (Fig. 1, lane 7). Chondroitinase ABC digestion of pericardial PG yielded a core protein preparation having an estimated Mr of 42 k (lane 10); exposure of this core protein to V8 protease converted it in a time-dependent manner (Fig. 1, lanes 12-14) to a 40kDa protease resistant polypeptide and a product that moves close to the dye front (less than 14kDa). For comparison, tendon PG II was analyzed under the same conditions and on the same gel as pericardial PG. Tendon PG II alone (Fig. 1, lane 6) appeared as a more heterogeneous preparation, with a lower mobility than pericardial PG (M r 145 k). After exposure to V8 protease (lane 8), a similar 40 kDa resistant protein resulted with the liberation of a long Alcian Blue stainable streak of considerably slower mobility. Chondroitinase ABC treatment of tendon PG yielded a core protein of nearly identical mobility to pericardial PG

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Fig.I. Gradient (5-15%) SDS-PAGE analysis of pericardial PG (PPG) and tendon PG (TPG) before and after chondroitinase ABC (C'ASE) and Vg protease (Vg) digestion. Intact PPG (lanes 1 and 5) was digested with Vg for 10 min (lane2), 30 min (lane3) and 60 min (lane 4). C'ASEdigested PPG (core protein, lane 10) is shown after 10 min (lane 12), 30 min (lane 13) and 60 min (lane 14) of Vg digestion. For comparison TPG is shown before (lane 6) and after 60 min digestion with C'ASE (core protein, lane 9), Vs (lane 8) or C'ASE followed by Vg, 60 min each (lane 11). Alkaline borohydride-liberated (OH-) GAG chain from PPG is shown in lane 7. Lane 15 - low molecular weight standard protein markers. The position of Alcian Blue-stained GAG chains liberated from PPG (open arrowheads) and from TPG (closed arrowheads between lanes 7 and 8) is shown. All lanes contain 251lg PG; the gel was stained with Coomassie Blue followed by Alcian Blue (see "Methods" for details).

core protein (Fig. 1, lane 9 vs. 10). Tendon PG core protein yielded the same V8 resistant 40 kDa protein (Fig. 1, lane 11).

Presence of Disulfide Bonds To determine whether there were any intra-chain disulfide bonds in the pericardial PG protein core, purified samples of PG and enzyme-digested preparations were analyzed by SDS-PAGE under reducing and non-reducing conditions (Fig. 2). The apparent mobility of the intact PG did not significantly change (compare lanes 1 and 4, Fig. 2). However, the V8 protease treated (compare lanes 2 and 5, Fig. 2) as well as the chondroitinase ABC-treated (lanes 3 and 6) products showed a decreased mobility under reducing conditions. An internal control was the bovine serum albumin present in the chondroitinase ABC preparation (the band at 66 kDa, lane 6) which displayed a decreased mobility under reducing conditions. These results demonstrate the presence of intramolecular disulfide bonds in the core protein of the pericardial PG.

Immunochemical Studies Immunological cross reactivity between tendon and pericardial small PGs has been initially shown by direct ELISA (not shown). The results of an immunoblotting experiment are shown in Fig.3. Pericardial and tendon PG's were digested with chondroitinase ABC and V8 protease, the digests were separated on a 5 -15% gradient gel

and then electroblotted. The liberated core protein (lane 3), V8 protease digested PG (lane 2) and the V8 proteasedigested core protein (lane 1) derived from pericardial PG reacted with the anti-tendon PG II antiserum. Little, if any immunological reactivity could be observed for the 12 kDa V8 protease product.

Localization ofthe Glycosaminoglycan Chain on the Protein Core

In an attempt to determine the location of the GAG chain on the pericardial PG core protein, the pericardial PG was digested with Cathepsin C which removes dipeptides sequentially from the N-terminus of proteins (Callahan et aI., 1972). Fig. 4 shows the time-course digestion of pericardial PG with Cathepsin C, as analyzed by SDS-PAGE. After 10 min, a protein band of identical mobility to the core protein (i. e., chondroitinase ABC deglycosylated PG) was produced. The first Alcian blue detactable GAG chains were seen after 20 min of digestion. After 40 min, almost the entire pericardial PG band disappeared with the concomitant accumulation of a 41-42 kDa protein band and a 32 -42 kDa GAG chain streak. After 120 min some of the 41-42 kDa protein seemed to have been degraded. These results indicate that pericardial PG contains only one GAG chain located close to the amino terminus of the core protein.

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Fig. 2. Gradient (5-15%) SDS-PAGE analysis of the behavior of pericardial PG and its chondroitinase ABC (C'ASE) and Vg protease products before and after reduction with beta-mercapto ethanol (ME). Pericardial PG (lanes 1 and 4) was digested 60 min with Vg protease (lanes 2 and 5) or C'ASE (lanes 3 and 6) and samples containing 25 lAg PG were loaded on the 5 -15% gel before (lanes 1-3) or after reduction (lanes 4-6) with 2% (w/v) ME. Coomassie Blue-stained gel. Low molecular weight standard markers are shown in lane 7. The 66 kDa band is bovine serum albumin from the C' ASE preparation.

Fig. 3. Immunochemical studies of pericardial PG. Pericardial PG and tendon PG were digested separately for 60 min with chondroitinase ABC (core protein, lanes 3 and 6, respectively) alone, with Vg protease alone (lanes 2 and 5) or with chondroitinase ABC followed by Vg (lanes 1 and 4). Aliquots containing approx. 15 g PG were separated by SDS-PAGE on a 5-15% gradient gel, electrotransferred onto an immobilon PDVF membrane and reacted with rabbit anti-tendon PG II antiserum. Localization of bound IgG was done using the Vectastain Kit.

a perifibrillar deposition of the antibody-PG complexes (Fig.6C).

Electron Microscopic Histochemistry of Proteoglycans Glycosaminoglycan Chain Size The alkaline borohydride liberated GAG chain from pericardial PG was eluted (Fig. 5) from a calibrated Sepharose CL 6B column at a Kav of 0.52 which, according to Wasteson (1971), corresponds to a Mr of about 18 kDa.

Light and Electron Microscopic Immunohistochemistry Frozen sections of pericardium reacted strongly with anti-tendon PG II antiserum (Fig. 6B) but remained totally unstained with preimmune rabbit serum (Fig.6A). Immunoelectron microscopy of pericardium using the same antiserum revealed electron-dense deposits associated primarily with pericardial collagen fibers (Fig. 6D) sometimes suggestive of an ordered arrangement (inset, Fig. 6D). Cross sections through pericardial collagen fibrils revealed

Ultrastructural localization of pericardial PG was achieved by fixing and processing pericardial tissue fragments in the presence of Cuprolinic Blue (Fig. 7). Pericardium fixed in the absence of the dye revealed an abundance of tightly packed collagen fibrils which displayed the typical type I collagen banding pattern (Fig. 7 A). Cuprolinic Blue revealed numerous collagen-associated filaments or dots (cross-sectioned PG filaments). Most of the PG-dye complexes were associated in a periodic fashion to the die bands of collagen fibers (Fig.7B-inset) and in some areas they strongly suggested a perifibrillar arrangement (arrows Fig.7B). When stained collagen fibers were viewed in a cross-section, extended filaments appeared to associate in a spike-like manner with the collagen fibrils (Fig. 7C). These results correlate well with the immunohistochemical data (Fig. 6).

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Fig. 4. Time-course digestion of pericardial PG with Cathepsin C (dipeptidyl aminopeptidase). Pericardial PG was incubated with Cathepsin C at an enzyme/PG ratio of 8 mU/flg. Aliquots of the PG-enzyme mixture were retrieved at designated time intervals and were applied to a 5-15% gradient SDS-PAGE gel. The gel was stained with Coomassie Blue followed by Alcian Blue. Open arrowheads (GAG) indicate the position of Alcian Blue-stained GAG chain(s) released by the enzyme. The position of chondroitinase ABC-treated PG (core protein) is indicated by the full arrow. STD-Iow molecular weight standard protein markers.

The average collagen fibril diameter was estimated by morphometric analysis of 100 fibrils (not shown). Almost 60% of measured fibrils ranged between 80 and 100 nm in diameter (mean 90 nm). Filaments that represent PG-Cuprolinic Blue complexes were estimated to have a mean length of 52 ± 4 nm (data not shown).

Discussion Structural Features A low molecular weight dermatan sulfate proteoglycan was isolated and purified from bovine parietal fibrous pericardium (Simionescu et al., 1988). In order to achieve a more complete characterization, pericardial PG was digested with Staph. Aureus Vs protease which was reported to cleave peptide bonds on the carboxyl terminal side of either Asp or Giu (Drapeau et aI., 1972). When the enzyme-PG ratio is held low (e. g. 0.06 mU/f.lg PG) the

Fig. 5. Gel filtration chromatographic profile of pericardial PG and alkaline borohydride-liberated GAG chain. Pericardial PG (-e-.e-) was applied to a calibrated Sepharose CL 6B column which was equilibrated in 0.2 M NaCl. Elution was performed at 9 mllh at room temperature. Every fraction was analyzed for uronic acids (expressed in flglml). Alkaline borohydride-liberated GAG chain (-0-0-) from pericardial PG was applied to the same column and eluted as above. Intact PG eluted at a Kav of 0.38 and the GAG chain at 0.52. enzyme attacks a major sensitive site, yielding the free GAG chain and a 40 kDa resistant fragment. The same results when the chondroitinase-liberated pericardial PG core protein is treated with Vs protease. A doublet of about 12 and 15 kDa appears after 30 min of digestion indicating a secondary cleavage site. This Vs protease digestion pattern is similar to that reported previously for PG-II from other tissues (Vogel and Fisher, 1986). Sequence analysis showed that the major V g protease cleaving site on the tendon PG II is located on the carboxy side of a Glu residue located 17 amino acids from the amino-terminus (Vogel et al., 1987). In order to relate bovine pericardial PG directly to tendon PG, a purified preparation of the latter was analysed before and after chondroitinase ABC and Vs protease digestions and compared on the same gel with pericardial PG products. Intact tendon PG appears as a slower migrating component compared to pericardial PG. This difference is probably accounted for by the GAG chain, which upon liberation from the core protein by V s protease behaves in a similar manner (i. e., slower mobility and larger heterogeneity than pericardial PG GAG chain). Gel filtration experiments indicated a Mr of about 18 kDa for the pericardial PG GAG chain which is significantly smaller than the reported (Vogel and Fisher, 1986) Mr of tendon PG II GAG chain (30-40 kDa).

Pericardial Proteoglycan

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Fig. 6. Light and electron microscopic immunohistochemistry of pericardial tissue using anti-tendon PG II antiserum. Light microscopy reveals a strong positive reaction of pericardium with the antiserum (B) while normal rabbit serum gives a negative reaction (A). Ultrastructurally, deposits which represent antigen-antibody complexes are found around the collagen fibrils (arrows) in a cross-section (C) and closely associated with the surface of the fibrils in a longitudinal section (D). Sometimes an ordered arrangement of deposits (arrowheads) can be seen (D, inset). Magnifications: A and B X 400; C, D and inset: X 60000.

The presence of disulfide bonds in the structure of bone PG II core protein was shown (Day et aI., 1987) from sequence analysis whereas chemical analysis of the skin PG II yielded a maximum of 3 disulfide bridges which were essential for core protein-collagen interactions (Scott et a\. , 1986). The SDS-PAGE behavior of the pericardial PG core protein and the V8 protease 40 kDa product before and after reduction with BME suggests the presence of intramolecular disulfide bonds in both the core protein and Vg protease-resistant product. Bovine serum albumin which is known to be stabilized by intrachain disulfide bonds (Sogami et aI., 1969) acted as a positive control, i.e. decreased its mobility after reduction. Immunological Properties Using ELISA, Heinegard et al. (1986) have shown that low molecular weight PG's from several sources crossreacted immunologically. Vogel et a\. (1986) demonstrated that the core proteins of PG's, isolated from tendon, cartilage and bone, as well as their V g protease-resistant fragment crossreacted by immunoblotting. The pericardial PG core protein as well as its Vg protease 40 kDa resistant fragment reacted also with anti-tendon PG on immunoblots. Immunohistochemical studies of the pericardial tissue revealed a cross reaction with the anti-tendon PG antibody.

GAG Chain Attachment Site The GAG chain attachment site on the core protein of the skin PG has been determined to be the closest Ser to the Nterminus (i. e., Ser-4), by Chopra et a\. (1985) utilizing Cathepsin C to remove sequentially dipeptides from the Nterminus. The N -terminal sequence Asp-Glu-Ala-Ser-GlyX-Gly was found to be shared by tendon (Vogel et aI., 1987), skin (Pearson et aI., 1983), bone and human fibroblasts (Day et aI., 1987) small PG core proteins; this environment around the Ser residue has been reported to be a recognition site for GAG chain attachment to PG core proteins (Day et aI., 1987). When pericardial PG was digested with Cathepsin C, SDS-PAGE analysis revealed a time-dependent release of the GAG and core protein very similar to that reported for skin PG. Ultrastructural Features The ultrastructural localization of PGs using Cuprolinic Blue according to the critical electrolyte concentration method (Scott and Orford, 1981; Scott and Haigh, 1985) has gained wide acceptance. Electron-dense filaments of up to 60 nm in length (considered to represent PG-dye complexes) were ·found to be associated regularly with the die band of the type I collagen fibrils in skin, tendon, cornea (Scott, 1986) and sclera (Young et aI., 1988). Cell surface heparan sulphate PG have been visualized with Cuprolinic Blue as filaments whose length corresponds with the length of core protein (Iozzo, 1984), as it has been shown for other

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Fig. 7. Transmission electromicrographs of pericardial collagen fibers stained with Cuprolinic Blue. A) control specimen fixed in the absence of the dye showing a longitudinal section of characteristically banded (type I) collagen fibrils (X 60,000); B) longitudinal section through a pericardial collagen bundle stained with Cuprolinic Blue (in 0.1 M MgCI 2 ). Notice the periodic association of PG-dye complexes with the collagen fibrils. In some areas, a perifibrillar array of stained filaments is evident (arrows). Fine interfibrillary filamentous networks sometimes associate with the PG's (double arrowheads) (X 60,000). Inset-higher magnification showing regularly spaced PGs (arrowheads) associated primarily with the die band of the typical collagen fibril pattern (X 100,000); C) cross-section through Cuprolinic Blue stained pericardial collagen fibrils showing predominantly extended perifibrillary PG filaments (X 60,000).

Pericardial Proteoglycan tissues (Van Kuppervelt et al., 1987). Staining of fibrous pericardium with Cuprolinic Blue reveals short filaments associated primarily with the die band; sometimes their disposition is suggestive of a spike-like pattern around the collagen fibril. The mean corrected length of the filaments is 52 ± 4.0 nm, which correlates well with the size of the GAG chain (18 kDa). These data are consistent with the immunohistochemical localization of pericardial PG indicating that the Cuprolinic Blue-stained filaments represent the same pericardial PG molecules as those characterized biochemically. As initially described by Heinegard (1986) the common features of PG II include a low molecular weight (80-120 kDa) and 1-2 chondroitin or dermatan sulfate GAG chains bound close to the N-terminus of a 42-45 kDa core protein. Still, tissue-specificity exists and it might reside in the nature and size of GAG chain(s) or different post-translational modifications of the core protein. Based on Heingard's proposed nomenclature, we suggest that the bovine pericardial PG be given the abbreviated designation PGSm II DS (pericardium). The bovine PG has structural features in common with the human PG reported by Krusius and Ruoslahti (1986). A number of functional features have been suggested for low molecular weight PG's including collagen fibril (Vogel et al., 1987) and/or cell binding interactions (Rosenberg et al., 1986), and mechanical (Daniels and Mills, 1988) or optical properties (Young et al., 1988). In tendon, for example, DSPG is mostly found associated with fibers that have to withstand tensional stresses whereas compressional regions are richer in high Mr chondroitin sulfate PG (Daniel and Mills, 1988). In spite of broad structural similarities to small molecular weight PG's isolated from other tissues, the molecule isolated from the pericardium has unique features, such as the size of its GAG chains and the fact that it is found in a tissue with unique functional properties, i. e., tensional and expansional stresses. It is also important to recognize that clinically the pericardium is closely related to a vital organ whose proper function is influenced by changes in the pericardium. An important consideration is the possible role of DSPG in calcification because it has been reported that the primary failure mode of artificial valves made of pericardium is through mineral deposition (Schoen et al., 1986). PG can presumably function as an ion-exchange matrix which under normal conditions maintains a proper calcium-phosphate equilibrium (Hunter, 1987). Although little is known about the factors that trigger calcification in connective tissues which do not normally calcify, the presence of a derma tan sulfate PG in pericardial tissue and its possible role in maintaining ion balance should provide areas for future studies.

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Acknowledgements The authors wish to thank Dr. Kathryn Vogel for her gift of purified tendon PG II, rabbit antiserum to PG II and valuable suggestions. We are also indebted to J. Minda for excellent technical assistance, and to Ms. Maryann Mason for typing the manuscript. This work was supported in part by grants AR-20553 and HL29492 (to N.A.K.) and CA-39481 andAG-5707 (to R. V.I.) from the National Institutes of Health.

References Blumenkrantz, N. and Asboe-Hansen, G.: New method for quantitative determination of uronic acids. Anal. Biochern.54: 484-490,1973. Callahan, P. X., McDonald, J. K. and Ellis, S.: Sequencing of peptides with dipeptidyl aminopeptidase I. Meth. Enzyrnol. 25: 282 - 298, 1972. Carlson, D.M.: Strutures and immunochemical properties of oligosaccharides isolated from pig submaxillary mucins. J. Bioi. Chern. 243: 616-626, 1968. Chopra, R.K., Pearson, C.H., Pringle, G.A., Fackre, D.S. and Scott, P.G.: Dermatan sulfate is located on serine-4 of bovine skin proteo-dermatan sulfate. Demonstration that most molecules possess only one glycosaminoglycan chain and comparison of amino acid sequences around glycosylation sites in different proteoglycans. Biochern. J. 232: 277 - 279,1985. Crofts, C.E. and Trowbridge, E. A.: The tensile strength of natural and chemically modified bovine pericardium. J. Biorned. Mat. Res. 22: 89-98, 1988. Daniel, J. C. and Mills, D. K.: Proteoglycan synthesis by cells cultured from regions of the rabbit flexor tendon. Conn. Tissue Res. 17: 215 - 230, 1988. Day, A.A., Mcquillan, c.1. Termine, J.D. and Young, M.R.: Molecular cloning and sequence analysis of the eDNA for small proteoglycans of bovine bone. Biochern. J. 248: 801-805, 1987. Drapeau, R. G., Boily, Y. and Houmard, J.: Purification and properties of an extracellular protease of staphylococcus aureus. J. Bioi. Chern. 247: 6720-6726, 1972. Hames, B. D.: An introduction to polyacrylamide gel electrophoresis. In: Gel Electrophoresis of Proteins - A Practical Approach, ed. by Hames, B.D. and Rickwood, D., IRL Press, Oxford, Washington, DC, 1984, pp.1-91. Heinegard, D., Franzen, A., Hedbom, E. and Sommarin, Y.: Common structures of the core proteins of interstitial proteoglycans. In: Function of the Proteoglycans, Wiley, Chichester (Ciba Foundation Symposium 124),1986, pp. 69-88. Hunter, G.K.: An ion exchange mechanism of cartilage calcification. Conn. Tissue Res. 16: 111-120,1987. Ionescu, M.l. and Tandon, A.P.: Ionescu-Shiley Pericardia I Xenograft Heart Valve. In: Tissue Heart Valves, ed. by Ionescu, M.l., Butterworth and Co. Ltd., London, 1979, pp. 201- 252. Iozzo, R.B.: Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface. J. Cell Bioi. 99: 403 -417, 1984. Ishihara, T., Ferrans, V.]., Jones, M., Bovyce, S. W. and Roberts, W. c.: Structure of bovine parietal pericardium and of unimplanted Donescu-Shiley pericardial valvalur bioprotheses. J. Thorac. Cardiovasc. Surgery81: 747-757, 1981. Krusius, T. and Ruoslahti, E.: Primary structure of an extracellular

310

D. Simionescu et al.

matrix proteoglycan core protein deduced from cloned eDNA. Proc. Natl. Acad. Sci. USA 83: 7683-7687, 1986. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T 4 • Nature 227: 680-685, 1970. Leak, L. V., Ferrans, V.J., Cohen, S.R., Eidbo, E.E. and Jones, M.: Animal model of acute pericarditis and its progression to pericardial fibrosis and adhesions: ultrastructural studies. Am. ]. Anatomy 180: 373-390, 1987. Pearson, C.H., Winterbottom, N., Fackre, D.S., Scott, P.G. and Carpenter, M.R.: The NHrterminal amino acid sequence of bovine skin proteodermatan sulfate. J. Bioi. Chem.258: 15101-15104,1983. Rosenberg, L. c., Choi, H. U., Poole, A. R., Lewandowska, K. and Culp, L.A.: Biological roles of dermatan sulfate proteoglycans. In: Functions of the Proteoglycans, Wiley, Chichester (Ciba Foundation Symposium 124), 1986, pp. 47 -68. Rosenberg, L.c., Choi, H. U., Tang, L.H., Johnson, T.L., Pa, S., Webber, c., Reiner, A. and Poole, A. R.: Isolation of dermatan sulfate proteoglycan from mature bovine articular cartilage. J. BioI. Chem. 260: 6304-6313, 1985. Schoen, F.]., Tsao, ]. W. and Levy, R.].: Calcification of bovine pericardium used in cardiac valve bioprostheses. Implications for the mechanisms of bioprosthetic tissue mineralization. Am. ]. Pathol.123: 134-145,1986. Scott, J. E.: Proteoglycan-collagen interactions. In: Functions of the Proteoglycans, Wiley, Chichester (Ciba Foundation Symposium 124), 1986, pp. 104-124. Scott, ]. E. and Haigh, M.: Proteoglycan-Type I collagen fibril interactions in bone and non-calcifying connective tissue. Bioscience Reports 5: 71-81, 1985. Scott, ]. E. and Orford, C. R.: Dermatan sulfate-rich proteoglycan associates with rat tail tendon collagen at the d band in the gap region. Biochem.J.197: 213-216, 1981. Scott, P.G., Winterbottom, N., Dodd, C.M., Edwards, E. and Pearson, C. H.: A role for disulfide bridges in the protein core in the interaction of proteodermatan sulfate and collagen. Biochem. Biophys. Res. Comm . 138: 1348-1354,1986.

Simionescu, D., Alper, R. and Kefalides, N.A.: Partial characterization of a low molecular proteoglycan isolated from bovine parietal pericardium. Biochem. Biophys. Res. Comm. 151: 480-486, 1988. Sogami, M., Petersen, H.A. and Foster, ].F.: The microheterogeneity of plasma albumins. V. Permutations in disulfide pairings as a probable source of microheterogeneity in bovine albumin. Biochem. 8: 49-58, 1969. T owbin, H., Staehelin, T. and Gordon,].: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proe. Natl. Acad. Sci. USA 76: 4350-4354, 1979. Van Kuppevelt, T.H.M.S.M., Butten, T.L.M. and Kuyper, C. M. A.: Ultrastructural localization of proteoglycans in tissue using cuprolinic blue according to the critical electrolytic concentration method: comparison with biochemical data from the literature. Histochem.]. 19: 520-526, 1987. Vogel, K. G. and Fisher, L. W.: Comparisons of antibody reactivity and enzyme sensitivity between small proteoglycans from bovine tendon bone and cartilage. ]. Bioi. Chem. 261: 11334-11340, 1986. Vogel, K. G., Koob, T.]. and Fisher, L. W.: Characterization and interaction of a fragment of the core protein of the small proteoglycan (PGII) from bovine tendon. Biochem. Biophys. Res. Comm.148: 658-663, 1987. Wasteson, A.: A method for the determination of the molecular weight and molecular weight distribution of chrondroitin sulfate.]. Chrom.59: 87-97, 1971. Young, R.D., Powell, ]. and Watson, P.G.: Ultrastructural changes in scleral proteoglycans precede destruction of the collagen fibril matrix in necrotizing sclereitis. Histopathol.12: 75-84,1988. . Dr. Nicholas Kefalides, Connective Tissue Research Institute, 3624 Market Street, Philadelphia, PA 19104, USA.