A Large Chondroitin Sulfate Proteoglycan - The Journal of Biological ...

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A Large Chondroitin Sulfate Proteoglycan (PG-M) Synthesized before. Chondrogenesis in the ... Following chon- droitinase AC I1 (or ABC) digestion, core molecules .... by its sensitivity to hyaluronate lyase (data not shown). The. 0.45-0.55 M ...
Vol. 261,No. 29,Issue of October 15.PP,,13517-13525 1986 Printed in Ij.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

A Large Chondroitin Sulfate Proteoglycan (PG-M) Synthesized before Chondrogenesis in theLimb Budof Chick Embryo* (Received for publication, April 24,1986)

Koji KimataS, Yasuteru Oike, Katsuko Tani, Tamayuki Shinomura, Masahito Yamagata, Masahiro Uritani,and SakaruSuzuki From the Department of Chemistry, Faculty of Science, Nagoya University, Nagoya 464, Japan

Extraction of stage 22-23 chick embryo limb buds that had been metabolically labeled with [SsS]sulfate yielded heparan sulfate proteoglycan, small chondroitin sulfateproteoglycan, and largechondroitin sulfate proteoglycan (designated PG-M). PG-M constituted over 60%of the total macromolecular [SsS]sulfates.It was larger in hydrodynamic size, richer in protein, and contained fewer chondroitin sulfate chains as compared to the predominant proteoglycan (PG-H, M , 1.5 x lo6)of chick embryo cartilage. The chondroitin sulfate chains were notable for their largesize (Mr 2 60,000) and high content of nonsulfated chondroitin units (about 20% of the total hexosamine). Hexosamine-containing chains corresponding in size to Nlinked and O-linked oligosaccharides were also present. The core protein was rich in serine, glutamic acid (glutamine), and glycine which together comprised about 38% of the total amino acids. Following chondroitinase AC I1 (or ABC) digestion, core molecules were obtained which migrated on sodium dodecyl sulfate gel electrophoresis as a doublet of bands with approximately M , = 550,000 (major)and 500,000, respectively. The M, = 550,000core glycoprotein was structurally different from the core glycoprotein (Mr = 400,000) of PG-H, as ascertained by trypticpeptide mapping and immunochemical criteria. Immunofluorescent localization of PG-M showed that theintensity of PG-M staining progressively became higher in the core mesenchyme region than in the peripheral loose mesenchyme, closely following the condensation of mesenchymal cells. Since the cell condensation process has been shown to begin with the increaseof fibronecI collagen concentration,thesimilar tinandtype change inPG-M distribution suggests that PG-M plays an important role in the cell condensation process by means of its interaction with fibronectin and type I collagen.

In the developing limb buds of chick embryo, muscle and cartilage cells differentiate from relatively uniform mesenchyme cells during a short, butspecific time period. Cartilage formation begins at stage 22-23 with a cellular condensation process in the core of the proximal half of the limb bud (1). The cellular condensation is associated with the deposition of type I collagen and fibronectin in the intercellular space (2).

* This work was supported by grants-in-aid for Cancer Research, Scientific Research, and Special Project Research from the Ministry of Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 To whom correspondence should be addressed.

As cartilage differentiation progresses, type I collagen is gradually replaced by type I1 collagen (2,3),fibronectin disappears (2,4), the rateof [35S]sulfateincorporation into proteoglycans becomes higher in the core mesenchyme region than in the surrounding loose mesenchyme region (5), and a cartilagespecific proteoglycan, PG-H,’ becomes detectable in cartilage primordia (6). Many investigators have described that limb mesenchyme cells synthesize sulfated proteoglycans and that the transition from mesenchyme to cartilage involves a change in proteoglycan biosynthetic pattern as assessed by rate zonal sedimentation ormolecular sieve chromatography (7-12). A prediction could be made, therefore, that not only type I collagen and fibronectin but also proteoglycan species unique to developing mesenchyme may participate in the cellular condensation process to promote chondrogenesis. Since we previously described the programed change in proteoglycan biosynthetic pattern during chondrogenesis i n vitro (11)and i n ouo (6), we were interestedin an explanation for this change. In the present work, we extend these previous studies by isolating and characterizing proteoglycans from stage 22-23 limb buds. The focus in this report is on a large size chondroitin sulfate proteoglycan, as it represents more than 60% of total macromolecular [35S]sulfateactivity. The accompanying paper (13) describes the production of a PG-”like molecule by chick embryonic fibroblasts in culture and its interaction with hyaluronic acid, fibronectins, collagens, and other matrix macromolecules. EXPERIMENTAL PROCEDURES* RESULTS

Proteoglycan Composition of Stage 22-23 Limb BudsAbout 230 mg of stage 22-23 limb buds dissected from 100 embryos were incubated in [35S]sulfate-containing medium for 3handthenextracted twice with 3 ml each of 4 M guanidine HCI in the presence of protease inhibitors. Negligible amounts of radioactivity remained in the pellet after the The abbreviations used are: PG-H, PG-Lb, and PG-Lt,proteoglycans isolated from 12-day-old chick embryo cartilage (for the structures, see Refs. 14-17);PG-M, the predominant proteoglycan isolated from chick embryo limb bud (see the text for details); SDS, sodium dodecyl sulfate; ELISA, enzyme-linked immunosorbent assay; GlcA, glucoronic acid; IdoA, L-iduronic acid. Portions of this paper (including “Experimental Procedures,” Tables I and 11, and additional references) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-1351, cite the authors, and include a check or money order for $4.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal thatis available from Waverly Press.


Mesenchyme Proteoglycan of Limb Buds


second extraction. The extracts were combined and passed over a Sephadex G-50 column to remove low molecular weight materials. About 2.12 x lo6 cpm of 35S or 0.64pmol (as hexuronate) of carbazole-positive material was recovered in the excluded fraction. The macromolecular fraction from the gel was partitioned by rate zonal sedimentationona glycerol gradient under dissociative conditions. The distribution of the incorporated [35S]sulfate(Fig. lA)indicates that the labeled material was resolved into two fractions (I and 11). When aliquots of each fraction were assayed for chondroitinase ABC-susceptible radioactive materialand for chondroitinase ABC-resistant radioactive material (see Ref. 3 in Miniprint for the assay method), the sedimentation diagrams shown in Fig. 1 ( B and C ) , respectively, were obtained. The diagrams show that almost all of the sulfate label of Fraction I was sensitive to chondroitinase ABC, whereas about 65% of the sulfate label of Fraction 11 was resistant to chondroitinase ABC. When PG-H(the predominant proteoglycan of 12-day-old chick embryo cartilage) was subjected to rate zonal sedimentation under the same condition, itssedimentation velocitywas higher than that of Fraction I (Fig. l A ; PG-H was found in the bottom fraction, as indicatedby the arrow). To isolate the chondroitinaseABC-resistant 35S-labeled material in Fraction 11, a portion of Fraction I1 was treated with chondroitinase ABC in the presence of protease inhibitors and then chromatographed on DEAE-Sephacel. The resistant material was eluted as asingle 35Speak at 0.4 M NaCl (see Miniprint). When the material thus isolated was treated

Staqe 22-23 limb buds (dissected fresh or metabolically radiolabeled) Extraction with 4M guanidineHCl/protease inhibitors followed by centrifugation

Precipitate Supernatant 2nd extraction and centrifugation

+ -

Supernatant Precipitate

Dissociative CsCl isopycnic centrifugation ( P , = 1.33 g/ml) Bottom two-fifths Dissociative rate zonal sedimentation on a glycerol gradient (see Fig. 1) Fraction I Addition of Triton X-100 to 0.2 % (w/v) followed by dialysis against 7 M urea/0.04 M Tris-HC1, p~ 7.4/0.2 % Triton X - 1 0 0 (solution A)


DEAE-Sephacel chromatography with 0.1 to 1.0 M NaCl gradient in solution A (see Fig. 3)

0.4-0.5 M NaCl fraction Precipitation with 3 volumesOf 95 % ethanol/l.3 % potassium acetate

16 12 -


FIG. 2. Extraction and purification of PG-M from chick embryo limb buds.


sequentially with alkali and Pronase, greater than 98% of the label was released as glycosaminoglycanssensitive to hepari0” tinase? indicating that the chondroitinase-resistant components in Fraction I1 are mainly heparan sulfate proteoglycans. 12 In the present study, these components were not analyzed further. The chondroitinase ABC-sensitive component in Fraction 8I, on the other hand, hasbeen isolated and characterized as a chondroitin sulfate proteoglycan distinct from hitherto known ,4proteoglycans of chick embryo cartilage, e.g. PG-H, PG-Lb, and PG-Lt (14-17) (for this reason, the mesenchyme chon0” droitin sulfate proteoglycan is termed “PG-M”). Theevidence C 8for this assignment follows. Purification of Unlabeled and Radiolabeled PG-”For further detailed characterization, unlabeled PG-M, ~ u l f a t e - ~ ~ s 4labeled PG-M (specific radioactivity, 3.3 x lo6 cpm/pmol of hexuronate), and hex~sanine-~H-labeled PG-M (specific ra10 20 30 O? I dioactivity, 8 x lo4 cpm/pmol of hexuronate) were prepared .* Tube No. (1 ml/tube) * as outlined in Fig. 2. The guanidine HCl extract of stage 22TOP Bottom FIG. 1. Profiles of [36S]sulfate-labeled proteoglycans sedi- 23 limb buds, either dissected fresh or incubated with radiomented on 10-35% linear glycerol gradients under dissocia- active precursor, was first subjected to dissociative isopycnic tive conditions (in 4 M guanidine HCl). The macromolecular CsCl centrifugation to remove the bulk of nonproteoglycan fraction (2 X lo6 cpm) from a Sephadex G-50column was subjected proteins. The high density fractions (bottom two-fifths of the to rate zonal sedimentation on the glycerol gradient. A , whole 35S- gradient) were pooled, dialyzed, and subjected to rate zonal labeled materials; B , chondroitinase ABC-sensitive 35S-labeledma- sedimentation on a glycerol gradient to remove the heparan



terials; C, chondroitinase ABC-resistant 36S-labeledmaterials. The arrow denotes the position of PG-H (standard). The brackets above curues indicate the fractions which were subsequently pooled for further analyses.

K. Kimata, M. Uritani, N. Hirose, T. Shinomura, and S. Suzuki, unpublished observations.

Mesenchyme Proteoglycan of Limb Buds


sulfate proteoglycan and other minor proteoglycans of relatively low sedimentation rates (see above). The faster sedimenting proteoglycan fractions (cf. Fig. 1, Fraction I) were pooled, dialyzed, and chromatographed on DEAE-Sephacel (Fig. 3). The sulfate label was eluted as a single peak at the NaCl concentration of 0.45-0.55 M, whereas the hexosamine label eluted as two peaks, one being co-eluted with the sulfate label and the othereluted earlier. The earlier eluting 3H peak with no sulfate label represents hyaluronic acid as assessed by its sensitivity to hyaluronate lyase (data not shown). The 0.45-0.55 M NaCl fractions were pooled, and proteoglycan was collected by ethanol precipitation. * o About 0.6pmol (ashexuronate) of PG-M was obtained from 1.7 g of limb buds dissected from 750 chick embryos at stage 22-23 (wet weight). 0 Characterization of PG-M-On a permicromole of hexuronTube No. (0.5ml / tube) ate basis, the content of core protein (as determined by the FIG. 4. Sepharose CL-ZB chromatography of the proteoglymethod of Lowry et al. (see Ref. 15 in Miniprint)) of PG-M fraction from DEAE-Sephacel column (see Fig. 3,brackwas 50 pg. This value is about 1.7 times the value of PG-H, can ets). Aliquots (2 ml) of the pooled fraction were chromatographed on the major proteoglycan of chick embryo cartilage. a Sepharose CL-2B column (1 X 68 cm) equilibrated and eluted with Fig. 4 shows the elution profiles of ~ulfate-~~S-labeled PG- 4 M guanidine HC1,0.2% (w/v) Triton X-100, 0.05 M Tris-HC1, pH M and hexo~amine-~H-labeled PG-M from Sepharose CL-2B 8.0, 10 mM EDTA. The elution profiles of [35S]sulfate-labeledsample columns under dissociative conditions. Both of the labels were (0)and [3H]glucosamine-labeledsample (0)are shown. For comparis also eluted with a K., of 0.12. Under the same conditions, sulfate- ison, the elution profile of s~lfate-~'S-labeled PG-H (standard) shown (X). V,,, void volume; V,, total column volume. 35S-labeledPG-H (standard) was eluted with a K., of 0.26, indicating that PG-M is larger in hydrodynamic size than PG-H. Thiswould appear to be incompatible with the observation that PG-M was lower in sedimentation velocity than PG-H (see above). However, the slower sedimentation of PGM is probably a combination of its lower density due to the high protein-to-chondroitin sulfate ratio and a much higher frictional coefficient due to a large, more open hydrodynamic size, in part the result of the much larger chondroitin sulfate chain (see below). The low degree of sulfation of the polysaccharide chains in PG-M may also be a reason for the low sedimentation velocity of PG-M relative to thatof PG-H. Characterization of Glycosaminoglycan Chain.~"ulfate-~~SLabeled PG-M and hexo~amine-~H-labeled PG-M were treated with alkaline/Pronase and alkaline borohydride, respectively, and the resultant carbohydrate chains were analyzed by molecular sieve chromatography. As Fig. 5 shows, the [35S]sulfate-labeledchains were eluted from a Bio-Gel A1.5m column as a peak with an estimated M, 2 60,000. This Tube No. (0.7ml I tube)



9;:. . .




FIG. 5. Bio-Gel A-1.5m chromatography of sulfated glycosaminoglycans from suZfate-s6S-labeledPG-M (0)and sulfateS6S-labeledPG-H (0)The . proteoglycan sample was treated successively with alkali and Pronase and then applied to a calibrated column (1 X 64 cm) of Bio-Gel A-1.5m eluted with 0.4 M ammonium acetate, pH6.0, at room temperature. The arrows denote the positions of M, = 40,000 chondroitin 4-sulfate and M , = 12,000 chondroitin 6sulfate (standards).

M, is about 3 times the average M, of the chondroitin sulfate chains obtained from PG-H. When the [3H]hexosamine-labeled carbohydrate chains from PG-M were analyzed by chromatography on Bio-Gel P10, about 79% of the hexosamine label was eluted in the excluded fraction as would be expected for large size glycosaFIG. 3. DEAE-Sephacel chromatography of Fraction I (the minoglycan chains, but about 11 and 5% of the label were faster sedimenting fraction from rate zonal sedimentation, eluted at and near positions expected for hyaluronate hexasee Fig. 1). Triton X-100 was added to 12 ml of pooled Fraction I saccharide and undecasaccharide, respectively (Fig. 6). The to 0.2% (w/v), and the mixture was dialyzed against solution A. The results indicate that PG-M contains nonsulfated, hexosadialyzed material was applied on a DEAE-Sephacel column (2 X 15 mine-containing oligosaccharides that are similar in size to cm) equilibrated with solution A. The column was washed with 100 ml of 0.1 M NaCl in solution A and then eluted with an increasing N-linked and 0-linked oligosaccharides previously described salt gradient from 0 to 1.0 M NaCl in solution A (400 ml). The elution in other proteoglycans (Refs. 18-20, among others). On a per profiles of [35S]sulfate-labeledsample (0)and [3H]glucosamine-la- chain length basis, the amount of [3H]hexosamine in the beled sample (0) are shown. hexasaccharide fraction is very high, suggesting that PG-M

Mesenchyme Proteoglycan of Limb Buds

13520 61

al 5 -





A, Coomassie


11 1 1

6, Fluorography

Blue staining

HA oligomer 108 6 4







2 1

2 3


1, 3


ill I 0




30 40 50 60 Tube No. ( 0.57ml I tube)

a m





FIG.6. Bio-Gel P-10 chromatographyofcarbohydrate The colchains releasedfrom [SH]glucosamine-labeled PG-M. umn (1 X 50 cm) was eluted with 0.4 M ammonium acetate at 4 "C. The arrows denote the positions of decasaccharide (HA,,,), octasaccharide (HA*),hexasaccharide (HA6),and tetrasaccharide (HA,) derived from hyaluronic acid by hyaluronidase digestion. contains a number of 0-linked oligosaccharides. The radiolabeled glycosaminoglycan chains were further analyzed with chondroitinases and keratanase. As assessed by sensitivity to keratanase, no sulfate label can be assigned to keratan sulfate. Relative digestibility of the glycosaminode glycan samples by chondroitinase ABC and chondroitinase front AC (Table I) indicated that the glycosaminoglycan chains in PG-M are undersulfated chondroitin sulfates containing at FIG.7. SDS-polyacrylamide gel electrophoresis of PG-M most 5% dermatan sulfate units. The and protein-enriched core molecules derived therefrom. Analysis of Core Protein-Amino acid analysis of purified compounds in 3.75% gel (A) and 5% gel ( B ) were visualized by PG-M revealed that serine, glutamic acid (glutamine), and Coomassie Blue staining and fluorography, respectively.Lane 1, glycine together comprised about 38%of the totalamino acids unlabeled PG-M lane 2, chondroitinase AC-11-treated PG-M label 3, AC-11-treated PG-H; lane 1', [?3]methionine-labeled (Table 11). The amino acid composition suggests that PG-M chondroitinase PG-M; lane 2 ' , as in lane 1' but treated with chondroitinase AC-11. is distinct from the proteoglycans (PG-H, PG-Lb, and PG- Arrowheads denote the positions of the following molecular weight Lt) previously isolated and characterized from chick embryo markers: a, laminin A chain (400,000); b, chick cellular fibronectin (240,000);c, laminin B chain (200,000);d, phosphorylase b (94,000); cartilage (14-17). For further characterization of the core protein of PG-M, e, bovine serum albumin (67,OO); f , ovalbumin (43,000).X,proteins stage 22-23 limb buds were metabolically labeled with [36S] from the chondroitinase AC-I1 preparation. methionine, and methi~nine-~~S-labeled PG-M was isolated as outlined in Fig. 2. Unlabeled PG-M, methi~nine-~~S-labeled with chondroitinase ABC but was removedwith chondroitinPG-M, and protein-enriched core moleculesderived therefrom ase AC I1 so that thechondroitinase ABC-treated core molewere analyzed by electrophoresis in SDS-polyacrylamide gels cule was larger in size than the chondroitinase AC 11-treated followed by either Coomassie Blue staining (Fig. 7A) or fluo- core molecule (14). Since the chondroitin sulfate chains are rography (Fig. 7B). Before chondroitinase ACI1 treatment, much fewer in PG-M than in PG-H (see above), it is possible the methionine label (PG-M) did not enter thegel ( l a n e 1'). that disaccharide units linked to thelinkage oligosaccharides, After chondroitinase ACI1 treatment, the methionine label if any, may not detectably alter the mobility of PG-M core was no longer at theorigin and a doublet of radioactive core molecules in the gel. protein bands appeared at positions corresponding to apparIt is also noteworthy that further treatment of these core ent M , = 550,000 and 500,000,respectively ( l a n e 2'). Pretreat- molecules with keratanase did not alter theirmobility on SDS ment of the core sample with 2-mercaptoethanol did not alter gel electrophoresis. The result suggests that PG-M differs the mobility of the doublet (not shown). CoomassieBlue from PG-H in lacking keratan sulfate chains: staining could visualize the M, = 550,000 component but not The unlabeled protein contained in the Coomassie Bluethe M , = 500,000 component ( l a n e 2), perhaps owing to the positive M , = 550,000 band from a dried polyacrylamide gel presence of thelatter at afar lower concentration. The was radioiodinated and digested with trypsin for 18 h. The keratan sulfate-carrying core molecule prepared from PG-H resultant tryptic peptides were displayed two-dimensionally by digestion with chondroitinase ACI1 (14, 17) had an ap- on a silica gel thin layer plate (Fig. 8, A and B ) . The profile of major peptide spots was entirely different from that of the proximate M, = 400,000 in the same gel ( l a n e 3). The mobility of the doublet of core protein bands was not M, = 400,000 core protein of PG-H (Fig. 8C). Although not significantly altered when PG-M was digestedwith chondroiMonoclonal antibody HM-110(raised against PG-H) that speciftinase ABC instead of chondroitinase AC I1 (data notshown). ically recognizes corneal and skeletal keratan sulfate did not react This is in contrast to our previous observations that, when with PG-M as ascertained by ELISA (Y. Shimomura, K. Kimata, N. PG-H was examined in a similar way, the sulfated disaccha- Maeda, Y. Oike, M. Yamagata, and S. Suzuki, unpublished observaride closest to the linkage oligosaccharides resisted digestion tions).

Mesenchyme Proteoglycan of Limb Buds


FIG. 8. Tryptic peptide maps of the core molecules from PG-M and PG-H. Core molecules were labeled with '%I in the gel slices (see Fig. 7A) and then digested with trypsin. The resultant peptides were displayed on a silica gel thin layer plateby electrophoresis in the first dimension and ascending chromatography in the second dimension. A, M,= 550,000 core molecule from PG-M an autoradiograph of the thin layer plate is shown. B, traced figure of the autoradiograph. C, M, = 400,000 coremoleculefrom PG-H (standard);a traced figure of the thin layer plate is shown.


8 . lmmunoblotting l6'x


Mr Origin-


500 550

600 200

1800 5400 Dilution of antibody




PG-M used in these experiments was prepared by passing over PG-H core protein-conjugated Sepharose 4B. In control experiments in which this absorption procedure was omitted, the results of Western blots were the same as above (data not shown), suggesting that thecore protein structures of PG-M and PG-H are extensively different. As demonstrated in the accompanying paper (13),the antibody is useful for immunoprecipitation of a PG-M-like proteoglycan from cellular fibronectin preparations and crude extracts of chick embryo fibroblasts. Hyaluronate Interaction-A solution of [35S]sulfate-labeled PG-M and unlabeled PG-H (carrier; this proteoglycan has no ability to bind to PG-M, as shown in Fig. 10B) in 4 M guanidine HCl was dialyzed to associative conditions in the absence or presence of hyaluronic acid and subjected to Cs2S04 gradient zonal rate sedimentation (Fig. 1OA). The presence of hyaluronic acid caused the aggregation of over 80% of the labeled proteoglycan as shown by a shift of the sedimentation profiles of the 35Slabel from the slowly sedimenting to the rapidly sedimenting band. When PG-H (carrier) was omitted from the reaction mixture, slower sedimenting aggregates including 35S-labeled PG-M were performed, perhaps owing to the fact that the amount of PG-M (1 nmol as hexuronate) relative to that of hyaluronic acid (5 nmol as hexuronate) was too small to form an aggregate saturated with proteoglycan molecules. In a control experiment (Fig. lOC),[3H]serine-labeledPG-Lb in 4 M guanidine HC1 was dialyzedto associative conditions in the presence of hyaluronic acid and PG-H. Cs2S04gradient zonal rate sedimentation showed that the presence of hyaluronic acid and PG-H caused no shift of the sedimentation profile of the 3H label. The results, taken together, indicate that PG-M contains a hyaluronic acid-binding site (for further binding properties of PG-M, see Ref. 13).

FIG. 9. Reactions of anti-PG-M antibodies with PG-H, PGLb, PG-Lt, and chick plasma fibronectin (FN).A, results of ELISA; B, immunoblots of PG-M and PG-H with chondroitinase ABC digestion followed by electrophoresis on a 5% polyacrylamide gel. The core proteins on the gels were transferred to nitrocellulose sheets and stained with anti-PG-M antibodies.



shown in thisfigure, PG-Lb (15)and PG-Lt(16)were mapped quite differently from PG-M, indicating that thecore proteins of PG-M, PG-H, PG-Lb, and PG-Lt aredissimilar. Since the unlabeled M , = 500,000 core molecule was yielded in an amount too small for tryptic peptide mapping, it could not be ascertained whether the appearance of the two core molecules reflects the existence of two different species of core protein or arises from processing or partial degradation of a single core protein. Immunochemical Studies-An antiserum was raised in a C ['HI ffi-Lb rabbit against PG-M. Antibodies to the proteoglycan were Pi-H detected by ELISA. A specific antibody to PG-M could be HA obtained from this antiserum by passing successively through a column of Sepharose 4B coupled with the protein-enriched core fraction from PG-H and a column of Sepharose 4B coupled with chick plasma fibronectin. The antibody thus 5 10 15 t obtained did not react with PG-H, PG-Lb, PG-Lt, and chick I Tube No. TOP n plasma fibronectin when examined by ELISA (Fig. 9A) (for FIG. 10. Interaction of proteoglycans with hyaluronic acid. reaction with cellular fibronectins, see Ref. 13).An antiserum PG-M on an associative against PG-H, on the other hand, did not cross-react with Sedimentation profile of sulf~te-~~S-labeled PG-M as detected by ELISA (data notshown). Moreover, the Cs2S04gradient afterdialysisto associative conditionsin the presence of hyaluronic acid (HA) and PG-H is shown (A, 0).Sedimentation antibody to PG-M reacted with Western blots of both the M, profiles on controls lacking PG-H (A, X) or hyaluronic acid (B, A), = 550,000 and 500,000 core molecule from PG-M but did not or both (A, 0 ) are also shown. Forcomparison, a sedimentation react with Western blots of the M , = 400,000 core molecule profile of ~erZne-~H-labeled PG-Lb after dialysis to associative confrom PG-H (Fig. 9B). It should be noted that theantibody to ditions in the presence of hyaluronic acid andPG-H is shown (C, A).

Mesenchyme Proteoglycanof Limb Buds


FIG. 11. Sections of chick embryo limb buds at stage 23 (A-1-3), stage 24 (B-1-3), stage 27 (C-2-3), and stage 37 (D-1-3). A-1, B-1, C-1, and D-1, phase-contrast micrographs; A-2, B-2, C-2, and 0 - 2 , staining with antiPG-M antibodies; A-3 and 0 - 3 , staining with anti-fibronectin antibodies. The tissue sections for staining with antifibronectin antibodies were treated with testicular hyaluronidase prior tothe staining with antibodies. B-3 and C-3, staining with anti-PG-H antibodies. Dashed lines indicate the distal end of limb bud. E, ectoderm; M , mesenchyme; Car, cartilage; Mus, muscle; Epi, muscle epimysium. Bur, 200 pm.

Changes in the Distribution Pattern of PG-M during Limb Bud Chondrogenesis-The distribution of PG-M in developing chick hind limb buds was studied by immunofluorescence with anti-PG-M antibody. At stage 23, no area of mesenchymal cell condensation has yet become apparent (Fig. 11, AI). Nevertheless, a significant increaseof the intensity of PGM staining can be seen in the core mesenchyme region of the limb bud compared to the surrounding mesenchyme (A-2). PG-M is also abundant in the ectoderm? The increase of PGM in thecore mesenchyme appears toprecede an increase of fibronectin (A-3).The first sign of PG-H appearance is seen at stage 24 (B-3).The PG-H fluorescence is strictly confined to thecartilage primordiacompared to the PG-M fluorescence (B-2).With further development of the cartilage primordia (from stages 25 to 27; C - I ) , an increase in the intensity of PG-M stainingcompared to the peripheral mesenchyme can be observed in thecartilage (C-2),but its concentration relative to thatof PG-H (C-3) falls off progressively. On day 11, a greatdecrease in the intensity of both PG-M and fibronectin compared to theperichondrium can be observed in the cartilage matrix (0-2;see Ref. 2 for the distribution of fibronectin). PG-M and fibronectin, however, persist in noncartilagenous tissues such as muscle epimysium (0-2 and 0-3).In parallel sections, no staining by anti-PG-H antibodies is observed in the noncartilagenous tissues (not shown). DISCUSSION

The above results indicate that thepredominant chondroitin sulfate proteoglycan (PG-M) synthesized in the mesenchyme of stage 22-23 chick embryonic limb buds is a unique .E When the ectoderm was separated from the mesenchyme and incubated with [35S]sulfate,incorporation of the label into a PG-M fraction was observed (K. Kimata, S. Ono, and S. Suzuki, unpublished observations).

species distinct from the proteoglycan (PG-H) synthesized in chick embryonic leg cartilages. In theformer, extremely large chondroitin sulfatechains of an average M , 2 60,000 (3 times larger than those of PG-H) and significantly undersulfated are attached toa large core glycoprotein of M , 550,000 (1.37 times larger than the core molecule from PG-H). PG-M is similar to PG-H in that it has an ability to interact with hyaluronic acid, but its amino acid composition, tryptic peptide profile, and immunological specificity are quite different from those of PG-H. Furthermore, PG-M differs from PG-H in lacking keratan sulfate and inhaving far fewer chondroitin sulfate chains percore protein. Earlier work from our laboratory (21) revealed the occurrence in chick embryo epiphyseal cartilage of two proteoglycan populations, PG-H (high M , fraction) and PG-L (low M , fraction), separated by sucrose gradient centrifugation. We have since demonstrated that PG-H resembles large proteoglycans found in various cartilages (14, 17) but that PG-L is composed of two distinct molecular species (PG-Lb and PGLt) with characteristic core proteins (15, 16, 22). The rate zonal sedimentation has also been used for analysis of [35S] sulfate-labeled proteoglycans synthesized by stage 22-23 chick embryo limb buds (8, 9, 11, 12). A notable difference between the sedimentation profiles of radioactive proteoglycans from chick embryo cartilage and limb bud was the appearance in the limb bud sample of a unique band with a sedimentation velocity between PG-H and PG-L. This component, referred to as Fraction I11 (€9, PCS-M (9), Fraction I1 (ll),or PGS(LM)-1 (12), corresponds in sedimentation velocity to that described here as PG-M. Although detailed information is as yet lacking about thelimb bud proteoglycans other than PG-M, it is clear that the observed difference between the sedimentation profiles of cartilage and limb bud proteoglycans results mainly from a difference in therelative

Mesenchyme Proteoglycan of Limb Buds amounts of two distinct species, PG-M and PG-H. Studies of [35S]sulfate-labeledproteoglycans synthesized by stage 22-23 limb bud mesenchyme cells have shown that, when the cells were cultured as apellet in chemically defined (serum-free) medium BGJb (77), the proteoglycans synthesized at the initiation of culture exhibited sedimentation profiles of the mesenchyme type, but theprofiles progressively became more like those of cartilage-type proteoglycans during 9 days of culture (11).The results suggest that transitions occur in the types of proteoglycan synthesized during limb bud cell cultures in the serum-free medium. De Luca et al. (IO), on the other hand, have studied cell layer-associated proteoglycans synthesized by stage 23-24 limb bud mesenchyme cells in culture. These authors showed that the major [35S]sulfate-labeledproteoglycan molecules synthesized by the cells in day 2 cultures differed from those synthesized in day 8 cultures inbeing smaller in hydrodynamic size, in containing keratan sulfate chains of smaller average M,, and in possessing no ability to interact with hyaluronic acid. Apparently, this proteoglycan fraction does not represent PG-M because the latter is larger than the cartilage-proteoglycan (PG-H)in hydrodynamic size (Fig. 4), does not contain keratan sulfate, and possesses an ability to interact with hyaluronic acid (Fig. 11).Since studies with antibodies made against PG-H have indicated the synthesis of the cartilage-type proteoglycan by stage 24 limb buds (6), it seems likely that the molecules detected inthe cell layers of day 2 cultures represent immature forms of the cartilage-type proteoglycan. As shown in the accompanying paper (13), preparations of cellular fibronectin from chick embryonic fibroblasts contain a PG-M-like proteoglycan. This proteoglycan was cross-reactive with antibodies made against PG-M, gave a doublet of M , = 550,000 and 500,000 core molecules on SDS gelsby chondroitinase digestion, has some ability to interact with hyaluronic acid, andcontained very large ( M , 2 60,000) chondroitin sulfate chainswith a high proportion of 6-sulfate (about 60%). All these results suggest that the chick embryo fibroblast proteoglycan and PG-M are identical. A similar large proteoglycan has also been described for 11-12-day-old chick embryonic leg muscle (23, 24). Relative to the predominant proteoglycan (PG-H) from chick embryonic leg cartilage, the skeletal muscle proteoglycan has a larger hydrodynamic size and contains longer chondroitin sulfate chains( M , 70,000) with a high proportion of 6-sulfate (about 90%). The core protein length of the skeletal muscle proteoglycan is slightly longer than that of the cartilage proteoglycan as visualized by electron microscopy with molecular spreading techniques (25).As shown by our immunofluorescence studies with antibodiesagainstPG-M (Fig. ll), astrongPG-M fluorescence is detected on the muscle epimysium in ll-dayold chick embryonic leg muscle. Although it remains to be established whether PG-M and the skeletal muscle proteoglycan are identical in core protein structure: the above observations strengthen the notion that PG-M is not restricted to the limb bud mesenchyme but is expressed by cells in more specialized, matured tissues. It should be noted in this respect that a large size chondroitin sulfate proteoglycan has been found in various noncartilagenous soft tissues including sclera (26), skin (27-29), aorta (30-32), smooth muscle (33), ligament(34),tendon(35),and developing bone mesenchyme (36). Heinegirdet al. (37) have recently shown that the large proteoglycans from tendon, sclera, and aorta are similar to Digestion of a large proteoglycan fraction from the chick embryonic leg muscle with chondroitinase ABC resulted in a major core protein band of M , = 450,000 plus a minor band of M, = 550,000 (Y. Yamagata, K. Kimata, and S. Suzuki, unpublished observations).


the large proteoglycan from cartilage in containing the structure typical for the hyaluronic acid-binding region and in displaying similar peptide patterns. This suggests that PG-M differs from the large proteoglycans of tendon, sclera, and aorta in core protein structure since the peptide pattern of PG-M is entirely different from that of the cartilage-type proteoglycan (Fig. 8). It has been found by immunofluorescent localization (2) that themesenchymal cell condensation and the formation of cartilage primordia in developing chick embryo limb buds (stages 23-27) are associated with the deposition of type I collagen and fibronectin in the intercellular space. The proteoglycan (PG-M) we have described shows a similar distribution (Fig. 11).Furthermore, a PG-M-likeproteoglycan synthesized by embryonic chick fibroblasts has been shown to possess some abilities to interact with collagens and fibronectins (13). The results suggest important roles of PG-M, type I collagen, and fibronectin in the early events in chondrogenesis. Besides these extracellular matrix components, hyaluronic acid has been suggested to participate in regulation of the cell condensation events preceding chondrogenesis and myogenesis(38-40). Since PG-M has hyaluronate-binding activity, this proteoglycan may modulate interactions among hyaluronic acid, fibronectin, and type I collagen, thereby participating in regulation of the condensation events. At the beginning of stage 28when chondrocytes in the middiaphysis become elongated in a direction perpendicular to the long axis of the cartilage, an additional proteoglycan species, PG-Lb, becomes detectable in the zone of elongated chondrocytes (6). The expression of genetically distinct proteoglycan species, PG-M, PG-H, and PG-Lb, in a well-defined temporal andspacial pattern may well be an important mechanism by which distinct morphologies are established. Acknowledgment-We wish to thank Dr. N. Takahashi, Nagoya City University, for the amino acid analysis. REFERENCES 1. Fell, H. B., and Canti, R. B. (1934) Proc. R. SOC.Lo&. B Biol. SCZ.116,316-349 2. Dessau, W., von der Mark, H., von der Mark, K., and Fischer, S. (1980) J. Embryol. Exp. Morphl. 57,51-60 3. Linsenmayer, T. F., Toole, B. P., and Trelstad, R. L. (1973) Deu. BWl. 35,232-239 4. Lewis, C. A., Pratt, R. M., Pennypacker, J. P., and Hassell, J. R. (1978) Deu. Biol. 6 4 , 31-47 5. Searls, R. L. (1965) Deu. Biol. 1 1 , 155-168 6. Shinomura, T., Kimata, K., Oike, Y., Maeda, N., Yano, S., and Suzuki, S. (1984) Deu. Bwl. 103, 211-220 7. Goetinck, P. F., Pennypacker, J. P., and Royal, P. D. (1974) Exp. Cell Res. 87, 241-248 8. Okayama, M., Pacifici, M., and Holtzer, H. (1976) Proc. Natl. Acad. Sci. U. S. A. 73,3224-3228 9. Kitamura, K., and Yamagata, T. (1976) FEBS Lett. 71,337-340 10. De Luca, S., HeinegHrd, D., Hascall, V. C., Kimura, J. H., and Caplan, A. I. (1977) J. Biol. Chem. 252, 6600-6608 11. Karasawa, K., Kimata, K., Ito, K., Kato, Y., and Suzuki, S. (1979) Deu. Biol. 70, 287-305 12. Royal, P. D., Sparks, K. J., and Goetinck, P. F. (1980) J. Biol. Chem. 255,9870-9878 13. Yamagata, M., Yamada, K. M., Yoneda, M., Suzuki, S., and Kimata, K. (1986) J. Biol. Chem. 2 6 1 , 13526-13535 14. Oike, Y., Kimata, K., Shinomura, T., Nakazawa, K., and Suzuki, S. (1980) Biochem. J. 191,193-207 15. Shinomura, T., Kimata,K., Oike, Y., Noro, A., Hirose, N., Tanabe, K., and Suzuki, S. (1983) J. Biol. Chem. 268,93149322 16. Noro, A., Kimata, K., Oike, Y., Shinomura, T., Maeda, N., Yano, S., Takahashi, N., and Suzuki, S. (1983) J. Biol. Chem. 268, 9323-9331 17. Oike, Y.,Kimata, K., Shinomura, T., Suzuki, S., Takahashi, N., and Tanabe, K. (1982) J. Bwl. Chem. 257,9751-9758

Mesenchyme Proteoglycan of Limb Buds


18. Lohmander, S. L., De Luca, S., Nilsson, B., Hascall, V. C., Caputo, C. B., Kimura, J. H., and Heinegcird, D. (1980) J. Bwl. Chem. 256,6084-6091 19. Nilsson, B., De Luca, S., Lohmander, S. L., and Hascall, V.C. (1982) J. Biol. Chem. 267,10920-10927 20. Takahashi, N., Ishihara, H., Tejima, S., Oike, Y., Kimata, K., Shinomura, T., and Suzuki, S. (1985) Biochem. J. 229, 561571 21. Kimata, K., Okayama, M., Suzuki, S., Suzuki, I., and Hoshino, M. (1971) Biochim. Biophys. Acta 237,606-610 22. Kimata, K., Oike, Y., Ito, K., Karasawa, K., and Suzuki, S. (1978) Biochem. Biophys. Res. Commun. 86,1431-1439 23. Carrino, D. A., and Caplan, A. I. (1982) J. Bwl. Chem. 2 5 7 , 14145-14154 24. Carrino, D. A., and Caplan, A. I. (1984) J. Biol. Chem. 2 6 9 , 12419-12430 25. Pechak, D. G., Carrino, D. A., and Caplan, A. I. (1985) J. Cell Biol. 100,1767-1776 26. Coster, L., and Fransson, L.-A. (1981) Biochm. J. 193,143-153 27. Damle, S. P., Kieras, F. J., Tzeng, W.-K., and Gregory, J. D. (1979) J. Biol. Chem. 254,1614-1620 28. Habuchi, H., Kimata, K., and Suzuki, S. (1986) J. Biol. Chem. 261,1031-1040

Supplementary uateria1 to: A LDxge Chondroitin Sulfate PrOteOglyCan IPG-MI Synthesized before Chondrogenesis in the Limb Bud O f Chick Embryo K. Kimata,


Oike, 1. Tani, T. Shinomura, u. Uritani, and 5 . Suruki





Materials Uaterials Used in this study and their 8 o u r c e ~were as follows: tissue culture materials were from Grand Island Biological co.; D-16-3H1glucosanine (32 Ci/umol) and sodium [12511iodide (carrier-free) were from the Radiochemical Centre, h e r s h a m ; L-[35Slmethlonlne (1106 Cl/mol) was from New England NUCleOIi 135Slsulfate (carrier-free) was from Japan Isotope AsBOCiation, Tokyo, Japan: x-ray film for 1251 autoradiography was from Eastman K d a k ; x-ray film r3H film) for fluorography was from LKB: Pronase was from Ktaken Seiyaku Co.. Tokyo ( a gift from Y . Kanekol: pepstatin wag from Institute of Microbiological Chemistry, Tokyo (a gift from T. Aoyagil; heparitinase, chondroitinase m C , chondroitinase AC. chondroitinase AC II, keratanase, &Ir40.000 chondroitin 4-sulfate. Idr 12,000 Chondroltm (-sulfate. hyaluronic acid, and Oligosaccharides derived therefromwere from Seikagaku KOgyO Co.. Tokyo (gifts from M. M i r u t m i ) ; Streptomyces hyaluronate lyase was from Amano Pharmaceutical Co., Nagoya l a gift from T. Ohyal: trypsin 133 units/mgl was from Boehringer Mmnheim Yamanowhi, Tokyo; b v m e serum albumin and testicular hyaluronidase were from sigma; Sephadex G-50, Sepharose 4B. SephaZOse CL-2B. D m - S e p h a c e l . and electrophoresis calibration kit (Er markers) were from P h a m a c i a Japan, Tokyo: Bio-Gel P-10 and Bio-Gel A-1.5m were from Bio-Rad laboratories1 completeand incomplete Freund's adjuvant were from Pifco; peroxid~.se-conjugated goat anti-rabbit IgG serum was from Hilee LaboratoIies: and fluorescein +hiocyanate-conjugated goat antibodiee to rabbit imunoqlobulinswere from Miles-Yeda, m h o b o t , Israel. The following materials were prepared by the indicated m e t b d b (unless otherwise indicated, the references cited in the miniprint section are typed at the end of the section): PG-H (14 in Typeset Part), PG-Lh 115 ln Typeset Part). and PG-Lt 116 in Typeset Part1 from chick embryo epiphyseal Cartilages; protein-enriched core mo1ecu1es derived from PG-H by chondroitinase ACII and keratanase digestion (14 and 17 in Typeset Part); Isulfate-35S1PG-H, [serine-3H1PG-H, and Inerine-3H1PG-Lb from Chick embryo epiphyseal cartilages that had been metabolically radrolabeled ( 1 4 and 15 in Typeset Part); Chick plasma fibronectln (13 in Typeset Part); laminin A-chain and 8-chain from the Engelbreth-Holm-swam tumor (1); rabbit antibodies to human plasma fibronectin (2). PG-H core protein-conjugated Sepharose 4B and chick plasma fibronectin-conjugared Sepharose 48 were prepared according to the lnbtruction of P h a m c i a . Tissue Culture and Radioisotopic Labeling Anterior and posterior llmb buds were obtained from stage 22-23 Chrck embryos. During dissection, the limb buds Were placed in Hanks' balanced S a l t solution supplemented with 5 1 ( v / v ) fetal calf serum, 0.005 % (v/vl decorblc acid. and 0.029 8 Lw/vl Iglutamine at room temperature until a11 the limbs were collected. After washing twice with Ham's F12 medium supplemented with 10 8 ("/VI fetal calf serum and 0.005 E W/vI ascorbic acid, the tissueswere incubated with either 135Sl*ulfate (0.12 rnCi/mll or [ 6 - 3 H l g l ~ ~ 0 8 ~ m i 1n0e. 1 mci/mll I" the same redium at 37'C for 5 h or 8 h, respectively. in a humidifled atmosphere containing 5 E Co2. The tissues were then washed with Ham's F12 medium. Wnen l'5Slmethionine was used as a preCUraOr. the tissues were treated as above, except that Eagle's minimal essential medium (minus methionine] substituted for the Ham's F12 nedium and incubation was carried out with 135Slmethionine (0.03 mCi/mll for 8 h. Extraction and Purification Of PrOteOglyCanb The 4 M guanidine HCl extraction procedure utilizing protease lnhlbitors (10 mH EDTA. 10 mM E~


fluoride, and 0.36 mM pepstatinl ethylmaleinide. 1 mM phenylmeth.Desulfony1 'C from the tissues at all was used to extract proteoglycans for 24 h at O

29. Carlstedt, I., Coster, L., and Malmstrom, A. (1981) Biochem. J. 197,217-225 30. Oegema, T. R., Jr., Hascall, V. C., and Eisenstein, R. (1979) J. Biol. Chem. 264,1312-1318 31. Kapoor, R., Phelps, C. F., Coster, L., and Fransson, L.-A. (1981) Biochem. J. 197,259-268 32. Salisbury, B. G. J., and Wagner,W. D. (1981) J. BWL Chem. 266,8050-8057 33. Chang, Y.,Yanagishita, M., Hascall, V. C., and Wight, T. N. (1983) J. Biol. Chem. 2 5 8 , 5679-5688 34. Pearson, C. H., and Gibson, G. J. (1982) Biochem. J. 201,27-37 35. Vogel, K. G., and Heinegird, D. (1985) J. Biol. Chem. 260,92989306 36. Fisher, L.W., Termine, J. D., Dejter, S. W., Jr., Whitson, S. W., Yanagishita, M., Kimura, J. H., Hascall, V. C., Kleinman, H. K., Hassell, J. R., and Nilsson, B. (1983) J. Bwl. Chem. 2 5 8 , 6588-6594 37. Heinegird, D., Bjorne-Persson, A., Coster, L., FranzBn, A., Gardell, s., Malmstrom, A., Paulsson, M., and Sandfalk, R., and Vogel, K. (1985) Biochem. J. 2 3 0 , 181-194 38. Singley, C. T., and Solursh, M. (1981) Deu. Biol. 84,102-120 39. Kosher, A., Savage, P., and Walker, H. (1981) J. Embryol. Exp. Morphol. 63,85-98 40. Knudson, C. B., and Toole, B.P. (1985) Deu. Biol. 112,308-318 Additional references are found on p. 13525.

t l m b . The crude extract was then fractionated, under dissociative Condlrions. by ueing con8ecutive rare zonal sedimentation on a glycerol gradient, csc1 isopycnic centrifugation, and/or DE=-Sephacel chromatography. as described under "Results". For separation Of heparain [35SlSulfate proteoglycans from chondroitin 135S1sulfateproteoglycans, Fraction I from rate zonal sedimentation C r f . Fig. 1) was desalted by precipitation with ethanol and then treated with chondroitinase ABCin the presence Of Protease inhibitors (14 in Typeset Part). Chondroitin Sulfate proteoglycans, but not heparan sulfate proreoglycans, were converted to protein-enriched core m01eCules by this procedure. Triton x-100 was added to the digest and the mixture vas dialyzed against 7 H urea/O.O4 M TriS-HC1. pH 7.4/0.2 8 Triton X - 1 0 0 lsolution AI. The dialyzed m t e r i o l YaB chromatographed On DEAESephacel with an increased salt gradient under the conditions indicated in the legend to Fig. 3. Heparan 135Slsulfate proteoglycans were eluted as a single 35S peak at 0.4 M NaC1, whereas core molecules derived from chondroitin sulfate proteoglycans elutedearlier. structur.1 ~ n a l y s i s o Proteoglycena f ~ ' ~ s l s u ~ f a r eor - [3H1glucosaninelabeled glycosaminog1ycan chainswere prepared from radiolabeled prOteOglyCan samples by treatment with alkali and then with Pxonase,as previously described (31. For analysis Of radiolabeled glycosaminoglycans. nonradioactive chondroitin sulfatewas added as a carrier to thealkali/Pronasetreated sample to give0.1 urn1 Of hexuronatdl x 10' cpm of 35s or 3H, and glycosaminoglycans were precipitated with 3 volumes of 9 5 8 ("/VI ethanol containing 1.3 E (w/vI p o t a s s i m acetate. Relative proportions of nomsulfated. 4-sulfated, and 6 - m l f a t e d disaccharide repeat units in the


glycosaminoglycan samples were determined by measuring "5 or 3H activities Of unsaturated disaccharide f r a w n t s released by digestion with chondroitinase AC and chondroitinase ABC ( 4 1 . Heparan [35Slsulfate WaB determined by degradation with heparitinase (5). After en'zymtic treatment (see Ref. 6 for details of the procedurel. aliquota O f the reaction mixtnrts were applled on Toyo NO. 50 filter paper and chromatographed in butyric acid/0.5 M ammonla ( 5 : ' . by volume) to separate degradation products from vndeqraded 35S-lakled materials remaining at the origin. The Dirnunt (cpml of heparan 135S1~ulfatewas estimated from the difference between the radioCictlvlty Of chondroitinase ABC-treated control and that Of the sample treated with both chondroitinase ABC and heparitinase (determined at the Origin of the chromatogram). For measurement Of keratan sulfate, glycosaminoglyccin samples were treated with keratanaseand the digests were analyzed by gel filtration. as previously described 17). For analysis o f glycoprotein-type OligosllCCharidee. I 3 H ] g l ~ ~ ~ ~ ~ i n e - l . b e lproteoglycan ed samples were treated for 48 h 181. The mixtures were then with 0.1 H NaBH,/O.OS H NaOH at 45'C neutmlized With glacial acetic acid. clarified by centrifugation, and s u b p c t e d to analysis by gel filtration. For estimation Of hydrodynamic Size Of carbohydrate chains, aliquotll of the radiolabeled carbohydrate samples obtainedas above were lyophilized. dissolved in a small volume Of 0.4 u ammonium acetate and applied to a BioG e l A-1.5m or Bio-Gel P-10 column whichwas equilibrated and eluted with 0.4 u a m m n i v m acetate. For analysis of amino acids, proteoglycan aamples were hydrolyzed with 6 n HC1 in sealed evacuated tubes at llODC for 18 h. Analysis was performed with an automatic amino acid analyzer IHitachi model 8351 by the m t h d of Spacknan 1.( 9 1 . For analysis of core proteins, protein-enriched core fractions were obtained from ploteoglycan s m p l e s by the methcd uaing chondroitinaise ABC. chondroitinase AC 11, and/or keratanase in the presence Of protease Inhibitors. as previously descl'lbed ( 1 4 in Typeset Part). The resulting Core molecules were recovered from the digests by ethanol precipitation, and subjected to SDS gel electrophoresis I" either 3.15 E (v/vl polyacrylamide gels (17 in Typeset Partl or 6 I [w/vl polyacrylamide separation gels with 3.75 8 (w/v) polyacrylcimlde stacking gel (101. For nonreduced core protein samples, 2 E ("/VI

Mesenchyme P r o ~ e o ~ ~ofy Limb c ~ n Buds SDS, 20 P glycerol, 10 M pethylmaleimide, 0.01 P ( W / V l bromphenol blue. and 5 0 nn Tris-HC1, pH 7.4, were added to test samples which were then heated at IOO'C for 1 min l o r 70'C for 30 mi") before application to the gels. Reduced samples were prepared similarly but in the presence of 5 m I v l v l 2-mercaptaethanol in place of X-ethylmaleimide. After electrophoiesis, the gels were stained vith C w m s s ~ eBlue and then subjected to fluorographyr as PreViOUSlY described 117 in lypeset Part). FOX tryptic peptlde mapping of proteoqlycan core proteins. a gel slice Containing a Coomassie Blue-stained core p r o t e m was labeled vith 12*1 in the presence of chloramine-T M d then treated wlth 25 Y q o f trypnln In 500 u l Of 50 mN NH,HC03. pH 8 . 4 , for 18 h at 37'C. The ~e6YltOlnttryptic peptides w r e displayed two dimensionally M a silica gel thin layer plate. as previously deliccibed 117 in Typeset Part). Hyaluronic Acid-binding Study with ProtBoglycans -The bindlng experiments were carried out essentially as deacribed by Hascall and Heineggrd 111) except that radiolabeled PG-n W ~ Bincubated with unlabeled hyaluronic acid in the presence of appropriate arwUnt8 ofanlabeled PG-H as a carrier lace "Results' for ita r o l e ) . The resulting proteoglycan-hyaluronate aggregates were reparated from prateoglycan monomer by rate zonal s e d i m n tation on a Cs250, linear gradient 1121. In a typical experiment, 2.000 cpn latout 1 nmol as haxuronatel of IsUlf.te-35S1PG-n in 4 n guanidine KC1 was m i x d vith 5 -1 10- hexuronate) o€ hyaluronic acid and 100 -1 1t.s hexuronata) of PG-8. and the solution vas dialyzed against 0.1 n sodium acetatelO.1 W Tria-HCl. pH 7.0/10 mN EDTAIIO mN ~-ethylnaleiralde. In control experimnts, PG-H or hyaluronic acid. O I both. vas omitted from the reaction mixture. When PG-Lb was examined for it8 ability to interact with hyaluronic as hexuronatel of laerine-3H1PG-Lb in 4 W guanidine acid. 3.200 cps 11.2 n-1 HC1 was treated ais above. Immunochemical Procedures Rabbit antiserm was prepared )io PC-M isolated folloving DE&€ SephaCFl chromatography l9Be "ResultS"1. A 20 ug ( a s protein1 of PG-n in 0.1 ml of phosphate-buffered saline we6 emulsified With an equal ~ l m of e Fzwnd's oomplete adjuvant and injected into a lymph node of the popliteal region. At tWo subsequent 2-veek intervals. 10 v g I d s protein) of the ismmoqen in 0.1 ml of phosphate-buffered saline emulsified ulth 0.1 1111 of incomplete Freund's adjuvant was inlected Into the Same site. The antibody titers were tested weekly by EZIPA 113). The serum was collected 3 week8 after the second boolter and passed over a c~lumnof chick plasma fibronectin-conju4ated Sepharone 48. and then a column of PC-H core protein-conjugated sepharose 4B. For Western blotting of proteoglycan core proteins, care protein bands on IDS-polyaerylbnide slab gels lsee above) were transferred to nitrocellulose sheets with Bio-Rad Trans Blot equipment. The sheets were washsd, incubated for 2 h atIaDm temperature with anti-PG-n antibodies appropriately diluted 11:2.5001 into 0.1 % l w / v ) bovine serum albumin in Paline. washed again. and stained vith peroxidase-conjugated goat anti-rabbit IgG serum. as previously described I16 in Typeset Part). POI innunofluorescent staining and microscopy. frozen sections ofchick emb?yO hind limba at stages 23 14 days]. 24 11.5 days), 27 15-5.5 daysl, and 37 I11 dayst were prepared. In some cases, air-dried sections were treated w i t h 0.5 m g h l testicular hyaluronidaae for 30 min at r w m t.nplrature and rinsed for 3 lain in phosphate-buffered saline three t i m e . me Sections w r e treated vith MtI-PO-H antibodies and then with fluorescein

13525 TRBLE I

COmpositiDn Of glycOsaminoglycan chains derlved from 135Slsulfate-labeled and 13H191ucosamine-labeled PG-H samples

Cotnpasition 5uga1 unit


a g 20.3

GlCA-GltlNAc GlcR-GnlNAC14-S041 GlcA-G~1NRcl6-so41 58.1 IdOA-G11NAC14-S04) Unidentifieds/

26.4 38.1 4.5 10.6

N&/ 25.8

8.1 8.0

%/ Per Cent O f total radioactivity, estimated from the yields Of the unsaturated Ollq0saCChBc~des Produced by digesticn vlth chondroitlnase AC and chondroitinase Aac. b/ Not determined because 155 would not label unsulfated carbohydrate units.

E/ The v a l u e s represent total radioactivity of chondroitinase products c0rrespondin.g to -nosaccharides, saturated disaccharides, and higher OligosaCchariden. TABLE I 1 Amino w i d composition of PG-n -na


irrothiosyan~te-conj"g*t*dgoat antibodies to rabbit immunoglobulins, (18 previously described 16 in lypeset Part). In Contcol experients. parallel sections vex-.? treated with n o m 1 rabbit imunoglobulin prepared from a noni-unircd rabbit. me treated Sections were viewed with nn olympus BH2RFL epi-illuminated fluoreaceme microscope. Other nethods Htxufonste dratemined by the promedure of Bitter and nuir I141 with glucuronolactone as a standard. Protein was determined by the method of Loxry et g. (151 with bovine serum a l b m i n as a standard.



Glx PI0 GlY

Ala CY. Val

Met I le Le"

Tyr Phe His LY 5 A W

residues/1000 residues 57 79 89 95 105 114 147 160 84 34 112 106 79 60 6 4 52 57 14 9 47 40 73 sa 11 21 32 37 21 16 16 44 14 19

%/ No corrections were made for the modification OfC y 5 and Lyr by pethylmaleimide during the extraction from ti5sues and for the destruction Of amino acids during HCI hydrolysis. %/ The data taken from Ref. 20 in Typeart Part. REFERENCES 1. Tmpl. R . . Rohde, H., Robey. P. G.. Rennard, 8 . R., Foldart, J.-M., and martin, G. R. I19791 J. Biol. Chem. 254, 9913-9937 2. Xmata. X.. Foidart. J.-P.. Pennypacker, J. P.. Kleinman. H. K., nartin, G. R . . and Hewitt. A. Tyl. 11982) Cancer Res. 42, 2384-2191 I. X i m t a , X . , OkayalM. X.. mhira. A., and SuIULi, 5 . (19741 J. 8101. Chem. 219, 1616-1653 4. Saito. H., Yaaagata, T.. end Suruki. S. I19681 J. B m l . Chem. 243, 1536-1542 5 . Linker, A . , and Hovingh. P. I19721 Methods Enzymol. 28, 902-911 6. Xirnaca. X . . HOnmd. X . . Okayama. M.. Oguri. K.. HOLumi, M., and Suzuki, S . 119831 Cancer Res. 43. 1347-1354 7. Nakasawa, X . , and suzuki. S . (19751 J. B i d . Chem. 250, 912-917 8 . De Luc.3, 5.. lohmander. L. S., NilsSOn. B., Hascall, v . c., and caplan. A . I. 119801 J. Biol. Chem. 255. 6077-6081 9. Spackman. D. H.. Stein, W . H.. and Moore, 5 . 119581 Anal. Chen. 10, 1190-1206 680-685 10- Laentli. U. K. 119701 Nature 1Lc-d.) 227. 11. Hascall. V. C.. and Helnegird. 0 . I19741 J. Biol. Chen. 249, 4232-4241 12. Ximta. X., XlmUra, J. H . 0 Thenar. E. 3 . -M. A,, Bairech, H. -J., Rannard, S . I., and Hascall, V . C. 119821 J. Biol. Chem. 251. 319-3826 13. Rennard. 5 . I . , Xirnata, K.. Desemund. 8.. Barrach. H. J., W l l ~ z e k , J., Ximura, J. X.. and Hascall. V. C. (19811 Arch. Bioshem. Biophya. 207, 399-406 14. Bitter. T., and nuir, x. n. 11962) anal. Biochem. 4, 330-334 15. Loxry, 0 . H.. Rosebrough, N. J., Farr. A . I,., and Randall. R. J. 119511 J. Biol. Chem. 193, 265-275