A Mapping Technique for Probing the Structure of Proteoglycan Core Molecules*

Our previous work showed that treatment of chick embryo cartilage proteoglycan (PG-H) with chondroiti-nase-AC I1 and keratanase yielded a protein-rich core fraction having enzymatically modified linkage oligosaccharides. The core sample has now been analyzed by tryptic peptide mapping, in which the isolated core sample contained in a single Coomassie blue-staining band from a dried slab gel is radioiodinated and treated with trypsin, and the resultant tryptic peptides are displayed two-dimensionally on a silica gel thin layer plate. The map thus obtained exhibited 22 major pep- tide spots, the resolution and location of which were reproducible. In order to identify regions of the core polypeptide from which the tryptic peptides are derived, PG-H was cleaved with clostripain under conditions that yield a hyaluronic acid-binding fragment with an apparent M, = 150,000 and chondroitin sulfate-peptide clusters of smaller molecular sizes. Although the peptide maps of the two size classes of clostripain fragments differed significantly from each other, the patterns of spots, as a whole, were extensively similar to those observed with the intact core molecule. These results have provided additional evidence that PG-H has a single, nonvariable core protein structure. In addition, the technique used here will provide a versatile method for the identification of genetic types in this increasingly complex family of matrix macro-molecules.

is due to a true heterogeneity in which distinct genetic types of core polypeptide are present or to a heterodispersity arising from post-translational processing events such as variable attachment of glycosaminoglycan chains or variable partial degradation of core polypeptide chains.
We have previously (8) presented an enzymatic procedure for selective removal of the bulk of the chondroitin sulfate and keratan sulfate of PG-H' without cleavage of peptide bonds. Upon zonal sedimentation on a sucrose gradient in 0.5% SDS, the resultant core preparation, PG(-CS,KS), gave a single sharp band with a sedimentation coefficient of 6 S, suggesting that PG-H is a single, albeit polydisperse, population of molecules. In the present work, the possibility has been examined more rigorously that the core molecule from PG-H may contain a single polypeptide. The method described here for probing the structure of the core polypeptide includes twodimensional peptide mapping of the enzymatically prepared core molecules in amounts as small as 5 pg, a technique which may also find application in establishing the presence of genetic variants of proteoglycan.

["'S]PG(-CS)K and [%]PG(-CS,KS)
were prepared from [sulfate-"'SS]PG-H as previously described (8). Chondroitinase-AC I1 (9), keratanase (endo-P-D-galactosidase from Pseudomonas sp.) (lo), and hyaluronic acid (M, 850,000) were products of Seikagaku Kogyo Co., Tokyo, and were kindly donated by Dr. T. Okuyama of the company. The commercial enzyme preparations are available from the above company or from Miles Laboratories, Elkhart, IN. Pepstatin (11) was a gift from Dr. H. Umezawa, Institute of Microbial Chemistry, Tokyo. The reagent is available commercially from Protein Research Foundation, Ina, Minoh, Japan, or from Sigma. Na ['251] iodide (carrier-free) was purchased from the Radiochemical Centre, Amersham, England; trypsin (33 units/mg) and clostripain were from Boehringer-Mannheim Yamanouchi, Tokyo; phenylmethanesulfonyl fluoride and bovine serum albumin (fraction V) were from Sigma; autoradiography enhancer ENJHANCE was from New England Nu-  clear; x-ray film for "S fluorography (Fuji RX) was from Fuji Film Co., Tokyo; x-ray film for Iz5I radioautography (X-Omat R) and Coomassie brilliant blue R-250 were from Eastman; Diaflo PM-10 membranes were from Amicon; Sepharose CL-2B, CL-4B, and CL-GB were from Pharmacia Japan, Tokyo; and acrylamide, N,N'-methylene bisacrylamide, and other chemicals for the preparation of the electrophoresis gels were from Nakarai Chemicals, Kyoto. Silica gel-coated thin layer plates (20 X 20 cm, 0.25-mm thickness) were from E. Merck, West Germany. A 1-cm width of silica gel layer was cut from each edge, and the plates were stored in a desiccator until use. Bovine serum albumin and RNA polymerase subunits a, b, and /3' , obtaiqed from Boehringer-Mannheim Yamanouchi were used as molecular weight markers in SDS-polyacrylamide gel electrophoresis. All of the other chemicals were of the highest purity commercially available.

Preparation of Core Molecules from PG-H by Digestion with
Chondroitinase-AC II and Keratanase-The preparative sequence is shown in Scheme 1. In each series of experiments, 125 pg (as protein) of purified PG-H was treated with 4 units of chondroitinase-AC I1 in the presence of 10 mM EDTA, 10 mM N-ethylmaleimide, 5 mM phenylmethanesulfonyl fluoride, and 0.36 mM pepstatin (hereinafter the mixture of the four reagents will be referred to as "protease inhibitor mixture"), under the conditions described in our previous paper (8). The reaction was terminated by adding SDS to a final concentration of 2% (w/w) followed by heating at 70 "C for 30 min. A 3-ml portion of the digest was applied to a Sepharose CL-4B column (1.5 x 91 cm) eluted with 0.2% (w/w) SDS, 40 mM Tris/HCl, pH 7.4, 5 mM EDTA at 4 "C. Fractions of 2.3 ml were collected and their absorbance at 280 nm or radioactivity content was determined. The core molecule, PG(-CS)*c, that eluted in the retarded fractions (see "Results"), was collected by precipitation with ethanol, and treated with keratanase (0.2 unit/l20 pg of core protein) in the presence of the protease inhibitor mixture, as previously described (8). The reaction was terminated and the digest chromatographed on a Sepharose CL-4B column, as described above. The core molecule, PG-(-CS,KS), that eluted in the retarded fractions (see "Results") was collected by precipitation with ethanol. A 100-pg (as protein) portion of the core sample was dissolved in 9 ml of a solution containing 0.2% (w/w) Triton X-100, 4 M guanidinium chloride, 50 mM Tris/HCl, pH 8.0, 10 mM EDTA, 2 mM phenylmethanesulfonyl fluoride, 10 mM Nethylmaleimide, and 0.36 mM pepstatin. CsCl was added to the solution to give a density of 1.25 g/ml. This solution was then subjected to isopycnic centrifugation at 4 "C for 50 h at 38,000 rpm (93,000 X g.") in a Hitachi RP-65T rotor. The gradients were each Procedure A (successive digestion) PG-H (Fig. 1, Track a ) . 1 Chondroitinase-AC I1 digestion Digest ( Fig. 1 . 1 Sepharose CL-4B chromatography (Fig. 3) Crude PG(-CS,KS) ( Fig. 1, Track g ) . 1 CsCl isopycnic centrifugation (Fig. 4) PG(-CS,KS) ( Fig. 1 I SCHEME 2. Preparative sequence of the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid and the chondroitin sulfate peptide clusters. fractionated into 10 fractions and their protein (12) or radioactivity content was determined. The core molecule, PG(-CS,KS), was recovered from the bottom fraction (se'e "Results") by precipitation with ethanol.
When PG-H (125 pg as protein) was treated with a mixture of chondroitinase-AC I1 and keratanase (Scheme 1, Procedure B), the procedures were as above except that 4 ml of an enzyme reagent containing 4 units of chondroitinase-AC 11,0.2 unit of keratanase, 0.1 M Tris/HCl, pH 7.4, and the protease inhibitor mixture was used and the reaction was carried out at 37 "C for 60 min.

Preparation of Proteoglycan Fragments by Clostripain Digestion-
The method which was devised for this purpose is based on the method of Caputo et al. (for Swarm rat chondrosarcoma proteoglycans) (13) with several modifications. The preparative sequence is shown in Scheme 2. All isopycnic centrifugations were at 10 "C for 50 h at 38,000 rpm (93,000 X gav) in a Hitachi RP-65T rotor. Cartilage samples were extracted two times with 9 volumes of 4 M guanidinium chloride in 50 m Tris/HCl, pH 8.0, containing the protease inhibitor mixture, as previously described (8). Hyaluronic acid, 1 mol (as uronic acid)/100 mol of total proteoglycan uronic acid, was added to the extracts, and the solution was dialyzed against 9 volumes of 0.5 M sodium acetate containing the protease inhibitor mixture. CsCl was added to the dialysis residue to give a density of 1.60 g/ml. The solution was then subjected to isopycnic centrifugation. The gradients were each fractioned and the bottom one-third fractions were collected. The pooled material was dialyzed against 100 volumes of 0.2 M sodium acetate containing the protease inhibitor mixture. Aliquots of this solution (2 pmol/ml as hexuronate) were subjected to rate zonal sedimentation on a Cs2S04 gradient (0.15 to 0.5 M in 0.2 M sodium acetate, pH 7.0) on a cushion of 1 ml of 2 M Cs2S04 in the same solvent (14). The gradients were centrifuged at 4 "C for 8 h at 22,500 rpm (63,000 X g.") in a Hitachi RPS-27-2 rotor. The proteoglycan aggregate fraction recovered in the bottom one-sixth of the gradient was dialyzed two times against 50 volumes of 0.1 M Tris/HCl, 0.1 M sodium acetate, pH 7.4, and concentrated to about 0.1 volume with an Amicon PM-10 filter; yield was 1.5 mg (as protein) or 50 pmol (as hexuronate) from 3 g of cartilages.
The proteoglycan aggregate fraction (5 pmol of hexuronate/ml) in the buffer was digested at 37 "C for 4 h with 2.5 units/ml of clostripain in the presence of 5 mM calcium chloride, 0.6 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, and 0.36 mM pepstatin. The reaction was terminated by adding iodoacetamide to a final concentration of 1 mM. CsCl was added to the reaction mixture to give a density of 1.50 g/ml. This solution was then subjected to isopycnic centrifugation. The gradients were partitioned into three equal fractions, and the bottom one-third (a) and top one-third ( b ) fractions were further purified as follows: (a) this fraction was concentrated to 0.3 volume with an Amicon PM-10 filter and applied to a Sepharose CL-2B column (1.2 X 50 cm) eluted with 0.5 M sodium acetate at 4 "C. The eluted fractions were assayed for hexuronate and protein. The chondroitin sulfate-peptide clusters recovered in the retarded fractions (17 ml) were pooled and mixed with an equal volume of 8 M guanidinium chloride. CsCl was added to the mixture to give a density of 1.47 g/ ml. This solution was then subjected to isopycnic centrifugation. The bottom one-third of the gradient was dialyzed successively against 0.5 M sodium acetate and water, and lyophilized; yield of chondroitin sulfate-peptide clusters was 840 pg (as protein) or 22 pmol (as hexuronate) from 1.5 mg (as protein) or 50 pmol (as hexuronate) of proteoglycan-link protein-hyaluronic acid aggregates. ( b ) The top one-third fraction from the clostripain digest was concentrated to 0.3 volume with an Amicon PM-10 filter and applied to a Sepharose CL-6B column (1.2 X 50 cm) eluted with 0.5 M sodium acetate at 4 "C. The complex of hyaluronic acid-binding region, link protein, and hyaluronic acid recovered in the excluded volume (6 ml) was dialyzed against water and lyophilized yield was 220 pg (as protein) or 0.4 pmol (as hexuronate) from 1.5 mg (as protein) or 50 pmol (as hexuronate) of proteoglycan-link protein-hyaluronic acid aggregates.
In order to isolate the hyaluronate-binding region, a 1-pmol (as hexuronate) portion of the complex with link protein and hyaluronic acid obtained as above was treated in 200 pl of an enzyme reagent containing 0.1 M Tris/HCl, pH 7.4, 30 mM sodium acetate, %(I the concentration of the protease inhibitor mixture, and 0.015 unit of chondroitinase-AC 11 at 37 "C for 2 h. The digest was then subjected to SDS-polyacrylamide slab gel electrophoresis, as described below. The same method was used to remove the bulk of the chondroitin sulfate chains from the chondroitin sulfate-peptide clusters. SDS-Polyacrylamide Slab Gel Electrophoresis-Samples (about 10 to 210 pg each as protein) were dissolved in 100 p1 of 50 mM Tris/ HCl, pH 7.4, containing 10 mM N-ethylmaleimide, 2% (w/w) SDS, 20% (w/w) glycerol, and 0.04% (w/v) bromphenol blue and were heated at 70 "C for 30 min before application to gels. When necessary, radioactive standards containing 400 to 20,000 cpm of " S were added to the mixtures before heating. Polyacrylamide slab gels, 3.75% crosslinked with 0.1% bisacrylamide (for the separation of core molecules) or 5% cross-linked with 0.15% bisacrylamide (for the separation of clostripain fragments), were prepared in 50 mM Tris, 50 mM glycine, 0.1% (w/w) SDS, pH 8.9, with 50 p1 of N,N,N',N'-tetramethylethylenediamine and 15 mg of ammonium persulfate in 40 ml of running gel (15). Electrophoresis was carried out in a Hoefer vertical slab gel electrophoresis unit at 10 mA for 1 h to load the samples and 30 mA until the tracking dye was near the end of the gel. Gels were fixed in 50% (v/v) methanol, 10% (v/v) acetic acid for 2 h, stained in 0.025% (w/v) Coomassie brilliant blue R-250,2596 (v/v) isopropanol 10% (v/ v) acetic acid for 2 h, and then destained in methanol/acetic acid/ water (1:1:8 by volume). For fluorographic detection of radioactive standards, the gels were treated with EN3HANCE, dried in a gel drier, and exposed to Fuji RX x-ray f i at -80 "C.
Tryptic Peptide Mapping-Radioiodination, trypsin digestion, and two-dimensional separation of peptides on thin layer plates were carried out by the method of Elder et al. (16) as modified by Yamaguchi et al. (17). Briefly, a Coomassie blue-stained protein band of unlabeled sample was cut out from the SDS-polyacrylamide electrophoresis slab gel (see above) and transferred to a glass tube. The gel slice was washed extensively with 25% isopropanol, then with 10% methanol to remove SDS and other contaminants, and the slice was dried under a heat lamp. The protein was then radioiodinated with 200 pCi of "'I for 1 h in the gel slice by the chloramine-T method, as described by Elder et al. (16). Approximate incorporation of ' " 1 was 2 X lo5 cpm/pg of protein. The gel slice was washed extensively with 10% methanol and dried, and then treated with 25 pg of trypsin in 0.5 ml of 50 mM NH~HCOJ, pH 8.0. After incubation for 18 to 24 h at 37 "C, the liquid in each tube was lyophilized, and the residue was redissolved in 2 0 4 of acetic acid/formic acid/H20 (3:1:16, by volume). A 3-to 6-4 portion of this solution was spotted on a silica gel-coated thin layer plate and peptides were resolved using electrophoresis in the fist dimension and ascending chromatography in the second dimension. Electrophoresis was carried out at 950 V for 80 min in acetic acid/formic acid/H20 (3:1:16, by volume) using a Pharmacia flat bed apparatus FBE 3000 with cooling at 4 "C. Chromatography was carried out in 1-butanol/pyridine/acetic acid/H20 (13:102:8, by volume) for about 5 h until the front reached the top. The plate was dried and peptides were located by radioautography with Kodak X-Omat R film.
Other Methods-Hexuronate was determined by the procedure of Bitter and Muir (18) with glucuronolactone as a standard. Protein was determined by the method of Lowry et al. (12) with bovine serum albumin (Fraction V) as a standard. Radioactivity was measured by spotting a sample solution on a paper disc (2.4-cm diameter); the disc was then dried in an oven at 50 "C and counted in an Aloka liquid scintillation spectrometer (Aloka Co., Tokyo) with the solvent system recommended by the manufacturer.

Preparation of Core Molecules from PG-H by Successive
Digestion with Chondroitinase-AC ZZ a n d Keratanase-Core molecules were obtained from PG-H by successive digestion with chondroitinase-AC I1 and keratanase, essentially as described in our previous paper (8). The preparative sequence is presented in Scheme 1 (Procedure A). At each step of the digestion and fractionation procedures, aliquots of the products were examined by SDS-polyacrylamide gel (3.75%) slab electrophoresis (Fig. 1). Since our previous studies (8) had indicated the presence of 35S in the core molecules, PG(-CS)Ac and PG(-CS,KS), prepared from [35S]sulfate-labeled PG-H, advantage was taken in the present study of the fact that core molecules derived from unlabeled PG-H can be readily distinguished from other proteins using the labeled compounds as internal or external standards. The presence of ["SS]sulfate residues in the PG(-CS,KS) preparation is due to the fact that although chondroitinase-AC I1 removes all the sulfated sugar units on the chondroitin sulfate chain, keratanase leaves about 10% of the sulfated sugar units on the keratan sulfate chain (8).
In the preparation of PG-H, no Coomassie blue-positive band was detected (Track a), although [s~lfate-~~S]PG-H (internal marker), detected by fluorography (Track k), was located at the origin; probably the core protein is masked by the preponderance of glycosaminoglycan chains. When PG-H was digested with chondroitinase-AC I1 and an aliquot of the digest was mixed with ["s]pG(-cS)~c and examined by gel electrophoresis (Fig. 1, Track b ) , a somewhat diffuse band staining with Coomassie blue was revealed in the position of standard ['%]PG(-CS)AC (cb Track I ) . The stained bands ( X ) ahead of the PG(-CS)A~ band can be attributed to proteins present in the chondroitinase-AC I1 preparation employed for digestion, because the same bands were present in a control gel containing enzyme alone (Track c).
In order to remove the contaminating materials which would interfere with subsequent digestion or fractionation procedures, the rest of chondroitinase digest of unlabeled  (Fig. 2 4 ) . When the unlabeled sample was chromatographed under the same conditions, UV (280 nm)absorbing fractions were diffuse and were not well separated (Fig. 2B). However, analysis on polyacrylamide gels of the column eluates indicated that the material in Area I is almost entirely composed of PG(-CS)Ac (Fig. 1, Track d) whereas Area I1 contains no PG(-CS)Ac. The material in Area I1 appears to be derived from the enzyme preparation, as the electrophoretogram (not shown) was essentially identical with The PC(-CS)A(. sample obtained as above was further treated with keratanase. When examined on polyacrylamide slab gels, the keratanase digest ( Fig. 1, Track e ) contained a highly diffuse band due to the proteins present in the enzyme preparation (see Track f for the pattern of the enzyme alone).
However, a single protein band, identified in Fig. 1, Track e,  as PC(-CS,KS), could be detected which was more compact and migrated more rapidly than the band from the parent molecule (Fig. 1, compare Trucks d and e). As the fluorogram (Track o ) shows, this component migrated with standard [%I In order to remove the contaminating proteins, the keratanase digest (unlabeled sample) was chromatographed on a column of Sepharose CL-4B. For comparison, a keratanase digest of [:'"S]PG(-CS)A~ was also run under the same conditions. From the ' "S profile of the digest of standard [:'%]-PG(-CS)AC (Fig. 3 A ) , it was expected that unlabeled PG(-CS,KS) product eluted in Fractions 30 to 42 ( Fig. 3B; Area I ) . That this is indeed the case was shown by analysis of the column eluates in Area I by polyacrylamide gels (cf. Fig. 1, Trackg). The fractions in Area 11, in contrast, produced a diffuse pattern without discrete bands (not shown).
When the material in Area I of Fig. 3B was recovered by ethanol precipitation and analyzed by gel electrophoresis, it was apparent that considerable purification of PC(-CS,KS) was realized (Fig. 1, compare Tracks e and g). However, the preparation still contained some of the proteins from the enzyme preparation which gave a diffuse band overlapping the PG(-CS,KS) band on gel electrophoresis. The material from Area I of Fig. 3R was therefore subjected to CsCl isopycnic centrifugation for further purification (Fig.  4R). For comparison, ["'SS]PG(-CS,KS) was centrifuged under the same conditions (Fig. 4A). Comparison of the sedimentation profiles in A and R indicated that most of the contaminating proteins are less dense than ["'"S]PG(-CS,KS). In fact, it was possible to obtain a PC(-CS,KS) preparation from the bottom fraction (Fraction 1) of CsCl isopycnic centrifugation (Fig. 4B) which showed, on subsequent gel electrophoresis, a sharp band of PG(-CS,KS) practically devoid of other protein bands (Fig. 1, Track h ) . The yield of PG(-CS,KS) expressed as protein measured by the method of Lowry et al. (12) was 65 pg from 120 pg of PG(-CS)/,(.. Although analysis on polyacrylamide gels indicated that a significant amount of PG-(-CS,KS) was present in Fraction 2 in the CsCl gradient (Fig.  4), the core molecule could not be isolated from this fraction because of the presence of a large amount of other proteins.

Preparation of PC(-CS,KS) by Simultaneous Digestion with Chondroitinase-AC II and Keratanase-The above pro-
cedure for preparing PC(-CS,KS) is both exacting and timeconsuming. Consequently, the need arose for the development of a simpler method which can more readily be applied to the routine preparation of core molecules from proteoglvcan samples. Such a method, based on the use of an appropriate mixture of chondroitinase-AC I1 and keratanase for the removal of glycosaminoglycan chains, has been devised. A graphic summary of this procedure is presented in Scheme 1 (Procedure €3). A separation of the PC(-CS,KS) product was achieved by Sepharose CL-4B chromatography followed by CsCl isopycnic centrifugation (the elution and sedimentation profiles were similar to those in Figs. 3 and 4, and are not shown here) to obtain an electrophoretic profile (Fig. 1 , Track  i ) that is comparable to the profile of the most purified sample obtained by Procedure A (Fig. 1

, compare Tracks h and i ) .
Attempts to remove contaminating enzyme proteins by still simpler methods have so far been unsuccessful; eg. omission of the gel chromatography step from the purification procedure resulted in incomplete removal of contaminating proteins on gel electrophoresis (Fig. 1, Trackj).
Analysis of the Core Molecules by Tryptic Peptide Mapping-To examine PG(-CS);W and PG(-CS,KS) by tryptic peptide mapping, slab gel electrophoresis of unlabeled core samples was carried out as above (cf Fig. 1, Tracks d, h, and  i ) and Coomassie blue-stained bands of PG(-CS)N, and PG(-CS,KS) (cf Fig. 1A) were cut out from the slab gel. The protein in each gel slice was radioiodinated and digested with trypsin for 18 to 24 h. The duration of tryptic digestion was chosen for further studies, as the peptide maps were most reproducible. The peptide products released from each gel slice were separated two-dimensionally on a silica gel-coated thin layer plate. Fig. 5 shows the radioautograms of tryptic peptide maps of PG(-CS)A(. and PG(-CS,KS). Fig. 6 shows the traced diagram of the radioautograms. Since no difference was detected between the maps of the PG(-CS,KS) samples obtained by Procedures A and R, only the map of the latter is shown. As many as 50 spots were produced after cleavage The core molecule samples were labeled with "'I in the gel slices and tryptic peptides were displayed on silica gel thin layer plates by electrophoresis in the first dimension and ascending chromatography in the second dimension. Radioautographs are shown. of the core molecules and all the major spots (numbered 1 to 22) were common to both the PG(-CS);\(. and PG(-CS,KS) samples. The patterns of the remaining minor spots were also very similar (over 90% were in common), although their radioactivities were too low to allow reliable comparison to be made between different protein samples.
In both the maps of PG(-CS),\(. and PG(-CS,KS), considerable amounts of labeled materials were found at the bottom of the plates. The occurrence of these relatively immobile components can be explained by assuming that they represent peptides of relatively large size or those bearing carbohydrate chains. The interpretation is consistent with the fact that the profiles of mobile peptide products were essentially the same regardless of whether or not a large portion of the keratan sulfate chains had been removed from the core molecule.
Comparison of "'Z-Peptide Maps of Fragments Produced by Clostripain Digestion of PG-H-Proteases have been used to fragment proteoglycan molecules selectively to define different functional regions for study. For example, Caputo et al.
(13) have shown that, after clostripain digestion of proteoglycan aggregates from Swarm rat chondrosarcoma, the hyaluronic acid-binding region remains bound with the link protein to hyaluronic acid while most of the core protein with covalently bound glycosaminoglycan chains is removed. Subsequent density gradient and molecular sieve techniques using dissociative solvents have been used to isolate the hyaluronic acid-binding region.
In order to identify regions of the core polypeptide from which the tryptic peptides are derived, core protein fragments of limit size were prepared from PG-H aggregates by a modification of the clostripain method, as outlined in Scheme 2 and in the legend to Fig. 7, and were separately subjected to tryptic peptide mapping (Figs. 8 and 9). In the fragmentation procedure with clostripain, the following two modifications are particularly important: 1) the addition of hyaluronic acid to the extract of cartilages to make up the natural deficiency of hyaluronic acid, and 2 ) the use of phenylmethanesulfonyl fluoride and pepstatin to prevent nonspecific cleavage of peptide bonds caused by other protease activities present in the clostripain preparation. When the exogenous supply of hyaluronic acid was omitted, the yield of proteoglycan-link proteinhyaluronic acid aggregates was reduced by about 50%. Omission of the protease inhibitors from the incubation with clostripain caused about 90% reduction in the yield of the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid.
Aliquots (40 pg as protein) of the fragment samples thus obtained were further treated with chondroitinase-AC I1 in the presence of protease inhibitors and applied to 5% polyacrylamide slab gels. The gels were stained with Coomassie blue (Fig. 7). Before chondroitinase-AC I1 digestion, neither the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid nor the chondroitin sulfate-peptide clusters penetrated the gel (Tracks a and c). In the case of the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid, it was noticed that the hyaluronic acid component forms a jelly-like layer at the origin of the slab gel,  thereby interfering with penetration of the protein components. After chondroitinase-AC I1 digestion, the hyaluronic acid-binding region, link protein, and hyaluronic acid complex produced two discrete bands corresponding in mobility to link protein and its fragment (Track h, .?FiK and 22K, respectively),' plus a somewhat diffuse band (150K) which represents a portion of the proteoglycan core protein containing the hyaluronic acid-binding region. The size dispersity of the 150K fragment is probably due to the presence of keratan sulfate chains (13). The stained band marked X represents proteins from the chondroitinase preparation.
The apparent size of the hyaluronic acid-binding region fragment is almost twice as large as that observed for bovine or rat proteoglycans (13). The reason for this discrepancy is unknown, but it is noteworthy that the 150K fragment could not be an intermediate produced by an incomplete cleavage of the core protein, because the time course of clostripain digestion has shown that the electrophoretic profiles of the fragments are very similar a t all time points from 4 to 6 h, whereas higher molecular weight intermediate profiles were obtained from a 2-or 3-h digest.
After chondroitinase-AC I1 digestion, the chondroitin sulfate-peptide clusters produced a diffuse weakly staining pattern (track d 50"sOK) without discrete bands. The feeble Coomassie blue reaction suggests that the 50-80K fragments contain a large number of covalently bound carbohydrate chains such as chondroitin sulfate remnants, intact keratan sulfate chains, and Nand 0-linked oligosaccharide chains.
' Note that the 35K and 22K samples were not reduced prior to gel electrophoresis. The link protein from rat chondrosarcoma has been shown to migrate either as a protein with an approximate M , = 35,000 to 38.000 (before reduction) or M , = 45,000 (after reduction) on the same gel (13). The 35K protein, but not the 22K protein, has been obtained directly from the aggregate of proteoglycan, link protein, and hyaluronic acid (Y. Oike. K. Kimata, T. Shinomura, S. Suzuki, N. Takahashi, and K. Tanabe, unpublished observations), suggesting that the 22K protein is derived from the 35K protein by cleavage with clostripain. FIG. 8. Tryptic peptide mapping of the hyaluronic acid-binding fragment and chondroitin sulfate-peptide clusters obtained from proteoglycan aggregates by clostripain digestion (Scheme 2) followed by gel electrophoresis (Fig. 7). The gel slices indicated by 250K (Trach b) and CPC (Track c ) in Fig. 7 were cut out and labeled with '"I, and tryptic peptides were displayed on silica gel thin layer plates by electrophoresis in the first dimension and ascending chromatography in the second dimension. Radioautographs are shown. FIG. 10. Two-dimensional pattern of a mixture of 1251-labeled tryptic peptides released from the gel slices of the hyaluronic acid-binding fragment (Fig. 7, Band 150K) and chondroitin sulfate-peptide clusters (Fig. 7, Band CPC). The combined sample was displayed on a silica gel thin layer plate by electrophoresis in the first dimension and ascending chromatography in the second dimension. Left, radioautograph; right, traced figure of the radioautograph. Spots that are numbered are clearly homologous in this map and the map of PG(-CS)AC or PC(-CS,KS) shown in Fig. 6.

roteoglycan Core Molecules 9757
Tryptic peptide mapping was performed with the gel slices of the hyaluronic acid-binding fragment (Band 150K, Track b) and chondroitin sulfate-peptide clusters (Band CPC, Track c). The radioautographs and traced figures are shown in Figs. 8 and 9. Because the maps of the 150K and chondroitin sulfate-peptide cluster fragments are quite different (of the 22 major spots, only three are common), it is apparent that there are considerable differences between these fragments in their polypeptide structures. However, comparison between the maps in Fig. 9 and the map of PG(-CS)A~ or PC(-CS,KS) in Fig. 6 indicates that there are many common spots between the intact core molecule and each of the clostripain fragments.
Of the 22 major spots detected in the map of PC(-CS,KS), 13 and 12 spots were produced from the hyaluronic acid-binding fragment and chondroitin sulfate-peptide clusters, respectively. Fig. 10 shows the result of mapping of a reconstructed core protein sample, in which the '2511-labeled peptides released from the 150K and chondroitin sulfate-peptide cluster gel slices were combined before mapping on the thin-layer plate. It is clear that the major spots (1 to 22) are homologous to those in the maps of the original core molecule (Fig. 5);' DISCUSSION The combined results of the variety of experiments described here and in the previous paper (8) constitute strong evidence that PC-H has a single, nonvariable core protein structure. In the previous study, the core fraction prepared from ['4C]serine-labeled PC-H by digestion with both chondroitinase-AC I1 and keratanase sedimented as a narrow band with a sedimentation coefficient of 6 S in velocity gradients.
In the present work, the use of SDS-polyacrylamide gel electrophoresis for resolution of core molecules and the tryptic peptide mapping procedure described by Elder et al. (16) permitted us to obtain further evidence to support the view that the core has a uniform structure. Thus, SDS-polyacrylamide gel electrophoresis of PC(-CS,KS) followed by Coomassie blue staining revealed a single narrow band (Fig. l). Radioiodination of the core molecule, followed by trypsin digestion, resulted in a highly reproducible peptide map (Fig.  5). Furthermore, clostripain cleavage of the proteoglycan into two size classes of fragments, followed by tryptic peptide mapping, indicated that, although the patterns of peptide spots of these fragments are different from each other (Fig.  8), the combined peptide patterns (Fig. 10) are very similar to those observed with the intact core molecules (Fig. 5). Should the core protein have been variable, the probability of obtaining such a combination of consistent results by chance would have been exceedingly small. Several recent reports have provided evidence that the newly synthesized core proteins of proteoglycans have a uniform size prior to the addition of glycosaminoglycan chains (19)(20)(21)(22). Since the unlabeled samples treated in the present work represent proteoglycan molecules accumulated in the cartilage matrix of 12-day chick embryos, our results suggest that there was no significant processing or degradation of the core protein during the time of accumulation of PC-H molecules in the matrix, although a highly selective cleavage of peptide bonds by specific peptidases could not be ruled out by the present data.
Since successful maps were obtained directly from the chondroitin sulfate-peptide clusters which were not treated with chondroitinase-keratanase and which did not penetrate the gel, it is expected that intact PC-H could be radioiodinated and mapped directly without recourse to either the enzyme digests or the SDS gel. Our preliminary experiments have indeed shown that the peptide map obtained directly from PC-H is very similar to that obtained from PC(-CS)Ac or PC(-CS,KS) (Y. Oike, K. Kimata, T. Shinomura, S. Suzuki, N. Takahashi, and K. Tanabe, unpublished observations).
The similarity of peptide maps of PG(-CS)AC and PG-(-CS,KS) (Fig. 5) suggests that the bound keratan sulfate groups do not significantly influence our analysis. This is consistent with the observations of Elder et al. (23) that carbohydrate-carrying peptides do not migrate in the apolar solvent used in the thin layer chromatography. Even after removal of the bulk of the glycosaminoglycan chains, the resultant core preparation, PG(-CS,KS), contains the residual linkage oligosaccharide regions of the digested chondroitin sulfate and keratan sulfate (8) and perhaps Nand 0-linked oligosaccharides (24). It should be stressed, therefore, that the peptide mapping technique may not detect variations of amino acid residues in carbohydrate-carrying peptide regions, although we do not have information concerning the precise distribution of the carbohydrate groups in the core molecule. Also to be considered is the fact that ' "1 reacts only with tyrosine, histidine, and phenylalanine (16), and therefore only the tryptic peptides containing these three amino acids could be visualized by radioautography. Thus, the extent of homology estimated from the number of '2sI-labeled tryptic peptides may not necessarily correspond to the extent of homology in the amino acid sequence. Despite these limitations, there is little doubt that only extensive homology in amino acid sequences gives extensive similarity in peptide maps. Two minor proteoglycan components (PG-Lt and PG-Lb), isolated from chick embryo cartilages (6), have recently been characterized by this mapping technique and further evidence for the uniqueness of their core proteins ~b t a i n e d .~ I t is likely that this technique will be useful for characterization of other proteoglycans in small amounts in tissues or in cell cultures.