identification of cyanogen bromide-fragments of the protein core of bovine nasal cartilage...

82 Biochimica et Biophysica Acta, 743 (1983) 82-90 Elsevier Biomedical Press

BBA31513

IDENTIFICATION OF CYANOGEN BROMIDE-FRAGMENTS OF THE PROTEIN CORE OF BOVINE NASAL CARTILAGE PROTEOGLYCAN MONOMER

F. BONNET, S. LE GLI~DIC, J.-P. PI~RIN, J. JOLLIES and P. JOLLES *

Laboratoire des Prot~ines, Universitb de Paris V (Unitb I N S E R M U-116 and E.R. CNRS No. 102), 45 rue des Saints-Pbres, F 75270 Paris Cedex 06 (France)

(Received August 23rd, 1982)

Key words: Cartilage; Proteoglycan; N-terminal sequence; CNBr fraction

Cyanogen bromide treatment of bovine nasal cartilage proteoglycan monomer gave rise to three major fractions (CN-1 to CN-3), isolated by Sepharose CL-6B chromatography. The uronate-rich fraction in the void volume (CN-1) digested with chondroitinase ABC (C treatment) yielded a fragment (CN-I C/6B) with a unique N-terminal sequence. The same fraction, when digested sequentially by chondroitinase ABC and trypsin (CT treatment), was resolved into two distinct fractions, CN-1 CT/6B-1 and CN-1 CT/6B-2. CN-I CT/6B-1 consisted in a keratan sulfate-rich region, representing the N-terminal moiety of the CN-I fraction; these data suggested, according to the model of the proteoglycan monomer structure described by Heinegard, D. and Axelsson, I. (1977) J. Biol. Chem. 252, 1971-1979, that its C-terminal moiety is localized at the end of the core bearing the chondroitin sulfate chains. CN-I CT/6B-2 contained two fragments from the chondroitin sulfate-bearing region: one of them has been submitted to Edman degradation. The CN-2 fraction upon cbondroitinase and trypsin treatments gave rise to a keratan-bearing region (CN-2 CT/6B-I) and a mannose-rich region (CN-2 CT/6B-2). After reduction and alkylation of CN-2, the N-terminal sequence of the isolated major fragment (CN-2 RA/6B-1) was determined. The CN-3 fraction revealed a pattern upon electropboresis similar to that of the cyanogen bromide-treated hyaluronic acid-binding region.

Introduction

The proteolycan monomer molecules, isolated from hyaline cartilage, are composed of a central protein core, constituted of three different major regions, the chondroitin sulfate- and keratan sulfate-enriched regions and the hyaluronic acid- binding region [1]. The polydispersity of these macromolecules is attributed to the variable length of the chondroitin sulfate chain enriched region [2-4]. This schematic representation is due to data mainly obtained after different enzyme treatments of these monomers [1,5].

* To whom correspondence should be addressed.

0167-4838/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

In this study, the major fragments from proteoglycan monomers, obtained after cyanogen bromide cleavage, were characterized and localized. A first series of data concerning their N-terminal sequences before or after chondroitinase-trypsin digestion are also reported.

Materials and Methods

Reagents. Cyanogen bromide, benzamidine hydrochloride hydrate, Suprapur cesium sulfate and Suprapur cesium chloride were from Merck. Guanidine hydrochloride was from Farmitalia. 6- Aminohexanoic acid, ethylenediaminetetraacetic acid (disodium salt), diphenyl carbamylchloride- treated trypsin (EC 3.4.21.4) and chondroitinase

83

ABC (EC 4.2.2.4) were from Sigma and hydrogen fluoride/pyridine from Fluka. Sepharose CL-6B and Sephacryl S-200 were from Pharmacia. All other reagents (analytical grade) were from Pro- labo, Labo Disc or Canalco.

Analytical procedures. Protein was determined by absorption measurements at 280 nm, and uronate by the carbazol procedure [6] with o- glucuronolactone as standard: no correction has been made for reaction of other sugars with this reagent. Analytical Cs2SO 4 density gradient centrifugation was performed in 0.05 M sodium acetate buffer, pH 5.8 [7]. Sodium dodecyl sulfate-polyacrylamide gel electrophoreses were performed according to Laemmli [8] (12% and 6% polyacrylamide, pH 8.9); protein bands were stainded with R-250 Coomassie brilliant blue. Amino acid and sugar analyses were performed as previously described [7]. Automated Edman degradation was carried out in a 890 C Beckman Sequencer by the 1 M quadrol single-cleavage method; the phenylthiohydantoin-amino acids were characterized by thin-layer chromatography (chloroform/methanol, 90: 10, v/v; pure chloroform) and by high-performance liquid chromatography (Waters-chromatograph, model ALC/GPC 204) on a 30 cm Waters #-Bondapak C18 column.

Preparative procedures Preparation of proteoglycan monomers. Proteo-

glycans were sequentially extracted as previously described [7]. The proteoglycan complex (A1KC1- guanidine-HC1) (O > 1.7 g/ml) was obtained after CsC1 density gradient centrifugation of the KCI- guanidine-HC1 extract. The proteoglycan monomers were isolated after CsC1/4 M guanidine-HC1 dissociative density gradient centrifugation of the proteoglycan complex.

Isolation of CN fragments from monomers (Scheme I). Samples of proteoglycan monomers (2 mg uronate/ml) were dissolved in 70% formic acid containing 2% cyanogen bromide (w/v) and held for 24 h at room temperature in the dark with continuous stirring. The samples were then diluted with 3 volumes of 30% acetic acid and con- centrated; they were submitted to chromatography on Sepharose CL-6B under dissociative conditions (4 M guanidine-HCl/0.05 M sodium acetate, pH 5.8).

For the study devoted to N-terminal sequence determinations, a large amount of cyanogen bromide-treated monomers (230 mg uronate equivalent) was submitted to associative CsC1 density gradient centrifugation (p = 1.7 g/ml, 35000 rpm, 48 h at 20°C). The gradients were collected in two equal parts: the lower part (p > 1.7 g/ml) was the A1 fraction, and the upper part (p < 1.7 g/ml) the A2 fraction. Only the A2 fraction was submitted to Sepharose CL-6B chromatography.

Isolation of chondroitinase ABC and trypsin- treated CNfragments. All chondroitinase ABC and trypsin digestions were performed at 37°C for 6 h in 0.1 M sodium acetate/0.1 M Tris-HC1, pH 7.3 buffer. The CN-fragments obtained from proteoglycan monomers were treated under identical conditions with 1 unit of chondroitinase ABC and 120 /~g trypsin/12 mg uronate equivalent. The resulting substances were isolated by Sepharose CL-6B chromatography.

Isolation of deglycosylated CN fragments after hydrogen fluoride (HF)/pyridine treatment. The larger reduced and alkylated CN-2 fragment (CN-2 RA/6B-1) and the keratan sulfate-enriched region from CN-1 (CN-1 CT/6B-1) were treated by HF/pyridine as described by Coudron et al. [9]. The resulting compounds were dialyzed, con- centrated and purified by Sepharose CL-6B gel chromatography.

Results and Discussion

Characterization of CN fragments from proteoglycan monomers (Scheme I)

Chromatography on Sepharose CL-6B. When cyanogen bromide-treated monomers (20 mg uronate equivalent) were chromatographed under dissociative conditions on Sepharose CL-6B, the elution profile (Fig. 1) showed three peaks: a uronate-rich component in the void volume (CN-I) and two peaks characterized by their absorption at 280 nm (CN-2 and -3). Analytical data for amino acids and hexosamines are given in Table I. The CN-1, CN-2 and CN-3 fractions contained 50, 30 and 20% of the total recovered proteins, respectively.

The amino acid composition of CN-1, with high serine, glutamic acid and glycine contents, was characteristic of a chondroitin sulfate-enriched re-

84

[ associative Cs2SO 4 gradient (Fig. 2)

Proteoglyean monomers I

cyanogen bromide

I ] associative CsC1 gradient *

Sepharose CL-6B chromatography (Fig. 1)

[ l void volume included fractions

CN-1 CN-2 CN-3

T I chondroitinase ehondrolt chondroitinase- reduction and

l trypsin l trypsin ~ alkylation

Sephaxose CL-6B ehromato- Sepharose CL-6B chroma- Sei~harose CL-6B Sepha'rose CL-6B graphy (CN-1 C/6B) tography (F i~ 4) chromatography chromatography (Fig. 3) I (Fig. 5) (Fig. 6)

ON-1 CT/6B-1 ON-1 CT/eB-2 ON-2 RA/6B-1

HF treatment HF t atment

SDS-polyacrylaz~nide gel electro- SDS-polyacrylamide !el electrophoresis (Fig. 7) phoresis (Fig. 7).

Scheme I. Cyanogen bromide fragmentation of proteoglycan monomers followed by enzymic digestions. * Associative CsCl gradient was only used for the purification of a large amount of CN fragments during the study devoted to N-terminal sequence analysis.

gion [10]; however, its high proline content also suggested the presence of a keratan sulfate- enriched region [11]. A low tyrosine content was observed in this fraction when compared to CN-2 and CN-3 fractions.

CN-1 CN-2 CN-3 t = t i = =

0.8 0.3

o.e .o 0.2

¢,1 0.4 < E

} /""

50 100 150 200 Vo Fractions ( 2.2 ml) Vt

Fig. I. Sepharose CL-6B gel f i l t rat ion (200 x 2.5 cm column) of the cyanogen bromide-treated proteoglycan monomers. Eluent: 0.05 M sodium acetate buffer, pH 5.8, containing 4 M guanidine hydrochioride. - - , uronate; . . . . . . , A2s o.

TABLE I

A M I N O ACID (RESIDUES/1000 RESIDUES) AND AMINO SUGAR (nmol /mg PROTEIN) COMPOSITIONS OF CN FRACTIONS FROM PROTEOGLYCAN MONO- MERS

Amino acids Fractions and amino sugars

CN- 1 CN-2 CN-3

Asp 74 80 98 Thr 46 85 59 Ser 141 65 76 Glu 141 137 112 Pro 123 92 79 Gly 131 93 78 Ala 43 92 84 Val 73 63 68 Ile 40 32 58 Leu 80 73 86 Tyr 6 37 49 Phe 44 55 23 Lys 23 17 32 His 14 13 35 Arg 21 66 63

GalNAc 23600 1200 6500 GIcNAc 1800 2000 700

A

E O~

E

0

o C- D

I O a0

<

B

1.0

0.5

\

\

X

1.7

1.6 " " E

1.5 a

i I I

1.4 I

5 10 15

F r a c t i o n s ( 2 m l )

Fig. 2. Distribution of CN fragments from proteoglycan monomers after centrifugation in CszSO 4 density gradient. (A) Cs2SO 4 density gradient (p--l.5 g/ml) of the cyanogen bromide-treated proteoglycan monomers. (B) Analytical 1270 (gels a, b, c) and 670 (gels a', b', c') SDS-polyacrylamide gel electrophoresis of the protein-enriched fractions (15-20, # <

85

The amino acid compositions of CN-2 and CN-3 were similar, the only differences concerned the higher proportion of glutamic acid in peak 2 and an inverse tyrosine/phenylalanine ratio.

When cyanogen bromide-treated monomers were submitted to CsC1 density gradient centrifugation before Sepharose CL-6B chromatography (preparative scale preparation), the A1 fraction contained the CN-1 compound and the A2 fraction the CN-2 and -3 components.

Density gradient centrifugation in Cs2SO 4. The cyanogen bromide-treated monomers (10 mg uronate equivalent) were submitted to a Cs2SO 4 density gradient centrifugation (p --- 1.5 g/ml). The majority of uronate banded at 1.55 g /ml (Fig. 2A), while untreated monomers banded at a lower density [7]. 50% of proteins (A280nm) initially banding with the untreated monomer were recovered in fractions of density below 1.43 g /m l (upper part of the gradient). This protein-enriched fraction was submitted to SDS-polyacrylamide gel electrophoresis (Fig. 2B). Several bands whose mo- bilities were similar to those of the cyanogen bromide-treated hyaluronic acid-binding region [12] were detected. A large amount of material remained, however, at the top of the 12% polyacrylamide gel and hardly penetrated in the 6% polyacrylamide gel. These two series of penetrating bands corresponded to the substances contained in the CN-3 and CN-2 fractions, respectively, characterized on Sepharose CL-6B in the preceding paragraph (data not shown).

Enzymic treatment of CN-1 and CN-2 fractions. The CN-1 fraction digested with chondroitinase ABC was chromatographed on Sepharose CL-6B (Fig. 3). One major peak (CN-1 C/6B) was recovered; this same CN-1 fraction when sequentially treated with chondroitinase ABC and trypsin gave rise, after chromatography on Sepharose CL- 6B, to two distinct peaks: CN-1 CT/6B-1 and CN-1 CT/6B-2 (Fig. 4), characterized by 280 nm absorbance and uronate determination, respectively; the A280 peak at the V 0 level contained less

1.52 g /ml ) in the absence (gels a and a') and in the presence (gels b and b') of 570 2-mercaptoethanol. The gels c and c' contained the protein markers (i , bovine serum albumin; 2, aldolase; 3, myoglobin) in the presence of 2-mercaptoethanol.

86

0.030

0.015

,,

so~ vo

CN-I C/r~

,

'i i / ' " I t , ',

I O 0

Fractions 0 .65 ml )

8

40

2 0 ~

I 150

vt

Fig. 3. Sepharose CL-6B gel filtration (250× 1.15 cm column) of the chondroitinase ABC-digested CN-I fraction. Eluent: 0.05 M sodium acetate buffer, pH 5.8, containing 4 M guanidine hydrochloride.

than 1% of the total protein. The amino acid composition of these two fragments were determined (Table II). These two fragments contained 33 and 46% of the total proteins of the CN-1 fraction, respectively. Peak CN-1 CT/6B-1 contained high quantities of glutamic acid, proline, phenylalanine and lysine, whereas aspartic acid, histidine and arginine contents were low and tyrosine present only in trace amounts. Fraction CN-1 CT/6B-2 characterized by the remaining uronate, contained large amounts of serine, glutamic acid and glycine, representing 50% of the total amino acids, reflecting a chondroitin sulfate- enriched region [10]. The amino acid compositions of these two fractions were similar to the keratan sulfate- and chondroitin sulfate-enriched regions, respectively, described by Heinegard and Axelsson [1 ], obtained after chondroitinase-trypsin digestion of the monomer.

The chondroitinase ABC- and trypsin-treated

TABLE II

A M I N O ACID (RESIDUES/1000 RESIDUES) A N D S U G A R ( n m o l / m g PROTEIN) COMPOSITION OF CHONDROITINASE-TRYPSIN-TREATED CN- 1

n.d., not determined.


CN-1 CT/6B-I CN-1 CT/6B-2

Asp 22 63 Thr 53 34 Ser 122 157 Glu 184 147 Pro 239 100 Gly 91 173 Ala 44 57 Val 43 79 Ile 27 44 Leu 49 92 Tyr 2 2 Phe 75 14 Lys 36 4 His 7 15 Arg 6 19

Xylose a n.d. n.d. Mannose 185 n.d. Galactose 4 800 2 700 GIcUA 900 1 700 GalNAc 1630 1440 GlcNAc 4 000 650 NeuNAc 500 n.d.

" The presence of an artefact at the level of xylose prevents this sugar determination in the cyanogen bromide-treated fractions.

(~o4

< i oo2 , !

i /

Vo

i / i

100 150

Fr~ct ions ( 1,5 m l )

Fig. 4. Sepharose CL-6B gel filtration (250x 1.15 cm column) of the chondroitinase ABC-trypsin-digested CN-i fraction. Eluent: 0.05 M sodium acetate buffer, pH 5.8, containing 4 M guanidine hydrochloride.

0 .04

<, ; 0 . 0 2

C,N- 2 C T/(SB~? CN-2 cr/~-2 CN- 2 CT/~3B- 3

5O~vo 1~o

/ \ / / /

/ /

A / /

q3o Fract ions (1.5 ml )

o 1'2o "~

4) "6 60 c 8

j i ~vt

Fig. 5. Sepharose CL-6B gel filtration (250 × 1.15 cm column) of the chondroitinase ABC-trypsin-digested CN-2 fraction. Eluent: 0.05 M sodium acetate buffer, pH 5.8, containing 4 M guanidine hydrochloride.

CN-2 fraction was chromatographed on Sepharose CL-6B (Fig. 5), and two fractions according to their galactose and glucosamine contents appeared to be keratan-enriched fragments (Table III): CN-2 CT/6B-1 and CN-2 CT/6B-2; they contained 10-15% and 35% of the total proteins present in the CN-2 fraction, respectively. The CN-2 C T/ 6B - 1 fraction whose Kay on Sepharose CL-6B was identical to that of the keratan sulfate-enriched fragment from CN-1 (CN-1 C T / 6 B - I ) presented amino acid and sugar compositions (Table III) similar to the latter (Table II). The CN-2 CT/6B-2 fraction whose Kav was identical to the chondroitin sulfate-bearing region from CN-1 (CN-1 CT/6B-2) presented an amino acid composition

TABLE III

AMINO ACID (RESIDUES/1000 RESIDUES) AND SUGAR (nmol/mg PROTEIN) COMPOSITIONS OF CHONDROITINASE-TRYPSIN-TREATED CN-2

n.d., not determined.


CN-2 CN-2 CN-2 CT/6B-I CT/6B-2 CT/6B-3

Asp 18 62 82 Thr 52 81 75 Ser 105 77 68 Glu 191 170 164 Pro 258 121 112 Gly 78 92 105 Ala 49 97 112 Val 51 84 63 Ile 27 33 24 Leu 41 56 73 Tyr 2 9 24 Phe 72 57 29 Lys 41 10 9 His 9 11 18 Arg 6 40 42

Xylose a n.d. n.d. n.d. Mannose trace 1 100 635 Galactose 7 970 6 235 1910 GIcUA 2260 1260 trace GalNAc 2 170 1440 510 GIcNAc 5 750 4 720 1510 NeuNAc 1 440 1100 310

a The presence of an artefact at the level of xylose prevents this sugar determination in the cyanogen bromide-treated fractions.

87

strikingly different from the keratan sulfate-enriched regions already described (CN-1 C T / 6 B - I and CN-2 CT/6B-1) (Tables II and III). A high mannose content appeared to be characteristic of the CN-2 CT/6B-2 fraction. Several laboratories have suggested that mannose is an integral part of the keratan sulfate structure [13,14]: recent data indicated that, at least for a part, mannose-rich structures were involved in N-linked oligosaccharides [ 15,16].

Electrophoretic studies of the higher molecular weight CN fragments after HF treatment. Upon reduction and alkylation, the CN-1 CT/6B-1 fraction did not liberate any penetrating band on analytical SDS-polyacrylamide gel electrophoresis. On the other hand, under similar treatment CN-2 gave rise to a major fragment of 150 + 50 kDa (value estimated by 3% SDS-polyacrylamide gel electrophoresis; not shown) with two minor components of lower molecular weights. The 150 kDa fragment was purified by Sepharose CL-6B chromatography (Fig. 6A and B); it was recovered in the first large peak (CN-2 RA/6B-1). The O-linked carbohydrates of CN-2 RA/6B-1 and CN-I CT/6B-1 fractions were removed by H F treatment [9]. After purification on Sepharose CL-6B, a single peak was recovered from both fractions. 75% and 95% of the proteins were recovered from CN-1 CT/6B-1 and CN-2 RA/6B-1 after this procedure, respectively. The apparent molecular weights of the HF-treated CN-1 CT/6B-1 and CN-2 RA/6B-1 fractions were estimated at 80000 + 10000 and 50000 ___ 10000 (mean values), respectively (Fig. 7). The amino acid compositions were identical to those of the untreated fragments. The hexosamines of the HF-treated CN-2 RA/6B-1 fraction were completely removed, when for fraction CN-I C T / 6 B - I the major part of glucosamine was removed, whereas 45% galactosamine remained. The presence of the remaining sugars in the HF-treated CN-1 CT/6B-1 fraction probably contributes to an overestimation of its molecular weight by SDS-polyacrylamide gel electrophoresis.

N-terminal sequences of CN-fragments from proteoglycan monomers (Table IV)

The fragments submitted to N-terminal sequence studies were prepared from 230 mg (uronate amount) proteoglycan monomers. After sequential

88

o..~

/

vo

cN-2 RA/~e-1 C~-Z RA/ee.2 CN-2 RA/ee 3

:!

i "-'"-"'.--__../ "\,.._./'i "X

~00 Free,ions (215 ml) Vt

a a' b b" c

Fig. 7. Analytical 8% SDS-polyacrylamide gel electrophoresis of the CN-! CT/6B-I (gels a and a') and the CN-2 RA/6B-I (gels b and b') fractions of the proteoglycan monomers, before (gels a and b) and after (gels a' and b') HF/pyridine treatment. Gel c contained the protein markers (see legend to Fig. 2).

Fig. 6. (A) Sepharose CL-6B filtration (250x 1.15 cm column) of the reduced and alkylated CN-2 fraction. Eluent: 0.05 M sodium acetate buffer, pH 5.8, containing 4 M guanidine hydrochloride. (B) Analytical 10% SDS-polyacrylamide gel electrophoresis of the reduced and alkylated CN-2 fraction after gel filtration. Gels a to c contained the fractions CN-2 RA/6B-I to CN-2 RA/6B-3, respectively. Gel d contained the protein markers (see legend to Fig. 2).

extraction, the proteoglycan monomer fraction isolated by 4 M guanidine-HCl had a uronate/protein ratio of 2.9; this value is closely related to that already published by Mason and Mayes [17], using the same sequential extraction procedure. Thus this proteoglycan monomer fraction contained ap- proximately 80 mg proteins. The yields of the various substances are indicated in Scheme II.

The N-terminal sequences of the chondroitinase ABC-treated CN-1 fraction (CN-1 C/6B) and of Its keratan sulfate-enriched region (CN-1 CT/6B- 1) were identical. According to the scheme proposed by Heinegard and Axelsson [1] for the proteoglycan monomer, indicating that the chondroi-

tin sulfate-bearing region was situated at one end of the monomer, the present data suggest that the C-terminal part of the whole molecule is located towards this chondroitin sulfate-bearing region. On dansylation the chondroitin sulfate-bearing region (CN-1 CT/6B-2) gave rise to two N-terminal amino acids: isoleucine and leucine. After chromatography on Sephacryl S-200 we purified a peptide with leucine as the unique N-terminal amino acid (data not shown), and a long N-terminal sequence (26 amino acids) was characterized. In positions 4 and 5 an inhomogeneity was observed, as a small quantity of glutamic acid was determined beside glycine and serine, respectively. Isemura and Ikenaka [18] determined short N- terminal sequences of pronase peptides extracted from bovine tracheal cartilage which were claimed to be linked to the chondroitin sulfate chain; others [19] described short peptides from bovine nasal proteoglycan monomer involved in chondroitin sulfate linkages. The major part of these peptides was present in our sequence, which in addition contains a repetition (residues 1 to 7 and 20 to 26).

The N-terminal sequences of the CN-2 RA/6B-

TABLE IV

N-TERMINAL SEQUENCES OF CN-FRACTIONS FROM PROTEOGLYCAN MONOMERS

X, as yet uncharacterized amino acid.

89

Fractions N-terminal sequences

l CN-1 C/6B Val- X-

1 CN-I CT/6B-l Val- X-

CN-1 CT/6B-l

after HF treatment

CN-1 CT/6B-2

(repurified, see text)

CN-2 RA/6B-I

CN-2 RA/6B-I

after HF treatment

IO Gln-Val-Gly-Pro-Gly-Val-Ala- Ala -Val-Pro-

10 Gln-Val-Gly-Pro-Gly-Val-Ala- Ala -Val-Pro-

1 10 20 Val -Thr a-Gln-Val-Gly-Pro-Gly-Val- Ala- Ala -Val-Pro-Ile-Gly-Glu-Glu-Thr-Thr-Ala- Ile-Pro-

3o Gly-Phe- Thr-Val-Glu-Pro-Glu-Glu- Lys -

I I0 20 Leu-Pro-Ser-(~l~ }-(s~ }-Gly-Pro-Glu-Val- Ser -gla-Ser-Gly-Val-Glu-Asp-Leu-Ser-(V~ 1)- Leu-Pro-

26 { s~r )-GIy-GIu-GIy- Pro -

I 7 Ser-Ser-Ala-Giy-Trp-Leu- Ala -

1 7 Ser-Ser-Ala-Gly- X-Leu-Ala-

a Only detected after HF treatment.

Proteoglycan m o n o m e r

(80 mg protein) l

CN-1 C N - 2 4 0 m g (a ) 2 4 m g (a )

I f chondroitinase- reduction and trypsin alkylafion

1 1 3 . 2 m g ( a ) 1 8 . 4 m g (a ) 2 . 4 m g (¢ )

7.5 mg (b, c) ~ 10.0 mg (b)

1 2 . 1 5 m g ( c ) ~ - ~

Scheme II. Yields of the fractions submitted to N-terminal sequence studies. (a) Amounts inferred from the analytical scale preparation (Fig. I). (b) Amounts effectively recovered during the preparative scale purification. (c) Amounts of fragments with a unique N-termlnal amino acid used for sequence studies. N-terminal amino acids are boxed.

1 samples s tud ied b e f o r e a n d a f t e r H F t r e a t m e n t

w e r e iden t i ca l e x c e p t fo r the t r y p t o p h a n res idue

( N o . 5), wh ich was n o t d e t e c t e d in the H F - t r e a t e d

f rac t ion .

C o n c l u s i o n

T h e s e p a r a t i o n a n d m o l e c u l a r c h a r a c t e r i z a t i o n

o f h igh m o l e c u l a r we igh t C N - f r a g m e n t s f r o m the

90

proteoglycan monomers are described in this study. The first fragment (CN-1) upon chondroitinase- trypsin treatment was cleaved into keratan sulfate- and chondroitin sulfate-bearing regions. A keratan sulfate-bearing region (CT- 1 CT/6B- 1) constituted the N-terminal part of the CN-1 fraction. This observation strongly suggests that the C-terminal end of the protein core of the proteoglycan monomer is the chondroitin sulfate-bearing region; con- sequently the hyaluronic acid-binding region might constitute its N-terminal end. In accordance with Keiser et al. [20], isoleucine and leucine were characterized as N-terminal amino acids of the chondroitin sulfate-bearing region (CN-1 CT/6B- 2). The repetitive sequence occurring in this fraction might reflect repeating sequences in the chondroitin-bearing region of the monomer according to a proposed model in a proteoglycan molecule [10]. The tryptic fragments (CN-1 CT/6B-1 and 6B-2) from the CN-1 fraction presented features (Kay and amino acid compositions) similar to those of the tryptic fragments from proteoglycan monomers previously described by Heinegard and Axelsson [1]. Upon chondroitinase-trypsin treatment the CN-2 fraction gave rise to beside a keratan-enriched fragment (CN-2 CT/6B-1), a mannose-rich fraction (CN-2 CT/6B-2). Lohmander et al. [16] localized mannose-rich oligosaccharides in a hyaluronic acid-binding region fragment from Swarm rat chondro- sarcoma proteoglycans. Thus the CN-2 CT/6B-2 fraction might be located in the hyaluronic acid- binding region vicinity.

Acknowledgements

The authors thank Miss. M. Rougeot and Mr. Ly Quan Le for their skilful technical assistance.

This research was supported by the INSERM (Unit6 U-116 and C.R.L. No. 82.3027), the CNRS (E.R. No. 102) and the Fondation pour la Re- cherche Mrdicale Franqaise.

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and Caplan, A.I. (1980)J. Biol. Chem. 255, 6077-6083 16 Lohmander, L.S., De Luca, S., Nilsson, B., Hascall, V.C.,

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identification of cyanogen bromide-fragments of the protein core of bovine nasal cartilage...

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