57

Biochimica et Biophysica Acta, 623 (1980) 57--68 © Elsevier/North-Holland Biomedical Press

BBA 38411

PROTEOGLYCAN COMPLEX AND PROTEOGLYCAN SUBUNIT POLYDISPERSITY

STUDY BY ISOPYCNIC CENTRIFUGATION IN CESIUM SULFATE DENSITY GRADIENTS

F. BONNET, J.-P. Pi~RIN and P. JOLLI~S *

Laboratory of Proteins, University of Paris V, 45, rue des Saints-Pbres, 75270 Paris Cedex 06 (France)

(Received July 31st, 1979)

Key words: Proteoglycan; Isopycnic centrifugation; Link protein; (Bovine nasal cartilage)

Summary

A true isopycnic centrifugation method for the study of the bovine nasal cartilage proteoglycan polydispersity is presented. The use of cesium sulfate as gradient forming salt instead of cesium chloride allowed proteoglycan banding without any sedimentation at the bottom of the centrifuge tube. Apparent buoyant densities of proteoglycan monomer and proteoglycan aggregate were different. The present method provides a useful tool for the study of proteo- glycan polydispersity and also allows us to follow the distribution of the link proteins in different proteoglycan extracts.

Introduction

The current model for hyaline cartilage proteoglycan monomer consists of a protein core bearing chondroitin sulfate and keratan sulfate side chains [1,2]. A protein rich region almost devoid of these sugar chains has been described as being responsible for the interaction of the monomer with hyaluronic acid to form the aggregated proteoglycan [3,4] stabilized by the link proteins [5,6]. In addition it has been suggested that 15--20% of the monomers in normal carti- lage were of smaller size than the average and were unable to interact with hyaluronic acid [7,8]. They were recovered in a low salt extract whereas the aggregates could only be extracted with high molarity of chaotropic solvents

* To whom correspondence should b e addressed .

58

such as guanidinium hydrochloride (Gdn. HC1) [8,9]. These data have been established with materials prepared by a now classical method using cesium chloride density centrifugation as initially described by Franek and Dunstone [10] and further developed by Hascall and Sajdera [11]. Unfortunately the apparent densities of the aggregates, monomers or low salt extracted proteo- glycans were so high in the presence of CsC1 that they concentrated for their major part at the bottom of the tube. Thus, this method did not allow a complete study of the proteoglycan polydispersity mainly due to the number and the length of the chondroitin sulfate chains [12]. Subfractionation of monomers in such a system was reported by Heineg~rd [13] but more than half of the uronate still sedimented in the densest fraction.

The differentiation of aggregates, monomers and low salt extracted proteo- glycans as a function of their buoyant densities has never been possible. Fol- lowing the data of Hearst et al. on DNA [14--16], Mashburn et al. [17] indi- cated in 1974 that hyaluronate, proteoglycan and related molecules have much lower densities in Cs2SO4 than in CsC1. The present paper deals with the use of cesium sulfate as gradient forming salt for the study of the proteoglycan poly- dispersity and the components distribution in the aggregate.

Materials and Methods

Reagent. Guanidinium hydrochloride (Gdn. HC1) was from Carlo Erba, guanidinium sulfate from Fluka, cesium chloride, cesium sulfate and benzami- dine hydrochloride hydrate were obtained from Merck, 6-aminohexanoic acid and ethylenediamine tetraacetic acid (disodium salt) from Sigma. Electropho- retic reagents were purchased from Canalco or Labo-Disc and all other reagents (analytical grade) were from Prolabo.

Analytical procedures. Proteins were determined by absorption measure- ments at 280 nm, uronate was determined by the carbazol procedure [18] with D-glucuronolactone as standard; no correction has been made for reaction of other sugars with this reagent. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoreses were performed according to Laemmli [19] (10% polyacryl- amide, pH 8.9); protein bands were stained with E-250 Coomassie brilliant blue and the gels were scanned at 550 nm by using a Beckman gel scanner. Gas chromatography was used for sugar analyses; for this purpose samples were treated according to Clamp [20] with 1.5 N HC1 in methanol. A Hewlett Packard (type 5710 A) gas chromatograph, equiped with a hydrogen flame ionization detector, was used for all analyses. Samples were assayed as trimeth- ylsilyl derivatives by continuous temperature programming (2°C/min)from 110--220°C; the temperature was maintained for 8 min at l l 0 ° C at the beginning and for 32 min at 220°C at the end of every analysis; nitrogen was used as carrier gas. Amino acid compositions were determined after total acid hydrolysis (HC1 6 N, 18 h, l l0°C, 0.2% 2-mercaptoethanol, under vacuum) with a Technicon amino acid Autoanalyzer.

Preparative procedures. Bovine nasal septa were obtained immediately after slaughter, dissected free of adhering tissues and ground to powder with a IKA.A.10 S mill (Janke and Kunkel) in liquid nitrogen (three grindings of 5 s interrupted by liquid nitrogen addition). The powder was then stored at--30°C

59

until use. Proteoglycans were extracted by stirring the powder (3.5 g) for 24 h at 4°C in different media in the presence of protease inhibitors {0.05 M benzamidine hydrochloride, 0.01 M ethylenediamine tetraacetate and 0.01 M 6-aminohexanoic acid); 15 vols of buffer were used per g of cartilage powder. The soluble extracted proteoglycans were recovered by centrifugation of the mixture for 1.5 h at 11 000 rev/min at 4°C in a Sorvall RC5 centrifuge (Rotor GSA) and exhaustively dialysed against distilled water containing the protease inhibitors. Directly Gdn. HCl-extracted proteoglycans were obtained by the action of 4 M Gdn • HC1 in 0.05 M sodium acetate, pH 5.8 buffer. Sequential extraction of the proteoglycans was performed by the action of 0.15 M KC1 in 0.05 M Tris-HC1, pH 7.0 buffer followed by the action of 4 M Gdn • HC1 in the 0.05 M sodium acetate, pH 5.8 buffer; this procedure provided the KC1- and KC1-Gdn • HCl-extracted proteoglycans respectively. All these substances were purified by CsC1 density gradient centrifugation under associative conditions (0.4 M Gdn • HC1, 0.05 M sodium acetate, pH 5.8 buffer, containing the pro, tease inhibitors). Centrifugation was carried out for 60 h at 15°C, 87 000 × g in a Beckman L2 65 B centrifuge (60 Ti rotor) with an initial density of 1.56 g/ml. The bottom fractions (density greater than 1.62 g/ml) yielded the Al fractions used for cesium sulfate density gradient centrifugation experiments. Monomers were directly prepared by submitting an aliquot part of the Gdn-HC1, KC1 and KC1-Gdn. HC1 .extracts t o CsC1 density gradient under dissociative conditions (4 M Gdn • HC1, 0.05 M sodium acetate, pH 5.8 buffer, initial density 1.50 g/ml) in the presence of the protease inhibitors. Centrifuga- tions were carried out as above; the bottom fractions (D1, density greater than 1.50 g/ml) yielded the Gdn • HC1, KC1 and KC1-Gdn • HC1 monomers respec- tively, which were exhaustively dialysed against distilled water. We want to point out that, during these various preparative steps, freezing or concentration of the samples was avoided and that no precipitation occurred in the fractions after CsC1 density gradient centrifugation.

Cs2S04 density centrifugations. Every proteoglycan preparation (directly Gdn .HCI-, KC1- and sequentially KC1-Gdn .HCl-extracted) isolated as described above was submitted to isopycnic centrifugations without any pro- tease inhibitor. The experimental conditions were chosen from preliminary results, in order to avoid the accumulation of the proteoglycans at the bottom of the tube in Cs2SO4 gradients (see Results). The proteoglycan concentration was roughly 0.5 mg/ml expressed as uronate. Every centrifugation was per- formed at 87 000 × g with a Beckman 60 Ti rotor for 60 h at 18 ° C. 23 frac- tions of 1.5 ml were collected from the bottom of the tubes and analysed for uronate and protein contents.

Results

Isolation of the A1 and D1 fractions Sequential extraction of the proteoglycans was carried out in order to char-

acterize aggregating (KC1-Gdn. HCl-extracted) and non-aggregating proteo- glycan~ (KCl-extracted). The direct dissociative extract (Gdn-HC1 extract) reflected the results observed during the study of KC1- and KC1-Gdn- HC1- extracted proteoglycans.

In the sequential extraction, KC1- and KC1-Gdn. HCl-extracted proteogly-

60

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61

cans contained 32 and 68% of the total extracted uronate. The uronate recov- eries in every A~ and D, fraction from the KCI-, KC1-Gdn • HC1- and Gdn • HC1- extracted proteoglycans when purified by CsC1 density gradient centrifugation (associative and dissociative conditions) were 90.2, 83.4 and 86.0% for A, and 98.0, 90.5 and 92.2% for D~ fractions, respectively.

Distribution of the proteoglycans in non dissociative Cs2S04 density gradients A, fractions. KCI-, KC1-Gdn- HC1- and Gdn-HCl-ex t rac ted proteoglycan

A1 fractions, when submit ted to Cs2SO4 density gradient centrifugation, in the absence of any dissociative agent such as guanidinium salts, were able to band in the centrifuge tube. Centrifugations were carried out in a 0.05 M sodium acetate, pH 5.9 buffer containing Cs2SO4 (0.635 g/ml) providing a density of 1.53 g/ml. The majori ty of the uronate of G d n . HC1- as well as of KC1- Gdn • HCl-extracted proteoglycans banded at a density of 1.50 g/ml, whereas for KCl-extracted proteoglycans it was at a higher density (1.58 g/ml) (Fig. 1). A faint uronate-rich denser material was present in both sequentially (KC1- G d n . HC1-) and directly (Gdn . HC1-) extracted proteoglycans (slightly more abundant in directly (Gdn • HCl-extracted ones). In directly (Gdn • HC1-) and sequentially (KC1-Gdn. HC1-) extracted proteoglycans a slight shift between uronate and protein distributions was noted: this observation was much more obvious for KCl-extracted proteoglycans.

DI fractions. 90, 83 and 91% of the uronate were present in the fractions with density values situated between 1.48 and 1.58 g/ml for directly (Gdn • HC1-), KC1- and sequentially (KC1-Gdn. HC1-) extracted proteoglycan mono- mer preparations, respectively. For directly (Gdn • HC1-) and sequentially (KC1- Gdn • HC1-) extracted proteoglycan monomer populations the apparent buoy- ant densities of the uronate containing material had higher values than those of the homologous proteoglycans recovered in the A~ fraction and slightly lower ones than those of the KCl-extracted proteoglycans A~ (Fig. 1).

Distribution of the link proteins in the At fractions The distribution of the link proteins was followed by submitting an aliquot

part of the 23 fractions recovered after Cs2SO4 density gradient centrifugation of the A~ fractions from Gdn • HC1, KC1 and KC1-Gdn • HC1 extracts to analyti- cal SDS polyacrylamide gel electrophoresis. The percentage distribution of the link proteins apparently followed the protein distribution in every gradient wi thout taking into account the presence of the proteoglycan core (Fig. 1). In the case of KCl-extracted proteoglycans the link proteins were predominantly characterized in the protein rich region which contained a low amount of uro- nate. The buoyan t density of the fraction containing the majority of the link proteins in the KCl-extracted proteoglycans was 1.45 g/ml whereas it was 1.50 g/ml in the KC1-Gdn • HCl-extracted proteoglycans. In the Gdn • HC1 extract a broader and intermediate distribution was observed. Sequentially (KC1-Gdn • HC1)-extracted proteoglycans (A~ fraction) contained 3.8 times more link pro- teins t~an the KCl-extracted A1 fraction.

Effect o f guanidinium salts on the distribution in Cs2804 gradients o f the A I fraction components from sequential extraction

When an AI fraction from the KC1-Gdn. HC1 extract was centrifuged in

62

1.2-

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Fig . 2 . Distr ibut ion o f uronic acid and prote in o f KCI (r ight) and sequent ia l ly K C I - G d n • HC1- ( le f t ) ex t rac t ed p r o t e o g l y c a n s ( recovered under associat ive c on d i t i o ns ) in a C s 2 S O 4 dens i ty gradient as a func- t i on o f the Gdn • HC1 m o l a r i t y in the centr i fugat ion m e d i u m . Results are expressed as percen t o f to ta l uronate ( e --) and as absorbance at 2 8 0 n m for prote in ( o o ) .

Cs2SO4 gradients with increasing concentrations of Gdn • HC1 at pH 5.9 (called heterogeneous system because of the presence of sulfate and chloride anions), we observed a progressive transfer of the uronate rich fraction towards a denser region of the gradient (Fig. 2). The density of this denser uronate rich fraction fitted exactly with the density value of the corresponding KC1-Gdn • HC1 pro- teoglycan monomers observed under identical conditions (Fig. 2 and 3). When increasing the Gdn • HC1 concentration, the uronate rich fraction from the AI

63

1.8

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Fig . 3. V a r i a t i o n o f the b u o y a n t d e n s i t y o f the u r on ate r ichest f r a c t i o n o f K C l - G d n • H C l - e x t r a c t e d p ro -

t e o g l y c a n m o n o m e r s as a f u n c t i o n o f t h e g u a n i d i n i u m i on c o n c e n t r a t i o n , o , Gdn • HCI , o , G d n 2 • H 2 S O 4.

KC1 extract always behaved as a single polydisperse population. In addition at 3 M Gdn • HC1 a polydisperse 280 nm absorbing material was observed in the gradient between the uronate rich fraction and the floating proteins which was absent from Ax in the KC1-Gdn • HC1 extract (Fig. 2).

In this heterogeneous system (pH 5.9; Cs2SO4, G d n . HC1) we noticed an increase in the apparent density of the uronate richest fraction of the KC1- Gdn • HC1 proteoglycan monomers when increasing the Gdn • HC1 concentra- tion (Fig. 3). However the increase of the apparent density of the uronate richest fraction of the same proteoglycan monomer preparation was much less obvious in a homogeneous system (Cs2SO4, Gdn2 • H2SO4) than in the hetero- geneous conditions (Fig. 3). The observation of an increase of the buoyant density as a function of Gdn • HC1 concentration was also made in the case of the KCNextracted proteoglycans (data not shown).

Chemical characterization o f the proteoglycans Sugar composition o f the proteoglycan A I fractions in the Cs2S04 density

gradient. The fractions containing at least 2% of the total uronate (Fig. 1) were considered for analyses. For every preparation the galactosamine/glucosamine molar ratio decreased as a function of decreasing density; the galactose content increased almost in parallel with glucosamine. These observations were consis- tent with a keratan sulfate enrichment as the proteoglycan fraction became less dense. At a density of 1.50 g/ml, a glucose rich fraction was characterized in KCl-extracted proteoglycans which was partially reflected in the directly (Gdn • HC1)-extracted proteoglycan fractions but was nearly absent from the KC1- Gdn • HCl-extracted proteoglycan fractions (Table I).

Amiao acid and sugar compositions o f the sequentially KCl-Gdn. HCl- extracted proteoglycan monomers. The fractions containing at least 2% of the total protein (Fig. 1 ) were considered for analyses. Sugar compositions of both sequentially (KC1-Gdn • HC1)-extracted proteoglycans AI and D1 fractions were

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4

Ph

e 3

3

36

3

8

39

4

1

42

4

2

43

4

3

43

4

2

Ly

s 2

1

22

2

3

23

2

6

27

3

0

34

3

6

40

4

3

H~

1

7

17

17

1

8

18

2

0

22

2

3

24

2

6

27

A

xg

3

4

37

3

8

40

4

2

44

4

6

48

4

9

50

5

0

Su

gar

Neu

tral

su

gar

s X

ylo

se

20

2

1

23

2

4

22

2

4

22

2

2

54

4

5

39

M

ann

ose

2

4

13

1

7

24

4

2

26

2

7

17

3

5

23

1

6

Gal

acto

se

93

1

08

1

12

1

29

1

34

1

57

1

69

1

62

1

65

1

80

2

30

G

luco

se

0 0

0 0

65

6

0 6

0 0

0

Aci

d s

ug

ar

Glu

cuzo

nic

aci

d

38

7

34

5

33

5

33

0

26

2

26

8

28

1

24

3

26

5

21

5

21

6

Am

ino

su

gar

s N

oA

c et y

lga

lac

tosa

min

e

40

2

44

2

41

3

38

9

34

0

36

9

34

9

36

7

34

9

32

8

29

1

N-A

ce

tylg

luc

osa

min

e

64

6

1

85

8

7

12

1

12

3

13

0

17

2

15

7

18

1

17

9

N-A

ce

tyln

eu

ram

inic

aci

d

I0

I0

15

1

7

24

2

7

22

II

5

28

2

9

Ga

lNA

c/G

IcN

Ac

6

.3

7.2

4

.9

4.5

2

.8

3.0

2

.7

2.1

2

.2

1.8

1

.6

67

very similar (enrichment in keratan sulfate as the density drops) (Table II). The amino acid composit ions presented in Table II reflected an important

serine and glycine content increase as a function of increasing density whereas the proline, tyrosine, lysine and arginine contents decreased.

Discussion

The great advantage of the use of Cs2SO4 instead of CsC1 as gradient forming salt was the absence of any uronate at the b o t t o m of the tube.

A relatively large amount of uronate (32%) was extracted with 0.15 M KC1; this result can be at tr ibuted to the length of the extraction time or to the powdery nature of the ground cartilage we used in comparison to that em- ployed by others [7,8]. In each case protease inhibitors were present in the extraction medium avoiding a degradation of the material. When only benzam- idine was used as inhibitor, the results were roughly the same. The behaviour of directly (Gdn • HC1-) extracted proteoglycans (A1 fraction) always appeared to be intermediate between those of KCI- and sequentially (KC1-Gdn. HC1)- extracted fractions taking account of the relative proport ions of the two latter proteoglycan populations. Thus we will mainly discuss the differences observed between the KC1 and KC1-Gdn • HCl-extracted proteoglycan A1 fractions.

It was obvious that in the A, fraction of the KC1-Gdn • HC1 extract the low density uronate rich material characterized in Cs2SO4 gradients corresponded to the aggregated proteoglycans described by others. This s tatement was sup- ported firstly by the distribution of the link proteins in the gradient; secondly by the difference of the buoyant density value of this material when compared to that of the corresponding proteoglycan monomers (D~), and thirdly by the uronate and protein distributions in the density gradients when dissociating agents were added. The aggregated proteoglycans presented a lower buoyant density than the corresponding monomers; this result might be due to a larger volume occupied by the aggregate than by the monomer for a similar uronate concentration. In this connection Hascall indicated a greater compressibility for the monomer than for the aggregate [21].

According to some authors, the KCl-extracted A, fraction should no t con- tain associated proteoglycans [8], however others found link proteins in KC1 extracts [22]. We characterized some link proteins in the A, fraction of the KC1 extract; they were embedded in a zone of the Cs2SO4 gradient suggesting their association with some dense molecules. When we compared the apparent densities of the link proteins containing fractions in KC1- and KC1-Gdn • HC1- extracted proteoglycans (in the absence of guanidinium salts) it was obvious that the link proteins were associated in the A~ fraction of the KC1 extract with components of lower densities than those to which they were bound in the AI fraction of the KC1-Gdn • HC1 extract.

The sugar analyses of KC1-Gdn • HCl-extracted proteoglycan aggregate and of the corresponding monomers presented no important difference in spite of the presence of hyaluronic acid and link proteins in the aggregate. The hyaluronic content in the fractions [4] and the sugar content of the link proteins [23] were too low for interfering with the results. Our data were in agreement with those of Heineg£rd [13] but gave a more detailed view of the proteoglycan

68

polydispersity as no material sedimented at the bottom of the tube. In the KC1 extract we characterized the presence of a fraction whose glucose content was very high. This result might reflect the presence of glycogen in this fraction more especially as the molar ratio of the other sugars remained unchanged when compared to similar KC1-Gdn. HCl-extracted proteoglycan fractions (with the exception of a slightly lower mannose content in the latter).

The Gdn • HC1 effect upon the apparent buoyant density of KC1-Gdn • HC1- extracted proteoglycan monomers in a Cs2SO4 gradient seemed to be primarily due to the contribution of C1- which competed with SO~- and caused a decrease in the net hydration of the proteoglycans. Mashburn et al. [17] found that, in CsC1 gradients, the addition of Gdn. HC1 caused a decrease in the buoyant density of the proteoglycans; they attributed this observation to a cation effect. In our case, Gdn ÷ contributed to dissociation but might have some additional effects which were masked by the anion effect. The possibility of isolating proteoglycans of better defined density is now available and makes possible a detailed study of the heterogeneity of other proteoglycan popula- tions.

Acknowledgements

The authors thank Miss M. Rougeot for skilful technical assistance. This research was supported by the C.N.R.S. (E.R. No. 102), the I.N.S.E.R.M. (group U-116) and the Fondation pour la Recherche m~dicale franqaise.

References

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