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Biochimica et Biophysica Acta, 532 (1978) 242--248 © Elsevier/North-Holland Biomedical Press

BBA 37842

ISOLATION AND CHEMICAL CHARACTERIZATION OF TWO DISTINCT "LINK PROTEINS" FROM BOVINE NASAL CARTILAGE PROTEOGLYCAN COMPLEX

F. BONNET, J.-P. PI~RIN and P. JOLLES

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

(Received August 3rd, 1977)

Summary

Bovine nasal cartilage proteoglycan aggregates have been dissociated and separated by dissociative density gradient centrifugation into proteoglycan sub- units and "link fraction". The latter contained mainly the two "link proteins" as shown by analytical sodium dodecyl sulfate-polyacrylamide gel electropho- resis. The two "link proteins" were then separated by preparative gel electro- phoresis under dissociative conditions. Molecular weight and amino acid com- position of both proteins are presented.

Introduction

Proteoglycan complex from bovine nasal cartilage consists of aggregated material which can be separated into distinct fractions by density gradient cen- trifugation in 4 M guanidinium chloride [1,2]. The proteoglycan subunit fraction recovered at the bottom of this gradient containschondroitin sulphateand keratan sulphate chains which are both covalently bound to a core protein. The top of this gradient contains compounds which are implicated in the reaggregation of proteoglycan subunits [3,4,5] (hereafter referred to as the "link fraction"). It has been demonstrated that the "link fraction" contains hyaiuronic acid and two major proteins which have been termed "link proteins" [5,6]. Although the reaggregation of proteoglycan subunits and hyaluronic acid in the absence of the "link fraction" has been reported [7,8]. Hascall and Heinegard [3] have shown that the proteoglycan complex was much more stable than proteoglycan subunits-hyaluronic acid mixtures. These data induced, as previously proposed [5], these authors to conclude that compounds present in the "link fraction", such as the "link proteins", seem necessary to stabilize the aggregate. Neverthe- less the stabilizing effect of purified "link proteins" has never been directly demonstrated.

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This communication, to our knowledge, is the first which presents a proce- dure for the purification of two distinct "link proteins". This was performed using preparative gel electrophoresis in dissociative conditions. The amino acid composition of each protein is also presented.

Materials and Methods

Reagents. Guanidinium chloride and cesium chloride were from Merck. Electrophoretic reagents were purchased from Canalco or Labo Disc and all other reagents (analytical grade) were from Prolabo. Densities were determined by pycnometry using a 200 pl constriction pipette.

Amino acid composition. The amino acid compositions were determined after total hydrolysis (6 M HC1, 0.05% 2-mercaptoethanol, l l0°C, 18 h, under vacuum) using a Technicon amino acid Autoanalyzer. Cyst(e)ine was deter- mined as cysteic acid after performic acid oxidation [9].

SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electro- phoresis was performed according to the method of Laemmli [10] (10% poly- acrylamide; pH 8.9).

For preparative electrophoresis, samples were dissolved in 0.0625 M Tris- HC1 buffer, pH 6.8, containing 2% SDS, 10% glycerol and bromophenol blue as marker dye.

For analytical electrophoresis the same buffer was used with or without the addition of 5% 2-mercaptoethanol; the presence or absence of this reducing agent will be indicated in the Results.

Proteins were stained with Coomassie brilliant blue R 250. Periodic acid- Schiff's reagent was used to characterize the presence of sugars in the protein bands [ I i ] .

Preparative SDS-gel electrophoresis was performed with the aid of a prepara- tive disc electrophoresis apparatus from Canalco (column P.D. 2/150). The heights of the separation and stacking gels were 8.5 cm and 2.5 cm respectively and the elution buffer was 0.3 M Tris-HCI, pH 8.3, containing 0.2% SDS. The applied current during this experiment was 5 mA and fractions (2 ml) were collected every 15 mn. After the elution of the bromophenol blue the absorption of the fractions at 280 nm was immediately determined.

Preparation of the "link fraction". Bovine nasal cartilage was sliced and ground in liquid nitrogen with the aid of a IKA-A-10 S mill (Janke and Kunkel) and the cartilage powder was extracted with 4 M guanidinium chloride, 0.05 M sodium acetate buffer, pH 5.8 (15 v/w) for 24 h at 4°C according to Sajdera and Hascall [1]. Insoluble material was removed by centrifugation for 1 h at 11 000 rev./min at 4°C (SorvaU RC-5, centrifuge rotor GSA).

Further steps in the purification procedure were performed according to Hascall and Sajdera [2] with minor modifications with regard to the starting density and fraction collection.

The soluble proteoglycans were reaggregated by dialysis of the supernatant against 9 volumes of 0.05 M sodium acetate buffer, pH 5.8 at 4°C for 24 h. CsC1 was added to obtain a starting density of 1.62--1.63 g/ml and the solution was centrifuged for 1 h at 35 000 rev./min at 20°C in a Beckman L2 65B cen- trifuge using a fixed angle rotor, type 60 Ti; the upper insoluble gel was

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removed. The contents of each tube were then mixed and the solutions were recentrifuged for 40 h in the same conditions as those described above.

The bottom fifth of each tube (density > 1.68 g/ml) was collected and pooled to yield the proteoglycan complex fraction. The matter was then sub- mitted to density gradient centrifugation under dissociative conditions. The concentration of guanidium chloride was brought to 4 M by the addition of a 7.5 M solution of the salt in 0.05 M sodium acetate buffer, pH 5.8, and the density was adjusted to 1.52--1.54 g/ml with solid CsC1. This solution was cen- trifuged for 40 h at 40 000 rev./min at 20°C using a SW 50.1 rotor. The upper sixth of each tube (density < 1.44 g/ml) was termed the "link fraction" and was subjected to analytical and preparative disc gel electrophoreses.

Results

Separation of the two "link proteins" The "link fraction", obtained by density gradient centrifugation under disso-

ciative conditions, comprised 20% of the proteins of the total material recovered from the gradient.

The application of analytical SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol to this "link fraction" indicated the presence of two major gel penetrating bands A and B (Fig. la). The relative proportion of protein A: protein B was determined by scanning densitometry of the gels at 660 nm and was found to be 2 : 3, respectively.

The "link fraction" was subjected to preparative SDS-polyacrylamide gel electrophoresis (Fig. 2): two peaks, 1 and 2, were observed. The relative propor-

Fig. I . Analyt ical gel e leetrophores l s o f the " l ink f rac t ion" and " l lnk proteins" . (a) in the absence o f (b) in the presence o f 2 -mercaptoe thano l . Coomassde-blue stained gels: 1 " l ink fract ion", 2 "Unk prote in A " and 3 " l ink prote in B". Periodic aeid-Sehlff 's reagent stained gels: 4 " l ink prote in A" and 5 " l ink pro- te in B".

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0.2~

C

o

0.2C

0.15

I 50

Fraction number

Fig. 2. E l u t i o n Pat tern o f the " l i n k f r a c t i o n " fo l l owing p repa ra t i ve disc gel e lec t rophoresds . E x p e r i m e n t a l c o n d i t i o n s w e r e descr ibed in the t e x t .

tion of the material in peak 1 : peak 2 was found to correspond very closely to the proportion of protein A : protein B indicated above.

Electrophoretic characterization of the two isolated "link proteins" and deter- mination of their molecular weight

The isolated material was subjected to analytical SDS-polyacrylamide gel electrophoresis in the presence and absence of 5% 2-mercaptoethanol as indicated in the Methods.

In the absence of 2-mercaptoethanol, the substance isolated in peak 1 following preparative SDS-polyacrylamide gel electrophoreses was found to correspond to protein A and similarly the substance isolated in peak 2 cor- responded to protein B (Fig. la).

In the presence of 2-mercaptoethanol, electrophoresis was carried out in the same conditions as those in the above experiment. Two major proteins were once more characterized, but their mobilities were reduced compared to the mobilities of proteins A and B as characterized in the absence of 2-mercapto- ethanol. However, two minor components were also detected possessing mobili- ties greater and smaller than those of the major proteins. By scanning densito- metry it was found that each of these two minor bands represented less than 5% of the proteins contained in bands A or B (Fig. lb).

The molecular weights for proteins A and B, after reduction, were calculated from the mobilities of the major bands and were found to be 44 500 + 1000 and 48500 +1000, respectively. Myoglobin (17200}, chymotrypsinogen (25 700), aldolase (40 000), catalase (60 000) and serum albumin (67 000) were used as markers.

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T A B L E I

A m i n o a c i d c o m p o s i t i o n o f t h e " l i n k f r a c t i o n " a n d " l i n k proteins". Values are given as a m i n o ac ids p e r 1 0 0 0 ; n .d . , n o t d e t e r m i n e d .

A m i n o ac id L i n k f r a c t i o n L i n k p r o t e i n L i n k p r o t e i n A B

Asp 1 0 5 1 0 3 1 0 5 T h r 61 4 6 4 5 S e r 6 6 71 8 2 Gin 1 0 4 9 2 9 7 P ro 8 0 4 1 3 5 Gly 88 115 118 A l a 83 7 5 71 ( C y s - ) n .d . 3 7 31 Val 71 6 6 63 Met 7 2 2 Ile 3 9 3 0 29 L e u 7 8 7 2 6 7 T y r 41 4 6 5 0 Phe 4 7 5 8 57 L y s 4 6 58 6 3 His 23 2 5 29 A r g 61 6 3 56

Amino acid composition o f proteins A and B Table I presents the amino acid compositions of proteins A and B, which

appear to be very similar. The differences observed between the amino acid compositions of the "link proteins" and the "link fraction" might be due to the presence of proteoglycans of low buoyant density in the latter [12] which have been eliminated during the purification procedure.

Sugar characterization in proteins A and B The two major bands characterized by polyacrylamide gel electrophoresis

in both reducing and non-reducing conditions were stainable with periodic acid-Schiff's reagent (Fig. la , lb). In addition, amino sugars were detected on the amino acid analysis chromatograms of both "link proteins". Unfortunately the quantities of "link proteins" we prepared proved to be insufficient to per- mit quantitative sugar content determinations to be made.

Discussion

According to previous observations of Keiser et al. [6] the presence of the two proteins A and B was demonstrated by SDS-polyacrylamide gel electro- phoresis of the lightest fraction (density <~ 1.44 g/ml) obtained from density gradient centrifugation of the proteoglycan complex in dissociative conditions. The use of preparative sodium dodecyl sulfate-polyacrylamide gel electro- phoresis allowed, for the first time, the separation of these two proteins. Treat- ment of both proteins (A and B) with 2-mercaptoethanol, in conditions similar to those used by Hascall and Heinegard [13], caused a decrease in their electro- phoretic mobilities. The molecular weights of proteins A and B, as determined in this paper, were in agreement with those previously reported by Hascall and Heinegard [13].

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Previous work by Keiser et al. [6] and Hascall andHeinegard [13] has shown that the lightest density fraction recovered after gradient centrifugation in dis- sociative conditions (the "link fraction") contained glycoproteins; however, the presence of sugars bound to the "link proteins" has never before been demon- strated. The periodic acid-~chiff's reagent positive reaction observed with pro- teins A and B in the presence and absence of 2-mercaptoethanol provided evi- dence that the two "link proteins" are glycoproteins.

Heinegard and Hascall [4] have characterized a T-G200-3 fraction from a chondroitinase-trypsin digest of their proteoglycan complex fraction they termed A1. They suspected that this T-G200-3 compound, which can be aggregated with hyaluronic acid, might be derived from one or both of the "link proteins". The mobility of this protein was similar to that of the smal- ler (a} of the two "link proteins" described by Hascall and Heinegard [13] (a and b "link proteins). They suggested [4] that T-G200-3 was equivalent to the a protein or was derived from it after a small modification, such as the removal of a small peptide from one end of the polypeptide. The possibility that it may have been derived from the larger b protein could not be ruled out however. We assume that the "link proteins" A and B are equivalent to the a and b bands they described. In fact the amino acid composition they presented for the T-G200-3 fraction was very similar to the amino acid compositions we have found for both the isolated "link proteins" A and B. Thus it is not possible for us to be certain at this stage whether one or both of the "link proteins" are capa- ble of binding to hyaluronic acid.

The other possible explanations for the fact that their T-G200-3 fraction contained only one protein band corresponding to their "link protein" a are (1) that some sugars from one or both of the proteins a and b may have been removed during digestion with chondroitinase and (2) that only one of the two proteins (b) may be more sensitive to digestion with trypsin.

In a recent paper Baker and Caterson [14] described a method for isolating "link proteins" freed of proteoglycan, but no separation of the A and B "link proteins" was achieved. In addition they detected a third "link protein". We have observed in some, but not in all analytical electrophoreses the presence of a similar faint band of material which migrates slightly more rapidly than "link protein" A.

Acknowledgements

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

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