connective tissues form distinct subgroups

Biochem. J. (1985) 230, 181-194Printed in Great Britain

181

The core proteins of large and small interstitial proteoglycans from variousconnective tissues form distinct subgroups

Dick HEINEGARD, Annika BJORNE-PERSSON, Lars COSTER, Ahnders FRANZEN, SvenGARDELL, Anders MALMSTROM, Mats PAULSSON, Rosmari SANDFALK and Kathryn VOGEL

Department of Physiological Chemistry, University of Lund, P.O. Box 94, S-221 00 Lund, Sweden

(Received 12 February 1985/17 April 1985; accepted 7 May 1985)

Large and small proteoglycans were separately isolated from a number of connectivetissues and compared to determine the extent of structural similarity. This was

studied by enzyme-linked immunosorbent assays and by the peptide patternsobtained when 125I-labelled proteoglycans were digested with trypsin. All the largeproteoglycans, i.e. from tendon, sclera, cartilage and aorta, appear to contain thestructure typical for the hyaluronic acid-binding region, both shown by enzyme-

linked immunosorbent assay and by content of peptides unique for this region. Theseproteoglycans also share other structural features of the protein core, as indicated byimmunological cross-reactivity and similar peptide patterns. The large proteoglycansfrom aorta in addition show the presence of unique structures both upon

immunoassay and with regard to peptide pattern. Among the small proteoglycans twogroups can be identified, although amino acid composition and protein core sizes are

grossly similar. One group consists of the small proteoglycans from aorta and cartilagehaving similar peptide maps and showing immunological cross-reactivity in enzyme-linked immunosorbent assay. The other distinctly different group consists of the smallproteoglycans from bone, cornea, sclera and tendon, which among them showidentity in enzyme-linked immunosorbent assay and similar peptide patterns.Proteoglycans from the two groups, however, show partial immunological cross-

reactivity.

In recent years a large number of apparentlydifferent proteoglycans have been isolated. Some,such as those containing heparan sulphate, appearto have a major function at the cell surface(Oldberg et al., 1979; Kjellen et al., 1981). Otherproteoglycans, containing chondroitin sulphate ordermatan sulphate, are primarily interstitial, i.e.localized in the extracellular matrix (for referencessee HeinegArd & Paulsson, 1984). It should bestressed, however, that there also appear to existdermatan and/or chondroitin sulphate proteo-glycans intercalated in the cell membrane and, onthe other hand, heparan sulphate proteoglycans inthe extracellular matrix, particularly in basementmembrane (Hassel et al., 1980).A fundamental body of knowledge on the

structure of the interstitial proteoglycans has beenobtained from studies of the large proteoglycans incartilage. These molecules have Mr values ofI x 106-3 x 106, but they can form large aggregateswith hyaluronic acid (Hardingham & Muir, 1972).The central core protein contains side-chain

Vol. 230

substituents of chondroitin sulphate, keratan sul-phate, 0-linked oligosaccharides and N-linkedoligosaccharides (for references see Heinegard &Paulsson, 1984). One large portion of the coreprotein contains all the glycosaminoglycan chains,i.e. the chondroitin sulphate-rich and keratansulphate-rich regions (Heineg'ard & Axelsson,1977), and the other major portion of the proteo-glycan, the hyaluronic acid-binding region (Heine-gard & Hascall, 1974), contains few substituents,i.e. only a few N-linked oligosaccharides(Lohmander et al., 1980).

Recent data show that there are two types ofaggregating cartilage proteoglycans, differing inelectrophoretic mobility on agarose/polyacryl-amide gels, in amino acid composition and withregard to side-chain constituents. Their proteincores show differences, as indicated by peptidepatterns of trypsin digests (Heinegard et al.,1985a).Other proteoglycans present in cartilage include

a population of non-aggregating large proteo-

D. Heinegard and others

glycans, apparently distinct from those aggregat-ing (Heinegard & Hascall, 1979) as judged fromamino acid composition, a limited immunologicalcross-reactivity and a smaller number of sidechains per protein.A fourth population of proteoglycans in carti-

lage appears to be completely unrelated to theother three. These molecules are much smaller,having an Mr of only 76000 (Heinegard et al.,1981). They contain one or two chondroitinsulphate side chains that are considerably largerthan those of the aggregating proteoglycans, i.e.weight-average M, values of about 35000 com-pared with 17000-18000. The core protein has anamino acid composition distinct in its very highcontent of leucine, aspartic acid/asparagine andglutamic acid/glutamine (Heinegard et al., 1981).It also contains some oligosaccharide substituents(Heinegard et al., 1981).

Other tissues contain predominant small proteo-glycans with many structural features similar tothose of the small cartilage proteoglycans. Thus Mrvalues of less than 100000 have been found forthose from bone (Franzen & Heinegard, 1984),sclera (Coster & Fransson, 1981) and tendon(Anderson, 1975; Vogel & Heinegard, 1985).These proteoglycans all have a protein core withhigh contents of leucine, aspartic acid/asparagineand glutamic acid/glutamine, being substitutedwith one or two very large chondroitin sulphate ordermatan sulphate chains as well as with oligo-saccharides. Similar small proteoglycans havebeen described in periodontal ligament (Pearson &Gibson, 1982), cervix (Uldbjerg et al., 1983), aorta(Salisbury & Wagner, 1981; S. Gardell, unpub-lished work), skin (Damle et al., 1982) and cornea(Axelsson & Heinegard, 1975). Typically, theprotein core prepared by chondroitinase digestionof all of these proteoglycans has a mobilityon sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis corresponding to an apparent M,of about 45 000-48000 when compared withglobular standard proteins.Many of these tissues also contain large proteo-

glycans with structural features resembling thoseof aggregating proteoglycans from cartilage. Ex-amples are proteoglycans from aorta (Salisbury &Wagner, 1981; S. Gardell, unpublished work),tendon (Anderson, 1975; Vogel & Heinegard,1985), sclera (Coster & Fransson, 1981), perio-dontal ligament (Pearson & Gibson, 1982) andskin (Damle et al., 1982). These proteoglycanshave high contents of serine, glycine and glutamicacid/glutamine. They all contain smaller glycos-aminoglycan side chains with weight-average Mrvalues of less than 20000. The side chains arechondroitin sulphate, and possibly in some casesdermatan sulphate with a high glucuronic acid/

iduronic acid ratio. Additional substituents are alarge number of 0-linked oligosaccharides. Fur-thermore a proportion of these proteoglycansappear to have the capacity to interact withhyaluronic acid, important for the formation ofproteoglycan aggregates (for references see Heine-gard & Paulsson, 1984).The present study was undertaken to show that

there are indeed groups of proteoglycans with verysimilar, if not identical, core proteins, although thetype of side chain may vary.

Materials and methodsPreparation ofproteoglycans and fragments thereof

Proteoglycans were extracted from bovine carti-lage, cornea, tendon, sclera, bone and aorta andpurified by using standard procedures as describedelsewhere (Axelsson & Heinegard, 1975; Heine-gard & Hascall, 1979; Coster & Fransson, 1981;Franzen & HeinegArd, 1984; Heinegard & Pauls-son, 1984; Heinegard et al., 1985a). The prepara-tion of corneal proteoglycans used was 50 P, i.e. theone containing dermatan sulphate (Axelsson &Heinegard, 1980).These procedures relied on extraction of pro-

teoglycans with 4M-guanidinium chloride, con-taining proteinase inhibitors, and subsequentpurification by CsCl-density-gradient centrifuga-tion, ion-exchange chromotagraphy in 7 M-ureaon DEAE-cellulose eluted with salt gradientsand gel chromatography. Hyaluronic acid-bindingregion and chondroitin sulphate peptides(AI.T.Al.CB.6B3) were prepared as described byHeinegard & Axelsson (1977). In all cases, puritywas checked by sodium dodecyl sulphate/poly-acrylamide-gel electrophoresis, where none of thesamples contained detectable non-proteoglycanproteins even when large amounts of sample wereapplied.Antibody production

Antibodies were raised in rabbits. Approx.0.5mg of proteoglycan was dissolved in 0.5mlof 0.15 M-NaCl/5 mM-sodium phosphate buffer,pH 7.4, and mixed with an equal volume ofFreund's complete adjuvant. Immunization wasdone subcutaneously in the neck of 6-month-oldrabbits. Booster doses of equal amounts of proteo-glycans in Freund's incomplete adjuvant weregiven monthly until sufficient antibody titre wasobtained, usually after one or two boosters.Rabbits were bled every 14 days by venesection inthe ear. Antibody titre was checked by enzyme-linked immunosorbent assay as described below.

ImmunoassayEnzyme-linked immunosorbent assay by the

procedure of Engvall & Perlman (1971), with some

1985

182

Core proteins of small and large interstitial proteoglycans

modifications (Heinegard et al., 1985b), wasperformed both for checking antibody titre and formeasuring immunological cross-reactivity as re-flected by the capacity of proteoglycans fromdifferent sources to inhibit in a particular assay.

It has previously been found important to usepoly(vinyl chloride) micro-titre plates rather thansuch of polystyrene when the enzyme-linkedimmunosorbent assay was performed with proteo-glycan antigens (Heinegard et al., 1985b). In thepresent work we used Dynatech M29 (DynatechLaboratories, Alexandria, VA, U.S.A.), which ingeneral gives good performance. Before assay allantigens were digested with chondroitinase ABC.Samples at 1 mg/ml in 0.1 M-Tris/HCl/0. 1 M-sodiumacetate buffer, pH 7.3, were digested with 0.01 unitof chondroitinase ABC for 4h at 37°C. Plates werecoated with 200uIl of antigen, dissolved in 4M-guanidinium chloride/5 mM-sodium acetate buffer,pH 5.8, at concentrations of 0.1 jug/ml (for hyalur-onic acid-binding region) and 1 ,ug/ml (for proteo-glycans), as indicated in the Figure legends foreach particular experiment. After incubation over-night at room temperature in a humidified cham-ber, the plates were extensively rinsed with 0. 15 M-NaCl/5 mM-sodium phosphate buffer, pH7.4,also containing 0.05% (w/v) Tween 20. Firstantibody (200 lu) in appropriate dilutions in 0. 15 M-NaCl/5mM-sodium phosphate buffer, pH7.4,containing 0.05% (w/v) Tween 20 was added. Afterincubation for 60min at room temperature theplates were rinsed and incubated with secondantibody, i.e. pig anti-(rabbit IgG) antibody(200pl) conjugated to alkaline phosphatase (OrionChemicals, Turku, Finland) at a dilution of 1:200.After 60min at room temperature the plates wererinsed and incubated with substrate solution(200 Il), i.e. p-nitrophenyl phosphate (SigmaChemical Co.) (1 mg/ml) in 1 M-diethanolaminebuffer, pH 10, containing 1 mM-MgCl2. The absor-bance at 405nm was measured with a Multiscanphotometer (Flow Industries) immediately aftersubstrate addition and again after a 60minincubation at room temperature. The increase incolour yield was taken as the value of enzymeactivity. The titre of first antibody giving anabsorbance value of about 1 was used for inhibi-tion assays. In these assays samples or standardsdiluted with incubation buffer were mixed with anequal volume of a dilution of the first antibody.The samples were incubated overnight at roomtemperature in polystyrene micro-titre plates, and200Ml portions of the mixture were added to thecoated wells in place of first antibody. Subsequentincubations were done as described above. Allanalyses were done in triplicates. In all casesinhibition curves for the reference, i.e. the antigenagainst which the antibodies were directed, and

the antigens to be compared were determined byincubating the samples in the same micro-titreplate.1 25I-labelling

Proteoglycan samples (or hya'luronic acid-bind-ing region) were labelled with 1251 by using thechloramine-T procedure essentially as described byGreenwood et al. (1963) with samples dissolved in6M-urea. The specific radioactivity of the proteo-glycans was 5000-20000 c.p.m./ng.

Peptide mapping1251-labelled samples (50000c.p.m.) were mixed

with 0.1 M-Tris/HCl buffer, pH 8.0, to give a finalconcentration of iOmM-Tris/HCI. Then 0.5!Ll ofa 1 mg/ml solution of 1-chloro-4-phenyl-3-tosyl-amidobutan-2-one ('TPCK')-treated trypsin(Sigma Chemical Co.) in the Tris buffer wasadded. Digestion was for 4h at 37°C.

Two-dimensional thin-layer high-voltage elec-trophoresis in pyridine/acetate buffer, pH 6.4,was done on silica plates (Merck, Darmstadt, WestGermany) in a Savant (New York, NY, U.S.A.)electrophoresis chamber. A plate (20cm x 20cm)was divided into two equal 1Ocm x 20cm strips,and two samples to be compared were each appliedto one such strip and simultaneously electrophor-esed for 40min with 50V/cm. After the plate hadbeen dried, chromatography in the second dimen-sion was developed with butanol/pyridine/aceticacid/water (6:5 :3 :1, by vol.) as described by Bates& Perham (1975). Autoradiography was done bysimply leaving the plate on top of an envelopecontaining a sheet ofAGFA-GEVAERT OSRAYM3 X-ray film for 2-3 days.

Electrophoresis on agarose/polyacrylamide gelsSamples were dissolved in 1% (w/v) sodium

dodecyl sulphate at 1 mg/ml. Portions containing20-50pg of proteoglycan were diluted with anequal volume of 0.02% Bromophenol Blue/60%(w/v) sucrose dissolved in electrophoresis buffer,pH6, and electrophoresed on agarose/polyacryl-amide slab gels essentially as described by McDe-vitt & Muir (1971). The procedure was modifiedsuch that the gels were cast in buffer containing0.1% (w/v) Triton X-100 (D. Heinegard, unpub-lished work).

Results and discussion

The proteoglycan preparations used in this studyhave been extensively characterized previously,with regard to molecular mass, size of glycos-aminoglycan side chains, carbohydrate compo-sition and amino acid composition. Their aminoacid compositions are given in Tables 1 and 2.

Vol. 230

183

D. Heinegard and others

Table 1. Amino acid compositions of large proteoglycansAmino acid composition (residues/1000 residues)

Cartilage

Tendon(Vogel &

Heinegard, 1985)

68631251639512266

61

40751329282032

Sclera(Coster &

Fransson, 1981)

96589213883110

658

62

41881643194537

Table 2. Amino acid compositions of small proteoglycansAmino acid composition (residues/1000 residues)

Cartilage Aorta Tendon Sclera BoneAmino (Heinegard (S. Gardell, (Vogel & (Coster & (Franzen &acid et al., 1981) unpublished work) Heinegard, 1985) Fransson, 1981) Cornea Heinegard, 1984)

Aspartic acid 129 136 145 123 136 128Threonine 39 44 46 49 47 45Serine 66 79 108 68 77 76Glutamic acid 98 100 108 122 103 105Proline 72 57 82 74 68 77Glycine 71 82 102 84 83 86Alanine 50 55 63 54 56 49Cysteine 19 - 9Valine 53 59 33 59 54 52Methionine 1 7Isoleucine 55 51 33 55 57 58Leucine 143 137 112 115 125 128Tyrosine 28 27 24 15 22 29Phenylalanine 34 36 30 34 34 32Histidine 36 30 26 25 30 31Lysine 64 61 61 76 77 69Arginine 44 46 27 32 31 36

The compositions of the large proteoglycansshow large similarities, both with regard to aminoacid compositions (Table 1) and with regard tohigh contents of galactosaminoglycans, primarilychondroitin sulphate. Furthermore, in some cases,a high content of 0-linked oligosaccharides hasalso been demonstrated (Heinegard et al., 1985a;

Vogel & Heinegard, 1985). The agarose/polyacryl-amide-gel electrophoresis of these indeed demon-strates further similarities (Fig. 1). All the samplesappear to be slightly heterogeneous, containingcomponents that differ somewhat in electro-phoretic mobilities. The migration is, however, inall cases similar to that of the aggregating proteo-

1985

Aminoacid

Chondroitinsulphate-rich(Heinegard

et al., 1985a)

Aspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineCysteineValineMethionineIsoleucineLeucineTyrosinePhenylalanineHistidineLysineArginine

Keratansulphate-rich(Heinegard

et al., 1985a)

675811915110412372

62

41742438122133

72621391389613668

57

41772133132028

Aorta(S. Gardell,unpublished

work)

941031251516310969

55

35571132233934

184


(a) (b)

Migration Migration

Fig. 1. Agarose/polvacrilarnide-gel electrophoresis of proteoglycansLarge proteoglycans (a) and small proteoglycans (b) at amounts of 20pg and 3Ojug respectively were electrophoresedon agarose/polyacrylamide gels. The tissue of origin is indicated in the Figure. The sample of large proteoglycansfrom cartilage represents a mixture containing both chondroitin sulphate (CS)-rich, keratan sulphate (KS)-rich andsmall proteoglycans.

glycans from cartilage (Heinegard et al., 1985a).Interestingly, the proportions of the two compon-ents vary somewhat from one tissue to another.The compositions of the small proteoglycans are

also rather similar within the group, but distinctfrom that of the large proteoglycans (Table 2). Thesmall proteoglycans contain large galactosamino-glycan side chains, have a core protein with anapparent M, of 45000-50000 and contain oligo-saccharides. There are, however, also differences.Some, like the one from cartilage, do not containsialic acid, and those from cartilage and aorta havesomewhat higher leucine contents than the others,indicating the presence of subgroups of relatedsmall proteoglycans. Agarose/polyacrylamide-gelelectrophoresis showed that all the small proteo-glycans had a similar electrophoretic mobility (Fig.1), although those from cornea gave a somewhatdifferent pattern. It is known, however, thatbovine comeal proteoglycans have a very lowcontent of sulphate (Axelsson & Heinegard, 1975),

possibly causing a higher degree of polydispersitywith regard to charge density and therefore electro-phoretic mobility.

In summary, the data suggest that there may bemore than one type of small proteoglycans,although all are similar according to severalstructural parameters.

Large proteoglycansImmunological cross-reactivity. To study the

presence of subgroups of the proteoglycans further,an immunochemical approach was taken. It shouldbe stressed that all antibodies used appear to bedirected against determinants in the core protein,since digestion of the proteoglycans with papaineliminated their antigenicity. Antibodies wereavailable to a mixture of the large cartilage proteo-glycans (Al.D1 fraction), to the large scleraproteoglycans and to large aorta proteoglycans.Since the two aggregating proteoglycans fromcartilage have been separated and purified (Heine-

Vol. 230

Cartilage

Aorta

Tendon

Sclera

Bone

Cornea

185

U).tl.r_

;ONL.It1.-t:.01.

CZ'n-t-D

'T

D. HeinegArd and others

gard et al., 1985a), each could be used as theantigen in assays employing an antibody raisedagainst a mixture. Altogether four assays, i.e. forthe cartilage chondroitin sulphate-rich, for thecartilage keratan sulphate-rich, for the sclera large,and for the aorta large proteoglycans were de-veloped, all showing good sensitivity (Fig. 2).Dilutions of the following antigens were tested forcapacity to compete in all the assays: chondroitinsulphate-rich and keratan sulphate-rich proteo-glycans from cartilage, large sclera, large aorta andlarge tendon proteoglycans as well as chondroitinsulphate peptides from cartilage proteoglycans. Inmost cases the preparations showed almost com-plete immunological identity, demonstrated by thesimilar slopes of the inhibition curves (Fig. 2).Notably, however, only the aorta proteoglycanreacted in the assay for this proteoglycan, indicat-

ing that these antibodies were directed againstsites not present in the other proteoglycans.Interestingly, the aorta proteoglycan gave inhibi-tion curves with a slope similar to that of theantigen used to raise the respective antibody in allthe other assays, showing that the aorta proteogly-cans also contain epitopes identical with those ofthe other proteoglycans. There were, however, alsosome other differences. The sclera proteoglycan, inthe assay for cartilage proteoglycans, showed adifferent slope than the others, and the tendonproteoglycan in the assay for the one from scleraalso gave a somewhat different slope. In summary,however, the striking feature of the large proteo-glycans is that they appear to share a largeproportion of the antigenic sites, showing that, atleast in part, they are of similar structure. Indeed,they also appear to contain similar hyaluronic acid-

0 0 oD m OmN14 C ~" N N N, E

0

0

0 00o LON - N "

ILO LON N

-.0

6 Ci 6 60

0

Proteoglycan (ug/ml)Fig. 2. Immunological cross-reactivity of large proteoglycans

Proteoglycans, i.e. cartilage, chondroitin sulphate-rich (a) at 1 pg/ml, cartilage keratan sulphate-rich (b) at 1 pg/ml,large sclera (c) at I pg/ml and large aorta (d) at 1 pg/ml, were coated on poly(vinyl chloride) micro-titre plates. Firstantibody, raised against aggregating proteoglycans from cartilage at a dilution of 1:1000 (a and b), raised againstlarge sclera proteoglycan at a dilution of 1:1600 (c) or raised against the large aorta proteoglycan at a dilution of1 :100 (d), was used and the assays of dilutions of chondroitinase ABC digested proteoglycans were performed asdescribed in the Materials and methods section. Aorta, large proteoglycan; -- *---, sclera, large proteo-

glycan; -, tendon, large proteoglycan;........., cartilage, chondroitin sulphate (CS)-rich proteoglycan; ,

cartilage, keratan sulphate (KS)-rich proteoglycan; ,----- chondroitin sulphate peptides (Al.TAl.CB.6B3, in

Heinegard & Axelsson, 1977) prepared from trypsin digests of cartilage proteoglycans. Zero inhibition is indicatedby a 0 symbol.

1985

0.9

0.7

0.5

0.3

0.111

:

0.9

0.7

0.5

0.3

0.1

(a) Coat: Cartilage, CS-rich (b) Coat: , 0* Cartilage, KS rich

(c) Coat: Sclera, large (d) Coat: Aorta, large

/',''--; j/~/:,,.1w L .-~~~~~~~~~~~7

L :,, -'- 1 -~~/-

186


binding regions. Several of the preparations oflarge proteoglycans contain molecules capable ofinteracting with hyaluronic acid to form aggre-

gates (Salisbury & Wagner, 1981; HeinegArd et al.,1985a; Vogel & HeinegArd, 1985; S. Gardell,unpublished work). In support, an assay for theisolated hyaluronic acid-binding region of bovinenasal-cartilage proteoglycans gave parallel inhibi-tion curves for all the antigens (Fig. 3), showingthat the antigenicity and therefore the structure ofthis region is similar in the proteoglycans. The poor

overall inhibition observed depends on the pres-ence of antibodies against neoepitopes first ex-

posed after isolation of the hyaluronic acid-bindingregion by trypsin digestion (Wieslander & Heine-gard, 1979), seen in the present study with proteo-glycans from nasal cartilage.

Peptide pattern. In the enzyme-linked immuno-sorbent assay only some structures in the mole-cules, i.e. those that are antigenic, will be detected.Therefore, although the extensive cross-reactivityshows that portions of the protein core are similarfrom one molecule to the other, there may never-theless be other less antigenic regions of the proteincore that are dissimilar. A more complete compari-son of regions of the protein core of proteoglycanscan, however, be obtained by way of studies ofpeptide patterns after proteinase treatment of the

0.9

0.7

0.5

0.3

0.1

0 0 LO 10) LO LO LOI (0 ODC4 .- N N LO r-

0 o

0

Proteoglycan (pg/ml)Fig. 3. Immunoassay of hyaluronic acid-binding region in

proteoglycansBovine nasal-cartilage proteoglycan hyaluronicacid-binding region (HABR) at 0.1 pg/ml was

coated on poly(vinyl chloride) micro-titre plates.Dilutions of chondroitinase ABC-digested proteo-glycans were assayed for contents of hyaluronicacid-binding region by using an antibody (dilution1:20000) raised against isolated hyaluronic acid-binding region. Curve forms are as defined in Fig. 2legend.

proteoglycan. The low content of protein, com-

bined with the high content of strongly polyanionicglycosaminoglycans of proteoglycans, does, how-ever, present difficulties in traditional peptideseparations by thin-layer high-voltage electro-phoresis followed by t.l.c. In order to detect thepeptides by staining with ninhydrin, such largeamounts of material have to be applied thatseparations are much impaired. Therefore in thepresent study we chose to label proteoglycans with1 251 and limit the study to such radioactivepeptides that could be detected with autoradio-graphy. The large number of labelled peptidesobserved, however (see below), should make thepatterns representative. The sensitivity in this caseis increased several thousandfold, and only a fewmicrograms of material is required for the wholeprocedure, including labelling with chloramine-T.The two populations of aggregating cartilage

proteoglycans gave similar peptide patterns, al-though a few peptides differed indicating minorstructural differences (Fig. 4). Some of the peptidesin both preparations were derived from thehyaluronic acid-binding region, shown for refer-ence (Fig. 4). Interestingly, many of the samepeptides were also identified in the large proteogly-cans from sclera and tendon, corroborating thestructural similarity of these molecules (Fig. 5).The large aorta proteoglycan showed a verydifferent pattern (Fig. 4), as would be expectedfrom the immunochemical data. The presence of astructurally very similar hyaluronic acid-bindingregion in these proteoglycans was also demon-strated, as indicated schematically in Fig. 5, wherethe blackened spots represent such peptides. Thelarge non-aggregating proteoglycans from carti-lage showed an entirely different peptide pattern,indicating their different nature.The data, then, indicate that large proteoglycans

containing both a hyaluronic acid-binding regionand other related peptide sequences are found inmany tissues. There are, however, also differences,most markedly in relative quantities of thepeptides. These different proportions of identicalpeptides in the proteoglycans may indicate thatthere are repetitive sequences of the core proteinor, simply, may represent an artifact of thelabelling procedure used.

Small proteoglycansImmunological cross-reactivity. Antibodies di-

rected against the small cartilage, the small tendon,the small sclera and the bone proteoglycans wereavailable and were used to develop sensitiveenzyme-linked immunosorbent assay for the re-

spective proteoglycan (Fig. 6). It is of note thatnone of the large proteoglycans react in the assayfor the small proteoglycan from the same tissue.

Vol. 230

/ ..~ ~ ~~~~~~~~~~~~~~....

/ .. ..* Coat: HABR

I II I

187

vBO


0

O o

x

A

(b) Cartilage,K S-rich

(d) Aorta, large

Z-cQCLm

CYF0-C1)

Fig. 4. Peptide patterns oJ trypsin-digested '25I-labelled large proteoglycans from cartilage and aortaPeptide patterns of trypsin-digested proteoglycans were developed on thin-layer plates of silica gel essentially as

described by Bates & Perham (1975). The + and - signs denote polarity during electrophoresis and the X denotesthe starting point. The samples were (a) cartilage, chondroitin sulphate (CS)-rich, (b) cartilage, keratan sulphate(KS)-rich, (c) hyaluronic acid-binding region (HABR) for reference and (d) aorta, large proteoglycans. Thosepeptides present in the isolated hyaluronic acid-binding region and identifiable in the other proteoglycans are

schematically shown in (e). The arrows indicate peptides only present in one of the cartilage proteoglycans.

1985

(a) Cartilage,CS-rich

(c) HABR

(e)

..:i_S:: ..

.^*:

*:

s-QWle

.....- ..

:i.s

+

188


(a) Sclera, large

(c) Cartilage,non-aggregating

Cz

O imqeO.oe

Sb

x0

A

(b) Tendon, large

(d) HABR

-cL-a0,D

E0L-)

Fig. 5. Peptide patterns of trypsin-digested I25I-labelled large proteoglycans from sclera, tendon and cartilage, non-aggregating

Peptide patterns of trypsin-digested proteoglycans were developed on thin-layer plates of silica gel essentially as*described by Bates & Perham (1975). The + and - signs denote polarity during electrophoresis and the X denotesthe starting point. The samples were (a) sclera, large, (b) tendon, large, (c) cartilage, non-aggregating, proteoglycansand (d) isolated hyaluronic acid-binding region (HABR) for reference. Those peptides identified in both the scleraand the tendon proteoglycan and common to cartilage, aggregating, proteoglycans are schematically shown in (e),where the filled spots indicate those peptides derived from the hyaluronic acid-binding region.

Vol. 230

*., ..

.S...e 0

189


N 0- Mo - - 0 0 0

0

0 0 01010 10 10

'4 C~ N N -O0O - 0 00o o 6o o o0i

0

Proteoglycan (ug/ml)

Fig. 6. Immunological cross-reactivity of small proteoglycansProteoglycans at 1 pg/ml, i.e. cartilage, small (a), bone (b), tendon, small (c), and sclera, small (d), were coated on

poly(vinyl chloride) micro-titre plates. First antibody (a) raised against the cartilage small proteoglycan was used atdilution 1: 1500, (b) raised against the bone proteoglycan was used at dilution 1: 2000; (c) raised against the tendonsmall proteoglycan was used at dilution 1:2500, or (d) against the sclera small proteoglycan was used at dilution1:1600. The assays for dilutions of chondroitinase ABC-digested small proteoglycans were done as described in theMaterials and methods section. Proteoglycans were:-......, cartilage, small; ---, aorta, small; , tendon,small;--*-, cornea, dermatan sulphate proteoglycan referred to as 50 P (Axelsson & HeinegArd, 1975);

*-- sclera, small; bone. For reference, dilutions of chondroitinase ABC-digested large proteoglycans

(0-0) from cartilage (a), from tendon (c) and from sclera (d) are included. Zero inhibition is indicated bya 0 symbol.

Neither do any of the small proteoglycans react inan assay for hyaluronic acid-binding region (resultsnot shown). It is apparent, then, that the smallproteoglycans are quite different in nature, furtherverified by peptide patterns, discussed below. Allof the small proteoglycans tested, i.e. cartilage,.aorta, tendon, cornea, sclera and bone, showedcapacity to inhibit in all assays (Fig. 6). Except forthe one from cartilage, the inhibition curves hadsimilar slopes indicating immunological identity.Those from bone, cornea, sclera and tendonshowed very similar inhibition on a weight basis,but the one from aorta gave less than 10% of thisinhibition, indicating that only a small proportionof the molecules carried the antigenic determinant.In the assay for the small cartilage proteoglycan,however, the aorta proteoglycan gave a similar

degree of inhibition, whereas the other proteo-glycans gave less pronounced inhibition. Takentogether, these data indicate that there are at leasttwo groups of closely related small proteoglycans,on one hand those from aorta and cartilage and onthe other hand those from bone, cornea, sclera andtendon.

Peptide pattern. Further support for the presenceof two groups of molecules was obtained from thepeptide patterns (Figs. 7 and 8). The smallproteoglycans from cartilage and aorta gave verysimilar patterns (Fig. 7), with at least ten peptidesfound in both, schematically shown in the Figure.On the other hand, the patterns observed for thesmall proteoglycans from bone, cornea, sclera andtendon were rather similar to each other but quitedistinct from those of aorta and cartilage proteo-

1985

0.9

0.7

0.5

0.3

0.1

0.9

0.7

0.5

0.3

0.1

(a) Coat: Cartilage, small (b) Coat: Bone

[X*//_ >* (d) Coat: Sclera. small

/ (c) CoatTendon, small

I I I I I I I ~~~~~~~~~~II I I1o o Lfu LN - N N.

190

In


La) Car-tilage.srmiall

(h) Aor-ta.siliall

W.

oC2

E

x~~~~~

Fig. 7. Peptide patterns of trtvpsin-digested 121I-labelledsmall proteog/vcans from cartilage and aorta

Peptide patterns of trypsin-digested proteoglycanswere developed on thin-layer plates of silica gelessentially as described by Bates & Perham (1975).The + and - signs denote polarity during electro-phoresis and the X denotes the starting point. Thesamples were (a) cartilage, small, and (b) aorta,small proteoglycans. For reference those peptidesidentified in both preparations are schematicallyshown in (c).

glycans. In this case also more than ten peptideswere found to be present in all four preparations.All the peptide patterns of the small proteoglycanswere different from those of the large proteo-glycans.

General discussionBoth the immunological cross-reactivities and

the chemical data from the peptide mapping showthat there are two groups of small proteoglycans,the molecules within each group having verysimilar core proteins. Interestingly, chemical datashow that the small proteoglycan from cartilageand aorta have somewhat higher leucine contentsthan do the other ones. The chemical differences

are consistent with the grouping of the molecules intwo classes. It is known, however, that they differwith respect to glycosaminoglycan substituent,being either chondroitin sulphate in the cartilage(Heinegard et al., 1981) and in the bone (Franzen &Heinegard, 1984) proteoglycans or dermatan sul-phate with high contents of iduronic acid in theaorta (S. Gardell, unpublished work), sclera (Cos-ter & Fransson, 1981) and tendon (Vogel &Heinegard, 1985) proteoglycans, and the cornealproteoglycan contains sparsely sulphated dermatansulphate chains with an intermediate iduronic acidcontent (Axelsson & Heinegard, 1975). All of theseproteoglycans contain very large galactosamino-glycan side chains compared with the large proteo-glycans. It appears that it is not the structure of thecore protein that determines the type of side chain,but rather the synthetic machinery of the cell oforigin, possibly its content of the necessaryepimerase.To probe further the identity of the core proteins

of the proteoglycans in the various groups, se-quence data or at least quantitative peptidepatterns representing all peptides are required.The presented data, however, do allow us tosuggest that there are classes of proteoglycans withsimilar core proteins, containing side-chain substi-tuents of either chondroitin sulphate or dermatansulphate. It appears that the core proteins in themolecules of a class are not identical, only closelysimilar.

Since much of the character of the proteoglycanis determined by the core protein, we suggest anomenclature for these proteoglycans (Table 3),stating group, PG-LA (large aggregating) for thelarge proteoglycans and PG-Sm (small) for thesmall ones, possibly subdividing the latter into twogroups, PG-SmI and PG-SmII representing thetype found in cartilage and aorta (PG-SmI) andthose in bone, cornea, sclera and tendon (PG-SmII),tissue of origin and possibly nature of glycosamino-glycan side chains, where all chains containingiduronic acid are called dermatan sulphate.The identification of large aggregating proteo-

glycans in many tissues is interesting. It is possiblethat a major difference in structure between thosefrom cartilage and those from fibrous connectivetissues is the relative proportion between 0-linkedoligosaccharides and keratan sulphate. Only thecartilage proteoglycans have to date been conclu-sively shown to contain keratan sulphate. Interest-ingly, the proportion of large proteoglycans ishigher in tissues exposed to compressive load, i.e.cartilage, distal compared with proximal flexortendon (Vogel & Heinegard, 1985) and aorta. It ispossible that the synthesis of these proteoglycansincreases in response to mechanical forces.

Another interesting group of proteoglycans are

Vol. 230

191

192

(a) Cornea

(c) Bone

(e)

0

0

°°0 t-Coo00 )

x


(b) Tendon,small

d

(d) Sciera, small

IC

Co0

E0

_-)

4-

Fig. 8. Peptide patterns of trypsin-digested ' 25I-labelled small proteoglycans from cornea, tendon, bone and scieraPeptide patterns of trypsin-digested proteoglycans were developed on thin-layer plates of silica gel essentially asdescribed by Bates & Perham (1975). The + and - signs denote polarity during electrophoresis and the x denotesthe starting point. The samples were (a) cornea, dermatan sulphate proteoglycan, referred to as 50 P (Axelsson &Heinegard, 1975), (b) tendon, small proteoglycan, (c) bone, major proteoglycan, and (d) sclera, small proteoglycan.Those peptides identified in all four samples are schematically shown in (e).

1985


Table 3. Suggested nomenclature for interstitial galactosaminoglvcan-containing proteoglycansProteoglycans that have not been subgrouped should be referred to as PG-L (large) and PG-Sm (small) respectively.Subgroups can be introduced by adding numbers as indicated.

PG-LAI-CS (cartilage)

-LAI

PG-LAI-DS

sent in tendon,irta)

PG-LAII-CS (cartilage)

-LAII

PG-LAII-DS

PG-LN-CS

PG-LN

PG-LN-DS

The aggregatingchondroitin sulphate-richproteoglycan incartilage

The aggregatingkeratan sulphate-richproteoglycan incartilage

The non-aggregatingproteoglycan in cartilage(probably also present intendon, sclera and aorta)

PG-SmI-CS (

.PG-SmI

PG-Sml-DS (

PG-Sm

cartilage) The small proteoglycanin nasal cartilage

(aorta) The small proteoglycanin aorta

(bone) The small proteoglycanin bone

(tendon),(sclera)(cornea)

The small proteoglycansin tendon, sclera andcornea

the large non-aggregating proteoglycans identifiedin nasal cartilage (HeinegArd & Hascall, 1979).Problems encountered in identifying this group ofproteoglycans in other tissues are different stabili-ties of the interaction of different proteoglycanswith hyaluronate, possibly an effect of minorstructural differences perhaps involving disul-phide bridges, as well as other modifications of theproteoglycans in the matrix. Furthermore, it is

likely that the cartilage non-aggregating proteo-glycan is a mixture of distinct molecules withfragments of the aggregating proteoglycans. Theidentification of this type of proteoglycan infibrous connective tissues requires better tech-niques for their fractionation.

Grants were from the Swedish Medical ResearchCouncil, Folksam's Yrkesskadors Stiftelse, Kock's Stif-

Vol. 230

193

194 D. Heinegard and others

telser, Osterlund's Stiftelse, Konung Gustaf V :s 80-Arsfond and the Medical Faculty, University of Lund.

ReferencesAnderson, J. C. (1975) Biochim. Biophys. Acta 379, 444-

455Axelsson, I. & HeinegArd, D. (1975) Biochem. J. 145,491-500

Axelsson, I. & Heinegdrd, D. (1980) Exp. Eye Res. 31,57-66

Bates, D. L. & Perham, R. N. (1975) Anal. Biochem. 68,175-184

Coster, L. & Fransson, L.-A. (1981) Biochem. J. 193,143-153

Damle, S. P., Coster, L. & Gregory, J. D. (1982) J. Biol.Chem. 257, 5523-5527

Engvall, E. & Perlman, P. (1971) Immunochemistry 8,871-874

Franzen, A. & Heinegard, D. (1984) Biochem. J. 224, 59-66

Greenwood, F. C., Hunter, W. M. & Glover, J. S. (1963)Biochem. J. 89, 114-123

Hardingham, T. E. & Muir, H. (1972) Biochim. Biophys.Acta 279, 401-405

Hassel, J. R., Robey, P. G., Barrach, H.-J., Wilczek, J.,Rennard, S. I. & Martin, G. R. (1980) Proc. Natl.Acad. Sci. U.S.A. 77, 4494-4498

HeinegArd, D. & Axelsson, I. (1977) J. Biol. Chem. 252,1971-1979

Heinegard, D. & Hascall, V. C. (1974) J. Biol. Chem. 249,4250-4256

Heinegard, D. & Hascall, V. C. (1979)J. Biol. Chem. 254,927-934

Heinegard, D. & Paulsson, M. (1984) in ExtracellularMatrix Biochemistry (Piez, K. & Reddi, H., eds.), pp.278-328, Elsevier, New York

Heinegard, D., Paulsson, M., Inerot, S. & Carlstrom, C.(1981) Biochem. J. 197, 355-366

HeinegArd, D., Wieslander, J., Sheehan, J., Paulsson, M.& Sommarin, Y. (1985a) Biochem. J. 225, 95-106

HeinegArd, D., Inerot, S. & Lindblad, G. (1985b) Scand.J. Lab. Clin. Invest. in the press

Kjellen, L., Pettersson, I. & H6ok, M. (1981) Proc. Natl.Acad. Sci. U.S.A. 78, 5371-5375

Lohmander, S., DeLuca, S., Nilsson, B., Hascall, V. C.,Caputo, C. B., Kimura, J. H. & Heinegard, D. (1980)J. Biol. Chem. 255, 6084-6091

McDevitt, C. & Muir, H. (1971) Anal. Biochem. 44, 612-622

Oldberg, A., Kjellen, L. & Hook, M. (1979) J. Biol.Chem. 254, 8505-8510

Pearson, C. H. & Gibson, G. J. (1982) Biochem. J. 201,27-37

Salisbury, B. G. J. & Wagner, W. D. (1981) J. Biol. Chem.256, 8050-8057

Uldbjerg, N., Malmstrom, A., Ekman, G., Sheehan, J.,Ulmsten, U. & Wingerup, L. (1983) Biochem. J. 209,497-503

Wieslander, J. & Heinegard, D. (1979) Biochem. J. 179,35-45

Vogel, K. & HeinegArd, D. (1985) J. Biol. Chem. in thepress

1985

connective tissues form distinct subgroups

Documents