THE JOURNAL OF BI~L~CICAL CHEMISTRY G 1990 by The Amencan Society for Biochemistry and Molecular Biology, Inc.

Vol. 265. No. 12. Issue of April 25, pp. 6992-6999, 1990 Printed in U.S. A.

Occurrence in Chick Embryo Vitreous Humor of a Type IX Collagen Proteoglycan with an Extraordinarily Large Chondroitin Sulfate Chain and Short cul Polypeptide*

(Received for publication, June 12, 1989)

Toshikazu Yadas, Sakaru Suzuki& Kunihiko Kobayashig, Miya KobayashiQ, Takeshi Hoshinos, Katsuyuki Horien, and Koji KimataS 11 From the $Znstitute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-11, Japan, the SDepartment of Anatomy, Nagoya University School of Medicine, Nagoya 466, Japan, and the lTokyo Research Institute, Seikagaku Kogyo Company, Tokyo, Japan

We have prepared a high buoyant density proteogly- can fraction from the vitreous humor of 13-day-old chick embryos. Using immunoblot analysis coupled with chondroitinase digestion, we demonstrate that the purified preparation is composed predominantly of type IX collagen-like chondroitin sulfate proteoglycan with an (Al chain M, -23,000 shorter than the known al in cartilage type IX. Also different from cartilage type IX is the size of the chondroitin sulfate chain attached to the (r2(IX) polypeptide; its M, is -350,000 indicating that it is -10 times larger in vitreous humor than in cartilage. Examination of vit- reous bodies at different developmental stages indi- cates that a transition occurs in the size of crl(IX) in a well defined temporal pattern; at about stage 31, a cartilage-type ~yl(1X) of M, 84,000 is the predominant species, whereas at stage 36 and thereafter, a M, 61,000 species appears with a concomitant disappear- ance of the M, 84,000 species.

Immunostaining for type IX collagen followed by electron microscopic observation of 13-day-old chick embryo vitreous humor reveals a regular D-periodic arrangement of vitreous type IX collagen proteoglycan along thin fibrils. It seems possible that the chondroitin sulfate chains of extraordinarily high viscosity and high molecular weight may extend away from the fi- brils, thus contributing to structural as well as func- tional properties of this unique matrix.

We previously isolated from chick embryo cartilage a minor proteoglycan component of relatively low buoyant density and called PG’-Lt (Kimata et al., 1978). Subsequently, we demonstrated that the core protein of PG-Lt is composed of three dissimilar collagenous polypeptides and that chondroi- tin sulfate is covalently linked to one of the polypeptides (Noro et al., 1983). It is now established that PG-Lt represents

* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture, Japan. A part of this work was done in the Department of Chemistry, Faculty of Science, Nagoya University, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addressed. ’ The abbreviations used are: PG, proteoglycan; SDS, sodium do-

decyl sulfate; PBS, phosphate-buffered saline (0.1 M, pH 7.2); HMW and LMW, high and low M, fragments, respectively, isolated from peptic digests of chick embryo cartilage type IX collagen.

an intact form of type IX collagen proteoglycan’ with the chain composition otl(IX), 02(1X), (u3(IX), of which the (uS(IX) chain bears chondroitin sulfate chain(s) (Vaughan et al., 1985; Bruckner et al., 1985; Huber et al., 1986; Konomi et al., 1986; McCormick et al., 1987; Huber et al., 1988).

In cartilage, it has been established that type IX molecules are localized in a periodic manner along cartilage collagen fibrils (Miiller-Glauser et al., 1986; Vaughan et al., 1988; Bruckner et al., 1988) and are cross-linked to type II collagen molecules within such fibrils (Eyre et al,, 1987; van der Rest and Mayne, 1988). Thus, type IX molecules on the fibrils have been implicated in interacting with each other and with other components of the matrix (Vasios et al., 1988). However, the role played by the glycosaminoglycan chains of type IX collagen proteoglycan is not precisely understood.

Embryonic chick cornea and vitreous humor have been shown to contain immunodetectable protein for type IX col- lagen (Fitch et al., 1988). Likewise, Ayad and Weiss (1984) have reported evidence for the occurrence in bovine vitreous humor of type IX collagen along with type II collagen. Not only these specific collagens but also some glycosaminogly- cans have been demonstrated in avian and mammalian vit- reous humor, e.g. chondroitin sulfate in chick embryo vitreous humor (Smith and Newsome, 1978) and hyaluronic acid in bovine vitreous humor (reviewed by Laurent and Fraser, 1986, among others). In an effort to understand the role played by the glycosaminoglycan side chains of type IX collagen proteo- glycan, we have investigated the nature, expression, and or- ganization of vitreous humor type IX collagen in the chick embryos at different developmental stages. Here we show that the vitreous humor type IX collagen molecule is unique in containing a short form of (rl(IX) polypeptide and an extraor- dinarily large chondroitin sulfate chain, the results suggesting a contribution of the structural modulation to the functional differences between cartilage matrix and the vitreous humor. We also give immunohistological data as to its longitudinal arrangement along thin fibrils in the vitreous humor. A pre- liminary account of this work has appeared (Yada et al., 1988).

Recently, a report of electron microscopic studies on the collagen fibrils of adult chicken vitreous humor has been

’ As shown in the present report, the type IX collagens (PG-Lt) of chick embryo cartilage and vitreous humor are different in both polypeptide and polysaccharide structures. Moreover, recent evidence indicates that some of the type IX collagen molecules of cartilage lack a chondroitin sulfate side chain (T. Yada, M. Arai, S. Suzuki, and K. Kimata, unpublished observation). To avoid nomenclatural confusion caused by the structural variation, the chondroitin sulfate- carrying form of chick embryo cartilage type IX collagen is referred to as “cartilage type IX collagen proteoglycan” in the present paper.

6992

by guest on February 10, 2018http://w

ww

.jbc.org/D

ownloaded from

Vitreous Humor Proteoglycan

presented by Wright and Mayne (1988). The results of these authors clearly show that the collagen fibrils are different from those in cartilage in that they are completely invested by a coat of chondroitinase-sensitive glycosaminoglycan which extends for a considerable distance from the fibrils. The difference in the morphological feature of the collagen fibrils can be accounted for by the results of biochemical analyses presented below.

6993

adjuvant was injected into a lymph node of the popliteal region of a rabbit. At three subsequent 2-weeks intervals, 0.2 mg of HMW in 0.5 ml of PBS, 0.05 mg of monophosphoryl lipid A plus 0.05 mg of trehalose dimycolate, 0.5 ml of Freund’s incomplete adjuvant was injected to the intradermal site. The antibody titer and specificity were examined by enzyme-linked immunosorbent assay (Rennard et al., 1981) and by immunoblotting (Towbin et al., 1979), respectively. The serum was collected 3 weeks after the third booster. The antisera were shown to react with both HMW and intact cartilage type IX or PG-Lt but not with collaeen tvnes I. II. III. IV. V. X. or XI.

EXPERIMENTAL PROCEDURES

Materials-Fertile eggs (White Leghorn) were obtained from a local supplier; DNase I, RNase A, bovine serum albumin, and phen- ylmethanesulfonyl fluoride were from Sigma; chondroitinase ABC, chondroitinase AC, shark cartilage chondroitin sulfate (average M, 43,000), and bovine tracheal cartilage chondroitin sulfate (average M, 15,000) were from Seikagaku Kogyo Co., Tokyo, Japan; Sepharose CL-4B was from Pharmacia Janan. Tokvo: Bio-Gel A-1.5m was from Bio-Rad Laboratories Japan: Tokyo;” complete and incomplete Freund’s adjuvant and skim milk were from Difco; horseradish per- oxidase-conjugated protein A was from E-Y Laboratories, San Mateo, CA; “‘I-protein A was from ICN Radiochemicals; x-ray film (Fuji- RX) was from Fuji Photo Film Co., Kanagawa, Japan; and Ribi adjuvant system (monophosphoryl lipid A and trehalose dimycolate) was from Ribi ImmunoChem Research Inc., Hamilton, MT.

Generous gifts of the following compounds are acknowledged: type III collanen (adult rat skin) from R. Hata. the Tokvo Medical and Dental College, Tokyo; type IV collagen (mouse Engelbreth-Holm- Swarm sarcoma) from M. Kato, in this laboratory; type V collagen (human placenta) from P. Bornstein, the University of Washington, School of Medicine, Seattle, WA; and a standard sample of the high molecular weight fragment derived from chick sternal cartilage type IX collagen bv aensin digestion (HMW) from R. Mavne. the Univer- sity of Alabama, Birmingham. Gollagens type I from rat tail tendon (Linsenmayer et al., 1978), types II, IX (HMW), and XI from chick embryo sternal cartilage (Reese and Mayne, 1981), and type X from mass chondrocyte cultures (Schmid and Linsenmayer, 1983), were prepared by the methods described in the indicated literature. Hya- luronic acid samples of different molecular weights were prepared from rooster comb by extraction with water followed by consecutive purification procedures including Pronase digestion and ethanol frac- tionation. To obtain samples of relatively small molecular weights, some preparations were subjected to either heating in an autoclave or digestion with testicular hyaluronidase before ethanol fractiona- tion. The average molecular weights of the resulting fractions were determined by the measurement of low angle laser light scattering according to Ueno et al. (1988). PG-H (a major PG species of chick embryo cartilage which is much higher in both molecular weight and buoyant density than PG-Lt and PG-Lb, Oike et al., 1980), PG-Lb (a second PG species of chick embryo cartilage which is smaller in size than PG-H, but higher in buoyant density than PG-Lt, Shinomura et al., 1983), and PG-Lt or cartilage type IX collagen proteoglycan (Noro et al., 1983) from chick embryo epiphyseal cartilage, and PG- M (a maior PG of chick embrvo mesenchvme and cultured fibroblast which is”different in core protein structure from PG-H, PG-Lt, and PG-Lb, Yamagata et al., 1986), were prepared as described.

Extraction and Separation of Vitreous Humor Proteoglycan-Fer- tile eggs were incubated for the periods indicated in individual exper- iments at 38 “C. Embryos were removed, rinsed in Hanks’ solution, and staged according to Hamburger and Hamilton (1951). The vit- reous humor obtained from embryos was homogenized in an equal volume of 8 M auanidine HCl. 0.1 M Tris-HCl. DH 8.0. 2 mM uhen- ylmethanesulfonyl fluoride, 2O’mM N-ethylmal&mide, 20 mM E’DTA, and the mixture was stirred at 4 “C for 12 h. The suspension was centrifuged at 4 “C for 30 min at 17,000 X g. The residue was discarded. CsCl was added to the clarified extract to give an initial density of 1.36 g/ml. A dissociative gradient was established by centrifugation in a Hitachi RP-50T2 rotor (145,800 X g, 10 “C, 48 h) and then fractionated from the bottom of the tubes. Hexuronate and protein contents in the fractions were determined by the carbazole method of Bitter and Muir (1962) and by the method of Lowry et al. (1951), respectively.

Antisera to Chick Embryo Sternal Cartilage HMW and Affinity- purified Antibodies to the 61-kDa Polypeptide of Vitreous Humor Proteoglycan-0.5 mg of HMW in 0.5 ml of 0.1 M PBS (pH 7.2), 0.05 mg of monophosphoryl lipid A plus 0.05 mg of trehalose dimycolate (Ribi adjuvant system) emulsified with 0.5 ml of Freund’s complete

From the anti-HMW antisera, antibodies specific for 61-kDa poly- peptide of vitreous humor proteoglycan (see “Results” for the prepa- ration of this compound) were affinity-purified as follows. A purified sample of the proteoglycan was treated with chondroitinase AC and subjected to SDS-polyacrylamide gel electrophoresis under reduced conditions (see below), followed by electrotransfer to a nitrocellulose membrane. The membrane was treated with 10% (w/v) skim milk in PBS and then with the anti-HMW antisera prepared as above. After washing with PBS. 0.05% (w/v) Tween 20 (five times). the 61-kDa band complexed with antibodies was cut out and eluted with 0.2 ml of 0.2 M glycine HCl, pH 2.4. The eluate was immediately neutralized with 0.02 ml of 1 M Tris-HCl, pH 9.0.

Analyses of Proteoglycan Core Polypeptides by SDS-Polyacrylamide Gel Electrophoresti-Polyacrylamide gel electrophoresis in the pres- ence of 0.1% (w/v) SDS was carried out in 5% (w/v) running gel (Laemmli, 1970) under nonreducing and reducing conditions. Before electrophoresis, proteoglycan samples were desalted by precipitation with 3 volumes of 95% (v/v) ethanol, 1.3% (w/v) potassium acetate at 0 “C (Kimata et al., 1974). To remove chondroitin sulfate chains, ethanol precipitates (about 40 pg each as hexuronate) were dissolved in 30 ~1 of 0.05 M Tris-HCl, pH 7.6,0.05 M sodium acetate containing 0.1 unit of chondroitinase ABC or chondroitinase AC and incubated at 37 “C for 2 h. Incubated mixtures and control samples were heated in a boiling water bath for 3 min. Proteoglycans and/or their core molecules were precipitated with ethanol (see above) and dissolved in the sample buffer by heating at 100 “C for 3 min. Reduction was carried out by adding 2-mercaptoethanol to a final concentration of 5% (w/v) in sample buffer. The molecular masses of the polypeptides were estimated with collagenous peptides as standards. After electro- phoresis, proteins and proteoglycans in gels were detected by staining with Coomassie Blue and Alcian Blue, respectively. For immunostain- ing, the proteins in a gel were transferred to a nitrocellulose mem- brane by the method of Towbin et al. (1979). The membrane was then incubated successively in the following solutions: 1) 10% (w/v) skim milk, PBS at 37 “C for 1 h; 2) anti-HMW antisera in PBS, 0.05% (w/v) Tween 20 (1:lOOO) or affinity-purified antibodies to the 61-kDa protein at room temperature for 2 h; 3) five changes of PBS, 0.05% (w/v) Tween 20; 4) horseradish peroxidase-conjugated protein A or iZ51-protein A in PBS, 0.05% (w/v) Tween 20 at room tempera- ture for 1 h; 5) five changes of PBS, 0.05% (w/v) Tween 20; 6) 0.05% (w/v) 4-chloro-1-naphthol, 0.01% (v/v) H,O, in PBS for 5 min to develop the color. For autoradiographic detection of bound lz51-protein A, the membranes were dried and exposed to Fuji RX x-ray film at -80 “C (Fig. 4).

Analyses of Glycosaminoglycans-Glycosaminoglycans were re- leased from proteoglycan samples by treatment with 0.2 M NaOH at room temperature for 24 h. After neutralization with acetic acid, the mixture was digested in 0.02 M Tris-HCl, pH 8.0, with Pronase (one- fiftieth the total amount of proteins) at 50 “C for 12 h. Glvcosami- noglycans were collected by ethanol precipitation, dissolvedin either 0.4 M ammonium acetate or 4 M auanidine HCl, 0.05 M Tris-HCl. DH 7.5, and chromatographed on a B&Gel A-1.5m column or a Sepharose CL-4B column equilibrated and eluted with sample buffer. Viscosities of glycosaminoglycans were measured in a Cannon-Manning micro- viscometer in 0.15 M phosphate buffer, pH 7.0, 0.2 M NaCl at 25 “C, according to Ueno et al. (1988).

For determination of disaccharide unit compositions of glycosa- minoglycans, chondroitinase digestion was performed as previously described (Saito et al., 1968), and separation of the products was carried out by high-performance liquid chromatography on a Licro- sorb-NH* column (0.26 x 25 cm) eluted with a linear gradient of NaH,PO* (Yoshida et al., 1989). The unsaturated disaccharide prod- ucts were detected and quantified by virtue of their absorbance at 232 nm.

Electron Microscopy-Vitreous bodies were dissected from 13.day- old chick embryos and immediately fixed in 0.01 M sodium periodate, 0.07 M lysine, 0.037 M phosphate, pH 6.2,2% (w/v) paraformaldehyde at 4 “C for 24 h. The specimens were dehydrated with a gradual

by guest on February 10, 2018http://w

ww

.jbc.org/D

ownloaded from

6994 Vitreous Humor Proteoglycan

increase of ethanol concentration at 5% (v/v)/h, cleaned in 1-butanol, and embedded in paraffin. Sections were placed on an egg white- coated glass slide, deparaffinized with xylene, washed with PBS, and incubated in a moist chamber with HMW antisera (1:lOO) in PBS, 1% (w/v) bovine serum albumin at room temperature for 1 h. The sections were washed with PBS (5 times) and then incubated with peroxidase-conjugated protein A (1:lOO) in PBS, 1% (w/v) bovine serum albumin at room temperature for 1 h. After washing with PBS, the sections were incubated with 0.05% (w/v) diaminobenzidine, 0.1% (w/v) H,O, in PBS for 5 min to develop the color. For light micros- copy, the immunolabeled sections were washed with distilled water, dehydrated, and mounted on a coverslip. For electron microscopy, the immunolabeled sections were postfixed with 1% (w/v) osmium tetroxide in PBS, embedded in Quetol-812 with Luft’s 5:5 preparation, and sectioned by a Sorval MT1 ultramicrotome using a diamond knife. The resultant ultrathin sections were stained with uranyl acetate and then with lead citrate by the method of Reynolds (1963), and examined through an electron microscope (Hitachi H-800). The longitudinal distance between immunodeposits was determined by measuring the mean distance between centers of the deposits on 193 independent thin fibrils.

RESULTS

Isolation of Vitreous Humor Proteoglycan-When the vit- reous humor obtained from 13-day-old (stage 39) chick em- bryos was extracted with 4 M guanidine HCl as outlined under “Experimental Procedures,” about 85% of the total carbazole reaction-positive material was brought into solution. The insoluble material was not studied further.3 However, it prob- ably represents so-called pecten, a major component of the vascular network in avian vitreous humor (Romanoff, 1960). The extract was partitioned by CsCl isopycnic centrifugation under dissociative conditions. As Fig. 1 shows, the carbazole reaction-positive material yielded three peaks a, b, and c, of which a and b were located in a lower one-third where little Lowry reaction-positive material was detected. When aliquots of each peak fraction were desalted by ethanol precipitation and assayed for chondroitinase ABC- or Streptomyces hyalu- ronidase-susceptible material, all of the carbazole reaction- positive material in peak a, lo-20% of the material in peak b, and approximately one-half of the material in peak c were digested with chondroitinase ABC, as assessed by the corre- sponding reduction in ethanol-precipitable, carbazole reac- tion-positive materials. None of the peak fractions, however, was digestible by Streptomyces hyaluronidase treatment. The resistant material in peak b probably represents DNA, as assessed by its strong adsorption at 260 nm, by its suscepti- bility to DNase I, and by its failure to react with RNase A (data not shown). The resistant material in peak c, on the other hand, was characterized as heparan sulfate in proteo- glycan form; successive treatment of peak c material with

3 Aliquots of the guanidine HCl extract and the residue were subjected sequentially to alkali treatment, Pronase digestion, and DNase I digestion. Subsequent ethanol precipitation yielded glycos- aminoglycan fractions free from most of the proteins and nucleic acid, which could interfere with the carbazole method. Based upon the hexuronate contents in the preparations, 88% of the total glyco- saminoglycans was extracted with 4 M guanidine HCl. Since, as shown in the later section, most, if not all, glycosaminoglycans in the vitreous were derived from type IX collagen proteoglycan, the results suggest that most of a type IX collagen proteoglycan were solubilized by the guanidine HCl. In addition, other aliquots of the guanidine HCl extract and the residue were dialyzed and then digested with pepsin, respectively. The digests were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-HMW antibodies. The stained band corresponding to HMW was only seen in the guanidine HCl extract, confirming the above suggestion. It is of note, however, that there were some antibody-positive materials in the origins of both samples, which may represent the occurrence of a small propor- tion of highly cross-linked subpopulation of type IX collagen in both fractions.

FRACTION NUMBER Bottom Top

FIG. 1. CsCl isopycnic centrifugation under dissociative conditions of the extract from 13-day-old chick embryo vit- reous humor. Sedimentation profiles of carbazole reaction-positive material (q expressed as hexuronate) and protein (0) are shown. The fractions (I, II, and III) were pooled for further analyses as indicated by the boxes. Carbazole reaction-positive peaks denoted a, b, and c from the bottom to the top of the tube, respectively, were served for enzymatic analysis of their compositions.

alkali and Pronase yielded a glycosaminoglycan sample which behaved as a mixture (about 1:l with respect to the color intensity in carbazole reaction) of chondroitin sulfate and heparan sulfate when examined by electrophoresis on a cel- lulose acetate membrane before and after treatment with chondroitinase AC or heparitinase, or both (data not shown). In the present study, the material in peak c was not investi- gated further.

To test the possible occurrence of a proteoglycan form of type IX collagen, the gradients were partitioned into three fractions, I, II, and III, as in Fig. 1, and aliquots of each pooled fraction were examined by SDS-polyacrylamide gel electro- phoresis (5% gel) before or after treatment with chondroiti- nase ABC (Fig. 2). Before enzyme treatment, fraction I did not give any Coomassie Blue-positive band in the running gel (lane a) but exhibited a strong Alcian Blue-positive band at the top of the gel (not shown). Chondroitinase ABC digestion resulted in the disappearance of the Alcian Blue-positive band with a concomitant appearance of a single Coomassie Blue- positive band at 270 kDa relative to collagen standards (lane b). The doublet near the gel front (indicated by the open arrowheads) can be attributed to proteins from the chondroi- tinase preparation, because the same bands were present in a control gel containing enzyme alone (not shown). Even when the enzyme-treated fraction I was subjected to SDS-electro- phoresis in a 5-20% (w/v) polyacrylamide gel, no appearance of any detectable core protein band other than the 270-kDa one was again observed (data not shown). In addition, when the pooled fractions were analyzed for chondroitin sulfate contents by the measurement of the hexuronate which was released from macromolecules by chondroitinase ABC diges- tion, 87, 12, and 1% of the total chondroitin sulfate were recovered in fractions I, II, and III, respectively, It is likely therefore that the Alcian Blue-positive material represents a large chondroitin (dermatan) sulfate proteoglycan with a core molecule corresponding in M, to the core of type IX collagen proteoglycan (270 kDa) and at least 87% of the total chon- droitin sulfate of the vitreous was derived from type IX collagen proteoglycan. Similar enzymatic treatment of frac- tion II also resulted in the appearance of a 270-kDa protein band (lane d) but its amount was far smaller than in fraction

by guest on February 10, 2018http://w

ww

.jbc.org/D

ownloaded from

Vitreous Humor Proteoglycan 6995

Coomassie Blue - staining gel

Mr abcdef

x10-3 qm

270. &&

.:. 1% g$ @ &j

B -,r

Immunoblot

ghijkl +origii

*front FIG. 2. SDS-gel electrophoresis (5% gels) and immunoblot

analysis of the fractions from CsCl isopycnic centrifugation (Fig. 1). The fractions (I, ZZ, and ZZZ) pooled as indicated in Fig. 1 were electrophoresed under nonreduced conditions on a 5% SDS- polyacrylamide gel and stained with Coomassie Blue (lanes a-f). Lanes g-l, result of staining nitrocellulose blots with anti-HMW antisera. The samples are: a, fraction I; b, as in a but treated with chondroitinase ABC; c, fraction II; d, as in c but treated with chon- droitinase ABC; e, fraction III; j, as in e but treated with chondroiti- nase ABC. Samples for lanes g, h, i, j, k, and 1 correspond to those for lanes a, b, c, d, e, and f, respectively. The closed arrowhead indicates position of type IX collagen triple polypeptides of 270 kDa; the open arrowheads indicate protein bands derived from the chondroitinase preparations.

I. In fraction III, no chondroitinase-sensitive component was detected (lane f).

To test whether the 270-kDa core protein is related to cartilage type IX collagen proteoglycan, samples were electro- phoresed as above and then transferred to nitrocellulose mem- branes. The membranes were stained using antisera raised against one of the triple-helical portions (HMW) of cartilage type IX collagen proteoglycan. As the immunostaining pat- tern (Fig. 2, lanes g-l) shows, the 270-kDa bands in lanes h and j (the band in lane j cannot be photographically repro- duced) but no other protein bands were immunoreactive to anti-HMW antisera.

The results, taken together, indicate that fraction I of the CsCl gradient (Fig. 1) contains a proteoglycan form of type IX collagen as a sole protein component, although its buoyant density (~1.52 g/ml) is much higher than the value reported for cartilage type IX collagen proteoglycan (51.34 g/ml, see Noro et al., 1983).

For further characterization of the vitreous humor proteo- glycan, about 50 ml of the vitreous humor collected fresh from 150 chick embryos were extracted and partitioned as above and the fractions with buoyant densities higher than 1.52 g/ ml (which were free of DNA) were pooled. The yield was about 2 mg/50 ml vitreous humor of 150 embryos; hereafter this preparation is referred to as “vitreous PG.”

Comparison between Vitreous PG and Cartilage Type IX Collagen Proteoglycan-Intact and chondroitinase AC-treated vitreous PG samples were analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing and reducing condi- tions (Fig. 3). The use of chondroitinase AC in place of the chondroitinase ABC in Fig. 2 was necessary to avoid an overlapping, under reduced conditions, of protein bands de- rived from the enzyme preparations with those from proteo- glycan samples. In agreement with the results of analyses using chondroitinase ABC (see above), intact vitreous PG did not enter the 5% gel (Fig. 3, lane a) whereas chondroitinase

Mr a b c d

x10-3 Mr x10-3 *rigIn

270.

4rcnt FIG. 3. SDS-gel electrophoresis (5% gels) of vitreous PG

and chondroitinase AC-treated vitreous PG. The samples for lanes a and b were electrophoresed under nonreduced conditions on a 5% SDS-polyacrylamide gel and stained with Coomassie Blue. The samples are: lane a, vitreous PG; lane b, as in a but treated with chondroitinase AC. Lanes c and d, result of gel electrophoresis of the same samples for lanes a and b, respectively, under reduced condi- tions. The loaded amount of reduced samples (lanes c and d) was twice as much as nonreduced samples (lanes a and b). Closed arrow- heads indicate the positions of type IX collagen triple polypeptide (270 kDa), al(IX) (84 kDa), a2(IX) and (u3(IX) (68 kDa), and a short form of 01(1X) (61 kDa); open arrowheads indicate proteins from the chondroitinase AC preparation.

AC-treated vitreous PG gave a single protein band at 270 kDa (Fig. 3, lane b). The bands indicated by the open arrowheads represent proteins from the enzyme preparation. After reduc- tion, both intact (lane c) and chondroitinase AC-treated (lane d) samples yielded a sharp band at 61 kDa, a somewhat broad band at 68 kDa, and a very weak band at 84 kDa. The color intensity of the 68kDa component was significantly higher in the chondroitinase-treated sample than in the nontreated sample, suggesting that a part of the 68-kDa band represents a polypeptide derived from glycosaminoglycan-bearing (Y chain. Consistent with this view, an Alcian Blue-positive band was detected at the top of the gel in lane c but not in lane d (data not shown).

When the single protein band at 270 kDa (lane b) was cut out and again subjected to the gel electrophoresis under reduced conditions, three bands at 84, 68, and 61 kDa with the different color intensities were observed (data not shown), similarly to those in Fig. 3, lane d, indicating that protein bands in the lane d were derived from the 270-kDa protein.

It is well established that cartilage type IX collagen proteo- glycan is a heterotrimer of three polypeptide chains cul(IX), (uS(IX), and a3(IX) with molecular mass 84,000, 68,000, and 68,000, respectively, and that the intact molecules contain chondroitin sulfate or dermatan sulfate covalently linked to the (uS(IX) chain. Therefore, the results in Fig. 3 suggest that, although vitreous PG is similar to cartilage type IX collagen proteoglycan, there are tissue-specific variations at the al(IX) chain leading to the appearance of a 61-kDa chain in place of the 84-kDa chain; hereafter they are referred to as al(IX)” kDs and CU~(IX)~~ kDa, respectively. The appearance of the weak band at 84 kDa in Fig. 3, lanes c and d, may reflect the fact that the vitreous humor preparation consists of a small amount of the cartilage-type component, ~Y~(IX)‘~~~~, in ad- dition to the major, vitreous humor-type component, a!l(IX)6lkna.

When chondroitinase AC-treated vitreous PG and cartilage type IX collagen proteoglycan were run under reduced con-

by guest on February 10, 2018http://w

ww

.jbc.org/D

ownloaded from

6996 Vitreous Humor Proteoglycan

ditions on an SDS gel and then examined by immunoblotting with anti-HMW antisera (Fig. 4), the 84, 68, and 61-kDa bands from vitreous PG (lane a) as well as the 84- and 68- kDa bands from cartilage type Ix collagen proteoglycan (lane b) were all immunoreactive to the antisera. When, however, the anti-HMW antisera were replaced by affinity-purified antibodies to the 61-kDa polypeptide (prepared with a nitro- cellulose membrane to which the 61-kDa polypeptide had been electrotransferred), the 68-kDa band corresponding to cu2(IX) and a3(IX) was no longer detected in both vitreous humor (lane c) and cartilage (lane d) samples. In contrast, the 84- and 61-kDa bands in the vitreous humor sample (lane c) as well as the al(IX)“4kDa band in the cartilage sample (lane d) showed significant reactivities, indicating that the 61-kDa polypeptide of vitreous PG is immunochemically re- lated to the (u~(IX)“~~~~ chains of cartilage type IX collagen proteoglycan and vitreous PG.

In addition to the difference in the polypeptide chains, a marked difference was observed when the glycosaminoglycan chains released from vitreous PG, cartilage type IX collagen proteoglycan, and other proteoglycans were compared on a Bio-Gel A-1.5m column. As Fig. 5 shows, t,he glycosaminogly- cans from vitreous PG were far larger in hydrodynamic size than the chondroitin sulfate or dermatan sulfate with M, ranging from 32,000 to 60,000 (Kimata et al., 1978; Yamagata et al., 1986), the samples prepared from chick embryo cartilage proteoglycans (PG-H, PG-Lb, and type IX collagen proteo- glycan/PG-Lt) and chick embryo fibroblast proteoglycan (PG-M). This behavior of the vitreous humor glycosamino- glycan chains could not be ascribed to their self-aggregation, as a similar elution pattern was demonstrated by chromatog- raphy on a Sepharose CL-4B column regardless of whether or not guanidine HCl (4 M) was present in the elution buffer (data not shown). By measurement using high-performance liquid chromatography on two TSK-Gel G6000PW columns straight forward connected (0.75 x 60cm), the average molec- ular weight of the vitreous humor glycosaminoglycan chains was estimated to be about 350,000 relative to hyaluronic acid standards with known molecular weights. A similar value was

a b cd Mr

x105 drigii

*front FIG. 4. Transfer blot analyses using anti-HMW antisera

and affinity-purified antibodies to the 61-kDa polypeptide. Vitreous PG and cartilage type IX collagen proteoglycan were treated with chondroitinase AC and then electrophoresed on a 5% SDS- polyacrylamide gel under reduced conditions. The result of staining nitrocellulose blots with anti-HMW antisera is shown in lanes a (vitreous PG) and b (cartilage type IX collagen proteoglycan). Lanes c (vitreous PG) and d (cartilage type IX collagen proteoglycan) show the result of staining with affinity-purified antibodies to the 61-kDa polypeptide to see whether the 61-kDa polypeptide is related to 84- kDa (ul(IX). The closed arrowheads indicate the positions of al(IX) (84 kDa), (uZ(IX) and/or (u3(IX) (68 kDa), and a short form of al(IX) (61 kDa). X, immunologically nonspecific bands.

VO M LbUH

I v

( 0.3mlltube) FIG. 5. Bio-Gel A-1.5m chromatography of the glycosami-

noglycans released from vitreous PG. Glycosaminoglycans ob- tained after alkali and Pronase treatment of vitreous PG were chro- matographed on a Bio-Gel A-1.5m column. J, positions for the gly- cosaminozlvcans of PG-H (H. M, 32.000). cartilage twe IX collagen proteogly&“n (Lt, M, 40,000), gnd P&Lb (Lb, M,-52,0bO) from cl&k embryo cartilage, and those of PG-M (M, M, 60,000) from chick embryo fibroblasts. V,, void volume. V,, total column volume.

TABLE I

Composition of glycosaminoglycans derived from vitreous PG and cartilage type IX collagen proteoglycan

Shown is the percentage of unsaturated disaccharides released by chondroitinase ABC or AC digestion.

Composition Disaccharide units

Vitreous humor Cartilage

% IdoA-GalNAc(4-SOJ 7 12 GlcA-GalNAc(4-SO.,) 17 46 GlcA-GalNAc(G-SOJ 63 21 GlcA-GalNAc 13 21

suggested when viscosities of the glycosaminoglycan samples were compared, the reduced viscosity of the vitreous humor sample was 6.5 dl/g compared to a value of 9.2 dl/g for hyaluronic acid with a light-scattering molecular weight 460,000. The molecular weight of the vitreous humor chon- droitin sulfate was calculated to be 330,000, based on its reduced viscosity. Far lower values, 0.45 and 1.4 dl/g were obtained with bovine cartilage chondroitin sulfate (molecular weight 15,000) and shark cartilage chondroitin sulfate (mo- lecular weight 43,000), respectively.

As Table I shows, analysis of the glycosaminoglycan sam- ples with chondroitinase ABC and AC indicates that the vitreous humor glycosaminoglycan is rich in chondroitin 6- sulfate units (63%), compared with the sample of cartilage type IX collagen proteoglycan at the same developmental stage which is rich in chondroitin 4-sulfate units.

Transition in crl (IX) Chain Size during the Development of Embryonic Chick Vitreous Humor-The demonstration in the 13-day-old (stage 39) embryonic chick vitreous humor of two different size otl(IX) polypeptides suggests that transitions may occur in the type of vitreous PG synthesized during the development of vitreous body. To test this possibility, the chain compositions of vitreous PG samples prepared from younger (stages 31 and 36) and older (stages 42 and 45) embryos and adult chicks (60 days old) were analyzed accord-

by guest on February 10, 2018http://w

ww

.jbc.org/D

ownloaded from

Vitreous Humor Proteoglycan

ing to the protocol used for the analysis of stage 39 sample. All the vitreous humor samples gave proteoglycan fractions with buoyant densities higher than 1.52 g/ml, suggesting the occurrence of proteoglycan molecules bearing very large chon- droitin sulfate chains. Analyses of the resultant proteoglycan fractions by immunoblotting using anti-HMW antisera (Fig. 6) indicate that stage 31 vitreous humor (lane a) contained a vitreous PG which showed, after chondroitinase AC digestion followed by mercaptoethanol reduction, an immunostaining pattern for ales kDa, ~u2(1X), and ~u3(1X) as predominant core peptides. At later stages, the vitreous humor showed a progressive increase of ,1(IX)61kDa with a concomitant de-

6997

crease of 0ll(1X)‘~ kDa, and the specimens from stage 42-45 chick embryos and 60-day-old chicks (lanes b-f) contained predominantly cxl(IX)‘l kDa, (~2(1X), and ~u3(1X).

D-Periodic Distribution of Vitreous PG along Thin Collagen Fibrils-A combination of immunological and electron micro- scopic methods was used to analyze the distribution of vitre- ous PG in 13-day-old embryonic chick vitreous humor. The sections immunolabeled with anti-HMW antisera followed by poststaining with uranyl acetate/lead citrate (Fig. 7a) showed that immunoreactive materials (vitreous PG; arrowheads) are distributed in a regular D-periodic arrangement of 66.9 nm (S.D. 8.7 nm, 193 fibrils) along individual thin fibrils (com- posed presumably of type II collagen) of a uniform diameter of 10 nm. These observations indicate that the epitopes of our polyclonal antibodies were not evenly distributed along a type IX collagen proteoglycan molecule in the fibril, as de- scribed by Bruckner et al. (1988) on human cartilage type IX collagen that had been stained with polyclonal antibodies raised against HMW + LMW. In Fig. 7a, clustered fibrils decorated by immunoreactive materials were frequently pres- ent, perhaps representing artifacts derived from individual thin fibrils by a shrinkage of vitreous humor during dehydra- tion procedures (apparent distances of immunoreactive ma- terials on these clustered fibrils are not included in the above data for D-periodicity). Control sections where anti-HMW antisera had been replaced by nonimmune sera revealed only thin and clustered fibrils with no periodically arranged ma- terials (Fig. 7b).

MrxlO-l a b c d e f

E w*r A.

FIG. 6. Results of transfer blot analyses using anti-HMW antisera to show a transition in the size of vitreous humor al(IX) polypeptide during chick embryo development. Vitre- ous PG samples prepared from the vitreous humor specimens at developmental stage of 31 (lane a), 36 (lane b), 39 (lane c), 42 (lane d), and 45 (lane e), and at 60 days (adult chick; lane f) were treated with chondroitinase AC and electrophoresed under reduced condi- tions as in Fig. 4. The result of staining nitrocellulose blots with anti- HMW antisera is shown (the top and bottom parts of the blots are not shown). ., positions for oll(IX) (84 kDa), 02(1X) and/or 03(1X) (68 kDa), and a short form of otl(IX) (61 kDa).

FIG. 7. Electron micrograph of immunolabeled fibrils in the vitreous humor of 13-day-old chick embryo. a, vitreous PG molecules (b) are visualized by the immunoperoxidase method using anti-HMW antisera; b, control using nonimmune serum in place of the HMW antisera; bar, 100 nm.

by guest on February 10, 2018http://w

ww

.jbc.org/D

ownloaded from

6998 Vitreous Humor Proteoglycan

DISCUSSION

The results presented here indicate that vitreous humor of chick embryos contains a modified form of type IX collagen proteoglycan which is characterized by the occurrence of a short form of cul(IX) polypeptide and by the attachment of extraordinarily large chondroitin sulfate chain(s).

Using probes specific for otl(IX) and (~2(1X) mRNAs, Svo- boda et al. (1988) have demonstrated that, although the size of a2(IX) mRNA is the same in chick embryo cornea as in cartilage, the crl(IX) mRNA is significantly smaller in the cornea than in the cartilage because 700 nucleotides are absent at the 5’ end of the mRNA encoding the major portion of the amino-terminal globular domain (NC4). It seems pos- sible that a similar, if not identical, alteration may have occurred in the mRNA for vitreous humor (Al polypeptide, leading to the synthesis of oll(IX)‘IkDa, a smaller form pre- sumably lacking the major portion the amino-terminal glob- ular domain (see below). It is noteworthy in this respect that, at early developmental stages (some time between stage 34 and 40), an epithelium (the neural retina) is responsible for the synthesis and secretion of collagens and glycosaminogly- cans into vitreous humor, whereas at later stages these com- ponents are being produced predominantly by a fibroblastic cell population (hyalocytes) within the vitreous matrix itself (Newsome et al., 1976; Smith and Newsome, 1978). It is possible that the observed transition of al(IX) polypeptide from the long to the short form is closely associated with the temporal and spatial transition in cell type during the devel- opment of chick embryo vitreous humor. Whether the fibro- blastic cells (hyalocytes) in the vitreous humor represent a population of cells that have emigrated from the neural retina remains to be established.

The most unusual feature of the present investigation is the demonstration in vitreous PG of chondroitin sulfate chains with an average molecular weight around 350,000, about 10 times the average value of ordinary chondroitin sulfate samples. The unusual chondroitin sulfate chain is detected as a covalently bound form in the 6%kDa polypeptide (presumably (~2(1X)). In cartilage type IX collagen proteogly- can, the chondroitin sulfate attachment site is the serine residue of the sequence Val-Glu-Gly-Ser-Ala-Asp contained in the nonhelical NC3 domain of the a2(IX) chain (Irwin et al., 1986; McCormick et al., 1987; Huber et al., 1988). Rotary shadowing of the fibrils prepared from chick embryo cartilage has revealed a D-periodic distribution of 35-40 nm long projections, each capped with a globular domain, correspond- ing to the amino-terminal globular and collagenous domains, NC4 and COL3, of type IX collagen proteoglycan (Vaughan et al., 1988). These authors suggest that, in cartilage matrix, type IX collagen proteoglycan must be distributed in a regular D-periodic arrangement along the fibrils, with the chondroitin sulfate either extending away from the fibril to potentially interact with other matrix components or interacting with molecules in the fibrils themselves. Our immunoelectron mi- croscopic examination of the vitreous humor of chick embryo also revealed a D-periodic distribution of vitreous PG along the fibril, thus suggesting that the distribution pattern of the chondroitin sulfate chains on collagen fibrils should princi- pally be identical in both vitreous humor and cartilage matrix. An intriguing question then arises as to whether the differ- ences between the vitreous humor and cartilage matrix in the size of al(IX) and chondroitin sulfate chain may contribute to the functional difference between these matrices.

The vitreous humor provides a transparent medium for the transmission of light as well as the maintenance of the spher- ical shape of the eye. In mammals, the vitreous humor is rich

in hyaluronic acid (reviewed in Laurent and Fraser, 1986, among others) which has the capacity to trap the flow of water, to exert an osmotic pressure, and to interfere with diffusion of macromolecular solutes (reviewed in Toole, 1976). These characteristics of hyaluronic acid can be related to various features of the vitreous humor, e.g. highly hydrated, swollen structure, low serum protein content, viscosity, os- motic pressure, transparency, and other mechanical proper- ties. Our studies reported here indicate that little or no hyaluronic acid is present in the vitreous humor of 13-day- old chick embryos and further suggest that the periodically arranged chondroitin sulfate chains of high molecular weight may provide a vitreous matrix for physicochemical support to maintain internal eye structure and function. It is noteworthy in this respect that a regular arrangement of keratan sulfate- and dermatan sulfate-proteoglycans along collagen fibrils has been demonstrated in cornea1 stroma of rabbit, rat, and cow (reviewed in Scott, 1988). The transparency of the cornea depends largely on the regular spacing of the collagen fibrils (Maurice, 1957), and it is thought that this regularity is maintained by the swelling pressure of the proteoglycans, which keeps the fibrils apart.

Another aspect to be considered is the orientation of the amino-terminal domain of otl(IX) in fibrils. Since the globular domain of cartilage (Al is reported to have a p1 value of 9.7 (Vasios et al., 1988), it could participate in ionic interac- tions with the polyanionic chondroitin sulfate chains attached to a2(IX) (see Laurent and Scott, 1964; Mathews, 1970, for the examples of chondroitin sulfate/protein interaction), thereby affording a force opposing chondroitin sulfate-in- duced osmotic swelling, hydration, viscosity, etc. If so, tran- sition of (Al polypeptide from the long to the short form would eliminate this force, driving the spherical expansion of the vitreous humor during development.

Using rotary shadowing procedures, Wright and Mayne (1988) have recently shown that the collagen fibrils prepared from adult chick vitreous humor are coated by glycosamino- glycan which could be removed by chondroitinase ABC diges- tion. The result contrasts with the previous observation with the collagen fibrils isolated from embryonic chick sternal cartilage which did not appear to have this glycosaminoglycan coat (Vaughan et al., 1988). The difference between the vit- reous humor and cartilage in the morphological feature of collagen fibrils may be interpreted as reflecting a difference between these two tissues in the size of chondroitin sulfate chains associated with the collagen fibrils. That the vitreous humor type IX collagen from adult chick has a very large chondroitin sulfate chain (just like the chain from embryonic chick vitreous humor) has been confirmed by our recent analysis.4 Another unique feature of the vitreous humor col- lagen fibrils of adult chick is the absence of the knob of NC4 domain in the type IX collagen-proteoglycan associated with the fibrils (Wright and Mayne, 1988). In cartilage, the NC4 knob was detected in every projection of type IX collagen- proteoglycan from the surface of the collagen fibrils (Vaughan et al., 1988). Apparently, these electron microscopic observa- tions are compatible with the hypothesis that the short form of &1(1X) found in the vitreous humor of chick embryo (and adult chick, see Fig. 6) may represent an ~ul(1X) chain lacking the NC4 domain which was never detected in chick cartilages.

Acknowledgments-We would like to thank Dr. M. Takehana and Dr. Y. Nakaiishi for helpful discussions, and M. Arai for his assist- ance in some of this work. We are also grateful to Dr. R. Mayne for providing helpful information.

4 T. Yada, M. Arai, S. Suzuki, and K. Kimata, unpublished obser- vation.

by guest on February 10, 2018http://w

ww

.jbc.org/D

ownloaded from

Vitreous Humc

REFERENCES

Ayad, S., and Weiss, J. B. (1984) Biochem. J. 218,835-840 Bitter, T., and Muir, H. M. (1962) Anal. Biochem. 4,330-334 Bruckner, P., Vaughan, L., and Winterhalter, K. H. (1985) Proc. Natl.

Acad. Sci. U. S. A. 82,2608-2612 Bruckner, P., Mendler, M., Steinmann, B., Huber, S., and Winter-

halter, K. H. (1988) J. Biol. Chem. 263, 16911-16917 Evre. D. R.. Anon. S.. Wu. J.. Ericsson. L. H.. and Walsh. K. A.

“(1987) FEBs’Lf&. 2io,337-341 ’ ’ Fitch, J. M., Mentzer, A., Mayne, R., and Linsenmayer, T. F. (1988)

Deu.Biol. 128,396-405 Hamburger, V., and Hamilton, H. (1951) J. Morphol. 88, 49-92 Huber, S., van der Rest, M., Bruckner, P., Rodriguez, E., Winterhal-

ter, K. H., and Vaughan, L. (1986) J. Biol. Chem. 261,5965-5968 Huber, S., Winterhalter, K. H., and Vaughan, L. (1988) J. Biol. Chem.

263, 752-756 Irwin, M. H., and Mayne, R. (1986) J. Biol. Chem. 261,16281-16283 Kimata, K., Okayama, M., Oohira, A., and Suzuki, S. (1974) J. Biol.

Chem. 249,1646-1653 Kimata, K., Oike, Y., Ito, K., Karasawa, K., and Suzuki, S. (1978)

Biochem. Biophys. Res. Commun. 85, 1431-1439 Kimata, K., Oike, Y., Tani, K., Shinomura, T., Yamagata, M., Uritani,

M., and Suzuki, S. (1986) J. Biol. Chem. 261, 13517-13525 Konomi, H., Seyer, J. M., Ninomiya, Y., and Olsen, B. R. (1986) J.

Biol. Chem. 261,6742-6746 Laemmli, U. K. (1970) Nature 227, 680-685 Laurent, T. C., and Fraser, J. R. E. (1986) in Function of the

Proteoglycans (Evered, D., and Whelan, J., eds) pp. 9-29, John Wiley & Sons, New York

Laurent, T. C., and Scott, J. E. (1964) Nature 202,661-662 Linsenmaver. T. F.. Gibnev. E.. Toole. B. P.. and Gross. J. (1978)

Exp. CeilRes. 116,470-474 ' ~

Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J Biol. Chem. 193,265-275

Mathews, M. B. (1970) in Chemistry and Molecular Biology of the Intercellular Matrix (Balazs, E. A., ed) pp. 115551169, Academic Press, New York

Maurice, D. M. (1957) J. Physiol. (Land.) 136, 263-286 McCormick, D., van der Rest, M., Goodship, J., Lozano, G., Nino-

miya, Y., and Olsen, B. R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,4044-4048

Muller-Glauser, W., Humbel, B., Glatt, M., Strauli, P., Winterhalter, K. H., and Bruckner, P. (1986) J. Cell Biol. 102, 1931-1939

lr Proteoglycan 6999

Newsome, D. A., Linsenmayer, T. F., and Trelstad, R. L. (1976) J. Cell Biol. 71, 59-67

Noro, A., Kimata, K., Oike, Y., Shinomura, T., Maeda, N., Yano, S., Takahashi, N., and Suzuki, S. (1983) J. Biol. Chem. 258, 9323- 9331

Oike, Y., Kimata, K., Shinomura, T., Nakazawa, K., and Suzuki, S. (1980) Biochem. J. 191, 193-207

Reese, C. A., and Mayne, k. (1981) Biochemistry 20, 5443-5448 Rennard. S. I.. Kimata. K.. Desemund. B.. Barrach. H. J.. Wilczek.

J., Kimura, J. H., and’Hascal1, V. C. (i98i) Arch. Blochem. Biophys: 207.399-406

Reynolds, E. S. (1963) J. Cell Biol. 17, 208-212 Romanoff, A. L. (1960) The Auian Embryo, pp. 396-404, The Mac-

millan Company, New York Saito, H., Yamagata, T., and Suzuki, S. (1968) J. Biol. Chem. 243,

1536-1542 Schmid, T. M., and Linsenmayer, T. F. (1983) J. Biol. Chem. 258,

9504-9509 Scott, J. E. (1988) Biochem. J. 252, 313-323 Shinomura, T., Kimata, K., Oike, Y., Noro, A., Hirose, N., Tanabe,

K., and Suzuki, S. (1983) J. Biol. Chem. 258, 9314-9322 Smith, G. N., and Newsome, D. A. (1978) Deu. Biol. 62,65-77 Svoboda, K. K., Nishimura, I., Sugrue, S. P., Ninomiya, Y., and

Olsen, B. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7496-7500 Toole, B. P. (1976) in Neuronal Recognition (Barondes, S. H., ed) pp.

275-329, Plenum Publishing Corp., New York Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Nutl. Acad.

Sci. U. S. A. 76, 4350-4354 Ueno, Y., Tenaka, Y., Horie, K., and Tokuyasu, K. (1988) Chem.

Pharm. Bull. 36,4971-4975 van der Rest, M., and Mayne, R. (1988) J. Biol. Chem. 263, 1615-

1618 Vasios, G., Nishimura, I., Konomi, H., van der Rest, M., Ninomiya,

Y., and Olsen, B. R. (1988) J. Biol. Chem. 263,2324-2329 Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1985) J. Biol.

Chem. 260,4758-4763 Vaughan, L., Mendler, M., Huber, S., Bruckner, P., Winterhalter, K.

H.. Irwin. M. H.. and Mavne. R. (1988) J. Cell. Biol. 106.991-997 Wright, D. W., and Mayne,k. (1988) J. &a.struct. Mol. Struct. Res.

100,224-234 Yada, T., Suzuki, S., Kimata, K., Kobayashi, K., and Hoshino, T.

(1988) Seikwaku 60,615 Yamagata, M.,-Yamada, K. M., Yoneda, M., Suzuki, S., and Kimata,

K. (1986) J. Biol. Chem. 261. 13526-13535 Yoshida, K., Miyauchi, S., Kikuchi, H., Tawada, A., and Tokuyasu,

K. (1989) Anal. Biochem. 177, 327-332

by guest on February 10, 2018http://w

ww

.jbc.org/D

ownloaded from

T Yada, S Suzuki, K Kobayashi, M Kobayashi, T Hoshino, K Horie and K Kimatapolypeptide.

with an extraordinarily large chondroitin sulfate chain and short alpha 1 Occurrence in chick embryo vitreous humor of a type IX collagen proteoglycan

1990, 265:6992-6999.J. Biol. Chem. 

  http://www.jbc.org/content/265/12/6992Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/265/12/6992.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on February 10, 2018http://w

ww

.jbc.org/D

ownloaded from