THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol . 260, No. lasue of September 15, PP. 11131-11139,19@5 Printed in U.S.A.

Purification and Characterization of Protease-resistant Secretory Granule Proteoglycans Containing Chondroitin Sulfate Di-B and Heparin-like Glycosaminoglycans from Rat Basophilic Leukemia Cells*

(Received for publication, December 7, 1984)

David C. Seldin, K. Frank Austen, and Richard L. Stevens From the Department of Medicine, Harvard Medical School and the Department of Rheumatology and Immunology, Brigham and Women’s Hospital, Boston, Massachusetts 021 15

Proteoglycans were extracted from nuclease-di- gested sonicates of los rat basophilic leukemia (RBL- 1) cells by the addition of 0.1% Zwittergent 3-12 and 4 M guanidine hydrochloride and were purified by sequential CsCl density gradient ultracentrifugation, DE52 ion exchange chromatography, and Sepharose CL-GB gel filtration chromatography under dissocia- tive conditions. Between 0.3 and 0.8 mg of purified proteoglycan was obtained from approximately 1 g initial dry weight of cells with a purification of 200- 800-fold. The purified proteoglycans had a hydrody- namic size range of M, 100,000-150,000 and were resistant to degradation by a molar excess of trypsin, a-chymotrypsin, Pronase, papain, chymopapain, col- lagenase, and elastase. Amino acid analysis of the pep- tide core revealed a preponderance of Gly (35.4%), Ser (22.5%), and Ala (9.5%).

Approximately 70% of the glycosaminoglycan side chains of RBL- 1 proteoglycans were digested by chon- droitinase ABC and 2790 were hydrolyzed by treatment with nitrous acid. Sephadex G-200 chromatography of glycosaminoglycans liberated from the intact mole- cule by &elimination demonstrated that both the ni- trous acid-resistant (chondroitin sulfate) and the chon- droitinase ABC-resistant (heparinbeparan sulfate) glycosaminoglycans were of approximately M, 12,000. Analysis of the chondroitin sulfate disaccharides in different preparations by amino-cyano high perform- ance liquid chromatography revealed that 9-29% were the unusual disulfated disaccharide chondroitin sulfate di-B (1dUA-2-SO4-+GalNAc-4-SO4); the remainder were the monosulfated disaccharide GlcUA+GalNAc- 4-504. Subpopulations of proteoglycans in one prepa- ration were separated by anion exchange high per- formance liquid chromatography and were found to contain chondroitin sulfate glycosaminoglycans whose disulfated disaccharides ranged from 9-49%. How- ever, no segregation of subpopulations without both chondroitin sulfate di-B and heparinbeparan sulfate glycosaminoglycans was achieved, suggesting that RBL- 1 proteoglycans might be hybrids containing both classes of glycosaminoglycans. Sepharose CL-GB chro- matography of RBL- 1 proteoglycans digested with chondroitinase ABC revealed that less than 7% of the molecules in the digest chromatographed with the hy-

*; This work was supported by Grants AI-07167, AI-22531, AM- 35984, HL-17382, and RR-05669 from the National Institutes of Health. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

drodynamic size of undigested proteoglycans, suggest- ing that at most 7% of the proteoglycans lack chon- droitin sulfate glycosaminoglycans.

RBL-1 cells stimulated with the calcium ionophore A23187 exocytosed proteoglycans, histamine, and B- hexosaminidase in comparable noncytotoxic dose-re- lated fashion, and regression analyses of the net per- centages released indicated that proteoglycans were in secretory granules. The exocytosed and retained pro- teoglycans were of similar size, buoyant density, and glycosaminoglycan composition, providing further evidence for a single pool of secretory granule proteo- glycans. The co-purification and co-localization of pro- teoglycans containing heparin/heparan sulfate glycos- aminoglycans and chondroitin sulfate di-B glycosami- noglycans introduces the possibility that this tumor cell polymerizes both classes of glycosaminoglycans onto a single peptide core. The intragranular proteoglycans of the RBL-1 cell, whether two separate classes or hybrid molecules, are distinguished from those of the rat serosal mast cell in being one-fifth the hydrody- namic size and having predominantly chondroitin sul- fate di-B glycosaminoglycans. However, intragranular proteoglycans of RBL- 1 cells and rat serosal mast cells possess the common properties of oversulfation, pro- tease resistance, and a preponderance of Gly and Ser in their peptide cores.

The RBL-ll cell line was established from a chemically generated rat basophilic leukemia (1). Its morphology varies with the rate of cell division, and it can resemble an undiffer- entiated sparsely granulated promyelocyte or a well-granu- lated mature basophil (2). The RBL-1 cell contains 0.1-1.0 pg/cell of histamine in secretory granules which can be re- leased in some sublines (3) by cross-linking of its well-char- acterized surface IgE receptors (4-10). AII tines are amenable

’ The abbreviations used are: RBL-I, rat basophilic leukemia; GnHC1, guanidine hydrochloride; HPLC, high performance liquid chromatography; UA, uronic acid; IdUA, iduronic acid GlcUA, glu- curonic acid; ADi-OS, 2-acetamido-2-deoxy-3-O-(fl-~-gluco-4-enepyr- anosyluronic acid)-D-galactose; ADi-4S, 2-acetamido-2-deoxy-3-0-(fl- D-gluco-4-enepyranosyluronic acid)-4-0-sulfo-~-galactose; ADi-6S, 2-acetamido-2-deoxy-3-O-(fl-~-gluco-4-enepyranosyluronic acid)-6- 0-sulfo-D-galactose; chondroitin sulfate A, GlcUA+GalNAc-4-S04; chondroitin sulfate B, IdUA4alNAc-4-S04; chondroitin sulfate di- B, IdUA-2-S04+GalNAc-4-SO4; chondroitin sulfate D, GlcUA-2- S044alNAc-6-S04; chondroitin sulfate E, GlcUA+GalNAc-4,6- diSO4; trisulfated chondroitin sulfate, IdUA-2 or 3-S04+GalNAc- 4,6-diSOI.

11131

11132 RBL-1 Proteoglycans

to activation by the calcium ionophore A23187 under the appropriate conditions, resulting in de novo generation and release of prostaglandins (11) and leukotrienes (12-14), as well as release of preformed mediators (15).

Rat (16-19) and mouse (20) serosal mast cells contain an extremely acidic intragranular heparin proteoglycan of M, 0.75-1 X lo6. Heparin proteoglycan is distinguished from extracellular matrix proteoglycans by having regions of gly- cosaminoglycans rich in an unusual trisulfated disaccharide, 1dUA-2-SO4-+GlcN,6-diSO4, by being resistant to proteolysis and by having a unique peptide core which is primarily a copolymer of Ser and Gly (18, 19). The intragranular proteo- glycan of the mouse interleukin 3-dependent mast cell derived in vitro from bone marrow (20, 21), fetal liver, and immune lymph node (22) is a chondroitin sulfate proteoglycan of about M, 250,000 possessing an unusual disulfated disaccharide, GlcUA-GalNAc-4,6-diSO4, termed chondroitin sulfate E, as a major glycosaminoglycan constituent.

The recognition of an oversulfated chondroitin proteogly- can in the secretory granule of the cultured mouse mast cell subclass suggested that other histamine-containing cells might contain the same or a homologous oversulfated non- heparin proteoglycan. RBL-1 cells maintained in vivo as a solid tumor were previously characterized in this laboratory as having heterogeneous cell-associated proteoglycans (23). In the present study, proteoglycans were obtained from the RBL-1 cell line maintained in tissue culture and purified to apparent homogeneity by ultracentrifugation and conven- tional chromatography techniques performed under dissocia- tive conditions. The hydrodynamic size of the purified proteo- glycans and glycosaminoglycans, the disaccharide composi- tion of the glycosaminoglycans, and the amino acid composi- tion of the purified proteoglycans were determined. Iono- phore-induced secretion experiments demonstrated an intra- granular localization of these proteoglycans. The RBL-1 pro- teoglycans share properties with rat serosal mast cell heparin proteoglycans of oversulfation and protease resistance, sug- gesting that these molecules are members of a class of intra- granular proteoglycans that fulfill related functional require- ments; however, each population of proteoglycans has a dis- tinct physicochemical composition characteristic of the par- ticular cell type.

MATERIALS AND METHODS

Cell Culture and Radiolabeling-Adherent RBL-1 cells were main- tained in 175-cm2 flasks containing 80 ml of Earle's minimal essential medium supplemented with 10% (v/v) fetal calf serum, 2 mM L- glutamine, 0.1 mM nonessential amino acids, 100 units/ml penicillin, and 100 pg/ml streptomycin (Grand Island Biological Co., Grand Island, NY) at pH 7.2 in a humidified 37 "C incubator with a 6% COn atmosphere. The medium was changed twice per week until the cells reached confluence (0.5-1.0 X 10' cells/flask). Cells were passaged by washing with Hanks' balanced salt solution without calcium or mag- nesium, incubating with 10 ml of a trypsin-EDTA solution (Gibco) for 10 min at 37 "C, and dividing the detached cells into 10 flasks with fresh medium. To radiolabel proteoglycans biosynthetically for subsequent purification and analysis, confluent cells were incubated with 100 pCi of [35S]sulfate (New England Nuclear)/ml of medium for 4 h at 37 "C. Ten flasks (approximately lo9 cells) were harvested, washed, sedimented at 400 X g, resuspended in 1 ml of 0.05 M sodium acetate, pH 6.0, and disrupted with 30 pulses of a Branson sonifier (Danbury, CT). In the standard procedure, intact nucleic acids, which were found to copurify with RBL-1 proteoglycans, were degraded by incubation of the cell sonicate with 100 units/ml ribonuclease A and 1000 units/ml deoxyribonuclease I (Sigma) for 30 min at 37 'C, after which MgSO, was added to a final concentration of 5 mM for a further 30-min incubation. The digestion mixture was then suspended in 0.1 ml of 0.05 M sodium acetate containing 1% (w/v) Zwittergent 3-12 detergent (Calbiochem-Behring), 0.1 M 6-aminohexanoic acid, 0.1 M sodium EDTA, 5.0 mM benzamidine HC1, and 1.0 mM sodium iodo-

acetamide, followed by 0.9 ml of 4 M GnHCl in the same buffer. In order to determine the rate of incorporation of [?S]sulfate into macromolecules, a sample of this nuclease-treated cell extract was diluted to 0.5 ml in 0.1 M Tris-HC1,O.l M sodium sulfate, 4 M GnHCI, pH 7.0 (TSG buffer) and chromatographed on a PD-10 gel filtration column (Pharmacia Fine Chemicals) equilibrated in the same buffer. Fractions (0.5 ml) were collected and mixed with 1 ml of ethanol and 10 ml of Hydrofluor (National Diagnostics, Somerville, NJ), and the radioactivity eluting in the void volume of the PD-10 columns was quantitated by &scintillation counting. To minimize the possibility of proteolytic degradation of the proteoglycans, in one preparation the nuclease digestion was omitted, the cell pellet was directly sus- pended in acetate buffer with detergent and inhibitors followed by 4 M GnHCI, and the extracted proteoglycans were isolated, purified, and analyzed as described below for proteoglycans extracted in the standard way from nuclease-treated cell sonicates.

Proteoglycan Purification-Extracts of lo9 RBL-1 cells were sub- jected to CsCl density gradient ultracentrifugation under dissociative conditions by adding CsCl in 0.05 M Tris-HC1, 4 M GnHCI, pH 7.0, to a starting density of 1.4 g/ml and centrifuging at 95,000 X g for 48 h at 17 "C. The centrifuged samples were frozen at -70 "C and cut into two equal fractions; the bottom half with the greater buoyant density was termed Dl , and the top half, D2. Twenty-five-microliter portions of each fraction were applied to PD-10 columns and eluted with TSG buffer. Fraction Dl, which contained the majority of the 35S-macromolecules, was dialyzed for 3 days against 0.1 M ammonium bicarbonate, lyophilized, resuspended in 2 ml of 0.05 M Tris-HC1, 4 M urea, pH 7.25 (TU buffer), and applied to a 1 X 10-cm column containing DE-52 cellulose (Whatman) equilibrated in the same buffer. The resin was washed with 20 ml of buffer and eluted with an 80-ml linear gradient to 1 M NaCl in the same buffer, and 1-ml fractions were collected. The absorbance at 280 nm and the conduc- tivity of the fractions were measured, and the radioactivity in 25 p1 of each was determined. Fractions containing 35S-macromolecules were pooled, dialyzed against 0.1 M ammonium bicarbonate, lyophi- lized, resuspended in 200 pl of TSG buffer, and applied to a 0.6 X 80- cm Sepharose CL-GB column. The column was eluted with TSG buffer by gravity flow at a rate of 2.5 ml/h, and 60 0.5-ml fractions were collected. The absorbance at 280 nm was measured, and the radioactivity in 20 p1 of each of the fractions was determined. The hydrodynamic size of the RBL-1 proteoglycans was estimated by calculating the K,,, and comparing it with values of proteoglycans of known hydrodynamic size chromatographed on Sepharose CL-GB. Fractions containing 36S-proteoglycans were pooled, dialyzed against 0.1 M ammonium bicarbonate, and lyophilized. The purification factor was calculated by comparing the ratio of 35S-macromolecules/dry weight of material, expressed as cpm/mg, in the starting material and in the purified proteoglycan preparations.

Protease Susceptibility of RBL-1 Proteoglycans-The susceptibility of RBL-1 proteoglycans to proteolytic degradation was evaluated using trypsin (204 units/mg), collagenase (178 units/mg) (Worthing- ton), wchymotrypsin (47 units/mg), papain (21 units/mg), chymo- papain (4.3 units/mg), elastase (34 units/mg) (Sigma), and Pronase (77 units/mg) (Calbiochem-Behring). Approximately 1 pg of purified proteoglycans (5000 cpm) was incubated with 5 pg of each enzyme in Hanks' balanced salt solution with calcium and magnesium (except papain, which was incubated in Hanks' balanced salt solution with 0.01 M EDTA and 0.05 M cysteine) at a final volume of 100 pl for 1 h at 37 "C; 100 p1 of TSG buffer was then added and the mixture was applied to the Sepharose CL-GB column. The elution profile of 36S- proteoglycans after incubation with each protease was compared with that of undigested controls. %3-Proteoglycans purified from Swarm rat chondrosarcoma chondrocytes (24) were digested in parallel and chromatographed on a Sepharose CL-4B column to evaluate the protease susceptibility of matrix proteoglycans.

Disaccharide Composition and Hydrodynamic Size of the Glycos- aminoglycans of RBL-I Proteoglycans-The composition of the gly- cosaminoglycans of the purified RBL-1 proteoglycans was determined by chemical and enzymatic degradation. The proportion of heparin and/or heparan sulfate was evaluated by susceptibility to nitrous acid treatment (20, 25, 26). One hundred pg of heparin carrier (Sigma) was mixed with 10,000 cpm of purified RBL-1 35S-proteoglycans in a volume of 100 pl. Dimethoxyethane (100 pl) and butyl nitrite (10 PI) were added, and the reaction was allowed to proceed at -20 "C for 16 h. As a control, 10,000 cpm of [3H]heparin (New England Nuclear) was incubated in parallel. Both reactions were halted by the addition of 7.5 pl of a saturated solution of sodium acetate and 250 p1 of TSG

RBL-1 Proteoglycans 11133

buffer. Hydrolysates were chromatographed on PD-10 columns in 4 M GnHCl to assess the degradation of glycosaminoglycans. The proportion of 35S-glycosaminoglycans that was chondroitin sulfate was determined by digestion of purified RBL-1 proteoglycans with chondroitinase ABC according to the procedure of Saito et al. (27). Radiolabeledproteoglycan (5,000 cpm) and 100 pg each of chondroitin sulfate A and C carriers were incubated with 0.4 unit of chondroitinase ABC (Miles Laboratories, Inc., Elkhart, IN) in the presence of 0.01 M sodium fluoride to inhibit contaminating sulfatases (28) for 1 h at 37 "C. The percentage of glycosaminoglycans digested to unsaturated disaccharides was assessed by PD-10 chromatography of the reaction mixtures. The proportion of those %-disaccharides that contained iduronic rather than glucuronic acid, i.e. those that were dermatan sulfate-like, was estimated by subtraction of the percentage of disac- charides generated by chondroitinase AC digestion, following the same protocol, from the percentage obtained by digestion with chon- droitinase ABC.

The composition of the chondroitin sulfate glycosaminoglycans was further analyzed by amino-cyano HPLC (29). 35S-Disaccharides liberated by chondroitinase ABC digestion were separated from un- degraded proteoglycans, enzyme, and contaminating macromolecules by an 80% ethanol extraction in which the digestion mixture was diluted with four volumes of absolute ethanol, cooled to 4 "C for 2 h, and centrifuged in a Beckman Microfuge at 8,000 X g for 5 min. The supernatant was decanted, dried over nitrogen, and resuspended in the HPLC solvent, which was 70% acetonitrile/methanol (31, v/v) and 30% 0.5 M ammonium acetate/acetic acid, pH 5.3, with an apparent final pH of 7.0. Chromatography was performed on a Rainin (Woburn, MA) gradient HPLC system controlled by an Apple IIe computer (Cupertino, CA). A 4.6 X 250-mm Partisil-10 PAC amino- cyano-substituted normal phase silica column, with a 4.6 X 25-mm precolumn containing the same packing (Whatman) was used for separating disaccharides. The ultraviolet absorbance of the column eluate was monitored continuously at 232 nm, the absorbance maxi- mum for unsaturated disaccharides, with a spectroMonitor D (LDC/ Milton Roy, Riviera Beach, FL). Peak analysis was performed by the Apple IIe in conjunction with a Gilson (Villiers le Bel, France) Data Master and accompanying software. Eluates containing radiolabeled disaccharides were collected for 0.5-min intervals and quantitated by @-scintillation counting in Hydrofluor. One-pg portions of ADi-OS, ADi-6S, and ADi-4S (Miles Laboratories, Inc.) were used routinely as calibration standards, and disaccharides generated from the follow- ing proteoglycans or glycosaminoglycans were used as reference po- lysulfated disaccharides: chondroitin sulfate di-B (the unsaturated disaccharide generated by chondroitinase ABC is ADi-diSB) from shark skin (30,31) and hagfish skin, which also contains a trisulfated chondroitin sulfate disaccharide (ADi-triS) (32); chondroitin sulfate D (ADi-diSD) from shark cartilage (33); and chondroitin sulfate E (ADi-diSE) from squid cranial cartilage (34, 35) or from mouse bone marrow-derived mast cells (20, 21). The retention times of these disaccharide standards were: ADi-OS, 5 min; ADi-GS, 6 min; ADi-4S, 7 min; ADi-diSD, 10 min; ADi-diSB, 14.5 min; ADi-diSE, 16.5 min; and ADi-triS, 21 min.

Glycosaminoglycans were liberated from 60,000 cpm of purified RBL-1 proteoglycans by 0-elimination in 30 pl of 0.5 M NaOH for 16 h at 4 "C followed by neutralization with 30 p1 of 0.5 M acetic acid. This glycosaminoglycan preparation was divided into three equal portions: one was untreated; one was subjected to the nitrous acid hydrolysis procedure, leaving the chondroitin sulfate glycosaminogly- cans intact; and one was subjected to chondroitinase ABC digestion, leaving the heparin-like glycosaminoglycans intact. An equal volume of 0.5 M sodium acetate, pH 6.5, containing 50 pg/ml heparin carrier (Sigma) was added to each sample, which was then chromatographed on a 0.7 X 100-cm column of Sephadex G-200 equilibrated with the acetate buffer containing 50 pg/ml heparin. Half-ml fractions were collected and analyzed for radioactivity. The molecular weights of the glycosaminoglycans were estimated by comparing the K.. values to published K., values for glycosaminoglycans of known molecular weight (36).

Anion Exchange HPLC of Purified RBL-I Proteoglycans-The Rainin/Apple IIe gradient HPLC system was used to perform anion exchange HPLC of purified RBL-1 proteoglycans. A 4.6 X 22-cm Aquapore AX-1000 column with a 3-cm guard column of the same packing (Brownlee Labs, Inc., Santa Clara, CA) was equilibrated with 200 ml of TU buffer containing 0.5 M NaCl at a flow rate of 1 ml/ min, which resulted in pump pressures of approximately 550 p.s.i. Five thousand cpm of purified RBL-1 proteoglycans (approximately

1 pg) dissolved in the same buffer was injected onto the column, which was washed for 20 min at the same flow rate. Proteoglycans were eluted with a linear gradient from 0.5-1 M NaCl in 50 min, followed by a 20-min wash at 1 M NaCl in TU buffer. The ultraviolet absorbance of the column eluate was monitored continuously at 280 nm on the spectroMonitor D. Fractions were collected for 1-min intervals with a Gilson 202 fraction collector, and the radioactivity in samples of each fraction was determined by 6-scintillation count- ing. The fractions containing radioactivity were pooled into three portions, based on increasing retention times of the column. The glycosaminoglycan content of each pool was determined by nitrous acid hydrolysis and chondroitinase ABC digestion followedby amino- cyano HPLC disaccharide analysis of replicate samples.

Gel Filtration of Chnndroitinme ABC-digested RBL-I Proteogly- cans-Purified 35S-proteoglycans (25,000 cpm) and 100 pg each of chondroitin sulfate A and C carriers were incubated with 0.4 unit of chondroitinase ABC in the presence of 0.01 M sodium fluoride for 1 h at 37 "C. TSG buffer was added and the mixture was applied to the Sepharose CL-GB column and eluted with TSG buffer as described. A control containing 25,000 cpm of undigested 35S-proteoglycans and carriers was also chromatographed.

Zonophore-stimulated Exocytosis of RBL-I Proteoglycans-RBL-1 cells were seeded in 24-well microtiter plates (Costar, Cambridge, MA) and grown to confluence. Fresh medium with or without 100 pCi of [35S]sulfate/ml was added to the cultures. After 4 h, the medium was removed and the cells were washed twice with Tyrode's buffer. Doses of ionophore A23187 (Calbiochem-Behring) from 0-20 p~ in 200 pl of Tyrode's buffer were added to wells containing Y3-labeled cells and to wells with unlabeled cells, each in quadruplicate. After a 15-min incubation at 37 "C, each supernatant was decanted and the cells were lysed by the addition of 200 p1 of Hz0 to each well in order to quantitate histamine (37), P-hexosaminidase (38), and lactate dehydrogenase (39) in the supernatants and cell pellets. The 36S- labeled cells were used to quantitate the amount of exocytosed 35S- proteoglycans as assessed by PD-10 chromatography and p-scintilla- tion counting of the cell supernatants and pellets. The mean per cent release [loo% X (amount in supernatant)/(amount in supernatant + amount in pellet)] of histamine, @-hexosaminidase, lactate dehydro- genase, and 35S-proteoglycans of the quadruplicate wells at each dose of calcium ionophore A23187 was calculated. The mean per cent release of each mediator in the absence of ionophore was subtracted from the mean per cent release value at each dose to obtain the mean net per cent stimulated release. The degree to which p-hexosamini- dase, proteoglycans, and histamine reside in the same pool in these cells was assessed by linear regression analysis of plots of all net per cent release values of &hexosaminidase or proteoglycans uerszu net per cent release values of histamine (40). A slope of 1 and a y intercept at the origin would indicate that two mediators reside in the same pool and are exocytosed from this secretory granule pool at the same rate.

To characterize secreted and retained proteoglycans, five 750-ml flasks of confluent RBL-1 cells were labeled with 100 pCi of [35S] sulfate/ml for 4 h at 37 "C, washed twice with Tyrode's buffer, and activated with 10 p~ A23187 in 10 ml of Tyrode's buffer/flask for 15 min at 37 "C. The supernatants were decanted, and the cells were lysed by adding 10 ml of distilled water to each flask. Cell superna- tants and pellets were pooled separately, and l ml of 0.05 M sodium acetate containing 1% (w/v) Zwittergent 3-12 detergent, 0.1 M 6- aminohexanoic acid, 0.1 M sodium EDTA, 5.0 mM benzamidine HCI, and 1.0 mM sodium iodoacetamide was added to both pools. The two pools were subjected to sonication for 30 s, dialyzed for 2 days against 0.1 M ammonium bicarbonate, lyophilized, and resuspended in 1.4 g/ ml Cscl in 0.05 M Tris-HC1,4 M GnHC1, pH 7.0, for density gradient ultracentrifugation. The ultracentrifuged samples were divided into two buoyant density fractions, D l and D2, and samples of each fraction were subjected to PD-10 chromatography to quantitate 3sS- macromolecules. The D l fractions, which contained the majority of the 35S-macromolecules from both the supernatants and pellets, were dialyzed against 0.1 M ammonium bicarbonate for 3 days and lyoph- ilized. These partially purified proteoglycan samples were used to assess the hydrodynamic size and glycosaminoglycan composition of the exocytosed and retained proteoglycans as described above.

Amino Acid Composition of the Peptide Core-Two samples of purified RBL-1 proteoglycans were used for analysis of the amino acid composition of the core peptide material. One mg of each sample of RBL-1 proteoglycans was subjected to acid hydrolysis in boiling HC1 for 24 h and analyzed for amino acid content on a Beckman

11 134 RBL-1 Proteoglycans

model 119CLW/126 amino acid analyzer. Integrated areas of optical absorbance were compared with areas obtained from standard amino acids to quantify relative amounts in the samples, which were con- verted to per cent of total amino acids.

RESULTS

Biosynthetic Labeling and Purification of Proteoglycans- RBL-1 cells maintained in tissue culture incorporated [35S] sulfate into macromolecules at a rate of 2800 f 1650 cpm/106 cells/h (mean +- S.D., n = 5). When nuclease-treated cell extracts were ultracentrifuged in CsCl at a starting density of 1.4 g/ml under dissociative conditions, 70.4 f 12.8% (mean +- S.D., n = 8) of the total cell-associated 35S-macromolecules were recovered in the D l fraction of the gradient. Because most protein, carbohydrate, and lipid contaminants are of lesser buoyant density than proteoglycans and appear in fraction D2, that fraction was discarded.

Fraction D l was dialyzed, lyophilized, and subjected to DE52 ion exchange chromatography. All of the 35S-macro- molecules eluted in a single sharp peak at a conductivity of 30-42 millisiemens, approximately equal to 0.4-0.65 M NaCl in this buffer (Fig. 1). Material with significant ultraviolet absorbance eluted from the column in a slightly earlier peak, overlapping with the 35S-proteoglycans. The fractions con- taining radioactivity were pooled, dialyzed, lyophilized, and chromatographed on Sepharose CL-GB. 35S-Macromolecules filtered as a single sharp peak with a K,, of 0.25 (Fig. 2), indicating a hydrodynamic size of approximately M, 100,000-150,000. Proteoglycans purified by this procedure from RBL-1 cell sonicates that were not treated with nu- cleases had the same buoyant density, charge characteristics, and hydrodynamic size; however, material that was presumed to be nucleic acid because of its ultraviolet absorbance ratio for 256 nm/280 nm of 1.4 coeluted with proteoglycans upon gel filtration. The nuclease procedure cleaved the nucleic acids to oligonucleotides, resulting in a shift of K,, from 0.25 to 0.9, well separated from proteoglycans (Fig. 2). The 35S-macro- molecules obtained after Sepharose CL-GB chromatography lacked material which could be detected by the Lowry assay (41) and were, therefore, deficient in aromatic amino acid- containing protein. These pooled 35S-macromolecules were

FRACJ/ONNUMBER FIG. 1. DE52 ion exchange chromatography in TU buffer

of SsS-proteoglycans obtained after nuclease digestion of RBL-1 cell extracts and dissociative CsCl density gradient ultracentrifugation. The 0.5-ml column fractions were screened for radioactivity, absorbance at 280 nm, and conductivity in milliSiemens (rnS).

FIG. 2. Sepharose CL-GB gel filtration chromatography in TSG buffer of pooled fractions 44-51 from the DE52 column depicted in Fig. 1. The 0.25-ml column fractions were screened for 36S radioactivity and ultraviolet absorbance at 280 nm.

F 3 cr, 9

3 1

0 io 20 30 40 50 60 70 FffACT/ON NUMffEff

FIG. 3. Protease susceptibility of purified RBL-1 proteogly- cans (A) and Swarm rat chondrosarcoma proteoglycans (B). Untreated (o”-o) or Pronase-treated (U) proteoglycans were applied to a Sepharose CL-GB (A) or 4B ( B ) column and eluted by gravity flow. The radioactivity in the 0.25-ml fractions was deter- mined.

considered to be purified proteoglycans. The dry weight of purified proteoglycans obtained from approximately 1 g dry weight ( lo9) cells ranged from 0.3-0.8 mg in four preparations, and purifications were calculated to be from 200-800-fold.

Protease Resistance of Purified RBL-1 Proteoglycans-Rep- licate samples of approximately 1 pg of purified 35S-proteogly- cans were incubated separately with an excess of each of seven proteases under appropriate conditions of pH and cation concentrations, and the individual digests were chromato- graphed on Sepharose CL-GB. The elution patterns of an undigested control sample and a sample treated with a molar excess of Pronase for 1 h were identical (Fig. 3A), indicating

RBL-1 Proteoglycans 11135

6 I B.

FRACTION NUMBER

FIG. 4. Degradation of glycosaminoglycans from purified RBL- 1 proteoglycans as assessed by PD-10 gel filtration chro- matography in TSG buffer after treatment with buffer (A) , chondroitinase ABC (B) , chondroitinase AC (C), or nitrous acid (D). Fractions (0.5 ml) were collected and subjected to p- scintillation counting.

that there was no detectable decrease in the hydrodynamic size of the RBL-1 proteoglycans. None of the enzymatic treatments resulted in an alteration in this elution profile. In contrast, [35S]sulfate-labeled chondroitin sulfate proteogly- cans from the Swarm rat chondrosarcoma chondrocyte, which chromatographed in the void volume of a Sepharose CL-4B column, were appreciably decreased in hydrodynamic size by digestion with Pronase (Fig. 3B) and with each of the other proteases when treated as described.

Glycosaminoglycan Side Chains of RBL-1 Proteoglycans- Purified RBL-1 proteoglycans were enzymatically digested with chondroitinase ABC or AC or were hydrolyzed with nitrous acid, and the percentage of disaccharides liberated by each procedure was quantitated by PD-10 chromatography. In the untreated control sample of 35S-proteoglycans all ra- dioactivity filtered in the void volume of a PD-10 column (Fig. 4A). Treatment with chondroitinase ABC resulted in 64% of the radioactivity being associated with disaccharides eluting in the included column volume (Fig. 4B), whereas treatment with chondroitinase AC liberated 45% of the radio- activity as disaccharides (Fig. 4C). Thirty-six per cent of the 35S-proteoglycans were hydrolyzed to oligosaccharides by ni- trous acid treatment (Fig. 40). These findings indicate that 64% of the glycosaminoglycans present in this preparation of proteoglycans were chondroitin sulfates of which 19% con- tained iduronic acid, whereas 36% were either heparin or heparan sulfate. Six different preparations of purified RBL-1 proteoglycans yielded material that was 71 f 9% (mean +_

S.D.) digested by chondroitinase ABC and 27 k 12% hydro- lyzed by nitrous acid.

The chondroitinase ABC-generated unsaturated disaccha- rides were analyzed by amino-cyano HPLC. 35S-Disaccharides eluted in a major peak at a retention time of 7 min, corre- sponding to ADi-4S, and a second peak at 14.5 min, corre- sponding to ADi-diSB (Fig. 5). Chondroitinase ABC digests of five different samples of purified RBL-1 proteoglycans re- vealed that 1 2 & 9% (mean +_ S.D.) of the disaccharides coeluted with the ADi-diSB standard. Since digestion with chondroitinase AC generated ADi-4S only, the disaccharide which eluted at the retention time of ADi-diSB contained iduronic acid and was presumed to be identical to the chon- droitin sulfate di-B disaccharide from other sources (29-31),

4s d

0 5 .FiEEAJTlOh' TIM€ ( ln i r~ , ;

FIG. 5. Partisil-10 amino-cyano HPLC of disaccharides generated from purified RBL-1 proteoglycans by chondroi- tinase ABC digestion followed by ethanol extraction. 0.5-ml fractions were collected and subjected to &scintillation counting. Retention times of commercial ADi-4s (4s) and ADi-diSB (dZB) from hagfish skin glycosaminoglycans, indicated by arrows, were 7 and 14.5 min, respectively. The retention time of ADi-diSz from squid cranial cartilage glycosaminoglycan was 16.5 min in this experiment.

and not the glucuronic acid-containing isomer. The glycosaminoglycans liberated from whole RBL-1 pro-

teoglycans by @-elimination chromatographed with a single broad peak of 0.44 KnV on Sephadex G-200, indicating a M, of 12,000 (Fig. 6). The chondroitin sulfate glycosaminoglycans, which remained intact after nitrous acid hydrolysis of the total glycosaminoglycans, and the heparin/heparan sulfate glycosaminoglycans, which remained intact after chondroitin- ase ABC digestion, had hydrodynamic sizes not significantly different from each other or from the untreated glycosami- noglycans (Fig. 6).

Anion Exhange HPLC of RBL-1 Proteoglycans-When 5000 cpm (approximately 1 pg) of purified RBL-1 proteoglycan was injected onto the AX-1000 anion exchange HPLC column, a broad peak containing 94% of the applied radioactivity was eluted (Fig. 7). No peak with ultraviolet absorbance at 280 nm was detected with the spectrophotometer set at a full scale sensitivity of 0.1 absorbance units. As indicated in the figure, the fractions containing radioactivity were divided into three portions, pools I, 11, and 111, in order of increasing retention times, and the glycosaminoglycans of the proteoglycans in each pool were analyzed by nitrous acid hydrolysis and chon- droitinase ABC digestion coupled with amino-cyano HPLC. More than 90% of the radiolabeled disaccharides and oligo- saccharides in each pool were identified (Table I), and it was observed that anion exchange HPLC did not separate proteo- glycans containing only chondroitin sulfate glycosaminogly- cans from proteoglycans containing only heparin/heparan sulfate glycosaminoglycans. However, the percentages of chondroitin sulfate disaccharides which were disulfated were greater in the pools of proteoglycans eluting from the column with longer retention times, being 9% in pool I, 22% in pool 11, and 49% in pool 111. Twenty-nine per cent of the chon- droitin sulfate disaccharides in the sample of purified proteo- glycans injected onto the AX-1000 column were ADi-diSB.

Gel Filtration of Chondroitinase ABC-digested RBL-1 Pro- teoglycans-The K., of a sample of purified 35S-proteoglycans mixed with 100 pg each of chondroitin sulfate A and C and rechromatographed on Sepharose CL-GB was 0.23 (Fig. 8). After incubation of a replicate mixture with chondroitinase

11136 RBL-1 Proteoglycans

FIG. 6. Sephadex (2-200 gel fil- tration chromatography of glycos- aminoglycans liberated from 60,000 cpm of purified RBL-1 pro- teoglycans by &elimination and subjected to no treatment (W); nitrous acid hydrolysis, leaving chondroitin sulfate glycosaminogly- cans intact (A-A); or chondroitin- ase ABC digestion, leaving heparin/ heparan sulfate glycosaminogly- cans intact (c".). Incomplete (3- elimination left some 35S-proteoglycan intact, which appeared at the void vol- ume ( Vo). Disaccharides and oligosac- charides liberated by chemical or enzy- matic treatment appeared near the total column volume ( Vt) .

i .2

I

.o e 1.0

8 2

0.8

3 \ 2 0.6

0.4 e (I, 0.2 2

%

0

VO +

20 40 60 80

4l 3 /' /"""

/'

RE TfNTlON TIMf (rnin)

FIG. 7. AX-1000 anion exchange HPLC of purified RBL-1 proteoglycans. Proteoglycans were injected onto the column in TU buffer with 0.5 M NaCl and eluted with a 20 mM/min gradient from 0.5-1.0~ NaCl followed by a 20-min wash at 1 M NaCl. Column fractions (0.5 ml) were screened for radioactivity. The ultraviolet absorbance at 280 nm was monitored continuously but did not exceed base-line. The broad peak of 35S-proteoglycans was divided into three pools (1, 11, and I l l ) as indicated.

TABLE I Disaccharide composition of proteoglycans from RBL-I cells separated

by AX-1000 anion exchange HPLC Disaccharide structure Pool I Pool I1 Pool 111

% % %

Heparin-like 15 19 19 Chondroitin sulfate 75 74 75 ADi-4s 91 78 51 ADi-diSe 9 22 49

ABC for 1 h at 37 "C, 65.8% of the 35S radioactivity appeared near V,, with a Kav of 0.91, representing [35S]chondroitin sulfate disaccharides. A peak containing 27.4% of the radio- activity appeared at a Kav of 0.43 and was presumed to be proteoglycan core peptide with heparin-like [35S]glycosami- noglycans attached. 6.7% of the radioactivity chromato- graphed with a K., of 0.23, suggesting that at most this percentage of the RBL-1 proteoglycans contained mainly heparin-like glycosaminoglycans which were not susceptible to chondroitinase ABC digestion and were not shifted in hydrodynamic size relative to the undigested control.

FRACTION NUMBER FIG. 8. Sepharose CL-GB gel filtration chromatography in

TSG buffer of purified RBL-1 proteoglycans (0- - -0) and purified RBL-1 proteoglycans digested for 1 h at 37 "C with chondroitinase ABC (u). The radioactivity in the 0.25-ml fractions was determined.

Exocytosis of Proteoglycans from RBL-1 Cells-The net per cent release of histamine, P-hexosaminidase, lactate dehydro- genase, and proteoglycans in response to increasing doses of calcium ionophore A23187 was determined (Fig. 9). The per- centages of histamine, P-hexosaminidase, and 35S-macromol- ecules spontaneously released during the 15-min incubation period at 37 "C were 0.5 f 0.4, 1.3 f 0.4, and 4.3 f 0.5% (mean f S.D., n = 4), respectively. The addition of increasing doses of ionophore resulted in the concomitant exocytosis of histamine, P-hexosaminidase, and proteoglycans, with maxi- mal release values of 31.6 f 3.0, 23.6 f 3.7, and 35.1 f 3.2% (mean f S.D., n = 4), respectively, at 20 p~ ionophore. Release of the cytosolic marker lactate dehydrogenase was detected only at this highest ionophore dose, at which 3.4 f 3.2% of this enzyme was released. Data from this and four similar dose response experiments were subjected to linear regression analysis of net per cent release of @-hexosaminidase uersus histamine (Fig. 1OA). For 25 data points, the line generated had a slope of 0.63 and a y intercept of 0.79%, with

RBL-1 PT

77 b4 40 t

0 5 i o i 5 20 p w I0NopHoRE

FIG. 9. Net per cent release of S6S-proteoglycan (U), histamine (M), @-hexosaminidase (A-A), and lactate dehydrogenase (A-A) from RBL-1 cells with increasing doses of calcium ionophore A23187. Points represent means of quadruplicates f S.D. Spontaneous release in this experiment was 4.3% for ?3-proteoglycan, 0.5% for histamine, and 1.3% for p-hex- osaminidase. The cytosolic marker lactate dehydrogenase was not detected in the cell supernatant except at the 20 PM dose of ionophore.

NET % RELEAS€ OF HISTAMIN€ FIG. 10. Linear regression analysis of the net per cent re-

lease of @-hexosaminidase (@-Hex) (A) and '"S-proteoglycans (86S-PG) (B) plotted against the net per cent release of hista- mine for 5 experiments (25 data points) and 4 experiments (20 data points), respectively.

the coefficient of determination, 9, equal to 0.93. Linear regression analysis of four dose-response experiments (20 data points) generated a line with a slope of 0.60, a y intercept of 4.5%, and an P of 0.72 for the net per cent release of 35S- proteoglycans uersw histamine (Fig. 10B).

Activation of 175-cm2 flasks of RBL-1 cells containing 35S- labeled proteoglycans with 10 pM calcium ionophore A23187 resulted in the exocytosis of 43% of the histamine, 31% of the P-hexosaminidase, and 20% of the 35S-proteoglycans without detectable release of lactate dehydrogenase. The secreted pro- teoglycans and the proteoglycans retained with the cells were partially purified in parallel by CsCl density gradient ultra- centrifugation for subsequent determination of hydrodynamic size and glycosaminoglycan composition. Eighty-three per cent of the exocytosed proteoglycans and 81% of the cell- associated proteoglycans were recovered in the high density D l fraction in the same ultracentrifuge run, and both popu-

*oteoglycans 11137

TABLE I1 Amino acid composition of the peptide cores of RBL-1 proteoglycans

and rat serosal and skin mast cell heparin proteoglycans Rat serosal mast cells

Rat skin RBL-l mast cells

(19) (18)

Asx Thr Ser Glx Pro GlY Ala Val Met Ile Leu TYr P he LYS His Arg

% 3.5 1.2

39.4 2.4 2.0

43.3 1.6 2.0 -

- - 1.2 2.0 1.2

% 4.8 2.5

22.5 6.6

35.4 9.5 2.4

1.3 3.3

5.6' 2.5 1.8 1.8

-

-

-

a Values are the mean of two separate preparations.

e May be artifactual since hexosamine sugars can cochromatograph Not detected.

with Phe in this system.

lations of proteoglycans were found to have Kay values of 0.22 upon Sepharose CL-GB gel filtration chromatography. Anal- ysis of the glycosaminoglycans of the exocytosed proteogly- cans revealed that 32% were degraded by nitrous acid and 68% were susceptible to chondroitinase ABC; of the chon- droitinase ABC-generated disaccharides, 15% were ADi-diSB and 85% were ADi-4S as analyzed by HPLC. The glycosami- noglycans of the retained proteoglycan were 8% susceptible to nitrous acid degradation and 80% digested by chondroitin- ase ABC; 12% of the chondroitin sulfate disaccharides were ADi-diSB and 88% were ADi-4s. In a second experiment in which 15% of the 35S-proteoglycans were released, 87% of the exocytosed and 63% of the retained proteoglycans were in the D l fraction of the CsCl gradient. Both populations of proteo- glycans had K., values of 0.2 upon Sepharose CL-GB chro- matography. The exocytosed and retained proteoglycans had glycosaminoglycans that were 73 and 69% chondroitinase ABC susceptible and 18 and 14% nitrous acid susceptible, respectively. Nine per cent of the disaccharides from the chondroitin sulfate glycosaminoglycans of the exocytosed pro- teoglycans and 11% of the disaccharides from the glycosami- noglycans of the retained proteoglycans were ADi-diSB.

Amino Acid Composition of the Peptide Core of RBL-1 Proteoglycam- Amino acid analysis was performed after acid hydrolysis on two different preparations of RBL-1 proteogly- cans, one purified with nuclease digestion of the cell sonicates and one purified without. The amino acid compositions of both of these preparations were in close agreement, indicating that the nuclease digestion step was not causing proteolysis of part of the peptide core. Consequently, the percentages of each amino acid in the two determinations were averaged (Table 11). The most common amino acid was Gly (35.4%), followed by Ser (22.5%), Ala (9.5%), and Glx (6.6%).

DISCUSSION

Proteoglycans from RBL-1 cells maintained in uitro were purified and characterized to allow comparison with previ- ously characterized protease-resistant Ser- and Gly-rich in- tragranular heparin proteoglycans of rat serosal and skin mast cells (16-19). Biosynthetically labeled proteoglycans were ex-

11138 RBL-1 Proteoglycans

tracted in the presence of protease inhibitors from nuclease- digested RBL-1 cell sonicates and purified by sequential CsCl density gradient ultracentrifugation, DE52 ion exchange chro- matography (Fig. l), and Sepharose CL-GB gel filtration chro- matography (Fig. 2). Single symmetrical peaks of 35S-macro- molecules were obtained in both chromatography steps, per- formed under dissociative conditions. The hydrodynamic size of these proteoglycans was M, 100-150,000. Measured as 35S- macromolecules/dry weight of starting material or final prod- uct, proteoglycans were purified 200-800-fold in four prepa- rations. RBL-1 cells maintained in tissue culture had approx- imately 0.5 pg/cell of proteoglycan.

The purified RBL-1 proteoglycans were comprised of both chondroitin sulfate (71 +. 9%, mean -+ S.D., n = 6) and heparin/heparan sulfate (27 _+ 12%, n = 6) glycosaminogly- cans, as determined by susceptibility to chondroitinase ABC and nitrous acid (Fig. 4). The dissacharides of the chondroitin sulfate glycosaminoglycans, analyzed by amino-cyano HPLC, consisted of ADi-4S and the unusual disulfated chondroitin disaccharide ADi-diSe (Fig. 5), previously identified in shark (30, 31) and hagfish (32) skin glycosaminoglycans. The rat serosal mast cell which has the trisulfated heparin disaccha- ride IdUA-2-S04+GlcN,6-diS0,, the mouse cultured mast cell with the disulfated chondroitin sulfate E disaccharide GlcUA+GalNAC-4,6-diS04, and the rat basophilic leukemia cell with chondroitin sulfate di-B (IdUA-2-S04+GalNAc-4- SO,) each synthesize a proteoglycan with a unique oversul- fated disaccharide. These disaccharide analyses provide a beginning for the classification of granulated histamine-con- taining cells based upon the structure of the intracellular proteoglycans.

Anion exchange HPLC was used to attempt to separate proteoglycans containing chondroitin sulfate di-B glycosami- noglycans from those containing heparin/heparan sulfate gly- cosaminoglycans (Fig. 7). Glycosaminoglycan analyses of three pools of proteoglycans eluting from an AX-1000 HPLC column in a single broad peak revealed each pool to contain similar proportions of chondroitin sulfate (74-75%) and hep- arin/heparan sulfate (1519%) glycosaminoglycans (Table I). However, proteoglycans were segregated based on the average sulfation of the chondroitin disaccharides, which were 9% ADi-diSB in the pool eluting with the shortest retention time, 22% in the second pool, and 49% in the pool eluting last. Upon Sepharose CL-GB chromatography of purified RBL-1 proteoglycans digested with chondroitinase ABC (Fig. 8), 65.8% of the radioactivity (the [35S]chondroitin sulfate disac- charides) eluted near V,, 27.4% eluted with a K., of 0.43 and was presumed to be core peptide with heparin or heparan sulfate glycosaminoglycans attached to them, and 6.7% of the radioactivity eluted with the same K., as the undigested pro- teoglycans. These findings suggest that a portion of the chon- droitin sulfate glycosaminoglycans and the heparin-like gly- cosaminoglycans reside on common peptide cores, with no more than 6.7% of the proteoglycans containing only or predominantly heparin-like glycosaminoglycans.

Proteoglycans were localized predominantly to the secre- tory granule of the RBL-1 cell by the noncytotoxic exocytosis of histamine, P-hexosaminidase, and proteoglycans in re- sponse to calcium ionophore (Figs. 9 and 10). There was no differential secretion of proteoglycans containing chondroitin sulfate di-B glycosaminoglycans and those containing hepa- rin/heparan sulfate glycosaminoglycans, since secreted and retained proteoglycans were found to have similar buoyant densities, K,, values on gel filtration, and glycosaminoglycan compositions. The correspondence in physicochemical prop- erties of proteoglycans purified from cell sonicates treated

with nucleases before the introduction of protease inhibitors, from cells directly extracted in the presence of protease inhib- itors with no nuclease treatment, and from calcium ionophore- activated cell supernatants indicates that the proteoglycans described in this study represent the major storage form of the secretory granule proteoglycan of the RBL-1 cells. The co-purification through conventional and high performance chromatography of intragranular protease-resistant proteo- glycans containing chondroitin sulfate and heparin-like gly- cosaminoglycans and the shift in hydrodynamic size of >93% of the radioactivity subsequent to chondroitinase ABC diges- tion is compatible with the existence of a population of predominantly hybrid proteoglycans substituted with approx- imately one-third heparin-like and two-thirds chondroitin sulfate glycosaminoglycans which have varying amounts of chondroitin sulfate di-B disaccharides.

Both the chondroitin sulfate and the heparin/heparan sul- fate glycosaminoglycans of RBL-1 proteoglycans are of ap- proximately M, 12,000 based on Sephadex G-200 gel filtration (Fig. 6), indicating that the proteoglycans have at most 10 glycosaminoglycan side chains. Rat serosal mast cell heparin proteoglycans have a hydrodynamic size of M, 750,000 and about 10 glycosaminoglycan chains of M, 75,000 each (19). Both RBL-1 cells and rat serosal or skin mast cells have peptide cores that are rich in Gly and Ser (Table 11). In contrast to protease-susceptible plasma membrane heparan sulfate proteoglycans (42) or extracellular matrix chondroitin sulfate proteoglycans (43), rat serosal mast cell heparin pro- teoglycans are highly resistant to proteolysis (17), as were the purified RBL-1 proteoglycans, which were not detectably degraded by a molar excess of trypsin, chymotrypsin, colla- genase, Pronase, papain, chymopapain, or elastase (Fig. 3). The presence of highly chargedglycosaminoglycan side chains that might limit the access of proteases to the peptide cores and the dearth of protease-sensitive basic or aromatic amino acids in the primary sequence of those cores probably contrib- ute to the protease resistance of intragranular proteoglycans.

Proteoglycans have been found in a number of immuno- and neurosecretory cells including mast cells, basophils (44), enterochromaffin cells (45), and platelets (46). These cells exocytose cationic amines such as histamine, serotonin, or catecholamines, as well as proteolytic and glycosidic enzymes. The functions of the anionic proteoglycans that are packaged into secretory granules with these mediators probably include preventing diffusion of small amines by ionic retention, bind- ing and intracellular inhibition of the large amounts of potent degradative enzymes, and pH and osmoregulation in the gran- ule. When the granule is exposed to the extracellular milieu, proteoglycans may control the rate of solubilization of media- tors into the tissue space. The high degree of sulfation and the protease resistance of intragranular proteoglycans may be important for preventing degradation in the granule microen- vironment. In addition, these releasable proteoglycans have been demonstrated to have important extracellular functions of their own. Heparin inhibits the coagulation cascade i n vivo (47). Both heparin and chondroitin sulfate E glycosaminogly- cans inhibit activation of the alternate complement pathway (48, 49) and initiate the Hageman factor-dependent contact activation pathway in vitro (50).

Acknowledgments-We thank Scott Adelman for excellent tech- nical assistance and Dr. Karl Schmid (Boston University Medical School, Boston, MA) for performance of the amino acid analyses.

REFERENCES 1. Eccleston, E., Leonard, B. J., Lowe, J. S., and Welford, H. J.

(1973) Nature New Biol. 244,73-76

RBL-1 Prc

2. Buell, D. N., Fowlkes, B. J., Metzger, H., and Isersky, C. (1976)

3. Barsumian, E. L., Isersky, C., Petrino, M. G., and Siraganian, R.

4. Kulczycki, A., Jr., Isersky, C., and Metzger, H. (1974) J. Exp.

5. Conrad, D. H., and Froese, A. (1976) J. Immunol. 116, 319-326 6. Kulczycki, A., Jr., McNearney, T. A., and Parker, C. W. (1976)

7. Isersky, C., Rivera, J., Mims, S., and Triche, T. J . (1979) J.

8. Morita, Y., and Siraganian, R. P. (1981) J. Immunol. 127,1339-

9. Isersky, C., Rivera, J., Triche, T. J., and Metzger, H. (1982) Mol.

10. Meyer, C., Wahl, L. M., Stadler, B. M., and Siraganian, R. P.

11. Levine, L. (1983) Biochem. Phurmacol. 32, 3023-3026 12. Parker, C. W., Falkenhein, S. F., and Huber, M. M. (1980)

13. Jakschik, B. A., Morrison, A. R., and Sprecher, H. (1983) J. Biol.

14. Orning, L., Hammarstrom, S., and Samuelsson, B. (1980) Proc.

15. Fewtrell, C., Lagunoff, D., and Metzger, H. (1981) Biochim.

16. Horner, A. A. (1971) J. Biol. Chem. 246, 231-239 17. Yurt, R. W., Leid, R. W. Austen, K. F., and Silbert, J. E. (1977)

J. Bid. Chem. 252, 518-521 18. Robinson, H. C., Horner, A. A,, Hook, M., Ogren, S., and Lindahl,

U. (1978) J. Biol. Chem. 253, 6687-6693 19. Metcalfe, D. D., Smith, J . A., Austen, K. F., and Silbert, J. E.

(1980) J. Biol. Chem. 255, 11753-11758 20. Razin, E., Stevens, R. L., Akiyama, F., Schmid, K., and Austen,

K. F. (1982) J. Biol. Chem. 257, 7229-7236 21. Razin, E., Ihle, J. N., Seldin, D., Mencia-Huerta, J.-M., Katz, H.

R., LeBlanc, P. A., Hein, A., Caulfield, J. P., Austen, K. F., and Stevens, R. L. (1984) J. Immunol. 132, 1479-1486

22. Razin, E., Stevens, R. L., Austen, K. F., Caulfield, J. p., Hein, A,, Liu, F.-T., Clabby, M., Nabel, G., Cantor, H., and Friedman, s. (1984) Immunology 52,563-575

23. Metcalfe, D. D., Wasserman, S. I., and Austen, K. F. (1979) Biochem. J. 185, 367-372

Cancer Res. 36, 3131-3137

P. (1981) Eur. J . Immunol. 11, 317-323

Med. 139,600-616

J. Immunol. 117,661-665

Immunol. 122, 1926-1936

1344

Immunol. 19,925-941

(1983) J. Immunol. 131,911-914

Prostaglandins 20, 863-886

Chem. 258,12797-12800

Natl. Acad. Sci. U. S. A . 77, 2014-2017

Biohys. Acta 644,363-383

24. Stevens, R. L., and Hascall, V. C. (1981) J. Biol. Chem. 256, 2053-2058

25. Cifonelli, J . A., and King, J. (1972) Carbohydr. Res. 21, 173-186

gteoglycans 11139

26. Stevens, R. L., and Austen, K. F. (1982) J. Biol. Chem. 257,253-

27. Saito, H., Yamagata, T., and Suzuki, S. (1968) J. Biol. Chem.

28. Handley, C. J., and Lowther, D. A. (1979) Biochim. Biophys. Acta

29. Seldin, D. C., Austen, K. F., Seno, N., and Stevens, R. L. (1984)

30. Seno, N., and Meyer, K. (1963) Biochim. Biophys. Acta 78, 258-

31. Akiyama, F., and Seno, N. (1981) Biochim. Biophys. Acta 674,

32. Seno, N., Akiyama, F., and Anno, K. (1972) Biochim. Biophys.

33. Seno, N., and Murakami, K. (1982) Carbohydr. Res. 103, 190-

34. Anno, K., Seno, N., Mathews, M. B., Yamagata, T., and Suzuki,

259

243 , 1536-1542

582,234-245

Anal. Biochem. 141,291-300

264

289-296

Acta 264,229-233

194

S. (1971) Biochim. B ~ O D ~ V S . Acta 237. 173-177 35. Kawai, Y., Seno, N., and Anno, K. (1966) J. Biochem. (Tokyo)

60,317-321 36. Wasteson, A. (1971) J. Chromatogr. 59, 87-97 37. Shaff, R. E., and Beaven, M. A. (1979) Anal. Biochem. 94, 425-

38. Robinson, D., and Stirling, J . L. (1965) Biochem. J . 107, 321-

39. Amador, E., Dorfman, L. E., and Wacker, W. E. (1963) Clin.

40. Schwartz, L. B., Lewis, R. A., Seldin, D., and Austen, K. F. (1981)

41. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

42. Carlstedt, Coster, L., Malmstrom, A., and Fransson, L.-A. (1983)

43. Heinegird, D., and Axelsson, I. (1977) J. Biol. Chem. 252, 1971-

44. Orenstein, N. S., Galli, S. J., Dvorak, A. M., Silbert, J . E., and

45. Margolis, R. U., and Margolis, R. K. (1972) Biochem. Pharmacol.

46. Barber, A. J., Kaser-Glanzmann, R., Jakahova, M., and Lfsch,

47. Howell, W. H., and Holt, E. (1918) Am. J . Physiol. 47, 328-341 48. Weiler, J. M., Yurt, R. W., Fearon, D. T., and Austen, K. F.

49. Wilson, J. G., Fearon, D. T., Stevens, R. L., Seno, N., and Austen,

50. Hojima, Y., Cochrane, C. G., Wiggins, R. C., Austen, K. F., and

430

327

Chem. 9,391-399

J. Immunol. 126,1290-1294

(1951) J. Biol. Chem. 193, 265-275

J. Biol. Chem. 258,11629-11635

1979

Dvorak, H. F. (1978) J. Immunol. 121, 586-592

22,2195-2197

E. F. (1972) Biochim. Biophys. Acta 286, 312-329

(1978) J. Exp. Med. 147,409-421

K. F. (1984) J. Immunol. 132,3058-3063

Stevens, R. L. (1984) Blood 63,3058-3063