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

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 dissociative 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 hydrodynamic 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 peptide core revealed a preponderance of Gly (35.4%), Ser (22.5%), and Ala (9.5%). Approximately of the side chains of were digested by chondroitinase and were chromatography

Proteoglycans were extracted from nuclease-digested 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 dissociative 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 hydrodynamic size range of M, 100,000-150,000 and were resistant to degradation by a molar excess of trypsin, a-chymotrypsin, Pronase, papain, chymopapain, collagenase, and elastase. Amino acid analysis of the peptide 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 chondroitinase ABC and 2790 were hydrolyzed by treatment with nitrous acid. Sephadex G-200 chromatography of glycosaminoglycans liberated from the intact molecule by &elimination demonstrated that both the nitrous acid-resistant (chondroitin sulfate) and the chondroitinase ABC-resistant (heparinbeparan sulfate) glycosaminoglycans were of approximately M, 12,000. Analysis of the chondroitin sulfate disaccharides in different preparations by amino-cyano high performance 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 preparation were separated by anion exchange high performance liquid chromatography and were found to contain chondroitin sulfate glycosaminoglycans whose disulfated disaccharides ranged from 9-49%. However, 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 chromatography of RBL-1 proteoglycans digested with chondroitinase ABC revealed that less than 7% of the molecules in the digest chromatographed with the hy-drodynamic size of undigested proteoglycans, suggesting that at most 7% of the proteoglycans lack chondroitin sulfate glycosaminoglycans.
RBL-1 cells stimulated with the calcium ionophore A23187 exocytosed proteoglycans, histamine, and Bhexosaminidase in comparable noncytotoxic dose-related fashion, and regression analyses of the net percentages released indicated that proteoglycans were in secretory granules. The exocytosed and retained proteoglycans were of similar size, buoyant density, and glycosaminoglycan composition, providing further evidence for a single pool of secretory granule proteoglycans. The co-purification and co-localization of proteoglycans containing heparin/heparan sulfate glycosaminoglycans and chondroitin sulfate di-B glycosaminoglycans 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 hydrodynamic size and having predominantly chondroitin sulfate di-B glycosaminoglycans. However, intragranular proteoglycans of RBL-1 cells and rat serosal mast cells possess the common properties of oversulfation, protease 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 undifferentiated sparsely granulated promyelocyte or a well-granulated mature basophil (2). The RBL-1 cell contains 0.1-1.0 pg/cell of histamine in secretory granules which can be released in some sublines (3) by cross-linking of its well-characterized surface IgE receptors (4-10). AII tines are amenable to activation by the calcium ionophore A23187 under the appropriate conditions, resulting in de novo generation and release of prostaglandins (11) and leukotrienes (12)(13)(14), as well as release of preformed mediators (15).
The recognition of an oversulfated chondroitin proteoglycan 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 nonheparin 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 conventional chromatography techniques performed under dissociative conditions. The hydrodynamic size of the purified proteoglycans and glycosaminoglycans, the disaccharide composition of the glycosaminoglycans, and the amino acid composition of the purified proteoglycans were determined. Ionophore-induced secretion experiments demonstrated an intragranular localization of these proteoglycans. The RBL-1 proteoglycans share properties with rat serosal mast cell heparin proteoglycans of oversulfation and protease resistance, suggesting that these molecules are members of a class of intragranular proteoglycans that fulfill related functional requirements; however, each population of proteoglycans has a distinct physicochemical composition characteristic of the particular cell type.

MATERIALS AND METHODS
Cell Culture and Radiolabeling-Adherent RBL-1 cells were maintained in 175-cm2 flasks containing 80 ml of Earle's minimal essential medium supplemented with 10% (v/v) fetal calf serum, 2 mM Lglutamine, 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 magnesium, 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 suspended 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 subjected 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 conductivity 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, lyophilized, resuspended in 200 pl of TSG buffer, and applied to a 0.6 X 80cm 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) (Worthington), wchymotrypsin (47 units/mg), papain (21 units/mg), chymopapain (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 36Sproteoglycans 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 Glycosaminoglycans of RBL-I Proteoglycans-The composition of the glycosaminoglycans 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 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 disaccharides generated by chondroitinase AC digestion, following the same protocol, from the percentage obtained by digestion with chondroitinase 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 undegraded 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 aminocyano-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 maximum 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 following proteoglycans or glycosaminoglycans were used as reference polysulfated 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 glycosaminoglycans 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 counting. 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 aminocyano HPLC disaccharide analysis of replicate samples.
Gel Filtration of Chnndroitinme ABC-digested RBL-I Proteoglycans-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 36Slabeled cells were used to quantitate the amount of exocytosed 35Sproteoglycans as assessed by PD-10 chromatography and p-scintillation 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 dehydrogenase, 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-hexosaminidase, 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 supernatants 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 6aminohexanoic 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 3sSmacromolecules. 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 lyophilized. 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 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 converted to per cent of total amino acids.

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-macromolecules 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 containing 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 nucleases 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-macromolecules obtained after Sepharose CL-GB chromatography lacked material which could be detected by the Lowry assay (41) and were, therefore, deficient in aromatic amino acidcontaining protein. These pooled 35S-macromolecules were   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 proteoglycans 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 radioactivity 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 radioactivity as disaccharides (Fig. 4C). Thirty-six per cent of the 35S-proteoglycans were hydrolyzed to oligosaccharides by nitrous acid treatment (Fig. 40). These findings indicate that 64% of the glycosaminoglycans present in this preparation of proteoglycans were chondroitin sulfates of which 19% contained 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% hydrolyzed by nitrous acid.
The chondroitinase ABC-generated unsaturated disaccharides were analyzed by amino-cyano HPLC. 35S-Disaccharides eluted in a major peak at a retention time of 7 min, corresponding to ADi-4S, and a second peak at 14.5 min, corresponding to ADi-diSB (Fig. 5). Chondroitinase ABC digests of five different samples of purified RBL-1 proteoglycans revealed 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 chondroitin sulfate di-B disaccharide from other sources (29-31), and not the glucuronic acid-containing isomer.
The glycosaminoglycans liberated from whole RBL-1 proteoglycans 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 chondroitinase ABC digestion, had hydrodynamic sizes not significantly different from each other or from the untreated glycosaminoglycans (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 chondroitinase ABC digestion coupled with amino-cyano HPLC. More than 90% of the radiolabeled disaccharides and oligosaccharides in each pool were identified (Table I), and it was observed that anion exchange HPLC did not separate proteoglycans containing only chondroitin sulfate glycosaminoglycans 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 chondroitin sulfate disaccharides in the sample of purified proteoglycans injected onto the AX-1000 column were ADi-diSB.
Gel Filtration of Chondroitinase ABC-digested RBL-1 Proteoglycans-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 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.  Exocytosis of Proteoglycans from RBL-1 Cells-The net per cent release of histamine, P-hexosaminidase, lactate dehydrogenase, and proteoglycans in response to increasing doses of calcium ionophore A23187 was determined (Fig. 9). The percentages of histamine, P-hexosaminidase, and 35S-macromolecules 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 maximal 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  (Fig. 10B).
Activation of 175-cm2 flasks of RBL-1 cells containing 35Slabeled 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 proteoglycans and the proteoglycans retained with the cells were partially purified in parallel by CsCl density gradient ultracentrifugation for subsequent determination of hydrodynamic size and glycosaminoglycan composition. Eighty-three per cent of the exocytosed proteoglycans and 81% of the cellassociated proteoglycans were recovered in the high density D l fraction in the same ultracentrifuge run, and both popu-*oteoglycans 11137 upon Sepharose CL-GB gel filtration chromatography. Analysis of the glycosaminoglycans of the exocytosed proteoglycans revealed that 32% were degraded by nitrous acid and 68% were susceptible to chondroitinase ABC; of the chondroitinase ABC-generated disaccharides, 15% were ADi-diSB and 85% were ADi-4S as analyzed by HPLC. The glycosaminoglycans of the retained proteoglycan were 8% susceptible to nitrous acid degradation and 80% digested by chondroitinase 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 proteoglycans had K., values of 0.2 upon Sepharose CL-GB chromatography. 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 proteoglycans and 11% of the disaccharides from the glycosaminoglycans 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 proteoglycans, 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 previously characterized protease-resistant Ser-and Gly-rich intragranular heparin proteoglycans of rat serosal and skin mast cells (16)(17)(18)(19). Biosynthetically labeled proteoglycans were ex-tracted in the presence of protease inhibitors from nucleasedigested RBL-1 cell sonicates and purified by sequential CsCl density gradient ultracentrifugation, DE52 ion exchange chromatography (Fig. l), and Sepharose CL-GB gel filtration chromatography (Fig. 2). Single symmetrical peaks of 35S-macromolecules were obtained in both chromatography steps, performed under dissociative conditions. The hydrodynamic size of these proteoglycans was M, 100-150,000. Measured as 35Smacromolecules/dry weight of starting material or final product, proteoglycans were purified 200-800-fold in four preparations. RBL-1 cells maintained in tissue culture had approximately 0.5 pg/cell of proteoglycan.
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 disaccharides) 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 proteoglycans. These findings suggest that a portion of the chondroitin sulfate glycosaminoglycans and the heparin-like glycosaminoglycans 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 secretory granule of the RBL-1 cell by the noncytotoxic exocytosis of histamine, P-hexosaminidase, and proteoglycans in response to calcium ionophore (Figs. 9 and 10). There was no differential secretion of proteoglycans containing chondroitin sulfate di-B glycosaminoglycans and those containing heparin/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 properties of proteoglycans purified from cell sonicates treated with nucleases before the introduction of protease inhibitors, from cells directly extracted in the presence of protease inhibitors with no nuclease treatment, and from calcium ionophoreactivated 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 proteoglycans containing chondroitin sulfate and heparin-like glycosaminoglycans and the shift in hydrodynamic size of >93% of the radioactivity subsequent to chondroitinase ABC digestion is compatible with the existence of a population of predominantly hybrid proteoglycans substituted with approximately 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 sulfate glycosaminoglycans of RBL-1 proteoglycans are of approximately 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 proteoglycans 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, collagenase, 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 contribute to the protease resistance of intragranular proteoglycans.
Proteoglycans have been found in a number of immunoand 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, binding and intracellular inhibition of the large amounts of potent degradative enzymes, and pH and osmoregulation in the granule. When the granule is exposed to the extracellular milieu, proteoglycans may control the rate of solubilization of mediators 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 microenvironment. In addition, these releasable proteoglycans have been demonstrated to have important extracellular functions of their own. Heparin inhibits the coagulation cascade in vivo (47). Both heparin and chondroitin sulfate E glycosaminoglycans inhibit activation of the alternate complement pathway (48,49) and initiate the Hageman factor-dependent contact activation pathway in vitro (50).