Abstract
Proteoglycans were extracted from nuclease-digested sonicates of 10(9) 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-6B 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 Mr 100,000-150,000 and were resistant to degradation by a molar excess of trypsin, alpha-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 27% were hydrolyzed by treatment with nitrous acid. Sephadex G-200 chromatography of glycosaminoglycans liberated from the intact molecule by beta-elimination demonstrated that both the nitrous acid-resistant (chondroitin sulfate) and the chondroitinase ABC-resistant (heparin/heparan sulfate) glycosaminoglycans were of approximately Mr 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 (IdUA-2-SO4----GalNAc-4-SO4); the remainder were the monosulfated disaccharide GlcUA----GalNAc-4-SO4. 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 heparin/heparan sulfate glycosaminoglycans was achieved, suggesting that RBL-1 proteoglycans might be hybrids containing both classes of glycosaminoglycans. Sepharose CL-6B chromatography of RBL-1 proteoglycans digested with chondroitinase ABC revealed that less than 7% of the molecules in the digest chromatographed with the hydrodynamic size of undigested proteoglycans, suggesting that at most 7% of the proteoglycans lack chondroitin sulfate glycosaminoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
From the Department of Medicine, Harvard Medical Schooland the Department of Rheumatologyand Immunology,Brigham and Women’s Hospital, Boston, Massachusetts 02115
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 a t 37 "C.TSG buffer was added and themixture was applied to the Sepharose CL-GB column and eluted with TSG buffer as described
Material with significant ultraviolet absorbance eluted from the column in a slightly earlier peak, overlapping with the 35S-proteoglycans.The fractions conchromatographed on Sepharose CL-GB. 35S-Macromolecules filtered as a single sharp peak with a K, of0.25 (Fig. 2), indicating a hydrodynamic size of approximately M, 100,000-150,000
Summary
Cell Culture and Radiolabeling-Adherent RBL-1 cells were maintained in 175-cm2flasks containing 80ml ofEarle'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 100pg/ml streptomycin (Grand Island Biological Co., Grand Island, NY) at pH7.2 in ahumidified 37 "C incubator with a 6% COn atmosphere. 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 a t 37 "C.TSG buffer was added and themixture was applied to the Sepharose CL-GB column and eluted with TSG buffer as described. The D l fractions, which contained the majority of the 35S-macromoleculesfrom both the supernatants andpellets, 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. 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
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