Synthesis of Chondroitin Sulfate E ...

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Razin, E., Cordon-Cardo, C., and Good, R. A. (1981) Proc. Natl. 8. Schrader ... Tertian, G., Yung, Y-P., Guy-Grand, D., and Moore, M. A. S. (1981) J. Zmmunol.

THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 258, No. 9, Issue of May 10, pp. 5977-5984 Prrnted in U.S.A .

Synthesis of Chondroitin Sulfate E Glycosaminoglycan ontop Nitrophenyl-P-D-xyloside and Its Localization to the Secretory Granules of Rat Serosal Mast Cells and Mouse Bone Marrow-derived Mast Cells* (Received for publication, October 26, 1982)

Richard L. Stevens$, Ehud Razin, K. Frank Austen, Ann Hein,and John P. Caulfield From the Departmentsof Medicine a n d Pathology, Harvard Medical School, a n d the Department of Rheumatology a n d Immunology, Brigham a n d Women’s Hospital, Boston, Massachusetts 02115

Nobuko Sen0 From the Departmentof Chemistry, Ochanomizu University, Tokyo, J a p a n

Karl Schmid and Fumiko Akiyama From the Departmentof Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

05669 from the National Institutesof Health and in part by a Milton Fund grant from Harvard University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence should be addressed at the Seeley G. Mudd Building, Room 626,250 Longwood Avenue, Boston,MA 02115.


Materials-Male BALB/c mice, C56BL/6J mice, C3H mice, and Sprague-Dawley rats (JacksonLaboratories, Bar Harbor, ME); RPMI 1640, fetal calf serum, penicillin, 2-mercaptoethanol, L-glutamine, nonessential amino acids, and Dulbecco’s modified Eagle’s medium


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Cultures of rat serosal mastcells and of mouse bone covalently bound heparin glycosaminoglycans. When incumarrow-derived mast cells of more than 97% purity, bated for short periods of time with the glycosaminoglycan which normally synthesize a heparinproteoglycan and acceptor p-nitrophenyl-P-D-xyloside, rat serosal mast cells a chondroitin sulfateE proteoglycan, respectively, continue to synthesize heparin glycosaminoglycan onto the were assessed for thespecies of 36S-glycosaminoglycan endogenous proteoglycan peptide core but produce a species The [“S] of chondroitin sulfate onto the exogenous acceptor (2). The polymerized onto p-nitrophenyl-8-D-xyloside. sulfate-labeled macromolecules were extracted, puriapparent lack of chondroitin sulfate synthesis under in vivo fied, and resolved into aproteoglycan-enriched fraction or normal in vitro culture conditions suggests that a biosynand a8-D-xyloside-linkedglycosaminoglycan-enriched thetic step before the addition of galactose, the second monofraction. Chondroitinase ABC digestion of these xylo- saccharide in the common glycosaminoglycan linkage site, side-linked chondroitin sulfate chains from both cultures followed by ascending thin layer chromatogra- determines whether heparinor chondroitin sulfate glycosamiphy, descending paper chromatography, and high volt- noglycan will be synthesized by these peritoneal mast cells. age electrophoresis revealed two equal molar disaccha- Although the intracellular location of the chondroitin sulfate rides possessing the mobility of 2-acetamide-2-deoxy- polymerized onto P-D-xylosidein rat serosal mast cell cultures 3-O-(~-~-gluco-4-enepyranosyluronic acid)-4-O-sulfo- was not determined, it was presumed to reside with heparin proteoglycan in the granules because it was notsecreted D-galactose and 2-acetamido-2-deoxy-3-O-~-~-gluco-4continually into the culture medium, as has been commonly enepyranosyluronic acid)-4-6-di-O-sulfo-~-galactose. Autoradiographic analysis of rat serosal mastcell cul- observed for connectiw tissue synthesizing cells (4-6). Mouse bone marrow cells differentiate in uitro, upon expotures exposed to [3H]xylosideindicated that themajority of isolated mast cells incorporated theradiolabeled sure to T cell-derived growth factors, into a relatively homoprecursor. Thatboth the 8-D-xyloside-linkedchondroi- geneous population of cells that resemble mast cells in terms tin sulfate E and chondroitin the sulfate E proteoglycan of the histologic staining of their intracellular granules and of mouse bone marrow-derived mast cells were local- their overall ultrastructural morphology (7-13). Their princiized to the secretory granules was indicated by their pal proteolgycan possesses chondroitin sulfate E glycosamiequal exocytosis from these cells when activated by noglycan side chains characterized by disaccharides rich in N calcium ionophore A23187. X-ray energy-dispersive acetylgalactosamine-4,6-disulfateratherthan heparin (3). analysis of normal and chronically fl-D-xyloside-treated When sensitized with monoclonal IgE, these cells respond to mouse bone marrow-derived mast cells detected mea- antigen-initiated activation with the release of the granule surable quantities of sulfur only in the secretory gran- markers, histamine, ,&hexosaminidase, and chondroitin sulules. Thus, both rat serosal mast cells and bone marfate E proteoglycan, and with the generation and release of row-derived mast cells have thecapacity to polymerize chondroitin sulfate E glycosaminoglycan onto a p-D- leukotrienes B4 and C4 (14,15). In order to analyze further xyloside acceptor, and thismonomeric glycosaminogly- the possible relationships of the bone marrow-derived mast can lacking a proteincore is capable of being directed cells differentiated in culture and the in vivo differentiated serosal mast cells, the chondroitinsulfate induced in rat to the secretorygranule. serosal mast cells by P-D-xylosidetreatment was compared in more detail to the highly sulfated chondroitin sulfate E glyRat (1, 2) and mouse (3) serosal mast cells synthesize in cosaminoglycan synthesized by normal and by P-D-xylosideprimary culture a proteoglycan of M , = 750,000 containing treated bone marrow-derived mast cells. In both populations of mast cells, the 35S-glycosaminoglycanspolymerized onto * This studywas supported in part by Grants AI-07722, AM-00775, the P-D-xyloside acceptor were chondroitin sulfateE and were AM-25960,AM-27270, HL-17382, HL-19717, GM-10374, andRRlocalized to the secretory granule.


Localization of Chondroitin Sulfate E in Mast Cell Granules


0 1 0


' The abbreviations used are: GnHCI, guanidine hydrochloride; FIG. 2. Descending paper chromatography of chondroitiADi-4S, 2-acetamido-2-deoxy-3-~-(~-~-gluco-4-enepyranosyluronic nase ABC digests of 35SS-glycosaminoglycans isolated from xyloside-treated rat m a s t cells. Chondroitinase ABC digests of ADi-6S, 2-acetamido-2-deoxy-3-0-(~-~acid)-4-O-su~fo-~-ga~actose; gluco-4-enepyranosyluronic acid)-6-O-sulfo-~-galactose; ADi-OS, 2- '%-glycosaminoglycans isolated from 1.0 mM /i"D-xyloside-treated rat acetamido-2-deoxy-3-0-(~-~-gluco-4-enepyranosyluronic acid)-D-ga- serosal mast cells ( l ) , of authentic squid chondroitin sulfate E (2), lactose; ADi-diSp, 2-acetamido-2-deoxy-3-0-(~-~-gluco-4-enepyranoand of authentic shark chondroitin sulfateD ( 3 ) .The paper was cut syluronic acid)-4,6-di-O-sulfo-~-galactose; ADi-diSr), 2-acetamido-2- along the dashed line and either stained withsilver nitrate (2 and 2) deoxy-3-~-(2-~-su~fo-~-~-g~uco-4-enepyranosy~uronic acid)-6-O-sul- or analyzed by autoradiography (1). The positions of standard disacfo-D-galactose. charides are indicated.

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with 4.5 g of glucose/liter (Grand IslandBiological Co., Grand Island, chloride, lyophilized, suspended in 250 pl of equilibration buffer, and NY); PD-IO gel filtration columns and SepharoseCL-4B (Pharmacia applied to replicate Sepharose CL-4B columns (0.6 X 120 cm). The columns, which were equilibrated in and eluted with 4 M GnHCI, 0.1 Fine Chemicals, Piscataway, NJ); ["S]sulfate (-4000 Ci/mol) (New England Nuclear, Boston, MA); Hydrofluor (National Diagnostics, M Tris-HCI, 0.1 M Na2SO.,, pH 7.0, containing 25 pg/ml of heparin Somerville, NJ); Whatman No. 1 chromatography paper (Whatman glycosaminoglycan carrier, resolved monomeric glycosaminoglycans Inc., Clifton, NJ); precoatedthinlayerchromatography cellulose from proteoglycans (2, 3). A sample of each 0.5-11-11column eluate sheets (EM Laboratories,Elmsford, NY); XAR-5x-ray film (Eastman fraction was added to 0.5 ml of70% (v/v) ethanol and 12.5 ml of Kodak, Rochester, NY); dimethyl sulfoxide and dimethylformamide Hydrofluor, and the radioactivity was quantitated on a Searle 6880 (J. T. Baker Diagnostics, Phillipsburg, NJ); pig mucosa heparin and P counter. The '"S-proteoglycan and ""S-glycosaminoglycan peaks p-nitrophenyl-P-D-xyloside(SigmaFineChemicals Inc., St. Louis, were pooled separately, dialyzed sequentially against water and 0.5 MO); calcium ionophore A23187, zwittergent 3-12 (Calbiochem, La M sodium acetate and then lyophilized. Jolla, CA); ultrapure GnHCI' (Schwarz/Mann, Spring Valley, NY); After the addition of chondroitin sulfateC (100 pg) and chondroitin whalecartilagechondroitinsulfate A, sharkcartilagechondroitin sulfate A (100 pg) carriers, radiolabeled proteoglycans andglycosamisulfate C, chondroitinase ABC, chondro-6-sulfatase, chondro-4-sulfanoglycans were digested at 37 OC for 30 min with 0.2 unit of chontase, ADi-4S, ADi-6S, and ADi-OS (Miles Laboratories, Elkhart, IN) droitinase ABC alone and with0.2 unit of chondro-6-sulfatase or 0.2 were obtained as noted. ADi-diSE and ADi-diSI) were prepared by unit of chondro-4-sulfatase or both sulfatases (3,22). Sodium fluoride chondroitinase ABC digestion of squid cartilage and shark cartilage (1m ~was ) added to samples that were incubated with chondroitinase chondroitin sulfate, respectively (16, 17).p-Nitrophenyl-P-D-xyloside ABC alone in order to inhibit any contaminating sulfatase activity wasradiolabeled by catalytic exchange with tritiated water (New (23). Digests were analyzed by ascending thin layer chromatography England Nuclear) and purified by reverse-phase high performance on precoatedcellulose plates (2,24), by descending paper chromatogliquid chromatography on an Altex model l00Ahigh performance raphy on WhatmanNo. 1paper (22), and byhigh voltage electropholiquid chromatograph (Altex, Berkeley, CA) withan ultrasphere ODS column and Hitachi model 100-40 on-line spectrophotometer. The column (4.6 x 150 mm) was run isocratically in 45% (v/v) dimethylformamide in 0.001 N acetic acid a t room temperature a t a flow rate of 0.5 ml/min. -ADi-OS Isolation, Culture, and Radiolabeling of Mast Cells-Rat serosal cells of >97% purity (1,2, 18) were resuspended at a concentration of -ADi-4S 1 X 10"/ml in Dulbecco'smodifiedEagle's mediumsupplemented with15 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, 10 mM N-[tris(2-hydroxymethyl)methyl-2-amino]ethanesulfonic acid, 10 mM 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid (pH 7.2), 100 --ADi-GS units/ml of penicillin, 100 units/ml of streptomycin, and 15% (v/v) heat-inactivated fetal calf serum (DMEM). Mouse mast cells were -ADi-diS differentiated from 1 X 10" BALB/c mouse bone marrow cells during +oligo 14 days of culture in 30 ml of 50% (v/v) enriched medium [RPMI ( ( 9 9 3 --origin 1640,supplemented with 10% (v/v) fetal calf serum, 50 p~ 2-mercaptoethanol, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 100 1 2 3 4 5 units/ml of penicillin, and 100 units/ml of streptomycin, pH 7.21 and in 50% (v/v) conditioned medium (3, 7). Conditioned medium was FIG. 1. Ascending thin layer chromatography of chondroitiobtained from 48-h concanavalin A (2 pg/ml)-stimulated splenocytes nase ABC digests of 35S-glycosaminoglycans isolated fromrat (10"/ml) fromC57BL/6J and C3H mice. After 14 days of culture, cells treated with increasing concentrations of xyloside. Rat >95%of the cellsweredefined as mast cells by metachromatic serosal mast cells were cultured without P-D-xyloside ( I ) , and with staining of granules with toluidine blue (3, 7) and differential cell 0.1 mM (2), 0.3 mM (31, 1.0 mM ( 4 ) . and 3.0 mM (5) P-D-xyloside, count after fixation and staining with Giemsa (14); 2.5 times the background, cans (Fig. 4). Chondroitinase ABC digestion of both the /?-Dwhereas sulfur was not greater than the background in the xyloside-linked "5S-glycosaminoglycans and "'S-proteoglycans I). The sulfursignal yielded two equal molar products whichco-migratedwith nucleus or the embedding medium (Table in/?-D-xyloside-treated cells was not significantlydifferent ADi-4S and ADi-diSE on descending paper chromatography. from that in untreated cells (Table I). Readings over granules Thus, the introduction of /?-D-xyloside into the culture methat did not contain dense cores and over extragranular cy- dium did not uncover a latent capacity of the mouse bone marrow-derived mast cells to polymerize additional types of toplasm gave spectra similar to the plastic background. Rat serosal mastcells (5 X lo6) were incubated with 25 pCi/ glycosaminoglycans. The capacityof the bone marrow-derived ml of ["Hlxyloside in 1.0 ml of DMEM in the presence or mast cell to respond to /?-D-xyloside the signal withan increase absence of 3 mM cold xyloside for 3 h at 37 "C followed by in chondroitin sulfate E synthesis permits two speculations. exhaustive washingin the presence of 3 mM /?-D-xyloside. First, the rate-limiting step in the synthesisof this istracellular One-fourth of each cell culture was subjected to extraction proteoglycan is not the polymerization of chondroitin sulfate and PD-10 gel filtration to confirm that >70% of the cell- but most likely the amount of protein core; and second, the associated radioactivity was macromolecular in size. Autora- lower molecular weight of the chondroitin sulfate E proteodiography of the remainder of each ["Hlxyloside-treated culglycan (2) relative to the heparinproteoglycan of rat serosal ture revealed incorporation of radioactivity in essentially ev- mast cells (1)is due to the nature of the peptidecores and the ery cell present in the purified rat serosal mast cell preparation length of the glycosaminoglycan side chains. (Fig. loa). In contrast, autoradiography of the serosal mast Autoradiography of VHIxyloside-treated rat serosal mast cells incubated with["Hlxyloside inthe presenceof 3 mM cold cells revealed radioactivity localized in the majority of cells xyloside failed to reveal radioactivityover any cell (Fig. lob). (Fig. lo), indicating that chondroitin sulfateE polymerization Thus, the preponderanceof purified rat serosal mast cells can is not limited to a small subpopulation of heparin-containing be induced by p-nitrophenyl-/?-D-xyloside treatment to syn- rat serosalmast cells. The subcellular localization of the thesize glycosaminoglycans onto the exogenous substrate. chondroitinsulfate E polymerized onto P-D-xyloside was sought using the cultured mouse bone marrow-derived mast DISCUSSION cells because of their fast doubling time (7), substantial rate Ratserosalmast cells cultured in the presence of the of proteoglycan and glycosaminoglycan synthesis (2), augexogenous glycosaminoglycan acceptor p-nitrophenyl-,8-D-xy- mented [:"S]sulfate incorporation in the presence of low con-


Localization Chondroitin of Sulfate

EMast Cell in Granules

Acknowledgments-We technical assistance.

thank Alex Zabik and Lloyd Klickstein for REFERENCES

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1536-1542 23. Handley, C. J., and Lowther, D. A. (1979) Biochim. Biophys.Acta 582,234-245 24. Mason, R. M., Kimura, J . H., and Hascall, V. C. (1982) J . Biol. Chem. 257, 2236-2245 25. Razin, E., Mencia-Huerta, J. M., Lewis, R. A., Corey, E. J., and Austen, K. F. (1982) Proc. Natl. Acad. Sci. U. S. A . 79, 46654667 26. Robinson, D., and Stirling, J. K. (1968) Biochem. J . 107,321-327 27. Schwartz, L.B., Austen, K. F., and Wasserman, S. I. (1979) J. Zmmunol. 123,1445-1450 28. Caulfield, J. P., Lewis, R. A., Hein, A., and Austen, K. F. (1980) J. Cell Biol. 85,299-311 29. Sher, A., Hein, A,, Moser, G., and Caulfield, J. P. (1979) Lab. Znuest. 41,490-499 30. Hein, A., and Caulfield, J. P. (1982) J. Cell Biol. 95, 397a 31. Yamagata, T., Saito, H., Habuchi, O., and Suzuki, S. (1968) J . Biol. Chem. 243, 1523-1535 32. Seno, N., Akiyama, F., and Anno, K. (1974) Biochim. Biophys. Acta 362,290-298

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9. Tertian, G., Yung, Y-P., Guy-Grand, D., and Moore, M. A. S. centrations of xyloside, ability to polymerize >75% of newly (1981) J. Zmmunol. 127,788-794 synthesized glycosaminoglycans onto theexogenous acceptor, and lack of selective degradation of P-D-xyloside-linked gly- 10. Nagao, K., Yokoro, K., and Aaronson, S. A. (1981) Science (Wash. D.C.) 212,333-335 cosaminoglycan. Ultrastructural analysis and x-ray microa- 11. Razin, E. A,, Cordon-Cardo, A,, Minick, C. R., and Good, R. A. nalysis did not reveal any difference between chronic p-D(1982) Exp. Hematol. 10, 524-532 xyloside-treatedbone marrow-derivedmast cells andun12. Nabel, G., Galli, S. J., Dvorak, A. M., Dvorak, H. F., and Cantor, H. (1981) Nature (Lond.)291,332-334 treated cells (Figs. 5 to 8). X-ray microanalysis revealed sulfur to be associated only with the electron-dense secretory gran- 13. Galli, S. J., Dvorak, A. M., Marcum, J. A., Ishizaka, T., Nabel, G., Simonian, H., Pyne, K., Goldin, J. M., Rosenberg, R. D., Cantor, ules or granule cores of both untreated and p-D-xylosideH., and Dvorak, H. F. (1982) J. Cell Biol. 95,435-444 treated cells (Fig. 9). As the ratio of exocytosed xyloside- 14. Razin, E., Mencia-Huerta, J.-M., Stevens, R. L., Lewis, R. A,, Liu, linked 35S-glycosaminoglycan to 35S-proteoglycanin response F.-T., Corey, E. J., and Austen, K. F. (1983) J . Exp. Med. 157, to calcium ionophore was similar to that present in unchal189-210 lenged j3-D-xyloside-treated cells (Fig. 4B),it is concluded that 15. Mencia-Huerta, J.-M., Razin, E., Ringel, E. W . , Corey, E. J., Hoover, D., Austen, K. F., and Lewis, R. A. (1983) J . Zmmunol., the low molecular weight chondroitin sulfate E side chain in press polymerized onto P-D-xyloside in mouse bone marrow-derived 16. Kawai, Y., Seno, N., and Anno, K. (1966) J. Biochem. (Tokyo) mast cells is directed to the secretory granules. Possible ex60,317-321 planations for this finding include the following. The nitro- 17. Suzuki, S., Saito, H., Yamagata, T., Anno, K., Seno, N., Kawai, phenylgroup of the P-D-xyloside is ableto mimic those Y., and Furuhashi, T. (1968) J. Biol. Chem. 234, 1543-1560 determinants on the proteoglycan peptide core which direct 18. Holgate, S. T., Lewis, R. A., and Austen, K. F. (1980) J. Zmmunol. 124,2093-2099 the proteoglycan into the secretorygranules; glycosaminoglycan polymerization and sulfation are late events in proteogly- 19. Kokski, I. R., Poplock, P. G., and Blaese, R. M. (1976) in Zn Vitro Methods in Cell Mediated and Tumor Immunity (Bloom, B. can synthesis, andall molecules containing covalently bound R., and David, J. R., eds) pp. 359-362, Academic Press, New glycosaminoglycan are already committed to a granule locaYork tion; or receptors present on the granule membranesspecifi- 20. Imura, J. H., Caputo, C. B., and Hascall, V. C. (1981) J. Biol. Chem. 256,4368-4376 cally recognize chondroitin sulfate E, and chondroitin sulfate E itself directs the nativeproteoglycan core and P-D-xyloside- 21. Hascall, V. C., andSajdera, S. W. (1969) J. Biol. Chem. 244, 2384-2396 linked glycosaminoglycan into the secretorygranule. 22. Saito, H., Yamagata, T., and Suzuki, S. (1968) J. Biol. Chem. 243,

Synthesis of chondroitin sulfate E glycosaminoglycan onto p-nitrophenyl-beta-D-xyloside and its localization to the secretory granules of rat serosal mast cells and mouse bone marrow-derived mast cells. R L Stevens, E Razin, K F Austen, A Hein, J P Caulfield, N Seno, K Schmid and F Akiyama J. Biol. Chem. 1983, 258:5977-5984.

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