Immunological Characterization of Secretory Proteins of Chromaffin Granules: Chromogranins A, Chromogranins B, and Enkephalin-Containing Peptides

The soluble proteins of bovine chromaffin granules can be resolved into about 40 proteins by two-dimensional electrophoresis. Use of several antisera enabled us to characterize most of these proteins with the immune replica technique. An antiserum against dopamine beta-hydroxylase reacted with one protein of Mr 75,000. Met-enkephalin antisera labeled eight proteins of Mr 23,000-14,000. A new method was developed to obtain highly purified chromogranin A for immunization. The antiserum reacted with chromogranin A and several smaller proteins of similar pI. This specific antiserum did not react with a second family of hitherto undescribed proteins, which we propose to call chromogranins B. An antiserum against these proteins was raised. It labeled several proteins ranging in Mr from 100,000 to 24,000 and focusing at pH 5.2. Subcellular fractionation established that chromogranins B are specifically localized in chromaffin granules of several species. They are secreted from the adrenal medulla during cholinergic stimulation. We conclude that apart from dopamine beta-hydroxylase chromaffin granules contain three families of immunologically unrelated proteins.     

Relationship of multiple forms of chromogranin

Chromogranin polypeptides of Mr 100,000, Mr 85,000, Mr 75,000, and Mr 65,000 have been detected in adrenal medulla chromaffin granules using anti-chromogranin antiserum. Monoclonal antibodies to this protein also detect the multiple molecular weight chromogranin polypeptides. Analysis of phosphorylated amino acids gives a value of 5 phosphoserine residues/mol of Mr 75,000 chromogranin polypeptide. Immunological analysis of dephosphorylated chromogranin shows that the anti-chromogranin serum reacts with both the phosphorylated and unphosphorylated forms of the protein. Each of the chromogranin polypeptides has been isolated using a combination of DEAE-cellulose chromatography and reverse phase high pressure liquid chromatography. Sequencer analysis of each protein revealed a high degree of amino acid identities at the amino terminal of these proteins. Amino-terminal Sequencer analysis of chromogranin fragments also provides evidence for a gene duplication event. Preliminary studies also show that chromogranin may be degraded in the chromaffin granules by a calcium-dependent mechanism.     

Chromogranin C: A Third Component of the Acidic Proteins in Chromaffin Granules

We characterized a group of acidic proteins of bovine chromaffin granules with an antiserum raised against a protein described by Rosa and Zanini [Eur. J. Cell Biol. 31, 94-98 (1983)] in pituitary gland. In adrenal medulla the proteins reacting with this antiserum are confined to chromaffin granules. Their largest component has a Mr of 86,000 and a pI of 5.0. In addition six proteins of lower molecular weight are recognized by this antiserum. In a cell-free system only one protein is synthesized that can be precipitated with this antiserum. The properties of these proteins are very similar to those of the previously described chromogranins A and B; however, there is no immunological cross-reaction between these protein groups. We suggest this third group of acidic proteins of chromaffin granules be named chromogranins C.     

Comparative Incorporation of Proenkephalin-Derived Peptides, Chromogranin A, and Dopamine β-Hydroxylase into Chromaffin Vesicles

The incorporation of enkephalin-containing peptides (ECPs) derived from proenkephalin into chromaffin vesicles was examined in primary cultures of adrenal medullary chromaffin cells. Cells were pulse-labeled with [35S]methionine and chased for periods up to 24 h. Chromaffin vesicles in cell homogenates were then fractionated by density gradient centrifugation and the presence of [35S]Met-enkephalin sequences in gradient fractions determined. 35S-ECPs were incorporated into particles suggestive of immature vesicles within 1-2 h after radiolabeling. Vesicle maturation, measured by co-equilibration of 35S-ECPs and total ECPs in the gradients, was complete within 9-12 h and was unaffected by treatments that increase proenkephalin synthesis. Incorporation of [35S]chromogranin A into chromaffin vesicles followed a similar time course, but 35S-labeled dopamine beta-hydroxylase was much more slowly incorporated, possibly reflecting differences in incorporation of membrane and soluble components. In summary, the data demonstrate that ECPs are rapidly sequestered in immature chromaffin vesicles, a process unaltered by changing rates of proenkephalin synthesis.     

Effect of secretagogues on chromogranin A synthesis in bovine cultured chromaffin cells. Possible regulation by protein kinase C.

Chromogranin A is a major component of storage granules in many different secretory cell types. After [35S]methionine labelling of proteins from cultured bovine chromaffin cells, chromogranin A was immunoprecipitated with specific antibodies, and the radioactivity incorporated into chromogranin A was determined and used as an index of its synthesis rate. Depolarization of cells with nicotine or high K+ evoked a Ca2+-dependent increase in chromogranin A synthesis, whereas muscarine, which does not evoke significant Ca2+ influx from bovine chromaffin cells, had no effect on chromogranin A synthesis. Forskolin, an activator of adenylate cyclase, affected neither the basal nor the nicotine-stimulated rate of chromogranin A synthesis. In contrast, 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, significantly enhanced the incorporation of radioactivity into chromogranin A. Sphingosine, an inhibitor of protein kinase C, abolished both nicotine-stimulated and TPA-induced chromogranin A synthesis. In addition, long-term treatment of chromaffin cells with TPA decreased protein kinase C activity and inhibited the nicotine-stimulated chromogranin A synthesis. These results suggest that protein kinase C may play an important role in the control of chromogranin A synthesis.     

Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization

Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. Core (protein) interactions seem to be responsible for the association of the proteoglycan with the extracellular matrix.     

Cell-associated proteoheparan sulfate from bovine arterial smooth muscle cells

Cell-associated proteoheparan sulfate has been isolated from bovine arterial smooth muscle cells preincubated with [35S]sulfate or a combination of [3H]glucosamine and [35S]methionine. The purified proteoheparan sulfate had an apparent Mr of 200,000 on calibrated Sepharose CL-2B columns. The glycosaminoglycan component (Mr approximately 30,000) was identified as heparan sulfate by its susceptibility to specific enzymatic and chemical degradation. After degradation of the proteoheparan sulfate by microbial heparitinase the resulting protein core had an apparent Mr of 92,000 on SDS-polyacrylamide gels. Its mobility was similar in the absence and presence of reducing agents indicating that the protein core consists of a single polypeptide chain. Pulse-chase experiments revealed that about 40% of the cell layer-associated proteoheparan sulfate was released into the medium, while the remainder was internalized and converted to smaller species through a series of degradation steps. Initially there was a proteolytical cleavage of the protein core generating glycosaminoglycan peptide intermediates with polysaccharides chains similar in size to the original. The half-life of the native proteoheparan sulfate was found to be about 4 h.     

Degradation of the heparin matrix of mast cell granules by cultured fibroblasts

The ability of cultured rat fibroblasts to phagocytose rat peritoneal mast cell granules has been previously demonstrated by light and electron microscopy. To determine if the heparin matrix of ingested granules could be degraded by fibroblasts after phagocytosis, the heparin within peritoneal mast cells was labeled with [35S]sulfate in vivo. The 35S-labeled rat peritoneal mast cells were purified and their granules were isolated and shown to contain [35S]heparin proteoglycan. Incubation of [35S]heparin proteoglycan-containing granules with cultured rat fibroblasts revealed internalization of radioactivity by the fibroblasts over the first 24 hr consistent with phagocytosis of the granules by these fibroblasts. The [35S]heparin proteoglycan internalized by the fibroblasts was shown to decrease in size over 72 hr indicating that the fibroblasts were capable of degrading the heparin within the ingested granules. Degradation of [35S]heparin proteoglycan within the fibroblast was accompanied by the appearance of free [35S]sulfate in the extracellular compartment. Similar findings were obtained using cultured human fibroblasts. These data demonstrate for the first time that both rat and human fibroblasts are not only capable of ingesting mast cell granules but also of degrading mast cell granule heparin proteoglycan. This ingestion and degradation of mast cell granules by fibroblasts may represent an important mechanism in the regulation of the biologic expression of heparin and other granule-associated mediators in immediate hypersensitivity reactions.     

Proteolytic processing of chromogranin A in cultured chromaffin cells

The prohormone chromogranin A is the major soluble component of secretory granules in chromaffin cells of adrenal medulla and in many other different endocrine cell types. The proteolytic processing of chromogranin A was studied in cultured bovine chromaffin cells using [35S]methionine to label proteins and a specific antibody to immunoprecipitate the native protein and its breakdown products. In resting cells, it was found that the degradation of chromogranin A is a slow process, since no degradation was observed after a 40 h incubation with radiolabelled methionine. Stimulation of cells with a single pulse or with successive pulses of nicotine did not significantly enhance the degree of proteolytic processing of chromogranin A. As it has recently been shown (Simon, J.P., Bader, M.F. and Aunis, D. Biochem. J. (1989) 260, 915-922) that protein kinase C may be involved in the regulation of chromogranin A synthesis, the possibility that prohormone processing may also be controlled by protein kinase C was examined using the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA). However, incubation of cells with TPA did not significantly modify chromogranin A processing, indicating that biosynthesis and proteolytic processing of chromogranin A are two distinctly regulated mechanisms. Glucocorticoids are known to exert regulatory control of chromaffin cell metabolism; however, incubation of cells with dexamethasone did not alter slow chromogranin A processing. Stimulation of labelled cells rapidly released newly synthesized chromogranin A into external medium. In addition, released chromogranin A was found to be actively processed into its 60 kDa and 43 kDa breakdown products. This extracellular proteolytic degradation mechanism may be of importance with regard to the function of chromogranin A as a prohormone.     

Evidence that leukosialin, CD43, is intensely sulfated in the murine T lymphoma line RDM-4.

Metabolic labeling of the murine T lymphoma cell line RDM-4 with [35S] sulfate results in intense incorporation into a cell-retained component of apparent Mr approximately 100,000. This macromolecule is identified as a glycoprotein by lectin chromatography. The sulfate is not incorporated as tyrosine sulfate. Release of the radiolabel by alkaline beta-elimination but not by endoglycosidase F is consistent with the sulfation of O- rather than N-linked oligosaccharides. The sulfated glycoprotein displays anomalous migration on SDS-PAGE in two respects: 1) the apparent Mr shifts from 115,000 to 87,000 on increasing the acrylamide concentration from 7 to 12%, and 2) on neuraminidase digestion migration is substantially reduced (apparent Mr 140,000). These properties indicate that the sulfated protein is both heavily glycosylated and extensively sialylated, and are characteristic of the lymphoid mucin, leukosialin (sialophorin, CD43). Specific labeling of the sialoglycoproteins of RDM-4 cells indicates that leukosialin, the most intensely labeled protein, comigrates with the sulfated protein on SDS-PAGE at varying acrylamide concentrations. Our data are therefore consistent with sulfation of at least some of the numerous O-linked oligosaccharides of this abundant glycoprotein in RDM-4 cells. No sulfation of CD43 in resting splenic T cells is observed.     

Biosynthesis of gastric mucus glycoprotein of the rat

We have studied the biosynthesis of rat gastric mucin in stomach segments using an antiserum against rat gastric mucin specific for peptide epitopes. Pulse-chase experiments were performed with [35S]methionine, [3H]galactose, and [35S]sulfate to label mucin precursors in different stages of biosynthesis, which were analyzed after immunoprecipitation. The earliest mucin precursor that could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a 300-kDa protein. The occurrence of N-linked "high-mannose" oligosaccharides on this protein was shown by susceptibility to degradation by endo-beta-N-acetylglucosaminidase H. This precursor could be labeled with [35S]methionine and not with [3H]galactose or [35S]sulfate. The 300-kDa precursor was converted into mature mucin after extensive glycosylation and sulfation. The mature mucin but not the 300-kDa precursor was in part secreted into the medium. Specific inhibition of sulfation with sodium chlorate had no effect on rate and amount of mucin secretion. In addition, we show that two core proteins are expressed in rats, slightly varying in Mr among individual animals.     

Proteoglycans synthesized by cultured mouse osteoblasts

Proteoglycan synthesis in nonmineralizing osteoblast cultures was investigated. Cultures were labeled with [35S]sulfate or [3H]serine, and proteoglycans were extracted from medium and cell layer with 4 M guanidine HCl. Labeled material was subjected to Sepharose CL-4B and DEAE-Sephacel chromatography and polyacrylamide gel electrophoresis. The size and composition of the glycosaminoglycan chains and the protein core size were determined. Two proteoglycan populations were isolated by Sepharose CL-4B chromatography: a minor excluded species with chondroitin sulfate chains of apparent Mr 25,000 and a smaller population (Kav = 0.43) accounting for 80% of the total labeled material. This small population resolved into two species by polyacrylamide gel electrophoresis. Both species contain dermatan sulfate chains of apparent Mr 40,000 and a core protein with Mr 45,000 on sodium dodecyl sulfate gels. With the exception of their glycosaminoglycan composition these species appear similar to those extracted from bone. In addition, high molecular weight hyaluronic acid and glycosaminoglycan peptides were found in cell extracts.     

Metastatic melanoma cell heparanase. Characterization of heparan sulfate degradation fragments produced by B16 melanoma endoglucuronidase

Heparan sulfate (HS), a prominent component of vascular endothelial basal lamina, is cleaved into large Mr fragments and solubilized from subendothelial basal lamina-like matrix by metastatic murine B16 melanoma cells. We have examined the degradation products of HS and other purified glycosaminoglycans produced by B16 cells. Glycosaminoglycans 3H-labeled at their reducing termini or metabolically labeled with [35S]sulfate were incubated with B16 cell extracts in the absence or presence of D-saccharic acid 1,4-lactone, a potent exo-beta-glucuronidase inhibitor, and glycosaminoglycan fragments were analyzed by high speed gel permeation chromatography. HS isolated from bovine lung, Engelbreth-Holm-Swarm sarcoma, and subendothelial matrix were degraded into fragments of characteristic Mr, in contrast to hyaluronic acid, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, keratan sulfate, and heparin which were essentially undegraded. Heparin, but not other glycosaminoglycans, inhibited HS degradation. The time dependence of HS degradation into particular Mr fragments indicated that HS was cleaved at specific intrachain sites. In order to determine specific HS cleavage points, HS prereduced with NaBH4 was incubated with a B16 cell extract and HS fragments were separated. The newly formed reducing termini of HS fragments were then reduced with NaB[3H]4, and the fragments hydrolyzed to monosaccharides by trifluoroacetic acid treatment and nitrous acid deamination. Since 3H-reduced terminal monosaccharides from HS fragments were overwhelmingly (greater than 90%) L-gulonic acid, the HS-degrading enzyme responsible is an endoglucuronidase (heparanase).     

Brefeldin A inhibits the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network.

The effects of brefeldin A (BFA) on membrane traffic between the trans-Golgi network (TGN) and the plasma membrane were investigated in intact PC12 cells and in a cell-free system derived from PC12 cells. In intact cells, BFA caused a virtually complete block of constitutive secretion, as indicated by the lack of release from, and accumulation in, the cells of a [35S]sulfate-labeled heparan sulfate proteoglycan (hsPG). Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that this block was due to the inhibition of formation of constitutive secretory vesicles (CSVs) from the TGN. BFA did not block the depolarization-induced release of [35S]sulfate-labeled chromogranin B (CgB) and secretogranin II (SgII) from secretory granules formed prior to the addition of the drug, showing that BFA does not block secretory granule fusion with the plasma membrane. The presence of BFA did, however, prevent the appearance of [35S]sulfate-labeled CgB and SgII in secretory granules, indicating that the drug inhibits the formation of secretory granules from the TGN. Evidence for a direct block of vesicle formation by BFA was obtained using a cell-free system derived from [35S]sulfate-labeled PC12 cells. In this system, low concentrations of BFA (5 micrograms/ml) inhibited the formation of the hsPG-containing CSVs and that of the SgII-containing secretory granules from the TGN to the same extent (50-60%) as, and in a non-additive manner with, the nonhydrolyzable GTP analogue GTP gamma S. Consistent with the inhibitory effects of BFA on vesicle formation from the TGN, BFA treatment of intact PC12 cells led to the hypersialylation of CgB, which presumably was due to the increased residence time of the protein in the TGN. In conclusion, our data are consistent with, and allow the generalization of, the concept that the BFA-induced block of anterograde membrane traffic results from the inhibition of vesicle formation from a donor compartment.     

Subcellular location of stimulus-affected endogenous phosphoproteins in the rat parotid gland

Rat parotid minces were labeled with [32P]Pi, stimulated with isoproterenol, homogenized in sucrose, and fractionated on continuous sucrose density gradients. We analyzed the resulting fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiograms were made from the gels. Comparison of fractions from control and isoproterenol-stimulated minces revealed seven phosphoproteins that were affected by isoproterenol. The subcellular location of these proteins was determined by comparing their distribution in the sucrose gradients with that of a number of enzymes that are characteristic of specific organelles. Isoproterenol decreased the phosphorylation of two cytoplasmic proteins (Mr 16,000 and 18,000) and increased the phosphorylation of a third (Mr 14,000). The phosphorylation of two endoplasmic reticulum proteins was increased by isoproterenol (Mr 20,500 and 22,500), as was an Mr 31,000 protein which was probably the S6 ribosomal protein. The phosphorylation of a secretory granule protein (Mr 24,000) was decreased by isoproterenol. We then developed a purification scheme for parotid secretory granules. By using this method, we demonstrated that the phosphorylation of the Mr 24,000 was also decreased by carbamylcholine. Granules purified by this method also contained a small number of other phosphoproteins whose phosphorylation was increased only by isoproterenol. Secretory granule-associated stimulus-affected phosphoproteins were found in the particulate fraction when the granules were hypotonically lysed, and were not extracted from the particulate fraction by washing with 0.6 M KCl.     

Calmodulin-binding proteins in granule and plasma membranes from bovine chromaffin cells

Calmodulin-binding proteins in chromaffin granule membrane and chromaffin cell plasma membranes have been investigated and compared. Chromaffin granules were purified by centrifugation over a 1.7 M sucrose layer. Plasma membranes were obtained in a highly purified form by differential and isopycnic centrifugation. Enzymatic determinations of 5'-nucleotidase, a generally accepted plasma membrane marker, showed a 40-50-fold enrichment as compared to the cell homogenate. Marker enzyme studies demonstrated only minimal contamination by other subcellular organelles. After solubilization with Triton X-100, calmodulin-binding proteins were isolated from chromaffin granule membranes and plasma membranes by affinity chromatography on a calmodulin/Sepharose 4B column. On two-dimensional polyacrylamide gelelectrophoresis a prominent protein (Mr = 65,000, pI ranging from 5.1 to 6) consisting of multiple spots, was present in the calmodulin-binding fraction from chromaffin granule membranes as well as from plasma membranes. Besides this 65 kDa protein both fractions had at least four groups of proteins in common. Also, proteins typical for either preparation were observed. In the calmodulin-binding protein preparations from chromaffin granule membranes a prominent spot with Mr = 80,000 and a pH ranging from 5.0 to 5.7 was present. This protein was enzymatically and immunologically identified as dopamine-beta-monooxygenase.     

Identification of the precursor protein to basement membrane heparan sulfate proteoglycans

The precursor protein of a basement membrane specific heparan sulfate proteoglycan has been identified as a 400,000 Mr polypeptide. Antibodies against large and small forms of this proteoglycan, isolated from a basement membrane (Engelbreth-Holm-Swarm, EHS) tumor, immunoprecipitated the same 400,000 protein from pulse-labeled EHS cells. The proteoglycan precursor protein was not recognized by antibodies against other basement membrane components or by antibodies to the cartilage proteoglycan. Furthermore, heparan sulfate proteoglycan purified from the EHS tumor blocked the immunoprecipitation of the precursor protein. Pulse-chase studies with [35S]methionine showed the precursor protein was converted to a proteoglycan. Pulse-chase studies with 35SO4 showed the large, low density proteoglycan appeared first and was degraded to a smaller, high density proteoglycan. We propose that the precursor protein is used after very little or no modification in the assembly of a large, low density heparan sulfate proteoglycan and that a portion of the population of these macromolecules are subsequently degraded to a smaller form.     

Different processing of chromogranin B into GAWK-immunoreactive fragments in the bovine adrenal medulla and pituitary gland

Chromogranin B 420-493 (GAWK)-like immunoreactivity (chromogranin B (420-493)-LI) was determined by radioimmunoassay using two different rabbit antisera, one raised against chromogranin B (420-436) (GAWK 1-17) (Ab420-436) and the other against chromogranin B 439-457 (GAWK 20-38) (Ab439-457), in bovine and human tissues. Chromogranin B (420-493)-LI was present in the bovine adrenal medulla chromaffin granules as well as in the anterior pituitary gland and was released from the cultured bovine chromaffin cells by stimulation with high K+ or nicotine. Chromogranin B (420-493)-LI present in the bovine tissues was detected using Ab420-436 but was not detected using Ab439-457. In the human tissues, chromogranin B (420-493)-LI was detected using Ab420-436 as well as Ab439-457. This suggests that the amino acid sequence of this region (chromogranin B 439-457) is different between human and bovine. On the gel permeation chromatography, chromogranin B (420-493)-LI was eluted at the void volume in the bovine adrenal medulla and at an apparent molecular weight of 4000 in the anterior pituitary gland. On the reverse-phase high-performance liquid chromatography, multiple peaks of chromogranin B (420-493)-LI was detected in the bovine adrenal medulla while one component of chromogranin B (420-493)-LI was found in the anterior pituitary gland. These results suggest that chromogranin B is processed into small fragments of chromogranin B (420-493)-LIs and that this processing is tissue-specific.     

The cell surface receptor for immunoglobulin E. Effect of tunicamycin on molecular properties of receptor from rat basophilic leukemia cells

Cell surface receptors for immunoglobulin E were isolated by repetitive affinity chromatography from rat basophilic leukemia cells biosynthetically labeled with L-[35S]methionine and D-[3H]mannose. Native immunoglobulin E receptor appeared as a very broad band in the 45,000 to 62,000 Mr region in sodium dodecyl sulfate polyacrylamide gels. However, from cells cultured in the presence of tunicamycin, a relatively narrow band with an apparent Mr of 38,000 was isolated. The 38,000 Mr band rebound to immunoglobulin E-Sepharose, was immunoprecipitated with antibodies to immunoglobulin E receptor, shared tryptic peptides with native receptor, and was labeled with L-[35S]methionine but not D-[3H]mannose, and thus appears to be immunoglobulin E receptor lacking N-linked oligosaccharides. It is demonstrated that N-linked oligosaccharides account for much of the apparent heterogeneity of native receptor in sodium dodecyl sulfate polyacrylamide gels and in two-dimensional gel electrophoresis. A receptor-associated protein with apparent Mr = 30,000, prominently labeled with L-[35S]methionine but not with D-[3H]mannose, did not have altered molecular properties when isolated from tunicamycin-cultured cells, and did not share tryptic peptides with receptor.     

Culture from mouse bone marrow of a subclass of mast cells possessing a distinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-acetylgalactosamine-4,6-disulfate

A differentiated population of cells with metachromatically staining granules and surface IgE receptors was obtained from mouse bone marrow cultured for 2 weeks in the presence of conditioned medium derived from concanavalin A-stimulated splenocytes. The cells were found to incorporate large amounts of [35S]sulfate into an intracellular 35S-labeled proteoglycan of Mr approximately 200,000 containing a maximum of seven glycosaminoglycan side chains (Mr = 25,000). After chondroitinase ABC treatment of density gradient-purified [3H] serine-labeled proteoglycan, the resulting core was Mr approximately 26,000 as assessed by gel filtration. Two-dimensional cellulose acetate electrophoresis of beta-eliminated 35S-labeled glycosaminoglycan revealed a single type of glycosaminoglycan that migrated at the position of oversulfated chondroitin sulfate E from squid cartilage. Chondroitinase ABC degradation of the 35S-labeled glycosaminoglycan yielded two cleavage products in approximately equal molar amounts which co-migrated in both descending paper chromatography and high voltage paper electrophoresis with a monosulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and a disulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-6-di-O-sulfo-D-galactose. The release of some free [35S]sulfate from the oversulfated disaccharide with either chondro-4-sulfatase or chondro-6-sulfatase and the complete desulfation by their combined action established that the oversulfated disaccharide contained N-acetylgalactosamine-4,6-disulfate. The 35S]labeled proteoglycan of these unique IgE receptor-bearing and histamine-containing cells, therefore, is composed of chondroitin sulfate E rather than heparin glycosaminoglycan, and thus is the first identification of such an intracellular localized proteoglycan in a mammalian cell.