Biochemical and functional characterization of proteoglycans isolated from basophils of patients with chronic myelogenous leukemia

Human basophils were obtained from three donors with myelogenous leukemia. Proteoglycans were labeled by using [35S]sulfate as precursor and were extracted in 1 M NaCl with protease inhibitors to preserve their native structure. [35S]proteoglycans filtered on Sepharose 4B with an average m.w. similar to that of a rat heparin proteoglycan that has an estimated m.w. of 750,000. The [35S]glycosaminoglycan side chains filtered with an average m.w. slightly smaller than a 60,000-m.w. glycosaminoglycan marker. The [35S]glycosaminoglycans were resistant to heparinase and susceptible to degradation by chondroitin AC lyase and chondroitin ABC lyase. The intact [35S]glycosaminoglycans chromatographed on DEAE Sepharose as a single peak eluting just before an internal heparin marker. These findings indicate that the [35S]glycosaminoglycans were made up only of chondroitin sulfates. No heparin was identified. The chondroitin sulfate disaccharides that resulted from the action of chondroitin ABC lyase on the basophil glycosaminoglycans consisted of 92% delta Di-4S, 6% delta Di-6S, and 2% disulfated disaccharides. The [35S]chondroitin sulfate proteoglycans were susceptible to cleavage with proteases and could be shown to be released intact from basophils during degranulation initiated by the calcium ionophore A23187. The basophil proteoglycans and glycosaminoglycans were capable of binding histamine in water, but not in phosphate-buffered saline, and had no anticoagulant activity.     

Dog mastocytoma proteoglycans: occurrence of heparin and oversulfated chondroitin sulfates, containing trisulfated disaccharides, in three cell lines

The cell-associated proteoglycans synthesized by three dog mastocytoma cell lines were isolated and their structural features compared. The lines were propagated as subcutaneous tumors in athymic mice for over 25 generations. In primary cell culture, all three lines incorporated [35S]sulfate into high molecular weight proteoglycans which were heterogeneous in size and glycosaminoglycan content. Two lines, BR and G, synthesized both a heparin proteoglycan (HPG) and a chondroitin sulfate proteoglycan (ChSPG) in different proportions. The third line, C2, synthesized predominantly a ChSPG with little or no detectable heparin. Gel filtration of the 35S-labeled HPG and ChSPG from the BR line on Sepharose CL-4B in dissociative conditions (4 M guanidine, Triton X-100) yielded a major polydisperse peak (Kav = 0.22) accounting for 70% of 35S activity. Under aggregating conditions (0.1 M sodium acetate) on Sepharose CL-4B, the BR proteoglycans eluted in the excluded volume. Proteoglycans from lines G and C2 also eluted in the void volume under nondissociative conditions, however the C2 line yielded additional fractions of smaller hydrodynamic size (Kav = 0.81) suggesting the presence of intracellular proteoglycan cleavage products or incompletely processed proteoglycans. As assessed by dissociative chromatography on Sepharose CL-4B, proteoglycans from the BR line were resistant to proteinase cleavage under conditions which degraded a rat chondrosarcoma proteoglycan. For all lines, glycosaminoglycans released by pronase/alkaline-borohydride had molecular weights ranging from 20,000 to 50,000 on gel filtration. For line BR, 75% of 35S-labeled glycosaminoglycans were degraded to oligosaccharides by nitrous acid, and the remaining 25% were degraded by chondroitinase ABC. Corresponding percentages for line G were 89% and 11%, and for line C2, 2% and 98%. Paper chromatography of the chondroitinase digestion products from lines BR and C2 showed products corresponding to unsaturated standards delta Di-diSB and delta Di-diSE, derived from the disaccharides IdoUA-2-SO4----GalNAc-4-SO4 and GlcUA----GalNAc-4,6-diSO4 respectively, in addition to smaller amounts of monosulfated disaccharides. Glycans from lines C2 and BR contained small quantities of a trisulfated disaccharide which was degraded to delta Di-diSB upon incubation with chondro-6-sulfatase. The results demonstrate the simultaneous presence of heparin and polysulfated chondroitin sulfate in dog mast cells of clonal origin.     

Intestinal mucosal mast cells from rats infected with Nippostrongylus brasiliensis contain protease-resistant chondroitin sulfate di-B proteoglycans

Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of [35S] sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the 35S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The cell-associated 35S-labeled proteoglycans were extracted from the MMC-enriched cell preparation by the addition of detergent and 4 M guanidine HCl and were partially purified by density gradient centrifugation. The isolated proteoglycans were of approximately 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. Analysis by high-performance liquid chromatography of chondroitinase ABC-treated 35S-labeled proteoglycans from these rat MMC revealed that the chondroitin sulfate chains consisted predominantly of disaccharides with the disulfated di-B structure (IdUA-2SO4----GalNAc-4SO4) and disaccharides with the monosulfated A structure (G1cUA----GalNAc-4SO4). The ratio of disaccharides of the di-B to A structure ranged from 0.4 to 1.6 in three experiments. Small amounts of chondroitin sulfate E disaccharides (GlcUA----GalNAc-4,6-diSO4) were also detected in the chondroitinase ABC digests of the purified rat MMC proteoglycans, but no nitrous acid-susceptible heparin/heparan sulfate glycosaminoglycans were detected. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain such a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched population of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leukemia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans as well as rat serosal mast cell heparin proteoglycans are all highly sulfated, protease-resistant proteoglycans.     

Co-sedimentation of chondroitin sulfate A glycosaminoglycans and proteoglycans with the cytolytic secretory granules of rat large granular lymphocyte (LGL) tumor cells, and identification of a mRNA in normal and transformed LGL that encodes proteoglycans

Rat large granular lymphocyte (LGL) tumor cell lines were analyzed for the presence of proteoglycans and glycosaminoglycans in their cytolytic secretory granules. When isolated rat LGL tumor cells were incubated in vitro for 1 to 3 hr with [35S]sulfate, and the 35S-labeled macromolecules were purified by density-gradient centrifugation, they filtered on Sepharose CL-4B columns predominantly as approximately 500,000 m.w. macromolecules. After 19 hr of incubation with [35S]sulfate, however, an 85,000 m.w. species predominated. Pulse-chase experiments revealed that the larger macromolecules were proteoglycans that with time were processed to glycosaminoglycan-sized macromolecules. As assessed by their susceptibility to chemical and enzymatic degradation and by high pressure liquid chromatography of the chondroitinase ABC-generated unsaturated disaccharides, the cell-associated rat LGL tumor cell proteoglycans bore almost exclusively chondroitin sulfate A glycosaminoglycans. Northern blot analysis using a gene-specific probe revealed that both normal peripheral blood and transformed rat LGL expressed the same approximately 1.3-kb mRNA that encodes the peptide core of the proteoglycans in the secretory granules of rat and mouse mast cells. In vivo radiolabeling of rat LGL tumor cells and isolation of their intact granules after nitrogen cavitation and density sedimentation established that glycosaminoglycans compartmentalized with cytolytic activity. Thus these negatively charged macromolecules may play a role in the regulation of the packaging and delivery of the cytolysins and basically charged serine proteases that have been identified in the cytolytic secretory granules of LGL.     

Identification of chondroitin sulfate E in human lung mast cells

Human lung mast cells (HLMC) enriched up to 99% purity by counter current elutriation and density gradient centrifugation were labeled with 35S-sulfate to determine cell-associated proteoglycans. The 35S-labeled proteoglycans were extracted by the addition of detergent and 4 M guanidine-HCl, and separated from unincorporated precursor by Sephadex G-50 chromatography. 35S-Proteoglycans chromatographed over Sepharose 4B with a Kav of 0.48. 35S-Glycosaminoglycans separated from the parent 35S-proteoglycans by beta-elimination and chromatographed over Sepharose 4B with a Kav of 0.63. Characterization of 35S-proteoglycans by chondroitin ABC lyase treatment revealed approximately 36% of the proteoglycan to be composed of chondroitin sulfates. Analysis by HPLC of component disaccharides liberated by chondroitin ABC lyase using an amino-cyano-substituted silica column indicated that the chondroitin sulfates consisted of the monosulfated A disaccharide (GlcUA----GaINAc4SO4) (75%) and the over-sulfated E disaccharide (GlcUA----GaINAc4,6-diSO4) (25%). Nitrous acid/heparinase-susceptible heparin proteoglycans accounted for approximately 62% of the total 35S-proteoglycans present in the HLMC. Proteoglycans remaining after exposure of the original proteoglycan extract to either heparinase or chondroitin ABC lyase were of similar size, suggesting that the majority of heparin and chondroitin sulfate glycosaminoglycans were on separate protein cores. Proteoglycans extracted from HLMC were protease insensitive. Hence, in addition to heparin proteoglycans, HLMC synthesize a hitherto unrecognized quantity of chondroitin sulfate E proteoglycans.     

Characterization of proteoglycans synthesized by rat thyroid cells in culture and their response to thyroid-stimulating hormone

A Fisher rat thyroid cell line was maintained in culture and the cells were labeled with [3H]glucosamine, [35S]sulfate, and [35S]cysteine to examine the synthesis of proteoglycans. 3H and 35S radioactivity from these precursors were incorporated into both chondroitin sulfate (CS) and heparan sulfate (HS) proteoglycans. CS proteoglycans were almost exclusively secreted into the medium while HS proteoglycans remained mainly associated with the cell layer. Single chain glycosaminoglycans released by papain digestion or alkaline borohydride treatment of either the CS or HS proteoglycans had average molecular weights of approximately 30,000 on Sepharose CL-6B chromatography. Both CS and HS proteoglycans were relatively small and contained only one or two glycosaminoglycans chains. 3H and 35S incorporation into both CS and HS proteoglycans were increased by thyroid-stimulating hormone (TSH) in a dose-dependent manner, which is in part explained by an adenylate cyclase-dependent mechanism as indicated by a similar effect in response to dibutyryl cAMP. TSH enhanced the incorporation of 35S into CS from [35S]cysteine about 1.5-fold and that from [35S]sulfate about 2-fold. This result demonstrated that the increased 35S incorporation from the [35S]sulfate precursor reflects an actual increase in sulfate incorporation and is not simply a result from an apparent increase in specific activity of the phosphoadenosine phosphosulfate donor. Analysis of disaccharides from chondroitinase digests revealed that the proportion of non-sulfated, 4-sulfated, and 6-sulfated disaccharides was not altered appreciably by TSH. These results, together with the disproportionate increase in 3H incorporation into CS from [3H]glucosamine, indicated that TSH increased the specific activity of the 3H label as well. Chase experiments revealed that CS proteoglycans were rapidly (t1/2 = 15 min) secreted into the medium and that the degradation of cell-associated proteoglycans was enhanced by TSH.     

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.     

Characterization of a human eosinophil proteoglycan, and augmentation of its biosynthesis and size by interleukin 3, interleukin 5, and granulocyte/macrophage colony stimulating factor

Human eosinophils were cultured for up to 7 days in enriched medium in the absence or presence of recombinant human interleukin (IL) 3, mouse IL 5, or recombinant human granulocyte/macrophage colony stimulating factor (GM-CSF) and then were radiolabeled with [35S]sulfate to characterize their cell-associated proteoglycans. Freshly isolated eosinophils that were not exposed to any of these cytokines synthesized Mr approximately 80,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 80,000 glycosaminoglycans. RNA blot analysis of total eosinophil RNA, probed with a cDNA that encodes a proteoglycan peptide core of the promyelocytic leukemia HL-60 cell, revealed that the mRNA which encodes the analogous molecule in eosinophils was approximately 1.3 kilobases, like that in HL-60 cells. When eosinophils were cultured for 1 day or longer in the presence of 10 pM IL 3, 1 pM IL 5, or 10 pM GM-CSF, the rates of [35S]sulfate incorporation were increased approximately 2-fold, and the cells synthesized Mr approximately 300,000 Pronase-resistant 35S-labeled proteoglycans which contained Mr approximately 30,000 35S-labeled glycosaminoglycans. Approximately 93% of the 35S-labeled glycosaminoglycans bound to the proteoglycans synthesized by noncytokine- and cytokine-treated eosinophils were susceptible to degradation by chondroitinase ABC. As assessed by high performance liquid chromatography, 6-16% of these chondroitinase ABC-generated 35S-labeled disaccharides were disulfated disaccharides derived from chondroitin sulfate E; the remainder were monosulfated disaccharides derived from chondroitin sulfate A. Utilizing GM-CSF as a model of the cytokines, it was demonstrated that the GM-CSF-treated cells synthesized larger glycosaminoglycans onto beta-D-xyloside than the noncytokine-treated cells. Thus, IL 3, IL 5, and GM-CSF induce human eosinophils to augment proteoglycan biosynthesis by increasing the size of the newly synthesized proteoglycans and their individual chondroitin sulfate chains.     

Biosynthesis of glycosaminoglycans by cultured mastocytoma cells

Biosynthesis of glycosaminoglycans by several lines of cultured neoplastic mouse mast cells was studied by incorporation of [³⁵S]sulphate (and in some cases [6-³H]glucosamine) into macromolecular materials found in both the cells and their growth media. Such intracellular and extracellular radioactively labelled materials (shown to be glycosaminoglycans by susceptibility to digestion with heparinase) were further characterized by ion-exchange chromatography and by digestion with testicular hyaluronidase and chondroitinase. All but one cell line produced chondroitin sulphate as the major sulphated glycosaminoglycan; the remainder of the glycosaminoglycan was heparin-like material. No [³H]hyaluronic acid was synthesized. Cells of a newly derived line, termed P815S, synthesized more glycosaminoglycan than the other lines. This glycosaminoglycan, found in both cells and growth medium, was almost entirely chondroitin 4-sulphate. No chondroitin 6-sulphate was found. The chondroitin 4-sulphate from the cells was shown by gel filtration to be smaller than the chondroitin 4-sulphate in the media of these cultures. This discovery of relatively high proportions of chondroitin 4-sulphate in these mastocytoma-derived cells is noteworthy, since mast cells have generally been considered to produce heparin as their major glycosaminoglycan.     

Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium

Bovine aortic endothelial cells were cultured in medium containing [3H]glucosamine and concentrations of [35S]sulfate ranging from 0.01 to 0.31 mM. While the amount of [3H]hexosamine incorporated into chondroitin sulfate and heparan sulfate was constant, decreasing concentrations of sulfate resulted in lower [35S]sulfate incorporation. Sulfate concentrations greater than 0.11 mM were required for maximal [35S]sulfate incorporation. Chondroitin sulfate was particularly affected so that the sulfate to hexosamine ratio in [3H]chondroitin [35S]sulfate dropped considerably more than the sulfate to hexosamine ratio in [3H] heparan [35S]sulfate. Sulfate concentration had no effect on the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. The ratios of sulfate to hexosamine in cell-associated glycosaminoglycans were essentially identical with the ratios in media glycosaminoglycans at all sulfate concentrations. DEAE-cellulose chromatography confirmed that sulfation of chondroitin sulfate was particularly sensitive to low sulfate concentrations. While cells incubated in medium containing 0.31 mM sulfate produced chondroitin sulfate which eluted later than heparan sulfate, cells incubated in medium containing less than 0.04 mM sulfate produced chondroitin sulfate which eluted before heparan sulfate and near hyaluronic acid, indicating that many chains were essentially unsulfated. At intermediate concentrations of sulfate, chondroitin sulfate was found in very broad elution patterns suggesting that most did not fit an "all or nothing" mechanism. Heparan sulfate produced at low concentrations of sulfate eluted with narrower elution patterns than chondroitin sulfate, and there was no indication of any "all or nothing" sulfation.     

Cell surface glycosaminoglycans of rat rhabdomyosarcoma lines with different metastatic potentials and of non-malignant rat myoblasts

Glycosaminoglycans of cultured nickel-induced rat rhabdomyosarcoma cell lines with different metastatic potentials and of non-malignant myoblasts, grown in the presence or in the absence of hydrocortisone, were studied comparatively. The newly formed [3H]glucosamine-labelled cell surface proteoglycans and glycosaminoglycans were separated by ion exchange chromatography and partially characterized. The overall incorporation of the label in the glycosaminoglycan fractions and the average molecular weight of the heparan and of the chondroitin sulfate proteoglycans was lower in the malignant cells than in the non-malignant L6 myoblasts. The strongly metastatic 9-4/0 parental line and the 6 subline were relatively richer in chondroitin sulfate and poorer in dermatan sulfate labels than the very weakly metastatic 8 subline and the L6 myoblasts. Hyaluronic acid and heparan sulfate labels were inversely related to the metastatic capacity of the cell lines studied. Hydrocortisone treatment induced an increase in the cell surface chondroitin and dermatan sulfate labels in the case of the strongly metastatic lines, and a decrease of the same parameters in the case of the weakly metastatic 8 line.     

Effects of heparan sulfate removal on attachment and reattachment of fibroblasts and endothelial cells

Human skin fibroblasts and calf aorta endothelial cells were grown as tissue culture monolayers in the presence of [35S]sulfate in order to label the glycosaminoglycan portions of proteoglycans for investigation of their role in cell attachment. The [35S]glycosaminoglycans were then selectively removed from the cell monolayers by the addition of various glycosaminoglycan-degrading enzymes. As previously described, in contrast to trypsin treatment none of these enzymes removed any cells from the culture plates. Incubation with a preparation from Flavobacterium heparinum left only small stubs of [35S]glycosaminoglycans on the cell monolayers, indicating that all the cell-surface proteoheparan [35S]sulfate and proteochondroitin [35S]sulfate was accessible to this enzyme preparation. The treatment did not change the amount or time of incubation with trypsin necessary for release of the cells from the monolayers. Thus, cell attachment was not weakened by removal of heparan sulfate or chondroitin sulfate. In contrast, neither fibroblasts nor endothelial cells in suspension would reattach in the presence of the F. heparinum preparation while reattachment occurred readily in the presence of chondroitin ABC lyase. This provides evidence that heparan sulfate, but not chondroitin sulfate, is involved in the process of cell attachment even though neither is necessary for maintaining attachment.     

Biosynthesis of glycosaminoglycans in the developing retina

The glycosaminoglycans of neural retinas from 5-, 7-, 10-, and 14-day chick embryos were labeled in culture with [³H]glucosamine and ³⁵SO₄, extracted, and isolated by gel filtration. The incorporation of label per retina into glycosaminoglycans increased with embryonic age, but that per cell and per unit weight of uronic acid decreased. Specific enzyme methods coupled with gel filtration and paper chromatography demonstrated that [³H]glucosamine incorporation into chondroitin sulfate increased between 5 and 14 days from 7 to 34% of the total incorporation into glycosaminoglycans. During this period, incorporation into chondroitin-4-sulfate increased relative to that into chondroitin-6-sulfate. Between 5 and 10 days, incorporation into heparan sulfate showed a relative decline from 89 to 61%. Incorporation into hyaluronic acid always represented less than 2% of the total. A twofold greater increase in galactosamine concentration than in glucosamine concentration in the glycosaminoglycan fraction between 7 and 14 days supports the conclusion that chondroitin sulfate was the most rapidly accumulating glycosaminoglycan. ECTEOLA-cellulose chromatography revealed a heterogeneity in the size and/or net charge of chondroitin sulfate and heparan sulfate. We conclude that incorporation of exogenous precursors into glycosaminoglycans in the chick retina decreases relative to cell number as differentiation progresses from a period of high mitotic activity to one of tissue specialization, and that it is accompanied by a net accumulation of glycosaminoglycan and a change in the pattern of its synthesis.     

Isolation and characterization of proteoglycans secreted by normal and malignant human mammary epithelial cells

The proteoglycans secreted by a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal human breast cell line (HBL-100). The physicochemical characteristics of these proteoglycans were established by hexosamine analysis, chemical and enzymatic degradations, and dissociative cesium chloride density gradient centrifugation, and by gel filtration before and after alkaline beta-elimination. Both cell lines secreted approximately 70% of the synthesized proteoglycans, which were composed of 20% heparan sulfate and 80% chondroitin sulfate proteoglycans. The MDA cell line secreted large hydrodynamic size (major) and small hydrodynamic size heparan sulfate proteoglycan. In contrast HBL cells secreted only one species having a hydrodynamic size intermediate to the above two. The chondroitin sulfate proteoglycans from MDA medium were slightly larger than the corresponding polymers from HBL medium. All proteoglycans except the small hydrodynamic size heparan sulfate proteoglycan from MDA medium were of high buoyant density. The proteoglycans of both cell lines contained significant proportions of disulfide-linked lower molecular weight components which were more pronounced in the proteoheparan sulfate polymers, particularly those from MDA medium, than in chondroitin sulfate proteoglycans. The glycosaminoglycans of heparan sulfate proteoglycans from MDA medium were more heterogeneous than those from HBL medium. The glycosaminoglycan chains of large hydrodynamic size heparan sulfate proteoglycans from MDA medium were larger in size than those from HBL medium while small hydrodynamic size heparan sulfate proteoglycans contained shorter glycosaminoglycan chains. In contrast to the glycosaminoglycans derived from chondroitin sulfate proteoglycans of both MDA and HBL medium were comparable in size. The heparan sulfate as well as chondroitin sulfate proteoglycans of both cell lines contained both neutral (di- and tetrasaccharides) and sialylated (tri- to hexasaccharides) O-linked oligosaccharides.     

Biosynthesis of glycosaminoglycans in the human colonic tumor cell line Caco-2: structural changes occurring with the morphological differentiation of the cells

The human colon cancer cell line Caco-2 cultured in vitro displayed morphological differentiation which was shown to be a growth-related event. We have investigated this phenomenon further in relation to the cell surface glycosaminoglycans produced by growing (5-day, i.e., prior to differentiation) and confluent (9-day, i.e., after morphological and functional differentiation) cultures. Neosynthesized [35S]glycosaminoglycans were purified on DEAE-cellulose; at confluency, they were bound more strongly to the column than the corresponding fractions from the growing cells. Analysis of Kav values of heparan sulfate and chondroitin sulfates from growing and confluent cells indicated an increase in chain length of both glycosaminoglycans in morphologically differentiated cells. Heparan sulfate was the main 35S-labeled glycosaminoglycan of the cell surface of both 5-day and 9-day cultures. Paper chromatography of the unsaturated disaccharides obtained by chondroitinase digestion showed that chondroitin sulfate chains were primarily 6-sulfated in the 2 studied extracts. Heparan sulfate chains were isolated as chondroitinase-resistant material and treated with nitrous acid. Analysis of N- and O-sulfate group-related radioactivity showed an increase in the amount of 35S-label in the form of N-sulfate groups and an increase in the O-35S-sulfation pattern in heparan sulfate from morphologically differentiated cells. Thus, the structural features of both chondroitin sulfates and heparan sulfate were significantly different when the growing cells became morphologically differentiated.     

Analysis of polysulfated chondroitin disaccharides by high-performance liquid chromatography

A high-performance liquid chromatography method for analyzing disaccharides derived from chondroitin sulfate glycosaminoglycans has been developed which employs a Whatman Partisil-10 PAC amino-cyano column and an acetonitrile/methanol/ammonium acetate solvent to resolve disulfated, monosulfated, and unsulfated disaccharides in a chromatographic run of less than 20 min. The single known trisulfated chrondroitin disaccharide can be eluted in an alternate solvent system containing the same mobile phase components in different proportions. Disaccharides were prepared for chromatography from glycosaminoglycans and proteoglycans of known compositions by digestion with chondroitinase ABC, with the exception of king crab cartilage glycosaminoglycan which was incubated sequentially with hyaluronidase and chondroitinase ABC. Disaccharides were extracted from the digestion mixtures in 80% ethanol, dried over nitrogen, resuspended in the HPLC solvent, and chromatographed at a flow rate of 1 ml/min. Unsaturated disaccharides in the column eluate were detected by continuous ultraviolet absorbance monitoring at 232 nm; alternatively, fractions were collected and assayed for uronic acid content or radioactivity. By utilizing the HPLC technique in conjunction with chondroitinase ABC and AC digestion and sulfatase hydrolysis, the epimeric structures of chondroitin sulfates E and H were confirmed. With this technique, rapid and reproducible analyses of chondroitin sulfate disaccharides generated from mouse mast cell proteoglycan and from glycosaminoglycans of squid cranial cartilage, shark skin, hagfish skin, and hagfish notocord were in close agreement with compositions obtained by other techniques.     

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-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)     

Biosynthesis of proteoglycans by rat embryo parietal yolk sacs in organ culture

The embryonic rat parietal yolk sac has been previously shown to synthesize a number of basement membrane glycoconjugates including type IV procollagen, laminin, and entactin. In this study, parietal yolk sacs were isolated from 14.5-day rat embryos and incubated in organ culture for 4-7 h with [35S]sulfate, [3H] glucosamine, and/or 3H-labeled amino acids, and the newly synthesized proteoglycans were characterized. The major [35S]sulfate-labeled macromolecule represented approximately 90% of the medium and 80% of the tissue radioactivity. It also represented nearly 80% of the total [3H]glucosamine-labeled glycosaminoglycans. After purification by sequential ion-exchange chromatography and isopycnic CsCI density gradient ultracentrifugation, size-exclusion high-performance liquid chromatography showed a single species with an estimated Mr of 8-9 X 10(5). The intact proteoglycan did not form aggregates in the presence of exogenous hyaluronic acid or cartilage aggregates. Alkaline borohydride treatment released glycosaminoglycan chains with Mr of 2.0 X 10(4) which were susceptible to chondroitinase AC II and chondroitinase ABC digestion. Analysis by high-performance liquid chromatography of the disaccharides generated by chondroitinase ABC digestion revealed that chondroitin 6-sulfate was the predominant isomer. The uronic acid content of the glycosaminoglycans was 92% glucuronic acid and 8% iduronic acid, and the hexosamine content was 96% galactosamine and 4% glucosamine. No significant amounts of N- or O-linked oligosaccharides were detected. Deglycosylation of the proteoglycan with chondroitinase ABC in the presence of protease inhibitors revealed a protein core with an estimated Mr of 1.25-1.35 X 10(5). These results indicated that the major proteoglycan synthesized by the 14.5-day rat embryo parietal yolk sac is a high-density chondroitin sulfate containing small amounts of copolymeric dermatan sulfate. Hyaluronic acid and minor amounts of heparan sulfate proteoglycan were also detected.     

Alteration of rat liver proteoglycans during regeneration.

Sepharose CL-6B column chromatography of crude extracts from the slices of regenerating rat livers after partial hepatectomy and sham-operated controls labeled with [35S]sulfuric acid revealed an enhancement of [35S]sulfate incorporation into proteoglycan fractions during regeneration. The 35S-labeled proteoglycans contained heparan sulfate (more than 80% of the total) and chondroitin/dermatan sulfate. The 35S-incorporation into both glycosaminoglycans increased to maxima 3-5 days after partial hepatectomy and decreased thereafter toward the respective control levels. When [35S]sulfuric acid was replaced by [3H]glucosamine, similar results were obtained. These results suggest that the maximal stimulation of proteoglycan synthesis in regenerating rat liver follows the maximal mitosis of hepatic cells 1-2 days after partial hepatectomy. The 35S-labeled proteoglycans from regenerating liver 3 days after partial hepatectomy and control were analyzed further. They were similar in chromatographic behavior on a gel filtration or an anion-exchange column and in glycosaminoglycan composition. Their glycosaminoglycans were indistinguishable in electrophoretic mobility. However, these proteoglycans were slightly but significantly different in their affinity to octyl-Sepharose and in the molecular-weight distribution of their glycosaminoglycans.     

Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of [3H]chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.