Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2032-2036, June 1975 Mucopolysaccharides Associated with Nuclei of Cultured Mammalian Cells (mouse melanoma/nuclear membrane/sulfated polysaccharides) V. P. BHAVANANDAN AND E. A. DAVIDSON Department of Biological Chemistry, The M. S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa. 17033 Communicated by Karl Meyer, March 3, 1975 ABSTRACT Mucopolysaccharides have been isolated, fractionated, and characterized from the nuclei of cultured B16 mouse melanoma cells grown in the presence of [3HJ- glucosamine and [35Slsulfate. Digestion of the nuclei with DNase followed by Pronase gave a mixture of complex car- bohydrates from which the mucopolysaccharides were iso- lated by precipitation with cetylpyridinium chloride. After fractionation by differential salt extraction and chro- matography on controlled pore glass bead columns, the components were identified by chemical and enzymatic methods. The major polysaccharide components were a family of high-molecular-weight chondroitin sulfates with different degrees of sulfation; a minor component has been characterized as heparan sulfate. The production of mucopolysaccharides by cultured cells and the effect of virus transformation on this has been demon- strated for several cell lines (1-5). A major portion of the mucopolysaccharide synthesized by the cells can be detected in the culture media and as intercellular material, not a surprising result, since mucopolysaccharides are considered to be extracellular components. In addition, mucopolysaccharide is detectable in cell pellets, although the nature of the associa- tion of this material with the cell has not been fully estab- lished. The presence of heparan sulfate as a surface component of several cell lines has been reported (3) but it is not clear whether the heparan sulfate-protein complex is an integral part of the plasma membrane of cells or is present as a cell coat (fuzz) immobilized by ionic interaction with cell mem- brane proteins. In this paper we present evidence for the presence of muco- polysaccharides associated with cell nuclei. The presence of anionic saccharides in the nucleus of mammalian cells may have considerable significance. While cell surface complex carbohydrates can be involved in determining antigenicity, intercellular recognition and adhesion, contact inhibition, and other surface-related phenomena, anionic saccharides of the nuclei are more likely to be involved in controlling nuclear and cytoplasmic events. Alternatively these components may have a common function in all membranes as structural elements maintaining organellar integrity or regulating membrane properties, such as transport of nutrients. MATERIALS AND METHODS B16 mouse melanoma cell line 3 and an amelanotic clone were propagated as described previously (5). Cells utilized for the isolation of nuclei were harvested in the late-log phase of growth at a density of about 15 X 106 cells per 16 oz (0.5 liter) bottle. In experiments requiring labeling of complex carbohydrates, the cells were cultured for 48 hr prior to harvest in sulfate-free medium containing per milliliter 10 MACi of [3H]glucosamine.HCl (New England Nuclear, 7.3 Ci/- mmol) and 50 MuCi of Na2aSO4 (New England Nuclear, 738 mCi/mmol). Cells were harvested by pouring off the media, washing the cell layer three times with serum-free medium, and treating with 0.01 M EDTA in calcium, magnesium-free phosphate- buffered saline (10 ml) at 370 for 5 min. Serum-free medium (10 ml) was added, and the cells were collected by centrifuga- tion and washed three times with the buffered saline by re- suspension and centrifugation. The viability of the cells was in the region of 85-95% as determined by trypan blue exclusion. Nuclei were prepared by two methods: one utilized 0.05% Triton X-100 in the buffer as described by Alwine et al. (6); the other method was essentially as described by Sakiyama and Burge (7) except that the nuclear pellet after the first gradient centrifugation was suspended in 4 ml of 0.2 M sucrose in buffer and layered on top of the gradient, and the centrifugation was repeated. The purification of nuclei was routinely monitored by phase contrast microscopy. Protein was determined according to Lowry et al. (8) with crystalline bovine serum albumin as the standard. DNA and RNA were determined by the modified Schmidt-Thannhauser method as described by Munro and Fleck (9) with calf thymus DNA and yeast RNA as standards, respectively. 5'-Nucleotidase was assayed by the method of Dewald and Touster (10) and acid phosphatase as described by Appelmans et al. (11). Succinic dehydrogenase was assayed by determining the reduction of cytochrome c (12) or of 2-p-iodophenyl-3-(p- nitrophenyl)-5-phenyl-2H-tetrazolium chloride (13). The isolation of the complex carbohydrate components was achieved by suspending the washed nuclei pellet in 0.01 M Tris * HCl buffer, pH 7.4, containing 0.01 M MgC12 and digest- ing with DNase. The digestion was carried out at 37° for a period of 24 hr with addition of 50 ,ug of DNase at time 0 and again after 12 hr. The digest was exhaustively dialyzed against distilled water and the nondialyzable portion was centrifuged (2000 X g, 10 min) and the radioactivity in an aliquot of the supernatant was measured. The pellet was resuspended in the supernatant (pH adjusted to 8.0) and digested with Pronase for 48 hr at 400 in the presence of toluene. The digest was centrifuged and the supernate was extensively dialyzed against 0.2 M NaCl followed by distilled water. Hyaluronic acid, chondroitin4-sulfate, and heparin (0.5 mg each) were added as carriers to the dialyzed digest and, after the solution was made 0.04 M in Na2SO4, 2% cetylpyridinium chloride (CPC) in 0.04 M Na2SO4 was added at 1 ml/10 ml, and the solution was mixed and allowed to stand at room tem- 2032 Abbreviations: CPC, cetylpyridinium chloride; CPG, controlled pore glass.
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Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2032-2036, June 1975
Mucopolysaccharides Associated with Nuclei of Cultured Mammalian Cells (mouse melanoma/nuclear membrane/sulfated polysaccharides)
V. P. BHAVANANDAN AND E. A. DAVIDSON
Department of Biological Chemistry, The M. S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa. 17033
Communicated by Karl Meyer, March 3, 1975
ABSTRACT Mucopolysaccharides have been isolated, fractionated, and characterized from the nuclei of cultured B16 mouse melanoma cells grown in the presence of [3HJ- glucosamine and [35Slsulfate. Digestion of the nuclei with DNase followed by Pronase gave a mixture of complex car- bohydrates from which the mucopolysaccharides were iso- lated by precipitation with cetylpyridinium chloride. After fractionation by differential salt extraction and chro- matography on controlled pore glass bead columns, the components were identified by chemical and enzymatic methods. The major polysaccharide components were a family of high-molecular-weight chondroitin sulfates with different degrees of sulfation; a minor component has been characterized as heparan sulfate.
The production of mucopolysaccharides by cultured cells and the effect of virus transformation on this has been demon- strated for several cell lines (1-5). A major portion of the mucopolysaccharide synthesized by the cells can be detected in the culture media and as intercellular material, not a surprising result, since mucopolysaccharides are considered to be extracellular components. In addition, mucopolysaccharide is detectable in cell pellets, although the nature of the associa- tion of this material with the cell has not been fully estab- lished. The presence of heparan sulfate as a surface component of several cell lines has been reported (3) but it is not clear whether the heparan sulfate-protein complex is an integral part of the plasma membrane of cells or is present as a cell coat (fuzz) immobilized by ionic interaction with cell mem- brane proteins.
In this paper we present evidence for the presence of muco- polysaccharides associated with cell nuclei. The presence of anionic saccharides in the nucleus of mammalian cells may have considerable significance. While cell surface complex carbohydrates can be involved in determining antigenicity, intercellular recognition and adhesion, contact inhibition, and other surface-related phenomena, anionic saccharides of the nuclei are more likely to be involved in controlling nuclear and cytoplasmic events. Alternatively these components may have a common function in all membranes as structural elements maintaining organellar integrity or regulating membrane properties, such as transport of nutrients.
MATERIALS AND METHODS
B16 mouse melanoma cell line 3 and an amelanotic clone were propagated as described previously (5). Cells utilized for the isolation of nuclei were harvested in the late-log phase of growth at a density of about 15 X 106 cells per 16 oz (0.5 liter) bottle. In experiments requiring labeling of complex
carbohydrates, the cells were cultured for 48 hr prior to harvest in sulfate-free medium containing per milliliter 10 MACi of [3H]glucosamine.HCl (New England Nuclear, 7.3 Ci/- mmol) and 50 MuCi of Na2aSO4 (New England Nuclear, 738 mCi/mmol).
Cells were harvested by pouring off the media, washing the cell layer three times with serum-free medium, and treating with 0.01 M EDTA in calcium, magnesium-free phosphate- buffered saline (10 ml) at 370 for 5 min. Serum-free medium (10 ml) was added, and the cells were collected by centrifuga- tion and washed three times with the buffered saline by re- suspension and centrifugation. The viability of the cells was in the region of 85-95% as determined by trypan blue exclusion.
Nuclei were prepared by two methods: one utilized 0.05% Triton X-100 in the buffer as described by Alwine et al. (6); the other method was essentially as described by Sakiyama and Burge (7) except that the nuclear pellet after the first gradient centrifugation was suspended in 4 ml of 0.2 M sucrose in buffer and layered on top of the gradient, and the centrifugation was repeated. The purification of nuclei was routinely monitored by phase contrast microscopy.
Protein was determined according to Lowry et al. (8) with crystalline bovine serum albumin as the standard. DNA and RNA were determined by the modified Schmidt-Thannhauser method as described by Munro and Fleck (9) with calf thymus DNA and yeast RNA as standards, respectively.
5'-Nucleotidase was assayed by the method of Dewald and Touster (10) and acid phosphatase as described by Appelmans et al. (11). Succinic dehydrogenase was assayed by determining the reduction of cytochrome c (12) or of 2-p-iodophenyl-3-(p- nitrophenyl)-5-phenyl-2H-tetrazolium chloride (13). The isolation of the complex carbohydrate components
was achieved by suspending the washed nuclei pellet in 0.01 M Tris * HCl buffer, pH 7.4, containing 0.01 M MgC12 and digest- ing with DNase. The digestion was carried out at 37° for a period of 24 hr with addition of 50 ,ug of DNase at time 0 and again after 12 hr. The digest was exhaustively dialyzed against distilled water and the nondialyzable portion was centrifuged (2000 X g, 10 min) and the radioactivity in an aliquot of the supernatant was measured. The pellet was resuspended in the supernatant (pH adjusted to 8.0) and digested with Pronase for 48 hr at 400 in the presence of toluene. The digest was centrifuged and the supernate was extensively dialyzed against 0.2 M NaCl followed by distilled water.
Hyaluronic acid, chondroitin4-sulfate, and heparin (0.5 mg each) were added as carriers to the dialyzed digest and, after the solution was made 0.04 M in Na2SO4, 2% cetylpyridinium chloride (CPC) in 0.04 M Na2SO4 was added at 1 ml/10 ml, and the solution was mixed and allowed to stand at room tem-
2032
Nuclear Mucopolysaccharides 2033
perature overnight. The precipitate was collected by centri- fugation (10,000 X g, 10 min) and washed three times with 0.2% CPC in 0.04 M Na2SO4 by resuspension and centrifuga- tion. The CPC precipitate was then fractionally extracted with 0.2, 0.4, 0.8, 1.2, 2.0 M NaCl utilizing 2 ml once and 1 ml four times for each extraction. The CPC in the extracts was removed by dialysis at 40-45° against 0.1 M NaCi fol- lowed by distilled water at 40 and the solutions were lyophi- lized.
Cellulose acetate electrophoresis was carried out as de- scribed earlier (5) but using pyridineacetic acid buffer, pH 3.5, at 10 mA for 20 min or 0.2 M calcium acetate, pH 7.0, at 5 mA for 3 hr. Paper electrophoresis was done on Whatman 3MM sheets in 0.5 N pyridineacetic acid buffer, pH 5.0, at 10 V/cm for 2-3 hr. Hexosamine in isotopically labeled com-
ponents was determined as described earlier (5). Sources of enzymes, isotopes, standard mucopolysac-
charides, and chemicals were as previously described (5). DNase was the electrophoretically purified grade from Worth- ington and was free of hyaluronidase and chondroitinase activities. Testicular hyaluronidase digestion was performed in 0.1 M sodium acetate buffer, pH 5.0, and 8 mM EDTA in
the presence of toluene and chloroform at 370 for 24 hr. Bacterial hyaluronidase digestion was performed in 0.1 M sodium acetate buffer, pH 5.0, at 370 for 24 hr. Chondroitinase and chondrosulfatase digestions were according to Suzuki and coworkers (14). Nitrous acid degradation was done by treating the sample in 160 Mu of water with 20,l of 3 M NaNO2 and 20 ul of glacial acetic acid at room temperature for 80 min. Excess nitrite was destroyed by addition of 50,l of 3 M glycine and after 60 min at room temperature, the product was
lyophilized. Liquid scintillation counting was performed on an Inter-
technique model SL36 spectrometer. Usually 1 ml aqueous
samples were mixed with 10 ml of the counting liquid con-
taining xylene and Triton X-114 (15).
RESULTS
The recoveries of protein and radioactivity in the nuclei were
5-10% of the total cellular protein and between 4 and 9% of the cell-associated 3IH and 35S activity. The DNA and RNA contents of the nuclei were 398 and 59 ,ug/mg of protein, respectively. The nuclei had less than 1% of the 5'-nucleotid- ase, a plasma membrane marker enzyme, detected in the original cell homogenate. There was no detectable activity of acid phosphatase (lysosomal enzyme marker) and succinic dehydrogenase (mitochondrial enzyme marker) in the nuclear preparations, with about 0.5 mg of protein per assay. Although high concentrations of detergent are known to solubilize the outer nuclear membrane (16), the concentration employed by us did not appear to affect the morphology of this structure. Nuclei prepared by the alternative procedure did not appear
as clean as those prepared with 0.05% Triton but the incor-
poration patterns of radioactivity into mucopolysaccharide and glycoprotein were similar for preparations obtained by the two methods.
After treatment of the nuclei with DNase and centrifuga- tion, about 90-95% of the radioactivity (3H and 35S) sedi- mented with the pellet. Pronase digestion of the pellet solubilized the labeled components; between 75 and 80% of the 3H and 35 activities in the nuclei were recovered in the
soluble fraction after the treatment. Thus the sulfated and
lo 11'u8. 25 3545 55 6500 0~~~~~~
2 IO0 In
FRACTION NUMBER
FIG. 1. Fractionation of Pronase-digested nuclear membrane pellet on a 1.5 X 115 cm controlled pore glass (CPG) 10-240 glass bead column with 0.5M CaCl2 as the eluting solvent. Fractions (4 ml) were collected and 50 ul aliquots were analyzed for radioac- tivity. Fractions 30-39 (Na), 40-47 (Nb), and 48-62 (Nc) were
combined, dialyzed against water, and lyophilized.
nonsulfated complex saccharides of the nuclei may be mainly associated with the nuclear membrane, which has been re-
ported to contain glycoproteins (17-19). The possibility that a minor portion of the complex carbohydrate of the nuclei is associated with lipids (glycolipids) cannot be excluded by these studies. Chromatography of the Pronase digest on a preparative
glass bead column gave the results illustrated in Fig. 1. The majority of the sulfate radioactivity and a small percentage of the tritium label appeared in the fast-moving higher molecular weight peak (Na) and represents components derived primarily from proteoglycans. The low-molecular- weight fraction (Nc) contains the bulk of the tritium label and is predominantly glycopeptide in nature. Analytical data of these two fractions are given in Table 1. The distribution of 3H and 35S radioactivity in the different
TABLE 1. Analytical data of various 3H- and 35S-labeled macromolecular fractions from nuclei*
Chondroi-
36S tinase ACt Fraction Mol. wt. 3H GlcN GaIN 3H M6S
Na 35,000+ - 46.0 54.0 64.3 89.5 Nc -15,000 - 88.3 11.7 ND ND 4a 35,000+ 4.3 4.9 95.1 81.8 92.0 4c 10,000 1.4 60.5 39.5 ND ND 8a 35,000+ 9.3 0 100 100 100 8b 1-45,000 6.9 48.6 51.4 56.0 60.0 12a 35,000+ 24.7 ND ND 85.4 86.2 12b '-'15,000 8.0 18.8 81.2 ND ND
ND is not determined. * Summary of analytical data for the different fractions de-
scribed in the text. The molecular weight values are based on the behavior of the labeled components relative to standard muco- polysaccharides during chromatography on CPG columns. The amino sugar values are as percentages of tritium label found for glucosamine and galactosamine.
t Percentage radioactivity eluting in the low-molecular-weight region on a CPG column after incubation with chondroitinase AC.
Proc. Nat. Acad. Sci. USA 72 (1975)
2034 Biochemistry: Bhavanandan and Davidson
60C
50C
40C
30C
30 40 60 FRACTION NUMBER
60
FIG. 2. (a) Fractionation of the 0.8 M NaCi extract of CPC- precipitable material from Pronase-digested nuclear membrane on a 0.9 X 95 cm column of CPG 10-240 glass beads. Fractions (1 ml) were collected and 50 jl was analyzed for radioactivity. The peak elution position of marker saccharides (HA, vitreous humor hyaluronic acid; CSA, porcine rib cartilage chondroitin-4-sulfate; and GlcUA, glucuronic acid) are indicated by the arrows. Frac- tions 29-35 and 36-55 were separately combined as peaks 8a and 8b, respectively. Figures (b) and (c) are elution patterns of peaks 8a and 8b after rechromatography on the same column; 700 IAl of 1 ml fractions were analyzed.
CPC fractions is given in Table 2. Over 90% of mS and about 5% of 3H activity are in salt extracts of molarity greater than 0.4 M and constitute the mucopolysaccharide content of nuclei. The CPC supernatant and the washings of the precipitate contain about 90% of the 3H activity and represent glyco- peptide components. A further 3% of the 3H activity present in the 0.2 M NaCl eluate is a sialoglycopeptide comparable to that isolated from the culture media of the cells (5). Char- acterization of these glycopeptides will be described in a subsequent report. Chromatography of the 0.8 N NaCl fraction which con-
TABLE 2. CPC precipitation and salt elution of 3H and 35S radioadtivity in Pronase digest of nuclei*
CPC- Molarity of NaCl for eluate solublet 0.2 0.4 0.8 1.2 2.0
3H 1548.3 58.3 28.8 28.3 5.9 2.5 (91.6) (3.4) (1.7) (1.7) (0.3) (0.15)
35S 5.3 3.1 16.9 96.6 21.0 1.0 (3.5) (2.1) (11.3) (64.4) (14.0) (0.67)
* Values are expressed in dpm X 10-3; the numbers within parentheses are the percentages. Results of a duplicate experi- ment were identical. Recovery of radioactivity was essentially quantitative.
t Material present in the CPC supernate combined with washes of the original CPC precipitate.
tained about 2/3 of. the sulfated polysaccharide associated with the nuclei is illustrated in Fig. 2a. Rechromatography of Fractions 8a and 8b gave the patterns shown in Fig. 2b and c. The hexosamine contents of these fractions are given in Table 1.
Fraction 8a. The chromatographic elution profiles of 8a after treatment with testicular and bacterial hyaluronidases and with chondroitinase AC are shown in Fig. 3. Control digestion of 8a with buffer alone and rechromatography gave a profile identical to that in Fig. 2b. The presence of galactosamine as the only hexosamine in this fraction and its complete suscep- tibility to testicular hyaluronidase and chondroitinase AC suggests that it consists of chondroitin-4 and/or 6-sulfate. This fraction was also susceptible to bacterial hyaluronidase but the fragments obtained were larger than tetrasaccharides, implying that the degree of sulfation is greater than 50%, since a polysaccharide with significantly less sulfation would give rise to di- and tetrasaccharides. The electrophoretic mobility of this component, in the two buffer systems tested, was consistent with that of a chondroitin sulfate having 0.5- 1.0 sulfate residues per disaccharide unit. Chondroitinase AC and chondroitinase AC plus chondro4-sulfatase digestion of this fraction followed by paper chromatography indicated that the sulfate ester group is predominantly in the 4-position of the galactosamine moiety.
After mild acid hydrolysis (0.04 N HC1, 1000, 90 min) of the 8a fraction, about 45% of the 3IS radioactivity migrated with inorganic sulfate on paper electrophoresis as well as on a Bio-Gel P2 column. It was also evident from these experi- ments that the polymer underwent considerable degradation. Both cartilage chondroitin-4-sulfate and hyaluronic acid treated in the same manner are degraded to fragments ranging from tetra-(major) to octa-saccharides, an observation not previously recorded. Previous findings from this laboratory (20) have established that about 35% of BSO42- was released from chondroitin-4-sulfate on hydrolysis with dilute acid under conditions comparable to those used in the present study. The possible presence of N-sulfate residues in this fraction
was considered even though the results of the chondroitinase AC and chondro-4-sulfatase digestions were not consistent with this. Treatment of the 8a fraction with nitrous acid, a reagent that causes deaminative cleavage of mucopolysac- charides containing sulfated amino groups (21), followed by chromatography on a CPG 10-240 column gave a profile identical to that in Fig. 2b, indicating that there were no such residues in this fraction. Gel filtration of Fraction 8a on the CPG 10-240 column indicated that this component has a molecular weight of 35,000 or greater, since it elutes in the void volume coincident with vitreous humor hyaluronic acid of molecular weight 35,000, whereas cartilage chondroitin-4- sulfate of molecular weight 15,000 elutes differently (Fig. 2). A high-molecular-weight chondroitin fraction isolated from the culture media of the same cells (B16 mouse melanoma) has been estimated to have a molecular weight of 97,000 i 8,800 by ultracentrifugation. (Bhavanandan and Davidson, unpublished results.) In order to determine whether the high molecular weight of Fraction 8a was due to incomplete Pronase digestion resulting in proteoglycan fragments rather than polysaccharide chains, we digested Fraction 8a with Pronase (two additions of 250 ug each at time 0 and 24 hr) for a total period of 72 hr and rechromatographed the product
Proc. Nat. Acad. Sci. USA 72 (1975)
D,
D.
)i
I
)i
Nuclear Mucopolysaccharides 2035
on a CPG 10-240 column. The elution profile was identical to that of the untreated sample (Fig. 2b), indicating that the high-molecular-weight nature of this fraction is probably not due to incomplete Pronase digestion. Soluble pig cartilage proteoglycan is completely degraded to polysaccharide chains of molecular weight about 15,000 on digestion with Pronase (22).
Fraction 8b. The labeled hexosamine content of this fraction was 51% galactosamine and 49% glucosamine (Table 1). Chromatography on CPG 10-240 gave the heterogenous elution profile shown in Fig. 2c; high-molecular-weight mate- rial is absent and the major portion of the fraction eluted in the 15,000-10,000 range. On treatment with chondroitinase AC and rechromatography on the same column, about 60% of the 3IS activity eluted in the low-molecular-weight region. Mild acid hydrolysis followed by paper electrophoresis of this fraction also gave results similar to those obtained with Fraction 8a; about 52% of 'IS radioactivity migrated with inorganic sulfate. Nitrous acid treatment of this fraction followed by chromatography on the CPG 10-240 column showed that 57.5% of -IS activity eluted in the low-molecular- weight region. These results suggest that this fraction is a mixture of heparan sulfate and chondroitin (4 or 6) sulfate of molecular weight similar to that of the cartilage polysaccha- ride. Chromatography of the 0.4 M and 1.2 M NaCl eluates on
the CPG 10-240 column gave the profiles shown in Fig. 4. Hexosamine and chondroitinase susceptibility data on Frac- tions 4a, 4c, 12a, and 12b are summarized in Table 1. Fractions 4a and 12a are high-molecular-weight chondroitin sulfate components, apparently the undersulfated and more fully sulfated homologs of Fraction 8a (see 35S/3H in Table 1). Fraction 4c contains lower molecular weight material com- posed of both amino sugars and sialic acid and is presumably sialoglycopeptide similar to that present in the 0.2 M NaCl eluate. There was insufficient material for complete charac- terization.
DISCUSSION
This study provides direct biochemical evidence for the association of anionic polysaccharides with mammalian cell nuclei. In the system investigated, a family of high-molecular- weight chondroitin-4-sulfates were the major nuclear muco- polysaccharides. The other mucopolysaccharides identified were heparan sulfate and a chondroitin sulfate of lower molecular weight. The presence of glycoproteins in cell nuclei has been pre-
viously reported by several workers (17-19) and is confirmed by our results. Based entirely on the tritium incorporation data (Table 2), it would appear that mucopolysaccharides are a minor component compared to glycoproteins; however, definitive information on the carbohydrate composition of nuclei must await mass analytical data. It is also essential to establish whether the association of mucopolysaccharides with the nuclei is a feature common to all mammalian cells. The physical properties and chemical architecture of the
anionic polysaccharides with heterogeneity of size and dis- tribution of functional groups (0-SO4; N-SO4; COOH) make them excellent candidates for a range of regulatory functions, including control of cationic environment at the nuclear membrane, transport selectivity into the nuclei, regulation of nuclear enzyme activity, and control of template activity in
2000
1500
150C
**,r ,
.f ,.
a
b
c
FRACTION NUMBER
FIG. 3. Chromatography of Fraction 8a on a 0.9 X 95 cm glass bead column after digestion with testicular hyaluronidase (upper), chondroitinase AC (middle), or bacterial hyaluronidase (lower). Fractions (1 ml) were collected and 700 ,uA analyzed for radioac- tivity. Abbreviations are as in Fig. 2.
chromatin. Several recent reports have suggested involvement of anionic polysaccharides in nuclear functions. Anionic…
Mucopolysaccharides Associated with Nuclei of Cultured Mammalian Cells (mouse melanoma/nuclear membrane/sulfated polysaccharides)
V. P. BHAVANANDAN AND E. A. DAVIDSON
Department of Biological Chemistry, The M. S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pa. 17033
Communicated by Karl Meyer, March 3, 1975
ABSTRACT Mucopolysaccharides have been isolated, fractionated, and characterized from the nuclei of cultured B16 mouse melanoma cells grown in the presence of [3HJ- glucosamine and [35Slsulfate. Digestion of the nuclei with DNase followed by Pronase gave a mixture of complex car- bohydrates from which the mucopolysaccharides were iso- lated by precipitation with cetylpyridinium chloride. After fractionation by differential salt extraction and chro- matography on controlled pore glass bead columns, the components were identified by chemical and enzymatic methods. The major polysaccharide components were a family of high-molecular-weight chondroitin sulfates with different degrees of sulfation; a minor component has been characterized as heparan sulfate.
The production of mucopolysaccharides by cultured cells and the effect of virus transformation on this has been demon- strated for several cell lines (1-5). A major portion of the mucopolysaccharide synthesized by the cells can be detected in the culture media and as intercellular material, not a surprising result, since mucopolysaccharides are considered to be extracellular components. In addition, mucopolysaccharide is detectable in cell pellets, although the nature of the associa- tion of this material with the cell has not been fully estab- lished. The presence of heparan sulfate as a surface component of several cell lines has been reported (3) but it is not clear whether the heparan sulfate-protein complex is an integral part of the plasma membrane of cells or is present as a cell coat (fuzz) immobilized by ionic interaction with cell mem- brane proteins.
In this paper we present evidence for the presence of muco- polysaccharides associated with cell nuclei. The presence of anionic saccharides in the nucleus of mammalian cells may have considerable significance. While cell surface complex carbohydrates can be involved in determining antigenicity, intercellular recognition and adhesion, contact inhibition, and other surface-related phenomena, anionic saccharides of the nuclei are more likely to be involved in controlling nuclear and cytoplasmic events. Alternatively these components may have a common function in all membranes as structural elements maintaining organellar integrity or regulating membrane properties, such as transport of nutrients.
MATERIALS AND METHODS
B16 mouse melanoma cell line 3 and an amelanotic clone were propagated as described previously (5). Cells utilized for the isolation of nuclei were harvested in the late-log phase of growth at a density of about 15 X 106 cells per 16 oz (0.5 liter) bottle. In experiments requiring labeling of complex
carbohydrates, the cells were cultured for 48 hr prior to harvest in sulfate-free medium containing per milliliter 10 MACi of [3H]glucosamine.HCl (New England Nuclear, 7.3 Ci/- mmol) and 50 MuCi of Na2aSO4 (New England Nuclear, 738 mCi/mmol).
Cells were harvested by pouring off the media, washing the cell layer three times with serum-free medium, and treating with 0.01 M EDTA in calcium, magnesium-free phosphate- buffered saline (10 ml) at 370 for 5 min. Serum-free medium (10 ml) was added, and the cells were collected by centrifuga- tion and washed three times with the buffered saline by re- suspension and centrifugation. The viability of the cells was in the region of 85-95% as determined by trypan blue exclusion.
Nuclei were prepared by two methods: one utilized 0.05% Triton X-100 in the buffer as described by Alwine et al. (6); the other method was essentially as described by Sakiyama and Burge (7) except that the nuclear pellet after the first gradient centrifugation was suspended in 4 ml of 0.2 M sucrose in buffer and layered on top of the gradient, and the centrifugation was repeated. The purification of nuclei was routinely monitored by phase contrast microscopy.
Protein was determined according to Lowry et al. (8) with crystalline bovine serum albumin as the standard. DNA and RNA were determined by the modified Schmidt-Thannhauser method as described by Munro and Fleck (9) with calf thymus DNA and yeast RNA as standards, respectively.
5'-Nucleotidase was assayed by the method of Dewald and Touster (10) and acid phosphatase as described by Appelmans et al. (11). Succinic dehydrogenase was assayed by determining the reduction of cytochrome c (12) or of 2-p-iodophenyl-3-(p- nitrophenyl)-5-phenyl-2H-tetrazolium chloride (13). The isolation of the complex carbohydrate components
was achieved by suspending the washed nuclei pellet in 0.01 M Tris * HCl buffer, pH 7.4, containing 0.01 M MgC12 and digest- ing with DNase. The digestion was carried out at 37° for a period of 24 hr with addition of 50 ,ug of DNase at time 0 and again after 12 hr. The digest was exhaustively dialyzed against distilled water and the nondialyzable portion was centrifuged (2000 X g, 10 min) and the radioactivity in an aliquot of the supernatant was measured. The pellet was resuspended in the supernatant (pH adjusted to 8.0) and digested with Pronase for 48 hr at 400 in the presence of toluene. The digest was centrifuged and the supernate was extensively dialyzed against 0.2 M NaCl followed by distilled water.
Hyaluronic acid, chondroitin4-sulfate, and heparin (0.5 mg each) were added as carriers to the dialyzed digest and, after the solution was made 0.04 M in Na2SO4, 2% cetylpyridinium chloride (CPC) in 0.04 M Na2SO4 was added at 1 ml/10 ml, and the solution was mixed and allowed to stand at room tem-
2032
Nuclear Mucopolysaccharides 2033
perature overnight. The precipitate was collected by centri- fugation (10,000 X g, 10 min) and washed three times with 0.2% CPC in 0.04 M Na2SO4 by resuspension and centrifuga- tion. The CPC precipitate was then fractionally extracted with 0.2, 0.4, 0.8, 1.2, 2.0 M NaCl utilizing 2 ml once and 1 ml four times for each extraction. The CPC in the extracts was removed by dialysis at 40-45° against 0.1 M NaCi fol- lowed by distilled water at 40 and the solutions were lyophi- lized.
Cellulose acetate electrophoresis was carried out as de- scribed earlier (5) but using pyridineacetic acid buffer, pH 3.5, at 10 mA for 20 min or 0.2 M calcium acetate, pH 7.0, at 5 mA for 3 hr. Paper electrophoresis was done on Whatman 3MM sheets in 0.5 N pyridineacetic acid buffer, pH 5.0, at 10 V/cm for 2-3 hr. Hexosamine in isotopically labeled com-
ponents was determined as described earlier (5). Sources of enzymes, isotopes, standard mucopolysac-
charides, and chemicals were as previously described (5). DNase was the electrophoretically purified grade from Worth- ington and was free of hyaluronidase and chondroitinase activities. Testicular hyaluronidase digestion was performed in 0.1 M sodium acetate buffer, pH 5.0, and 8 mM EDTA in
the presence of toluene and chloroform at 370 for 24 hr. Bacterial hyaluronidase digestion was performed in 0.1 M sodium acetate buffer, pH 5.0, at 370 for 24 hr. Chondroitinase and chondrosulfatase digestions were according to Suzuki and coworkers (14). Nitrous acid degradation was done by treating the sample in 160 Mu of water with 20,l of 3 M NaNO2 and 20 ul of glacial acetic acid at room temperature for 80 min. Excess nitrite was destroyed by addition of 50,l of 3 M glycine and after 60 min at room temperature, the product was
lyophilized. Liquid scintillation counting was performed on an Inter-
technique model SL36 spectrometer. Usually 1 ml aqueous
samples were mixed with 10 ml of the counting liquid con-
taining xylene and Triton X-114 (15).
RESULTS
The recoveries of protein and radioactivity in the nuclei were
5-10% of the total cellular protein and between 4 and 9% of the cell-associated 3IH and 35S activity. The DNA and RNA contents of the nuclei were 398 and 59 ,ug/mg of protein, respectively. The nuclei had less than 1% of the 5'-nucleotid- ase, a plasma membrane marker enzyme, detected in the original cell homogenate. There was no detectable activity of acid phosphatase (lysosomal enzyme marker) and succinic dehydrogenase (mitochondrial enzyme marker) in the nuclear preparations, with about 0.5 mg of protein per assay. Although high concentrations of detergent are known to solubilize the outer nuclear membrane (16), the concentration employed by us did not appear to affect the morphology of this structure. Nuclei prepared by the alternative procedure did not appear
as clean as those prepared with 0.05% Triton but the incor-
poration patterns of radioactivity into mucopolysaccharide and glycoprotein were similar for preparations obtained by the two methods.
After treatment of the nuclei with DNase and centrifuga- tion, about 90-95% of the radioactivity (3H and 35S) sedi- mented with the pellet. Pronase digestion of the pellet solubilized the labeled components; between 75 and 80% of the 3H and 35 activities in the nuclei were recovered in the
soluble fraction after the treatment. Thus the sulfated and
lo 11'u8. 25 3545 55 6500 0~~~~~~
2 IO0 In
FRACTION NUMBER
FIG. 1. Fractionation of Pronase-digested nuclear membrane pellet on a 1.5 X 115 cm controlled pore glass (CPG) 10-240 glass bead column with 0.5M CaCl2 as the eluting solvent. Fractions (4 ml) were collected and 50 ul aliquots were analyzed for radioac- tivity. Fractions 30-39 (Na), 40-47 (Nb), and 48-62 (Nc) were
combined, dialyzed against water, and lyophilized.
nonsulfated complex saccharides of the nuclei may be mainly associated with the nuclear membrane, which has been re-
ported to contain glycoproteins (17-19). The possibility that a minor portion of the complex carbohydrate of the nuclei is associated with lipids (glycolipids) cannot be excluded by these studies. Chromatography of the Pronase digest on a preparative
glass bead column gave the results illustrated in Fig. 1. The majority of the sulfate radioactivity and a small percentage of the tritium label appeared in the fast-moving higher molecular weight peak (Na) and represents components derived primarily from proteoglycans. The low-molecular- weight fraction (Nc) contains the bulk of the tritium label and is predominantly glycopeptide in nature. Analytical data of these two fractions are given in Table 1. The distribution of 3H and 35S radioactivity in the different
TABLE 1. Analytical data of various 3H- and 35S-labeled macromolecular fractions from nuclei*
Chondroi-
36S tinase ACt Fraction Mol. wt. 3H GlcN GaIN 3H M6S
Na 35,000+ - 46.0 54.0 64.3 89.5 Nc -15,000 - 88.3 11.7 ND ND 4a 35,000+ 4.3 4.9 95.1 81.8 92.0 4c 10,000 1.4 60.5 39.5 ND ND 8a 35,000+ 9.3 0 100 100 100 8b 1-45,000 6.9 48.6 51.4 56.0 60.0 12a 35,000+ 24.7 ND ND 85.4 86.2 12b '-'15,000 8.0 18.8 81.2 ND ND
ND is not determined. * Summary of analytical data for the different fractions de-
scribed in the text. The molecular weight values are based on the behavior of the labeled components relative to standard muco- polysaccharides during chromatography on CPG columns. The amino sugar values are as percentages of tritium label found for glucosamine and galactosamine.
t Percentage radioactivity eluting in the low-molecular-weight region on a CPG column after incubation with chondroitinase AC.
Proc. Nat. Acad. Sci. USA 72 (1975)
2034 Biochemistry: Bhavanandan and Davidson
60C
50C
40C
30C
30 40 60 FRACTION NUMBER
60
FIG. 2. (a) Fractionation of the 0.8 M NaCi extract of CPC- precipitable material from Pronase-digested nuclear membrane on a 0.9 X 95 cm column of CPG 10-240 glass beads. Fractions (1 ml) were collected and 50 jl was analyzed for radioactivity. The peak elution position of marker saccharides (HA, vitreous humor hyaluronic acid; CSA, porcine rib cartilage chondroitin-4-sulfate; and GlcUA, glucuronic acid) are indicated by the arrows. Frac- tions 29-35 and 36-55 were separately combined as peaks 8a and 8b, respectively. Figures (b) and (c) are elution patterns of peaks 8a and 8b after rechromatography on the same column; 700 IAl of 1 ml fractions were analyzed.
CPC fractions is given in Table 2. Over 90% of mS and about 5% of 3H activity are in salt extracts of molarity greater than 0.4 M and constitute the mucopolysaccharide content of nuclei. The CPC supernatant and the washings of the precipitate contain about 90% of the 3H activity and represent glyco- peptide components. A further 3% of the 3H activity present in the 0.2 M NaCl eluate is a sialoglycopeptide comparable to that isolated from the culture media of the cells (5). Char- acterization of these glycopeptides will be described in a subsequent report. Chromatography of the 0.8 N NaCl fraction which con-
TABLE 2. CPC precipitation and salt elution of 3H and 35S radioadtivity in Pronase digest of nuclei*
CPC- Molarity of NaCl for eluate solublet 0.2 0.4 0.8 1.2 2.0
3H 1548.3 58.3 28.8 28.3 5.9 2.5 (91.6) (3.4) (1.7) (1.7) (0.3) (0.15)
35S 5.3 3.1 16.9 96.6 21.0 1.0 (3.5) (2.1) (11.3) (64.4) (14.0) (0.67)
* Values are expressed in dpm X 10-3; the numbers within parentheses are the percentages. Results of a duplicate experi- ment were identical. Recovery of radioactivity was essentially quantitative.
t Material present in the CPC supernate combined with washes of the original CPC precipitate.
tained about 2/3 of. the sulfated polysaccharide associated with the nuclei is illustrated in Fig. 2a. Rechromatography of Fractions 8a and 8b gave the patterns shown in Fig. 2b and c. The hexosamine contents of these fractions are given in Table 1.
Fraction 8a. The chromatographic elution profiles of 8a after treatment with testicular and bacterial hyaluronidases and with chondroitinase AC are shown in Fig. 3. Control digestion of 8a with buffer alone and rechromatography gave a profile identical to that in Fig. 2b. The presence of galactosamine as the only hexosamine in this fraction and its complete suscep- tibility to testicular hyaluronidase and chondroitinase AC suggests that it consists of chondroitin-4 and/or 6-sulfate. This fraction was also susceptible to bacterial hyaluronidase but the fragments obtained were larger than tetrasaccharides, implying that the degree of sulfation is greater than 50%, since a polysaccharide with significantly less sulfation would give rise to di- and tetrasaccharides. The electrophoretic mobility of this component, in the two buffer systems tested, was consistent with that of a chondroitin sulfate having 0.5- 1.0 sulfate residues per disaccharide unit. Chondroitinase AC and chondroitinase AC plus chondro4-sulfatase digestion of this fraction followed by paper chromatography indicated that the sulfate ester group is predominantly in the 4-position of the galactosamine moiety.
After mild acid hydrolysis (0.04 N HC1, 1000, 90 min) of the 8a fraction, about 45% of the 3IS radioactivity migrated with inorganic sulfate on paper electrophoresis as well as on a Bio-Gel P2 column. It was also evident from these experi- ments that the polymer underwent considerable degradation. Both cartilage chondroitin-4-sulfate and hyaluronic acid treated in the same manner are degraded to fragments ranging from tetra-(major) to octa-saccharides, an observation not previously recorded. Previous findings from this laboratory (20) have established that about 35% of BSO42- was released from chondroitin-4-sulfate on hydrolysis with dilute acid under conditions comparable to those used in the present study. The possible presence of N-sulfate residues in this fraction
was considered even though the results of the chondroitinase AC and chondro-4-sulfatase digestions were not consistent with this. Treatment of the 8a fraction with nitrous acid, a reagent that causes deaminative cleavage of mucopolysac- charides containing sulfated amino groups (21), followed by chromatography on a CPG 10-240 column gave a profile identical to that in Fig. 2b, indicating that there were no such residues in this fraction. Gel filtration of Fraction 8a on the CPG 10-240 column indicated that this component has a molecular weight of 35,000 or greater, since it elutes in the void volume coincident with vitreous humor hyaluronic acid of molecular weight 35,000, whereas cartilage chondroitin-4- sulfate of molecular weight 15,000 elutes differently (Fig. 2). A high-molecular-weight chondroitin fraction isolated from the culture media of the same cells (B16 mouse melanoma) has been estimated to have a molecular weight of 97,000 i 8,800 by ultracentrifugation. (Bhavanandan and Davidson, unpublished results.) In order to determine whether the high molecular weight of Fraction 8a was due to incomplete Pronase digestion resulting in proteoglycan fragments rather than polysaccharide chains, we digested Fraction 8a with Pronase (two additions of 250 ug each at time 0 and 24 hr) for a total period of 72 hr and rechromatographed the product
Proc. Nat. Acad. Sci. USA 72 (1975)
D,
D.
)i
I
)i
Nuclear Mucopolysaccharides 2035
on a CPG 10-240 column. The elution profile was identical to that of the untreated sample (Fig. 2b), indicating that the high-molecular-weight nature of this fraction is probably not due to incomplete Pronase digestion. Soluble pig cartilage proteoglycan is completely degraded to polysaccharide chains of molecular weight about 15,000 on digestion with Pronase (22).
Fraction 8b. The labeled hexosamine content of this fraction was 51% galactosamine and 49% glucosamine (Table 1). Chromatography on CPG 10-240 gave the heterogenous elution profile shown in Fig. 2c; high-molecular-weight mate- rial is absent and the major portion of the fraction eluted in the 15,000-10,000 range. On treatment with chondroitinase AC and rechromatography on the same column, about 60% of the 3IS activity eluted in the low-molecular-weight region. Mild acid hydrolysis followed by paper electrophoresis of this fraction also gave results similar to those obtained with Fraction 8a; about 52% of 'IS radioactivity migrated with inorganic sulfate. Nitrous acid treatment of this fraction followed by chromatography on the CPG 10-240 column showed that 57.5% of -IS activity eluted in the low-molecular- weight region. These results suggest that this fraction is a mixture of heparan sulfate and chondroitin (4 or 6) sulfate of molecular weight similar to that of the cartilage polysaccha- ride. Chromatography of the 0.4 M and 1.2 M NaCl eluates on
the CPG 10-240 column gave the profiles shown in Fig. 4. Hexosamine and chondroitinase susceptibility data on Frac- tions 4a, 4c, 12a, and 12b are summarized in Table 1. Fractions 4a and 12a are high-molecular-weight chondroitin sulfate components, apparently the undersulfated and more fully sulfated homologs of Fraction 8a (see 35S/3H in Table 1). Fraction 4c contains lower molecular weight material com- posed of both amino sugars and sialic acid and is presumably sialoglycopeptide similar to that present in the 0.2 M NaCl eluate. There was insufficient material for complete charac- terization.
DISCUSSION
This study provides direct biochemical evidence for the association of anionic polysaccharides with mammalian cell nuclei. In the system investigated, a family of high-molecular- weight chondroitin-4-sulfates were the major nuclear muco- polysaccharides. The other mucopolysaccharides identified were heparan sulfate and a chondroitin sulfate of lower molecular weight. The presence of glycoproteins in cell nuclei has been pre-
viously reported by several workers (17-19) and is confirmed by our results. Based entirely on the tritium incorporation data (Table 2), it would appear that mucopolysaccharides are a minor component compared to glycoproteins; however, definitive information on the carbohydrate composition of nuclei must await mass analytical data. It is also essential to establish whether the association of mucopolysaccharides with the nuclei is a feature common to all mammalian cells. The physical properties and chemical architecture of the
anionic polysaccharides with heterogeneity of size and dis- tribution of functional groups (0-SO4; N-SO4; COOH) make them excellent candidates for a range of regulatory functions, including control of cationic environment at the nuclear membrane, transport selectivity into the nuclei, regulation of nuclear enzyme activity, and control of template activity in
2000
1500
150C
**,r ,
.f ,.
a
b
c
FRACTION NUMBER
FIG. 3. Chromatography of Fraction 8a on a 0.9 X 95 cm glass bead column after digestion with testicular hyaluronidase (upper), chondroitinase AC (middle), or bacterial hyaluronidase (lower). Fractions (1 ml) were collected and 700 ,uA analyzed for radioac- tivity. Abbreviations are as in Fig. 2.
chromatin. Several recent reports have suggested involvement of anionic polysaccharides in nuclear functions. Anionic…
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