Inhibition of heparan sulfate synthesis by chlorate.
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Animals↗
Biomedical subjects
H E Conrad
Publications and source records attributed to H E Conrad.
Degradation of heparan sulfate by nitrous acid.
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Amino Sugars↗
Heparins designed to specifically inhibit platelet interactions with von Willebrand factor.
BACKGROUND: Platelet interactions with the injured vessel wall may contribute significantly to the early and late failures of many cardiovascular interventions; the adhesive protein von Willebrand factor (vWF) is thought to play an important role. Previously, we demonstrated that heparin interfered with platelet/vWF hemostatic mechanisms by binding to vWF within the proteins's domain responsible for binding the platelet vWF receptor, glycoprotein Ib. The purpose of the present study was to develop and refine heparins with greater potency to inhibit platelet/vWF interactions. METHODS AND RESULTS: Immobilized synthetic peptides based on a known heparin-binding domain of vWF were used to yield novel fractions of standard heparin that demonstrated a sevenfold increase in their ability to inhibit vWF-dependent platelet agglutination and vWF/platelet binding. The high vWF affinity heparin showed enhanced anti-factor Xa activity but comparable activated partial thromboplastin time activity. Chemical modification of a standard heparin by periodate oxidation and borohydride reduction enhanced its ability to inhibit platelet/vWF interactions by threefold, while eliminating more than 90% of its activated partial thromboplastin time and anti-factor Xa activity. Affinity chromatography of the chemically modified heparin yielded a heparin with an eightfold higher inhibitory potency than the original heparin. CONCLUSIONS: Subspecies of heparin can be developed with significantly enhanced potency to inhibit vWF/platelet interactions. The vWF-inhibiting property of heparin can be dissociated from its antithrombin-binding activity. Based on a growing understanding of heparin/vWF interactions, combinations of affinity separations and chemical modifications could be designed to yield heparins uniquely suitable for prevention of arterial thrombosis.
Anticoagulants↗
Heparin and its non-anticoagulant analogues inhibit human keratinocyte growth without inducing differentiation.
In addition to its anti-coagulant effect, heparin inhibits the growth of several types of cells. Recent studies suggest that heparin inhibition of proliferation of cultured human keratinocytes, results primarily from interaction with keratinocyte-generated, heparin-binding autocrine growth factors. In this study, we evaluated whether non-anticoagulant heparin analogs, and oligosaccharide fragments of heparin, retain the growth-inhibitory properties of whole heparin on human keratinocytes. Second-passage neonatal keratinocytes were grown in serum-free keratinocyte growth medium, and the effect of heparin analogs was studied in the absence of exogenous growth factors using keratinocyte-conditioned medium. Cell proliferation was assessed by measurement of both DNA content and [3H]-thymidine incorporation. The addition of heparin inhibited the conditioned medium-stimulated keratinocyte proliferation in a dose-dependent manner, with 80% inhibition at or above 10 micrograms/ml. Moreover, heparin was not toxic to keratinocytes (as detected by propidium-iodide fluorescence and by retention of normal protein synthetic rate) and it did not induce terminal differentiation (as measured by cornified envelope formation). Furthermore, heparin stimulated protein secretion by keratinocytes without altering rates of protein synthesis. The growth-inhibitory effects of heparin oligosaccharides were directly proportional to their chain length. The hexasaccharide unit represented the minimum requirement for inhibition, whereas decasaccharide units demonstrated nearly equivalent growth inhibition to native heparin. Finally, two non-anticoagulant heparin analogs were equipotent with heparin in inhibiting autocrine-induced keratinocyte growth. These studies show that the growth-inhibitory activities of heparin are independent of the anticoagulant effects and that decasaccharides contain the optimal oligosaccharide chain length for the antiproliferative effect in human keratinocytes.
Anticoagulants↗
Type X collagen does not bind to matrix vesicles.
Cultured chick embryo tibial hypertrophic chondrocytes released matrix vesicles and Type X collagen into the culture medium. When the culture medium was filtered through a 0.1 micron nitrocellulose filter, both the matrix vesicles, measured as alkaline phosphatase, and the Type X collagen were retained quantitatively. None of the other collagen types in the culture medium was retained on the filter. Dissolution of the matrix vesicles on the filter in detergent solutions resulted in quantitative solubilization of the Type X collagen also. These results suggested that the Type X collagen was intimately associated with the matrix vesicles. However, when membrane filters that were composed of materials other than nitrocellulose, and that had a range of pore sizes, were used to filter the culture medium, the ratios of total matrix vesicles to total Type X collagen retained on the filters ranged from 53 (polysulfone membranes) to 0.3 (nitrocellulose-cellulose acetate membranes). Thus it is concluded that the quantitative retention of matrix vesicles and Type X collagen on 0.1 micron nitrocellulose filters was due to true filtration of the matrix vesicles and to selective adsorption of the Type X collagen. Removal of the noncollagenous extensions from the Type X collagen by brief pepsin or trypsin treatment converted the Type X collagen to its 45 kDa collagenous domain, which was no longer retained on nitrocellulose filters, suggesting that the adsorption of the Type X collagen on the filters was through one or both of its noncollagenous extensions. When the culture medium was subjected to ultracentrifugation to pellet the matrix vesicles, 98% of the membrane-associated alkaline phosphatase (matrix vesicles) was pelleted but only 15-20% of the Type X collagen was recovered in the pellet. These results indicate that matrix vesicles and Type X collagen are not the associated products of hypertrophic chondrocyte.
Alkaline Phosphatase↗
Structural analysis of periodate-oxidized heparin.
Treatment of heparin with HONO at pH 1.5 cleaves the polymer at N-sulfated, but not at N-acetylated GlcN residues, and yields di- and tetrasaccharides. The GlcNSO3 residues at the sites of cleavage are converted into anhydromannose (AMan) residues. Reduction of heparin cleavage products with NaB3H4 yields mixtures of di- and tetrasaccharides with reducing terminal [3H]anhydromannitol residues. The identification and quantification of these oligosaccharides by HPLC procedures have been described. These procedures have been used to determine the rates of periodate oxidation of the susceptible unsulfated GlcA and IdoA residues in heparin by measuring the disappearance of the di- and tetrasaccharides that contain GlcA and IdoA. Complete oxidation with IO4- results in the total loss of the unsulfated uronic acid-containing oligosaccharides, but kinetic studies reported here show that IdoA is oxidized much more rapidly than the major fraction of the GlcA under all reaction conditions. As the pH is lowered from 7 down to 3, the overall rate of the oxidation slows markedly, but the relative rates of GlcA and IdoA oxidation do not change. The slow rate of oxidation of GlcA residues at all pH's yields oxidation products early in the reaction progress in which all of the unsulfated IdoA residues are oxidized while 70-80% of the pH 1.5 nitrous acid-releasable GlcA----AMan(3,6-(SO4)2) are retained. The anticoagulant activity (APTT) of the partially oxidized product is reduced from 170 IU/mg to 38 IU/mg. Further studies show that the GlcA residue in the antithrombin III binding pentasaccharide is oxidized much more rapidly than the bulk of the GlcA residues in heparin. The results suggest that heparin contains GlcA----AMan(3,6-(SO4)2) sequences that lie outside of the antithrombin-binding pentasaccharide.
Antithrombin III↗
Coordinate inhibition of alkaline phosphatase and type X collagen syntheses by 1,25-dihydroxyvitamin D3 in primary cultured hypertrophic chondrocytes.
The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (2.3 x 10(-12) - 1.4 x 10(-6) [M]) on alkaline phosphatase, collagen, and cell proliferation were examined in primary cultured hypertrophic chondrocytes prepared from the distal epiphyseal growth plate of the tibias of 12-day chick embryos. 1,25(OH)2D3 showed time- and dose-dependent inhibitory effects on the alkaline phosphatase and collagen levels. The inhibition of alkaline phosphatase activity became detectable at 2 x 10(-11) [M] and reached 10% of control at 10(-7) [M]. The concentration of 1,25(OH)2D3 giving a 50% inhibition of the enzyme level was approximately 3 x 10(-10) [M]. Of the two extracellular collagen pools, a cell-associated matrix pool showed a more dramatic decrease (to 10% of control) than a culture medium pool (to 50% of control) at increased 1,25(OH)2D3 concentrations. The degree of inhibition was different for each type of chondrocyte-specific collagen (types II, IX, X, and XI). Types II and IX were inhibited in a parallel manner to only 60-80% of control. On the other hand, types X and XI were more greatly reduced up to 10% of control, and their dose-dependent inhibitory curves were similar to that of alkaline phosphatase. On cell proliferation, 1,25(OH)2D3 had a biphasic effect: stimulation at 10(-10)-10(-8) [M] and inhibition at higher levels. The results revealed the significant involvement of 1,25(OH)2D3 in the metabolism of two probable calcification-related products, alkaline phosphatase and type X collagen.
Alkaline Phosphatase↗
Heparin oligosaccharides enhance tissue-type plasminogen activator: a correlation between oligosaccharide length and stimulation of plasminogen activation.
The rate of plasminogen (Pg) activation by tissue-type Pg activator (t-PA) is enhanced by heparin-derived oligosaccharides. Kinetic analysis of the effects of heparin oligosaccharides, ranging in size from di- to dodecasaccharides, on Pg activation demonstrates that stimulation of the reaction is dependent on the size of the heparin oligosaccharides. Di- and tetrasaccharides enhance the activation through 2-fold increases in kcat and 4-fold decreases in Km. Hexasaccharide and larger oligosaccharides stimulate the reaction by increasing the kcat by as much as 4-fold, but do not affect the Km. Previous experiments have shown that lipoprotein(a) [Lp(a)] inhibits Pg activation by t-PA, but only in the presence of a template which enhances t-PA activity such as fibrinogen fragments or intact heparin. Similiarly, Lp(a) inhibits the enhancement of t-PA activity by the larger heparin oligosaccharides but has no effect on t-PA activity in the presence of di- and tetrasaccharides. The results of this study when considered with our previous observations (Edelberg & Pizzo, 1990) suggest that the enhancement in Pg activation by the smaller oligosaccharides is mediated exclusively via binding to t-PA while the larger oligosaccharides may interact with both t-PA and Pg. Furthermore, studies of Pg activation in the presence of both heparin oligosaccharides and fibrinogen fragments demonstrate that t-PA is stimulated preferentially by fibrinogen fragments.
Amino Acid Sequence↗
Correlations between heparan sulfate metabolism and hepatoma growth.
A rat hepatoma cell line (Gershenson et al., Science, 170:859-861, 1970) contains a dynamic steady-state pool of free heparan sulfate (HS) chains in the nucleus that increases in amount when growing cells reach confluence (Fedarko and Conrad, J. Cell Biol., 102:587-599, 1986). In logarithmically growing cells labeled with 35SO4(2-) steady-state levels of [35SO4]HS in the nucleus are altered by a variety of culture conditions. Rapidly dividing cells (doubling time = 18-22 h) growing under optimized conditions had steady-state levels of nuclear HS within the range of 40-50 pmol 35SO4 in nuclear HS/10(6) cells. The steady-state levels of nuclear HS were lowered by several changes in culture conditions, including 1) additions of 1 mM p-nitrophenyl-beta-D-xyloside, 0.25-0.5 mM (+)-catechin, 0.5 ng/ml transforming growth factor beta, 20 ng/ml phorbol-12-myristate-13-acetate, 1 mM dibutyryl cAMP, or 1 mM inositol-2-PO4; 2) decreased levels of D-glucose; or 3) deletions of serum, insulin, or inositol. In all cases lowering of the nuclear HS level was accompanied by an increase in the cell doubling times, suggesting a correlation in which nuclear HS levels must be optimized for maximal growth rates. When cells cultured under optimal growth conditions reached confluence, the level of nuclear HS increased threefold and the cells stopped dividing. The same culture conditions that lowered the steady-state levels of HS in the logarithmically growing cells prevented this rise in the nuclear HS as the cells reached confluence and resulted in loss of contact inhibition and overgrowth of the confluent cultures. These observations suggest a second correlation in which elevated nuclear HS levels are found when cell growth is inhibited at confluence; prevention of this rise results in continued growth. Consistent with this correlation between elevated nuclear HS and reduced growth rates, it was observed that addition of either 0.5 microgram/ml hydrocortisone or 0.05 microgram/ml retinoic acid to the culture medium of logarithmically growing cultures resulted in increases in steady-state levels of nuclear HS that were accompanied by increased cell doubling times. The two agents that increased the levels of nuclear HS in logarithmically growing cultures had little effect on levels of nuclear HS in confluent cells or on contact inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Control of cell division in hepatoma cells by exogenous heparan sulfate proteoglycan.
The effects of cell surface heparan sulfate proteoglycan (HSPG) prepared from log and confluent monolayers of a rat hepatoma cell line on hepatoma cell growth were studied. When HSPG isolated from confluent cells was added exogenously to log phase cells, it was internalized and free heparan sulfate (HS) chains appeared transiently in the nucleus. Concurrently, the growth of the treated cells was inhibited, but the cells resumed logarithmic growth as the level of nuclear HS fell, and the cells grew to confluence and became contact inhibited. When HSPG prepared from log-phase hepatoma cells was added exogenously to log phase cells, it was internalized but very little of the internalized HS appeared in the nucleus, and there was no change in the rate of cell growth. However, when the rate of cell growth was reduced by culture of the cells in serum- and insulin-deficient medium, HSPG prepared from log-phase cells stimulated the growth rate of these slow-growing cells. The cell cycle dependency of HSPG uptake and growth inhibition was studied in cultures synchronized by a thymidine/aphidicolin double block. When [35SO4]HSPG from confluent cells was added to synchronized cells just as they were released from the second block, a portion of the [35SO4]HSPG was internalized and [35SO4]HS appeared in the nucleus. However, at mitosis the [35SO4]HS disappeared almost completely from all of the cellular pools, and after mitosis, more of the [35SO4]HSPG was taken up and [35SO4]HS reappeared in the nucleus and remained in the nucleus until the cells divided again. When cultures were released from the aphidicolin block, both control and HSPG-treated cells progressed through the S, the G2, and the M phases of the cell cycle. However, the length of the G1 phase of the cycle was increased in the HSPG-treated cells. The treated cultures then progressed through the second S, G2, and M phases. Thus, the inhibition of cell division occurred in the G1 phase of the cell cycle, prior to the G1/S boundary. Addition of the HSPG to the synchronized cultures just after the first mitosis resulted in an immediate arrest of the cell cycle in G1.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
The disaccharide composition of heparins and heparan sulfates.
Heparin and heparan sulfate can be cleaved selectively at their N-sulfated glucosamine residues by direct treatment with nitrous acid at pH 1.5. These polymers can also be cleaved selectively at their N-acetylated glucosamine residues by first N-deacetylating with hydrazine and then treating the products with nitrous acid at pH 4. These procedures have been combined and optimized for the conversion of these glycosaminoglycan chains into their disaccharide units. A modified hydrazinolysis procedure in which the glycosaminoglycans were heated with hydrazine:water (70:30) containing 1% hydrazine sulfate gave rapid rates of N-deacetylation and minimal conversion of the uronic acid residues to their hydrazide derivatives. Under these conditions, N-deacetylation was complete in 4 h and the beta-eliminative cleavage of the polymer chains that occurs during hydrazinolysis (P. N. Shaklee and H. E. Conrad (1984) Biochem. J. 217, 187-197) was eliminated. Treatment of the N-deacetylated polymer with nitrous acid at pH 3 for 15 h at 25 degrees C then gave simultaneous cleavage at the N-unsubstituted glucosamine residues and the N-sulfated glucosamine residues. These deamination conditions minimized, but did not eliminate, the side reaction in which nitrous acid-reactive glucosamine residues undergo ring contraction without glucosaminide bond cleavage. Thus, the disaccharides were obtained in a yield of 90% of those originally present in the glycosaminoglycan chains. Since the ring contraction side reaction occurs randomly at the diazotized glucosamine residues, the disaccharides formed in the pH 3 nitrous acid reaction were recovered in proportions equal to those in the original glycosaminoglycan chain.(ABSTRACT TRUNCATED AT 250 WORDS)
Chromatography, Gel↗
Structure of heparan sulfate and dermatan sulfate.
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Animals↗
Analysis of oligosaccharides from heparin by reversed-phase ion-pairing high-performance liquid chromatography.
An ion-pairing high-pressure liquid chromatography procedure was developed for analysis of mixtures of oligosaccharides generated by nitrous acid cleavage of heparin. Oligosaccharides were eluted from a Hi-Chrom 5S ODS (C18) column using mixtures of acetonitrile and buffers containing 40 mM ammonium phosphate and 1 mM tetrabutylammonium phosphate. Isocratic conditions were developed for optimal separation of a number of individual disaccharides and tetrasaccharides that were characterized previously (M.J. Bienkowski and H.E. Conrad (1985) J. Biol. Chem. 260, 356-365). These isocratic conditions were then coupled to obtain gradient elution conditions for the ion-pairing separations of mixtures of disaccharides and mixtures of tetrasaccharides. A comparison of the elution profiles obtained in the ion-pairing chromatography procedure with profiles obtained by anion-exchange high-pressure liquid chromatography profiles showed markedly better overall resolution by the ion-pairing procedure. As a result of this improved resolution, the new procedure showed the presence of previously unidentified products in the heparin oligosaccharide mixtures.
Chromatography, High Pressure Liquid↗
Involvement of phosphatidylinositol and insulin in the coordinate regulation of proteoheparan sulfate metabolism and hepatocyte growth.
A rat hepatocyte cell line was cultured in Higuchi's medium with fetal calf serum and insulin and labeled with 35SO2/4-. The cells were treated with a number of ligands to displace the heparan 35SO4 proteoglycan (HSPG) from the pericellular matrix. Maximum release was obtained with D-mannose-6-PO4 (50 mM), D-glucose-6-PO4 (50 mM), myo-inositol-2-PO4 (2-5 mM), myo-inositol hexaphosphate (2-5 mM), and DL-myo-inositol-1-PO4 (1-2 mM). D-myo-Inositol-1,3,4-(PO4)3 (1 mM) and L-myo-inositol-1-PO4 (2 mM) were intermediate in their ability to release the cell surface HSPG, whereas heparin (2 mg/ml), yeast phosphomannan (4 mg/ml), D-xylose-1-PO4 (50 mM), D-glucose-6-SO4 (50 mM), and myo-inositol hexasulfate (5 mM) were ineffective. When 35SO2/4- was added to cell cultures, the total cell surface HSPG increased linearly, but the percentage of the total cell surface [35SO4]HSPG that was released by myo-inositol-PO4 increased with time during the labeling period, reaching a maximum of 65% after 5 h. When cells were labeled for 12 h without insulin in the medium, the maximum amount of cell surface HSPG that was released by myo-inositol-PO4 was reduced to 30%. However, when cells labeled in the absence of insulin were treated with phosphatidylinositol-specific phospholipase C and then myo-inositol-PO4, the release of the cell surface [35SO4]HSPG was increased to 73%. When the [35SO4]HSPG that was released from the cell surface by treatment with myo-inositol-PO4 was added to cultures of unlabeled hepatocytes, it was taken up very rapidly and a portion of the internalized HSPG was converted to free heparan SO4 chains which appeared in the nucleus. Uptake was Ca2+- and Mg2+-independent. The amount of [35SO4]HSPG taken up was markedly reduced when the myo-inositol-PO4-releasable [35SO4]HSPG was pretreated with trypsin, thermolysin, alkaline borohydride, or alkaline phosphatase. When the cells were grown in inositol-deficient medium or in the presence of myo-inositol-PO4, the amount of heparan SO4 found in the nucleus was markedly reduced, and the cells no longer exhibited contact inhibition. These effects of myo-inositol deficiency on the growth and nuclear heparan SO4 were accentuated by addition of LiCl to the cultures to prevent phosphatidylinositol synthesis from the endogenous myo-inositol-PO4.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Transport of heparan sulfate into the nuclei of hepatocytes.
Monolayer cultures of a rat hepatocyte cell line shown previously to accumulate a nuclear pool of free heparan sulfate chains that are enriched in sulfated glucuronic acid (GlcA) residues (Fedarko, N.S., and Conrad, H.E., (1986) J. Cell Biol. 587-599) were incubated with 35SO4(2-), and the rate of appearance of heparan [35S]sulfate in the nuclei was measured. Heparan [35S]sulfate began to accumulate in the nuclei 2 h after the administration of 35SO4(2-) to the cells and reached a steady state level after 20 h. Heparan [35S]sulfate was lost from the nuclei of prelabeled cells with a t1/2 of 8 h. Chloroquine did not inhibit the transport of heparan sulfate into the nucleus, but increased the t1/2 for the exit of heparan sulfate from the nucleus to 20 h and led to a doubling of the steady state level of nuclear heparan sulfate. Heparan [35S]sulfate which was obtained from the medium or from the cell matrix of a labeled culture and which contained only low levels of GlcA-2-SO4 residues was incubated with cultures of unlabeled cells, and the uptake of the exogenous heparan [35S]sulfate was studied. At 37 degrees C the cells took up proteoheparan [35S]sulfate and transported about 10% of the internalized heparan [35S]sulfate into the nucleus, where it appeared as free chains. The heparan [35S]sulfate isolated from the nucleus was enriched in GlcA-2-SO4 residues, whereas the heparan [35S]sulfate remaining in the rest of the intracellular pool showed a corresponding depletion in GlcA-2-SO4 residues. At 16 degrees C, where endocytosed materials do not enter the lysosomes, the cells also transported exogenous proteoheparan [35S]sulfate to the nucleus with similar processing. Thus, the metabolism of exogenous heparan sulfate by hepatocytes follows the same pathway observed in continuously labeled cells and does not involve lysosomal processing of the internalized heparan sulfate.
Animals↗
Xylosylation and glucuronosylation reactions in rat liver Golgi apparatus and endoplasmic reticulum.
We have studied in rat liver the subcellular sites and topography of xylosylation and galactosylation reactions occurring in the biosynthesis of the D-glucuronic acid-galactose-galactose-D-xylose linkage region of proteoglycans and of glucuronosylation reactions involved in both glycosaminoglycan biosynthesis and bile acid and bilirubin conjugation. The specific translocation rate of UDP-xylose into sealed, "right-side-out" vesicles from the Golgi apparatus was 2-5-fold higher than into sealed right-side-out vesicles from the rough endoplasmic reticulum (RER). Using the above vesicle preparations, we only detected endogenous acceptors for xylosylation in the Golgi apparatus-rich fraction. The specific activity of xylosyltransferase (using silk fibroin as exogenous acceptor) was 50-100-fold higher in Golgi apparatus membranes than in those from the RER. Previous studies had shown that UDP-galactose is translocated solely into vesicles from the Golgi apparatus. In these studies, we found the specific activity of galactosyltransferase I to be 40-140-fold higher in membranes from the Golgi apparatus than in those from the RER. The specific translocation rate of UDP-D-glucuronic acid into vesicles from the Golgi apparatus was 10-fold higher than into those from the RER, whereas the specific activity of glucuronosyltransferase (using chondroitin nonasaccharide as exogenous acceptor) was 12-30-fold higher in Golgi apparatus membranes than in those from the RER. Together, the above results strongly suggest that, in rat liver, the biosynthesis of the above-described proteoglycan linkage region occurs in the Golgi apparatus. The specific activity of glucuronosyltransferase, using bile acids and bilirubin as exogenous acceptor, was 10-25-fold higher in RER membranes than those from the Golgi apparatus. This suggests that transport of UDP-D-glucuronic acid into the RER lumen is not required for such reactions.
Animals↗
The disaccharides formed by deaminative cleavage of N-deacetylated glycosaminoglycans.
Chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and keratan sulphate were N-deacetylated by treatment with hydrazine and then cleaved with HNO2 at pH 4.0, and the resulting products were reduced with NaB3H4. This reaction sequence cleaved the glycosaminoglycans at their N-acetyl-D-glucosamine or N-acetyl-D-galactosamine residues, which were converted into 3H-labelled 2,5-anhydro-D-mannitol (AManR) or 2,5-anhydro-D-talitol (ATalR) residues respectively. The end-labelled disaccharides, composed of D-glucuronic acid (GlcA), L-iduronic acid (IdoA) or D-galactose (Gal) and one of the anhydrohexitols, were identified as follows: both chondroitin 4-sulphate and chondroitin 6-sulphate gave GlcA----ATalR(4-SO4), GlcA----ATalR(6-SO4), IdoA----ATalR (4-SO4) and GlcA(2-SO4)----ATalR(6-SO4); dermatan sulphate gave IdoA----ATalR(4-SO4), GlcA----ATalR(4-SO4), GlcA----ATalR(6-SO4)----IdoA(2-SO4)ATalR(4-SO4) and IdoA----ATalR (4,6-diSO4); keratan sulphate gave Gal(6-SO4)----AManR(6-SO4), Gal----AManR(6-SO4), Gal(6-SO4)----AManR and Gal----AManR. Several additional disaccharides were generated by treatment of the uronic acid-containing disaccharides with hydrazine to epimerize their uronic acid residues at C-5. A number of these disaccharides were found to be substrates for lysosomal sulphatases and glycuronidases. Methods were developed for the separation of all of the disaccharide products by h.p.l.c. The rate of N-deacetylation of chondroitin 4-sulphate by hydrazinolysis was significantly lower than the rate of N-deacetylation of chondroitin 6-sulphate or chondroitin. Dermatan sulphate was N-deacetylated at an intermediate rate. The relative amounts of disaccharides obtained from chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate under optimum hydrazinolysis/deamination conditions were comparable with the amounts of the corresponding products released from the polymers by chondroitinase treatment.
Chondroitin Lyases↗
A unique heparan sulfate in the nuclei of hepatocytes: structural changes with the growth state of the cells.
Growing and confluent cultures of a rat hepatocyte cell line were labeled with 35SO4(2-) and the heparan sulfate in the culture medium, the pericellular matrix, the nucleus, the nuclear outer membrane, and the remaining cytoplasmic pool was purified by DEAE-cellulose chromatography. The heparan sulfate in all pools from the confluent cells was bound more strongly on the DEAE-cellulose column than the corresponding pools from the growing cells. Gel filtration of each pool before and after beta-elimination showed that the heparan sulfate from the nuclear and nuclear membrane pools was composed of primarily free chains, whereas the heparan sulfate in all of the other pools was a mixture of proteoglycans and free chains. The heparan sulfate in each pool was cleaved with nitrous acid to obtain mixtures of di- and tetrasaccharides. Analysis of these mixtures showed that the structural features of the heparan sulfates in each pool were different and were altered significantly when the growing cells became confluent. The nuclear-plus-nuclear membrane pools represented 6.5% and 5.4% of the total cell-associated heparan sulfate in the growing cells and the confluent cells, respectively. The structural features of the heparan sulfate in the two nuclear pools were very similar to each other, but were markedly different from those of the heparan sulfate from the other pools or from any previously described heparan sulfate or heparin. The most unusual aspect of these structures was the high content of beta-D-glucuronosyl(2-SO4)----D-glucosamine-N,O-(SO4)2 disaccharide units in these sequences. The mode of biosynthesis and delivery of these unusual sequences to the nucleus and the potential significance of these observations are discussed.
Animals↗