Department of Cell and Molecular Biology 1, Lund University, S-221 00 Lund, Sweden
Received on September 9, 1999; revised on November 29, 2000; accepted on January 3, 2000.
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Abstract |
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Key words: brefeldin A/glypican/heparan sulfate/nitric oxide/suramin
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Introduction |
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When synthesis is completed, PGs are guided by their core protein to different sites, such as intracellular storage vesicles, the extracellular matrix, or the cell surface. Glypicans, which acquire their GPI-anchor in the ER, are found at the surface of many cell types. The exact function of the lipid-anchor is not clear. In polarized cells, it may serve to direct glypican to the apical surface. Many GPI-anchored proteins are concentrated to caveolae, specific plasmalemmal structures believed to be involved in endo-/transcytosis and signaling (for review, see Anderson, 1998). Cell-surface associated HS-containing PGs (HSPG) support a wide range of biological functions, including growth control, cell adhesion, endo- and transcytosis and anticoagulant activity (for references, see Mertens et al., 1992
; Misra et al., 1994
; Taipale and Keski-Oja, 1997
; Lindahl et al., 1998
; Bernfield et al., 1999
).
Normal human vascular endothelial cells produce a variety of membrane-bound HSPGs, mainly syndecans but also glypican-1 (Mertens et al., 1992). It has also been reported that endothelial PGs carry HS-chains with N-unsubstituted glucosamines (GlcNH2) which serve as recognition and bindings sites for L-selectin (Norgard-Sumnicht and Varki, 1995
). By using a specific monoclonal antibody that recognizes these GlcNH2 residues, van den Born et al. (1995)
detected an uneven distribution of this epitope among renal basement membranes.
The metabolic turnover of membrane-bound HSPGs follows different routes. Transmembrane intercalated forms are either internalized by endocytosis and degraded stepwise in endosomes or lysosomes, or cleaved proteolytically and shed into the extracellular space (Yanagashita and Hascall, 1992; Bernfield et al., 1999
). Glypicans can, in addition, be cleaved at the phosphate-inositol bond by phosphatidylinositol-specific phopholipase C resulting in release of the PG from its lipid-anchor (Schmidtchen et al., 1990
; David, 1993
). Recycling of a phospholipase C-resistant variant of fibroblast glypican has been proposed (Fransson et al., 1995
). This proposal was based on the observation that a HSPG with the properties of glypican was still being HS-chain radiolabeled when cells were treated with brefeldin A (BFA), an inhibitor of transport of newly-made core protein from the ER to the Golgi (Klausner et al., 1992
, and references therein). Furthermore, cell-surface HSPG tagged with biotin reappeared radiolabeled after incubation of cells with [35S]sulfate in the presence of BFA. It was suggested that the glypican variant recycles via endosomes to the trans-Golgi compartment. During recycling HS-chains were partially degraded and resynthesized on the remaining stubs, and the reprocessed PG was then returned to the cell surface (Edgren et al., 1997
). Suramin, which inhibits both internalization and degradation of HS (Nakajima et al., 1991
; Voogd et al., 1993
, and references therein), resulted in accumulation of glypican-like HSPG (Fransson et al., 1995
).
Results obtained in previous studies on PGs synthesized by human umbilical vein endothelial cells suggested a nonenzymatic, autodegradation of HS in cell extracts (Lindblom et al., 1989; Lindblom, 1991
; Fransson et al., 1998
). Vilar et al. (1997)
showed that endothelial cells can generate sufficient amounts of NO (and subsequently nitrite) to support degradation of exogenously added HS. We have now used a vascular endothelial cell-line (ECV 304) and the drugs BFA and suramin to arrest endogenously formed HSPGs at various stages of their turnover and recycling. Our results indicate that simultaneous manipulations of NO-formation or nitrite-deprivation affects the degradation of HS and the recycling of glypican-1.
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Results |
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In suramin-treated cells, intermediate-size, HS-lyase sensitive products accumulated (Figure 1C,F). One portion of the material was sensitive to alkali-treatment, whereas another was not (Figure 1F, dashed line) indicating that the material consisted of both free HS-fragments and HS attached to protein. The average size of the suramin-arrested HS-material (protein-bound as well as unbound) was somewhat larger (Kav 0.7) than that of the HS-oligosaccharides (Kav 0.9) isolated from untreated cells (Figure 1D). When BFA and suramin were combined, the result was the same as with BFA alone (data not shown). Hence, the BFA-block should precede that of suramin.
Glypican-1 proteoglycan accumulates in BFA-treated cells
The nature of the BFA-arrested HSPGs was determined by immunodetection after electrophoresis of the core proteins and by direct immunoisolation from the cell extracts. To be able to trace the material during purification, PGs were metabolically labeled with [35S]sulfate. Cell-associated PG was extracted from the cell layer, recovered by passage over DEAE-cellulose, and purified by gel permeation chromatography on Superose 6 (as in Figure 1E) followed by ion-exchange chromatography on Mono Q (Figure 2A). PG material (see bar) was pooled and digested with HS lyase followed by SDSPAGE. Western blot using immunostaining with monoclonal antibodies S1 or 2E9 specific for glypican-1 and syndecan-1/3, respectively, was then performed (David et al., 1990; Mertens et al., 1992
). The result (see inset in Figure 2 A) showed one core protein (66 kDa) reacting with glypican-1 antibody and another one (125 kDa), which was either that of syndecan-3 or aggregated syndecan-1 core protein (Bernfield et al., 1999
).
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The BFA-arrested material is a precursor of the suramin-arrested material
Pulse-labeling of BFA-treated cells with [35S]sulfate followed by extraction, precipitation with Alcian blue and SDSPAGE indicated that the accumulation of PG increased up to 12 h and then remained relatively constant (data not shown). In a pulse-chase experiment, cells were incubated with [35S]sulfate for 12 h and then chased in either drug-free or suramin-containing nonradioactive medium. Cells were extracted and aliquots were chromatographed on Superose 6 (Figure 3). After 24 of chase in drug-free medium, prelabeled PG material had been converted to intermediate-size products eluting in fractions 2938 (Figure 3B, solid line) and after 42 h most of this had disappeared (Figure 3C, solid line). Throughout the chase, a minor portion of PG-material remained undegraded. Oligosaccharides, expected to elute in fractions 4044, were not seen. No PG or oligosaccharide could be found in the medium (data not shown). However, during chase in suramin-containing medium, both intermediate-size products and oligosaccharides accumulated (Figure 3B,C, dashed lines). Hence, suramin can block further degradation of HS-material released from the BFA-block.
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Inhibitors of nitrite and nitrite-generation affect proteoglycan and oligosaccharide production
Nitrite is formed by oxidation of NO which, in turn, is produced by decomposition of arginine catalyzed by NO-synthases, the constitutive form cNOS and the inducible form iNOS (for review, see Wink et al., 1996). NO is unstable and either directly converted to nitrite or stored as protein-bound S-nitrosothiols. The latter release NO non-enzymatically in a process catalyzed by Cu2+ (for review, see Williams, 1996
). To explore whether NO-derived nitrite was involved in HS-degradation, cells were pre-exposed to the NO-synthase inhibitors N-methyl-arginine (inhibits both forms of NOS), N-nitro-arginine (inhibits preferentially cNOS) or aminoguanidine (inhibits preferentially iNOS) or to the nitrite-destroying agent ammonium sulfamate. Cells were then incubated with radiosulfate in the low-arginine medium 199 in the continued presence of the respective compounds. As shown in Figure 5AE treatment with these compounds reduced generation of HS-oligosaccharides (fractions 4045). The order of effectiveness was N-methylarginine = sulfamate > aminoguanidine > N-nitroarginine. There was, however, no concomitant increase in the radiolabeling of HSPG (fractions 2025), as was seen when cells not deprived of nitrite were treated with BFA (Figure 5F).
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To test whether nitrite was also involved in the degradation of HS-chain fragments and oligosaccharides, radiosulfate-labeled suramin-arrested material was chased in nonradioactive medium with or without sulfamate and the products were analyzed as in Figure 3. The turnover of HS-degradation products was unaffected by the presence of sulfamate (data not shown). However, as shown above, supply of nitrite appears necessary to sustain formation of the BFA-arrested HSPG, perhaps by facilitating HS-chain extension. Hence, we tested whether HS-chain extension and formation of the BFA-arrested HSPG could be restored in nitrite-deprived cells when NO-donor was supplied.
Formation of BFA-arrested proteoglycan in nitrite-deprived cells is restored by NO-donor
Cells were deprived of nitrite by treatment with a combination of aminoguanidine (to inhibit iNOS), sulfamate (to destroy nitrite), and neocuproine (to inhibit release of NO from S-nitrosothiols). The cells were then chased in fresh, BFA-containing medium with [35S]sulfate and increasing concentrations of NO-donor (sodium nitroprusside). [35S]PG was isolated from the cell extracts by ion exchange chromatography. As seen in Figure 6A (solid circles) formation of BFA-arrested HSPG was gradually stimulated in the presence of increasing concentrations of NO-donor. Maximal stimulation occurred at 300 µM nitroprusside or higher. The stimulated PG-formation reached the same level as in cells not deprived of nitrite and not treated with NO-donor (open circles).
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Subconfluent cell cultures were metabolically labeled with [35S]methionine/cysteine until cells were confluent (usually within 24 h) and then radiolabeling was continued for another 24 h in the presence of aminoguanidine, sulfamate, and neocuproin to minimize the endogenous nitrite concentration. Cells were then chase-labeled overnight in fresh BFA-containing medium with [3H]glucosamine (to label the HS backbone) and in the absence or presence of 300 µM sodium nitroprusside as NO-donor. Radiolabeled PG was recovered from the cell extracts by passage over DEAE-cellulose and then chromatographed on Superose 6 (Figure 6B). It is seen that very little [3H]PG was formed in unstimulated cells (dashed line), whereas a large [3H]PG peak was obtained from stimulated cells (solid line).
To obtain HSPG that could be used for core protein analysis, further purification by ion exchange chromatography on MonoQ was necessary in order to remove some non-PG protein (Figure 6C,D). The [35S]methionine/cysteine-labeled PG recovered from (C) pulse-labeled and (D) chase-labeled cells were thus isolated (see bars in Figure 6C,D), HS-chains were removed by digestion with HS lyase and [35S]core proteins were subjected to SDSPAGE (see inserts in Figure 6C,D). After the pulse, a HSPG with a core protein of approx. 120 kDa was seen (Figure 6C). After the chase in medium containing BFA and NO-donor, an additional HSPG with a core protein of ~70 kDa appeared (Figure 6D). This size corresponds to that of glypican (David et al., 1990; Schmidtchen et al., 1990
).
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Discussion |
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Degradation of HS-side chains could take place in several stages (Figure 7). We propose an initial partial endoglycosidic degradation on the nonreducing side of the GlcNH2 moieties of HS, releasing relatively large HS-chain fragments, and gradually generating a truncated PG with the GlcNH2 moieties near the nonreducing end of the stubs. Endoglycosidases (heparanase) capable of partially depolymerizing HS chains of HSPG have been demonstrated in many cell types, including endothelial cells (Godder et al., 1991). In most cases, these endoheparanases cleave ß-D-glucuronidic linkages which would generate truncated HSPG with stubs containing nonreducing terminal glucosamine residues (for an extensive list of references, see Sandbäck-Pikas et al., 1998
). We have previously observed that HS oligosaccharides generated by endoglycosidic cleavage in fibroblasts had been cleaved at sites located close to the heparinase I cleavage sites, i.e., at the nonreducing side of IdoUA(2-OSO3) (Schmidtchen and Fransson, 1994
). Recent studies confirm this observation. CHO cell- (Bai et al., 1997
) or human hepatoma- and platelet-derived endoheparanases (Sandbäck-Pikas et al., 1998
), which are not specific for the type of N-substitution of the adjacent glucosamine, have a requirement for 2-O-sulfate on neighboring hexuronic acid residues located on the reducing side of the cleavage site () in, e.g., the following sequence: -GlcNR-GlcUAGlcNR-HexUA(2-OSO3)-GlcNR- where R is an unspecified substituent and HexUA could be IdoUA. Cleavage of a glucuronidic linkage a short distance from the non-reducing side of a GlcNH2 would thus generate the non-reducing terminal sequence GlcNR-HexUA(2-OSO3)-[GlcNR-HexUA]n-GlcNH2- in the core protein stubs (Figure 7). When n is small or zero subsequent deaminative cleavage at the reducing side of GlcNH2 would result in an undetectable/marginal effect on overall chain/stub size. It is possible that these short non-reducing terminal GlcNH2-containing "telosaccharides" are cleaved off when sufficiently high concentrations of nitrite have been generated endogenously in a mildly acidic compartment providing fresh acceptor sites for HS chain extension (Figure 7). The "telosaccharides" may contain unexpected features, such as 3-O-sulfation. It is intriguing that certain isoforms of 3-O-sulfatase (e.g. 3-OST-3) recognize IdoUA(2-OSO3)-GlcNH2 repeats (Shukla et al., 1999
). It should be added that in fibroblasts, there may be no need for NO-derived nitrite as fibroblast HS chains do not appear to contain much GlcNH2 (Fransson et al., 1995
).
The intracellular location of the proposed events has not been specifically addressed. However, NO, the precursor of nitrite and thus a potential regulator of glypican recycling, may be formed both in caveolae and in endosomes. Constitutive endothelial NO-synthase (cNOS/eNOS) targets to the cytoplasmic side of caveolae when made lipophilic by acylation (Garcia-Cardenas et al., 1996; Shaul et al., 1996
). However, NOS appears to be less active when it is associated with caveolin, the structural scaffolding protein of caveolae (Feron et al., 1998
; Prabhakar et al., 1998
). Nevertheless, Vilar et al. (1997)
have shown that endothelial cells in culture are capable of generating sufficient amounts of NO (and subsequently nitrite) to support degradation of exogenously added HS. Oxidation of NO to nitrite increases exponentially with NO-concentration (Wink et al., 1996
). Therefore, effects on HS turnover may be seen when the amount of NO has reached a threshold level (e.g., >300 µM) in the local subcellular environment. This may occur when recycling glypican passes through the endosomal compartment where NOS should be fully active. In this way NO could regulate the level and structure of cell-surface HS. Specific structures in glypican HS-chains contribute to the anticoagulant potency of the intact vascular endothelium by binding/activation of antithrombin III (Mertens et al., 1992
). It is thus interesting that prolonged inhibition of NO-synthase decreases the expression of anticoagulant HS on endothelial cells (Irokawa et al., 1997
). Recycling glypican may also be involved in internalization and recycling of HS-binding growth factors and enzymes (reviewed in Bernfield et al., 1999
) or polyamines (Belting et al., 1999
).
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Materials and methods |
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Cell culture and radiolabeling
ECV 304 (a transformed vascular endothelial cell line) was cultured as monolayers in Dulbeccos Modified Earles Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml) in an incubator with humidified atmosphere and 5% CO2 at 37°C. Confluent cells were preincubated for 1 h in labeling medium supplemented with 2 mM glutamine and serum. The labeling medium was low-sulfate (0.05 mM) MgCl2-containing DMEM with serum. When NO-synthase inhibitors/nitrite-quenchers were tested, cells were incubated in a low-arginine medium (M 199). Drugs used were: BFA (10 µg/ml) or suramin (0.2 mM). Radioactivity was measured by ß-scintillation.
Extraction and isolation of cell-associated proteoglycans
After the incubations, medium was collected and pooled with two washings with ice-cold PBS (0.137 M NaCl, 3 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.5). Cells were extracted with 0.10.2 ml/cm2 dish of 0.15 M NaCl, 10 mM EDTA, 2% (v/v) Triton X-100, 10 mM KH2PO4, pH 7.5, 5 µg/ml ovalbumin containing 10 mM N-ethylmaleimide, and 1 mM diisopropylphosphoro-fluoridate on a slow shaker at 4°C for 10 min. Isolation and purification of PG was performed according to two different methods. Procedure I included chromatography on DEAE-cellulose, Superose 6, and Mono Q, and procedure II consisted of Alcian Blue precipitation (Björnsson, 1993) followed by electrophoresis (SDSPAGE).
In procedure I extracts were mixed with 1.3 vol. of 7 M urea, 10 mM Tris, pH 7.5, 0.1% Triton X-100, 10 mM NEM and passed over a 1 ml column of DE-53 equilibrated with 6 M urea, 0.5 M NaOAc, pH 5.8, 5 µg/ml ovalbumin, 0.1% Triton X-100. After sample application, the columns were washed successively with 10 ml portions of (1) equilibration buffer (see above); (2) 6 M urea, 10 mM Tris, pH 8.0, 5 µg/ml ovalbumin, 0.2% Triton X-100; and (3) 50 mM Tris pH 7.5. Bound material was eluted with 5 x 1 ml 4 M guanidine-HCl, 50 mM NaOAc, pH 5.8, 5 µg/ml ovalbumin, 0.2% Triton X-100. Radioactive fractions were pooled, precipitated with 5 vol of 95% ethanol and 100 µg of dextran as carrier, overnight at 20°C. Samples were centrifuged in a Beckman JS-7.5 at 4000 r.p.m. and 4°C for 45 min and dissolved in 4 M guanidine-HCl, 50 mM NaOAc, pH 5.8, 0.2% Triton X-100. They were then subjected to gel permeation FPLC on Superose 6 at a flow rate of 0.4 ml/min in the same buffer. Radioactivity was determined and fractions were pooled. Some fractions were chromatographed on Mono Q after buffer-change on Centricon 30. The buffer used was 7 M urea, 10 mM Tris, pH 8.0, 0.1% Triton X-100 and the gradient was between 0.3 M NaCl (fraction 10) to 1.2 M NaCl (fraction 70) in the same buffer. Radioactive PGs were pooled and precipitated as above.
Immunoisolation
Cell medium was decanted, and the cell layer was washed with PBS and solubilized in PBS containing 0.1% (w/v) SDS, 0.5 % (v/v) Triton X-100, 0.5 % (w/v) sodium deoxycholate by passage up and down a glass pipette at 4°C for 10 min. After addition of PMSF (a 1/1000 dilution of a saturated solution in ethanol), the medium and cell extract were treated with protein A Sepharose CL-4B (1/100) for at least 1 h on a slow shaker at 4°C. The supernatant was collected and treated with anti-glypican-1 antiserum (diluted 1/200) at 4°C overnight. Immune complexes were recovered on protein A Sepharose, which was washed six times with 0.15 M NaCl, 10 mM Tris, pH 7.4 containing 0.2% (v/v) Tween 20. Bound material was released by boiling in SDS-buffer and subjected to PAGE.
Degradation procedures
GAG chains were released from PG by treatment with 0.5 M NaOH, 0.1 M NaBH4 at room temperature overnight. Samples were neutralized with HOAc, freeze-dried, and redissolved for analysis by gel-permeation chromatography on Superose 6 as above. Cleavage of N-unsubstituted glucosamines was carried out with nitrous acid at pH 3.9 (Lindahl et al., 1973). HS-chains were also degraded with HS lyase (3 mU/ml) in the presence of proteinase inhibitors as described (Fransson et al., 1995
). Digestions were terminated by heating at 100°C for 1 min. The volume of samples was reduced and buffer changes were made by centrifugation in Centricon 30. Material was recovered by ethanol precipitation, dissolved in SDS-buffer and analyzed on SDSPAGE. Carrier protein (ovalbumin) and HS or dextran (50100 µg) were added prior to each purification and degradation step.
SDSPAGE
Radiolabeled PG and HS lyase-treated PG were dissolved in SDS-buffer consisting of 5% (w/v) SDS, 20% (v/v) glycerol, 4 mM EDTA, 0.04% bromphenol blue, 125 mM TrisHCl, pH 6.8, and 10% (v/v) ß-mercaptoethanol. The samples were boiled for 2 min before loading. For immunostaining gels were equilibrated in transfer buffer (92 mM glycine, 0.01 M Tris, pH 8.3, 20% metanol) for 30 min. Transfer to PVDF-membrane was carried out overnight at 4°C and a constant voltage of 20 V. After a 1 h exposure to PBS containing 10% milk (3% fat), the membrane was incubated with 20 µg/ml Mab S1 (anti-glypican) or 20 µg/ml Mab 2E9 (anti-syndecan 1+3) in PBS containing 5% milk (washing buffer) for 2 h. The membrane was rinsed twice for 5 min with washing buffer and further incubated for 1 h with horseradish peroxidaseconjugated anti-mouse IgG diluted 1:5000 in washing buffer. After three washings in washing buffer and two washings in TBS (100 mM Tris, 0.9% NaCl, pH 7.5), the membrane was finally developed with Supersignal substrate for chemiluminiscence and autoradiographed.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Bai,X., Bame,K.J., Habuchi,H., Kimata,K. and Esko,J.D. (1997) Turnover of heparan sulfate depends on 2-O-sulfation of uronic acids. J. Biol. Chem., 272, 2317223179.
Belting,M., Persson,S. and Fransson,L.-Å. (1999) Proteoglycan involvement in polyamine uptake. Biochem. J., 338, 317323.[ISI][Medline]
Bernfield,M., Götte,M., Park,P.W., Reizes,O., Fitzgerald,M.L., Lincecum,J. and Zako,M. (1999) Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem., 68, 729777.[ISI][Medline]
Björnsson,S. (1993) Simultaneous preparation and quantitation of proteoglycans by precipitation with Alcian Blue. Anal. Biochem., 210, 282291.[ISI][Medline]
David,G. (1993) Integral membrane heparan-sulfate proteoglycans. FASEB J., 7, 10231030.
David,G., Lories,V., Decock,B., Marynen,P., Cassiman J.-J. and Van den Berghe,H. (1990) Molecular cloning of a phosphatidylinositol-anchored membrane heparan sulfate proteoglycan from human lung fibroblasts. J. Biol. Chem., 267, 48704877.
Edgren,G., Havsmark,B., Jönsson,M. and Fransson L.-Å. (1997) Glypican (heparan sulfate proteoglycan) is palmitoylated, deglycanated and reglycanated during recycling in skin fibroblasts. Glycobiology, 7, 103112.[Abstract]
Feron,O., Saldana,F., Michel,J.B. and Michel,T. (1998) The endothelial nitric-oxide synthase-caveolin regulatory cycle. J. Biol. Chem., 273, 31253128.
Fransson,L.-Å., Karlsson,P. and Schmidtchen,A. (1992) Effects of cycloheximide, brefeldin A, suramin, heparin and primaquine on proteoglycan and glycosaminoglycan biosynthesis in human embryonic skin fibroblasts. Biochim. Biophys. Acta, 1137, 287297.[ISI][Medline]
Fransson,L.-Å., Edgren,G., Havsmark,B. and Schmidtchen,A. (1995) Recycling of a glycosylphosphatidyl-inositol-anchored heparan sulfate proteoglycan (glypican) in skin fibroblasts. Glycobiology, 5, 407415.[Abstract]
Fransson,L.-Å., Belting,M., Edgren,G., Jönsson,M., Mani,K., Schmidtchen,A. and Wiik,P. (1998) Degradation and reprocessing of heparan sulfate in recycling glypican (heparan sulfate proteoglycan). Trends Glycosci. Glycotechnol., 10, 8194.[ISI]
Garcia-Cardenas,G., Oh,P., Liu,J., Schnitzer,J.E. and Sessa,W.C. (1996) Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling. Proc. Natl. Acad. Sci. USA, 93, 64486453.
Godder,K., Vlodavsky,I., Eldor,A., Weksler,B.B., Haimovitz-Friedmann,A. and Fuks,Z. (1991) Heparanase activity in cultured endothelial cells. J. Cell Physiol., 148, 274280.[ISI][Medline]
Humphries,D.E., Sullivan,B.M., Aleixo,M.D. and Stow,J.L. (1997) Localization of human heparan glucosaminyl N-deacetylase/N-sulfotransferase to the trans-Golgi network. Biochem. J., 325, 351357.[ISI][Medline]
Hunziker,W., Whitney,A. and Mellman,I. (1991) Selective inhibition of transcytosis by BFA in MDCK cells. Cell, 67, 617627.[ISI][Medline]
Irokawa,M., Nishinaga,M., Ikeda,U., Shinoda,Y., Suematsu,M., Gouda,N., Ishimura,Y. and Shimada,K. (1997) Endothelial-derived nitric oxide preserves anticoagulant heparan sulfate expression in cultured porcine endothelial cells. Atherosclerosis, 135, 917.[ISI][Medline]
Klausner,R.D., Donaldson,J.G. and Lippincott-Schwartz,J. (1992) BFA: insight into the control of membrane traffic and organelle structure. J. Cell Biol., 116, 10711080.[ISI][Medline]
Lindahl,U., Bäckström,G., Jansson,L. and Hallén,A. (1973) Biosynthesis of heparin II. Formation of sulfamino groups. J. Biol. Chem., 248, 72347241.
Lindahl,U., Kusche-Gullberg,M. and Kjellén,L. (1998) Regulated diversity of heparan sulfate. J. Biol. Chem., 273, 2497924982.
Lindblom,A. (1991) Structure of endothelial cell proteoglycans. Characterization of biosynthetic and degradative components in tissue culture. Ph.D. thesis, Lund University, Sweden.
Lindblom,A., Carlstedt,I. and Fransson,L.-Å. (1989) Identification of the core proteins in proteoglycans synthesized by vascular endothelial cells. Biochem. J. 261, 145153.[ISI][Medline]
Mertens,G., Cassiman,J.-J., Van den Berghe,H., Vermylen,J. and David,G. (1992) Cell surface heparan sulfate proteoglycan from human vascular endothelial cells. J. Biol. Chem., 267, 2043520443.
Misra,K.B., Kim,K.C., Cho,S.Y., Low,M.G. and Bensadoun,A. (1994) Purification and characterization of adipocyte heparan sulfate proteoglycans with affinity for lipoprotein lipase. J. Biol. Chem., 269, 2383823844.
Nakajima,M., DeChavigny,A., Johnson,C.E., Hamada,J., Stein,C.A. and Nicolson,G.L. (1991) Suramin. A potent inhibitor of melanoma heparanase and invasion. J. Biol. Chem., 266, 96619666.
Norgard-Sumnicht,K. and Varki,A. (1995) Endothelial heparan sulfate proteoglycans that bind to L-selectin have glucosamine residues with unsubstituted amino groups. J. Biol. Chem., 270, 1201212024.
Prabhakar,P., Thatte,H.S., Goetz,R.M., Cho,M.R., Golan,D.E. and Michel,T. (1998) Receptor-regulated translocation of endothelial nitric-oxide synthase. J. Biol. Chem., 273, 2738327388.
Sandbäck-Pikas,D., Li,J.-P., Vlodavsky,I. and Lindahl,U. (1998) Substrate specificity of heparanases from human hepatoma and platelets. J. Biol. Chem., 273, 1877018777.
Schmidtchen,A. and Fransson,L.-Å. (1994) Analysis of heparan sulfate chains and oligosaccharides from proliferating and quiescent fibroblasts. A proposed model for endoheparanase activity. Eur. J. Biochem., 223, 211221.[Abstract]
Schmidtchen,A., Sundler,R. and Fransson,L.-Å. (1990) A fibroblast heparan sulfate proteoglycan with a 70 kDa core protein is linked to membrane phosphatidylinositol. Glycoconjugate J., 7, 563572.[ISI][Medline]
Seidenbecher,C.I., Richter,K., Rauch,U., Fässler,R., Garner,C.C. and Gundelfinger,E.D. (1995) Brevican, a chondroitin sulfate proteoglycan of rat brain, occurs as secreted and cell surface glycosylphosphatidylinositol-anchored isoforms. J. Biol. Chem., 270, 2720627212.
Shaul,P.W., Smart,E.J., Robinson,L.J., German,Z., Yuhanna,I.S., Ying,Y. Anderson,R.G.W. and Michel,T. (1996) Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J. Biol. Chem., 271, 65186522.
Shukla,D., Liu,J., Blaiklock,P., Shworak,N.W., Bai,X., Esko,J.D., Cohen,G.H., Eisenberg,R.J., Rosenberg,R.D. and Spear,P.G. (1999) A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry. Cell, 99, 1322.[ISI][Medline]
Smart,E.J., Ying,Y.S., Conrad,P.A. and Anderson,R.G. (1994) Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation. J. Cell Biol., 127, 11851197.[Abstract]
Taipale,J. and Keski-Oja,J. (1997) Growth factors in the extracellular matrix. FASEB J., 11, 5159.
van den Born,J., Gunnarsson,K., Bakker,M.A.H., Kjellén,L., Kusche-Gullberg,M., Maccarana,M., Berden,J.H.M. and Lindahl,U. (1995) Presence of N-unsubstituted glucosamine units in native heparan sulfate revealed by a monoclonal antibody. J. Biol. Chem., 270, 3130331309.
Vilar,R.E., Ghael,D., Li,M., Bhagat,D.D., Arrigo,L.M., Cowman,M.K., Dweck,H.S. and Rosenfeld,L. (1997) Nitric oxide degradation of heparin and heparan sulfate. Biochem. J., 324, 473479.[ISI][Medline]
Voogd,T.E., Vansterkenburg,L.M., Wilting,J. and Janssen,L.H.M. (1993) Recent research on the biological activity of suramin. Pharmacol. Rev., 45, 177203.[ISI][Medline]
Williams,D.L.H. (1996) S-Nitrosothiols and role of metal ions in decomposition to nitric oxide. Methods Enzymol., 268, 299308.[ISI][Medline]
Wink,D.A., Grisham,M.B., Mitchell,J.B. and Ford,P.C. (1996) Direct and indirect effects of nitric oxide in chemical reactions relevant to biology. Methods Enzymol., 268, 1231.[ISI][Medline]
Yanagashita,M. and Hascall,V.C. (1992) Cell surface heparan sulfate proteoglycans. J. Biol. Chem., 267, 94519454.