Elliott P. Joslin Research Laboratory, Joslin Diabetes Center and the Departments of Medicine and Biological Chemistry, Harvard Medical School, Boston, MA 02215, USA
Received on May 5, 2000; revised on June 2, 2000; accepted on June 6, 2000.
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Abstract |
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Key words: sulfated N-linked oligosaccharides/influenza virus hemagglutinin/sulfotransferases/subcellular localization/glycoprotein processing sequence
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Introduction |
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The apically directed HA glycoprotein of the influenza virus can serve as an attractive model for inquiries into the sulfation process as the multiple complex N-linked oligosaccharides of this envelope constituent (Keil et al., 1985; Mir-Shekari et al., 1997
) do not contain other anionic groups due to the intracellular action of the viral neuraminidase (Basak et al., 1985
; Kaverin and Klenk, 1999
). Furthermore, studies with influenza virusinfected kidney cells have indicated that the sulfate substituents on the HA glycoprotein occur on C-3 of the galactose and C-6 of the N-acetylglucosamine in the N-acetyllactosamine branches, as well as on C-6 of the outer N-acetylglucosamine of the di-N-acetylchitobiose core (Karaivanova and Spiro, 1998
).
Although it has been suggested from experiments employing brefeldin A that sulfation of N-linked oligosaccharides is a late biosynthetic event (Sampath et al., 1992; Karaivanova and Spiro, 1998
), the sequence and subcellular location of the various sulfation steps in relation to the processing of the carbohydrate units has not yet been defined.
In view of the structural information already available in regard to the HA sulfation sites in the mature influenza virus produced by infected MDCK cells, we have undertaken an evaluation of the temporal course of sulfate addition by studying this glycoprotein in subcellular fractions separated by Nycodenz gradient centrifugation subsequent to pulse-chase labeling with [35S]sulfate and have correlated these findings with the intracellular localization of the in vitro determined sulfotransferases and various oligosaccharide processing enzymes. Our findings indicate that sulfation of the N-linked oligosaccharides at even the more internally located sites begins only after endo H resistance has been established and continues on into the TGN.
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Results |
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Effect of endoglycosidase treatment on radiolabeled HA from Nycodenz gradient separated subcellular fractions
While PNGase digestion of [35S]sulfate-labeled HA (peak 2) resulted in a complete release of its radiolabel, this protein was resistant to endo H treatment (Figure 3). These observations indicated that the sulfate in HA was associated with N-linked oligosaccharides and that these carbohydrate units had undergone substantial processing prior to their sulfation. In contrast, the [3H]mannose-labeled HA from a 30 min pulse was susceptible to the action of endo H as would be anticipated if it was still situated in the ER compartment (Figure 3).
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Subcellular distribution of sulfotransferases
Examination of the two major sulfotransferases involved in the biosynthesis of the HA N-linked oligosaccharides by Nycodenz gradient centrifugation revealed that the distribution of the PAPS:Gal-3-O-sulfotransferase was similar to the galactosyltransferase (Figure 6, upper panel), with the enzyme activity occurring in both the Golgi and the TGN regions. In contrast the PAPS:GlcNAc-6-O-sulfotransferase was localized almost exclusively in the Golgi fractions (Figure 6, lower panel), where its peak was shifted to a slightly denser region than the Gal-3-O-sulfotransferase. No evidence for these sulfotransferases was detected in the ER region and furthermore, assay for the recently described enzyme which sulfates 3'-sialyl-N-acetyllactosamine on C-6 of the galactose residue (Spiro and Bhoyroo, 1998) indicated no discernible activity throughout the gradient (data not shown).
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Discussion |
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Consistent with the specificities of the known stepwise assembly of the N-acetyllactosamine branches of complex oligosaccharides, we observed that the transferases involved in the attachment of sulfate groups onto the N-acetylglucosamine and galactose residues of these chains distributed in a different manner on the Nycodenz gradient. The PAPS:GlcNAc-6-O-sulfotransferase which acts on terminal N-acetylglucosamine in ß-linkage to mannose, migrated in the gradient to a position compatible with the medial/trans Golgi, suggesting that sulfation of this sugar is completed before the glycoprotein reaches the TGN. In contrast, the PAPS:Gal-3-O-sulfotransferase, which is specific for terminal galactose in ß14 linkage to N-acetylglucosamine, was found in both Golgi and TGN locations. Since ß-galactosylation of the oligosaccharide branches must precede the sulfation catalyzed by the latter enzyme, the presence of the 3-O-sulfotransferase in both subcellular locations is consistent with the occurrence of the UDP-Gal:GlcNAc galactosyltransferase in these two sites in MDCK cells. The presence of Gal-3-O-sulfotransferase in the TGN would be particularly important in influenza virus infected cells since sialic acid residues of the HA envelope glycoprotein are known to be released intracellularly through the action of the viral neuraminidase (Basak et al., 1985; Kaverin and Klenk, 1999
), thereby providing additional galactose sites for 3-O-sulfation; in this instance, the sulfotransferase would most likely be in competition with the
13-galactosyltransferase, as galactose residues in
-linkage to the N-acetyllactosamine chains have been found in canine proteins (Spiro and Bhoyroo, 1984
) and indeed terminal
-galactosyl groups have been reported to occur on HA complex oligosaccharides from influenza virus infected Madin-Darby bovine kidney cells (Mir-Shekari et al., 1997
). However, since we find that the radiolabel in the Gal-3-SO4 is greater than that in GlcNAc-6-SO4 it would appear that the terminal ß-Gal groups are more available to the Gal-3-O-sulfotransferase than terminal N-acetylglucosamine is to the GlcNAc-6-O-sulfotransferase; the latter residues are probably rapidly covered by an active UDPGal:GlcNAc ß14 galactosyltransferase. Moreover it has been shown that even when the N-acetylglucosamine has been substituted with sulfate it still can serve as an acceptor for the ß14 galactosyltransferase (Spiro et al., 1996
).
Although a sulfate substituent has been reported to occur on the outer N-acetylglucosamine of the di-N-acetylchitobiose core of the N-linked oligosaccharides (Merkle et al., 1985), where it has been located on the C-6 of this sugar residue (Karaivanova and Spiro, 1998
), no specific transferase involved in this attachment has yet been identified. It was however apparent from our pulse-chase, endo H, and hydrazine-nitrous acid fragmentation studies, that this internally located substituent was not attached in a pre-Golgi compartment. This would be in agreement with studies employing glycosidase inhibitors which indicated that this internal sulfation must be proceeded by processing with ER enzymes (Merkle et al., 1985
).
Studies employing brefeldin A in a bovine endothelial cell line (Sampath et al., 1992), as well as in LLC-PK1 cells (Karaivanova and Spiro, 1998
) have indicated that sulfation of N-linked oligosaccharides is a late event, possibly occurring in the TGN. Our investigations employing an alternate approach to determine the subcellular locale for the attachment of these substituents indicated that while this process does take place in the TGN to a certain extent, substantial sulfation already occurs in the medial/trans Golgi cisternae.
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Materials and methods |
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Radiolabeling of cells
Plates of the influenza virusinfected MDCK cells were washed with sulfate-free DMEM and after a preincubation for 15 min in 2 ml of this medium, the cells were metabolically radiolabeled with 450 µCi/plate of [35S]sulfate (carrier-free, DuPont, New England Nuclear) and incubation was performed at 37°C for varying periods of time. At end of the incubation, the medium was removed and the plates were washed with PBS containing 2 mM unlabeled sodium sulfate. In a number of instances a 30 min pulse was followed by a 60 or 90 min chase in the presence of 2 mM of the unlabeled sulfate. The infected cells were also radiolabeled with [2-3H]mannose (24.3 Ci/mmol; DuPont-New England Nuclear) by incubation with 1.8 mCi/plate of this substrate in glucose-free medium for 30 min; at the end of the incubation the cells were washed with PBS containing 2 mM mannose.
Separation of subcellular organelles by Nycodenz gradient centrifugation
Radiolabeled cells were scraped from the plates in the PBS solution and following recovery by centrifugation for 10 min at 600 x g, they were homogenized in 0.8 ml of 0.25 M sucrose containing 10 mM Tris acetate, pH 7.4, by 20 passages through a 25 gauge needle. After centrifugation at 200 x g for 10 min in an Eppendorf microfuge, the pellet was rehomogenized in 200 µl of the sucrose solution, recentrifuged and the combined supernatants (approximately 1 ml) were applied to the top of a gradient consisting of 1.1 ml layers of 28, 23, 21, 19, 17, 15, 13, 11, and 9% (w/v) Nycodenz (Sigma). For this gradient the required concentrations of Nycodenz were obtained by dilution of a 28% (w/v) stock solution, as described by Rickwood et al. (1982). The gradients were allowed to diffuse overnight at room temperature before application of the samples; centrifugation was then carried out in an SW40 rotor (Beckman Instruments) for 3 h at 37,000 rpm with no brake and unloaded from the top. Refractive index measurements were made to determine the Nycodenz density (Rickwood et al., 1982
). Gradient fractions were concentrated using Millipore Ultrafree filters and the Nycodenz and sucrose were removed by repeated filtration using buffer appropriate for the enzyme assays; aliquots of the concentrated fractions were also taken for examination by SDS-PAGE.
Marker enzyme assays
The separation of the subcellular fractions achieved by the Nycodenz gradient centrifugation, which has previously been utilized to fractionate mouse liver subcellular organelles (Graham et al., 1990) and to separate ER and Golgi fractions (Gilbert et al., 1998
), was determined by the measurement of a number of enzymes. UDP-galactose:N-acetylglucosamine galactosyltransferase was assayed as previously described (Lubas and Spiro, 1987
), while endomannosidase and glucosidase II activities were determined by the release of glucosylmannose or glucose, respectively, from 14C-labeled Glc1Man9GlcNAc substrate (Hiraizumi et al., 1993
). To measure sialyltransferase, the incorporation of radioactivity from CMP-[3H]NeuAc (DuPont, New England Nuclear, 100,000 dpm, 3 nmol) into sialic acidfree fetuin (200 µg) was measured in a total volume of 50 µl of 0.1 M phosphate, pH 6.8, containing 0.15% Triton X-100 after an incubation of 60 min at 37°C. The protein was recovered by precipitation with ice-cold 5% (w/v) phosphotungstic acid in 0.5 M HCl and after extensive washing, the pellets, dissolved in 1 N NaOH, were transferred to vials and subsequent to neutralization with 2 N acetic acid were submitted to scintillation counting. Protein analyses were carried out by the dye-binding technique of Bradford (1976)
.
Assay of sulfotransferases
PAPS:galactose-3-O-sulfotransferase (Kato and Spiro, 1989) and PAPS: N-acetylglucosamine-6-O-sulfotransferase (Spiro et al., 1996
) were assayed under conditions previously described by incubating the gradient fractions with 0.7 µCi of [35S]PAPS (2.35 Ci/mmol; DuPont-New England Nuclear) and 80 nmol of N-acetyllactosamine or GlcNAcß1
6Man
1-O-Me at 30°C for 90 min and 3 h, respectively; each enzyme was assayed on equal aliquots of the gradient fractions. The samples were deproteinized by the addition of ethanol and subsequently desalted and separated from radiolabeled substrate by chromatography on charcoal/Celite columns as described previously (Kato and Spiro, 1989
; Spiro et al., 1996
). After thin layer chromatography of the material eluted from the columns on cellulose-coated plates, the sulfated saccharide products ([35S]Gal(3-SO4)ß14GlcNAc and [35S]GlcNAc(6-SO4)ß16Manß1-O-Me) were detected by fluorography and after elution with water were quantified by liquid-scintillation counting.
Hydrazine/nitrous acid treatment of glycopeptides from sulfated HA protein
After preparative SDS-PAGE of the various gradient fractions, the location of the HA protein was determined by fluorography and the gel segments containing it were cut out and subjected to Pronase digestion for 72 h with several additions of the enzyme as previously described (Edge et al., 1990). The glycopeptides were separated by filtration on Bio-Gel P-2 (Karaivanova and Spiro, 1998
), dried and then treated with hydrazine for 28 h at 100°C followed by nitrous acid degradation and NaBH4 reduction (Edge and Spiro, 1985
). After passage of the samples through Dowex 50 (H+ form) and removal of the boric acid as methyl borate, the sulfated saccharides were separated by thin layer chromatography on cellulose-coated plates. 3H-labeled disaccharide standards were prepared as described previously (Spiro and Bhoyroo, 1988
).
SDS-PAGE
Electrophoresis was carried out in SDS on 10% polyacrylamide gels (1.5 mm thick) which were overlaid by 3.5% stacking gels, according to the procedure of Laemmli (1970). Radioactive components were visualized by fluorography at 80°C using X-Omatic AR film after treatment with ENHANCE (Du PontNew England Nuclear).
PNGase and endo H digestions
Aliquots of the concentrated gradient fractions were taken to dryness on a Speed-Vac (Savant Instruments, Holbrook, NY) and were then boiled for 3 min in 25 µl of buffer containing 0.5% SDS and 0.1 M 2-mercaptoethanol. The denatured proteins were then incubated in a 125 µl volume with either 0.6 U of PNGase (PNGase F, Oxford GlycoSystems) or 10 mU endo H (Genzyme) for 30 h and 48 h, respectively, at 37°C in the presence of aprotinin (10 U/ml), phenylmethylsulfonyl fluoride (2 mM) and toluene. The composition of the buffer for the PNGase digestion was 60 mM Tris/HCl, pH 8.6, 6 mM EDTA, 1.0% (v/v) Nonidet P40, 0.1% SDS and 20 mM 2-mercaptoethanol, while that for the endo H treatment was 0.2 M sodium citrate, pH 5.2, 0.1% SDS and 20 mM 2-mercaptoethanol. At the end of the digestions the samples, as well as control incubations without enzyme, were examined by SDS-PAGE followed by fluorography.
Thin layer chromatography
Chromatography was performed on plastic sheets precoated with cellulose (0.1 mm thick, Merck) in pyridine/ethyl acetate/water/acetic acid, 5:5:3:1. A wick of Whatman 3MM paper was clamped to the top of the plate during chromatography; components were revealed by fluorography.
Radioactivity measurements
Liquid scintillation was carried out in Monofluor (National Diagnostics) with a Beckman LS7500 instrument. Components on electrophoretic gels and thin layer chromatographic plates were detected by fluorography at 80°C with the use of X-Omatic AR film (Eastman Kodak) after treatment with ENHANCE (New England Nuclear) or spraying with a scintillation mixture containing 2-methylnaphthalene (Spiro and Spiro, 1985), respectively. The amount of radioactivity present in electrophoretic gel bands was determined by densitometry of the fluorographs using a model 300A Molecular Dynamics densitometer (Sunnyvale, CA).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Bernstein,H.B. and Compans,R.W. (1992) Sulfation of the human immunodeficiency virus envelope glycoprotein. J. Virol., 66, 69536959.[Abstract]
Bradford,M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248254.[ISI][Medline]
Brändli,A.W. and Simons, K (1989) A restricted set of apical proteins recycle through the trans-Golgi network in MDCK cells. EMBO J., 8, 32073213.[Abstract]
Brockhausen,I. and Kuhns,W. (1997) Role and metabolism of glycoconjugate sulfation. Trends. Glycosci. Glycotechnol., 9, 379398.[ISI]
Chege,N.W. and Pfeffer,S.R. (1990) Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi cisternae from the trans-Golgi network. J. Cell Biol., 111, 893899.[Abstract]
Compans,R.W. and Pinter,A. (1975) Incorporation of sulfate into influenza virus glycoproteins. Virology 66, 151160.[ISI][Medline]
Daniels,P.U. and Edwardson,J.M. (1988) Intracellular processing and transport of influenza virus envelope proteins in Madin-Darby canine kidney cells. Biochem. J., 252, 693700.[ISI][Medline]
Edge,A.S.B. and Spiro,R.G. (1985) Structural elucidation of glycosaminoglycans through characterization of disaccharides obtained after fragmentation by hydrazine-nitrous acid treatment. Arch. Biochem. Biophys., 240, 560572.[ISI][Medline]
Edge,A.S.B., Kahn,C.R. and Spiro,R.G. (1990) Insulin receptor carbohydrate units contain poly-N-acetyllactosamine chains. Endocrinology 127, 18871895.[Abstract]
Gilbert,A, Jadot,M., Leontieva,E., Wattiaux-De Coninck,S. and Wattiaux,R. (1998) F508 CFTR localized in the endoplasmic reticulum-Golgi intermediate compartment in cystic fibrosis cells. Exp. Cell Res., 242, 144152.[ISI][Medline]
Graham,J.M., Ford,T. and Rickwood,D. (1990) Isolation of the major subcellular organelles from mouse liver using Nycodenz gradients without the use of an ultracentrifuge. Anal. Biochem., 187, 318323.[ISI][Medline]
Gravotta,D., Adesnik,M. and Sabatini,D.D. (1990) Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins. J. Cell Biol., 111, 28932908.[Abstract]
Griffiths,G. and Simons,K. (1986) The trans Golgi network: sorting at the exit site of the Golgi complex. Science, 234, 438443.[ISI][Medline]
Hiraizumi,S., Spohr,U. and Spiro,R.G. (1993) Characterization of endomannosidase inhibitors and evaluation of their effects on N-linked oligosaccharide processing during glycoprotein biosynthesis. J. Biol. Chem., 268, 99279935.
Karaivanova,V.K. and Spiro,R.G. (1998) Sulphation of N-linked oligosaccharides of vesicular stomatitis and influenza virus envelope glycoproteins: host cell specificity, subcellular localization and identification of substituted saccharides. Biochem. J., 329, 511518.[ISI][Medline]
Kato,Y. and Spiro,R.G. (1989) Characterization of a thyroid sulfotransferase responsible for the 3-O-sulfation of terminal ß-D-galactosyl residues in N-linked carbohydrate units. J. Biol. Chem., 264, 33643371.
Kaverin,N.V. and Klenk,H.-D. (1999) Strain-specific differences in the effect of influenza A virus neuraminidase on vector-expressed hemagglutinin. Arch. Virol., 144, 781786.[ISI][Medline]
Keil,W., Geyer,R., Dabrowski,J., Dabrowski,U., Niemann,H., Stirm,S. and Klenk,H.-D. (1985) Carbohydrates of influenza virus. Structural elucidation of the individual glycans of the FPV hemagglutinin by two-dimensional 1H n.m.r. and methylation analysis. EMBO J., 4, 27112720.[Abstract]
Laemmli,U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
Lubas,W.A. and Spiro,R.G. (1987) Golgi endo--D-mannosidase from rat liver: a novel N-linked carbohydrate unit processing enzyme. J. Biol. Chem., 262, 37753781.
Lucocq,J.M., Brada,D. and Roth,J. (1986) Immunolocalization of the oligosaccharide trimming enzyme glucosidase II. J. Cell Biol., 102, 21372146.[Abstract]
Merkle,R.K., Elbein,A.D. and Heifetz,A., (1985) The effect of swainsonine and castanospermine on the sulfation of the oligosaccharide chains of N-linked glycoproteins. J. Biol. Chem., 260, 10831089.
Mir-Shekari,S.Y., Ashford,D.A., Harvey,D.J., Dwek,R.A. and Schulze,I.T. (1997) The glycosylation of the influenza A virus hemagglutinin by mammalian cells. A site-specific study. J. Biol. Chem., 272, 40274036.
Nilsson,T., Pypaert,M., Hoe,M.H., Slusarewicz,P., Berger,E.G. and Warren,G. (1993) Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells. J. Cell Biol., 120, 513.[Abstract]
Pinter,A. and Compans,R.W. (1975) Sulfated components of enveloped viruses. J. Virol., 16, 859866.[ISI][Medline]
Prehm,P., Scheid,A. and Choppin,P.W, (1979) The carbohydrate structure of the glycoproteins of the paramyxovirus SV5 grown in bovine kidney cells. J. Biol. Chem., 254, 96699677.[ISI][Medline]
Rabouille,C., Hui,N., Hunte,F., Kieckbusch,R., Berger,E.G., Warren,G. and Nilsson,T. (1995) Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides. J. Cell Sci., 108, 16171627.
Rickwood,D., Ford,T. and Graham,J. (1982) Nycodenz: a new nonionic iodinated gradient medium. Anal. Biochem., 123, 2331.[ISI][Medline]
Sampath,D., Varki,A. and Freeze,H.H. (1992) The spectrum of incomplete N-linked oligosaccharides synthesized by endothelial cells in the presence of brefeldin A. J. Biol. Chem. 267, 44404455.
Shilatifard,A., Merkle,R.K., Helland,D.E., Welles,J.L., Haseltine,W.A. and Cummings,R.D. (1993) Complex-type N-linked oligosaccharides of gp120 from human immunodeficiency virus type 1 contain sulfated N-acetylglucosamine. J. Virol., 67, 943952.[Abstract]
Skelton,T.P., Hooper,L.V., Srivasta,V., Hindsgaul,O. and Baenziger,J.U. (1991) Characterization of a sulfotransferase responsible for the 4-O-sulfation of a terminal ß-N-acetyl-D-galactosamine on asparagine-linked oligosaccharides of glycoprotein hormones. J. Biol. Chem., 266, 1714217150.
Spiro,R.G. and Bhoyroo,V.D. (1984) Occurrence of -D-galactosyl residues in the thyroglobulins from several species. Localization in the saccharide chains of the complex carbohydrate units. J. Biol. Chem., 259, 98589866.
Spiro,M.J. and Spiro,R.G. (1985) Effect of anion-specific inhibitors on the utilization of sugar nucleotides for N-linked carbohydrate unit assembly by thyroid endoplasmic reticulum vesicles. J. Biol. Chem., 260, 58085815.[Abstract]
Spiro,R.G. and Bhoyroo,V.D. (1988) Occurrence of sulfate in the asparagine-linked complex carbohydrate units of thyroglobulin. Identification and localization of galactose 3-sulfate and N-acetylglucosamine 6-sulfate residues in the human and calf proteins. J. Biol. Chem., 263, 1435114358.
Spiro,R.G. and Bhoyroo,V.D. (1998) Characterization of a spleen sulphotransferase responsible for the 6-O-sulphation of the galactose residue in sialyl-N-acetyl-lactosamine sequences. Biochem. J., 331, 265271.[ISI][Medline]
Spiro,R.G., Yasumoto,Y. and Bhoyroo,V. (1996) Characterization of a rat liver Golgi sulphotransferase responsible for the 6-O-sulphation of N-acetylglucosamine residues in ß-linkage to mannose: role in assembly of sialyl-galactosl-N-acetylglucosamine 6-sulphate sequence of N-linked oligosaccharides. Biochem. J., 319, 209216.[ISI][Medline]
Sugumaran,G., Katsman,M. and Silbert,J.E. (1992) Effect of brefeldin A on the localization of chondroitin sulfate synthesizing enzymes. Activities in subfractions of the Golgi from chick embryo epiphyseal cartilage. J. Biol. Chem., 267, 88028806.
Taatjes,D.J., Roth.,J., Shaper,N.L., Shaper,J.H. (1992) Immunocytochemical localization of ß1,4 galactosyltransferase in epithelial cells from bovine tissues using monoclonal antibodies. Glycobiology, 2, 579589.[Abstract]
Torii,T., Fukuta,M. and Habuchi,O. (2000) Sulfation of sialyl N-acetyllactosamine oligosaccharides and fetuin oligosaccharides by keratan sulfate Gal-6-sulfotransferase. Glycobiology, 10, 203211.
Wagner,M., Rajasekaran,A.K., Hanzel,D.K., Mayor,S., Rodriguez-Boulan,E., (1994) Brefeldin A causes structural and functional alterations of the trans-Golgi network of MDCK cells. J. Cell Sci., 107, 933943.