Sulfation of the N-linked oligosaccharides of influenza virus hemagglutinin: temporal relationships and localization of sulfotransferases

Mary Jane Spiro and Robert G. Spiro1

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The occurrence of sulfate substituents on several positions of glycoprotein N-linked oligosaccharides prompted us to determine the subcellular localization and temporal relationships of the addition of these anionic groups employing as a model system the hemagglutinin (HA) produced by influenza virus-infected Madin-Darby canine kidney (MDCK) cells. It became apparent from a study of the HA glycoprotein in subcellular fractions resolved by Nycodenz gradient centrifugation following pulse-chase radiolabeling that sulfation of the complex N-linked oligosaccharides occurs only after they have been processed to an endo-ß-N-acetylglucosaminidase–resistant state and have reached the medial/trans Golgi and the trans Golgi network (TGN), with the former carrying out most of the sulfation activity. Hydrazine/nitrous acid/NaBH4 treatment of the HA from the subcellular fractions indicated that C-3 of the galactose as well as C-6 of the N-acetylglucosamine residues of the N-acetyllactosamine chains became sulfated in these post ER fractions, as did the C-6 of the outer N-acetylglucosamine of the di-N-acetylchitobiose core. Consistent with the specificities of the stepwise assembly of the N-acetyllactosamine branches, we observed that the 3'-phosphoadenosine 5'-phosphosulfate (PAPS):GlcNAc-6-O-sulfotransferase migrated in the gradient to a medial/trans Golgi position while in contrast the PAPS:Gal-3-O-sulfotransferase was found in both Golgi and TGN locations. In accordance with the concept that ß-galactosylation must precede the sulfation catalyzed by the latter enzyme, we observed the presence of UDP-Gal:GlcNAc galactosyltransferase in both these sites in the MDCK cells. The presence of the Gal-3-O-sulfotransferase in the TGN is particularly important in the influenza virus-infected cells, as it makes possible the addition of terminal anionic groups after removal of the sialic acid residues by the viral neuraminidase.

Key words: sulfated N-linked oligosaccharides/influenza virus hemagglutinin/sulfotransferases/subcellular localization/glycoprotein processing sequence


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
It has now been well established that the N-linked oligosaccharides of a number of diverse soluble and membrane glycoproteins undergo sulfation as a structural modification (for review, see Brockhausen and Kuhns, 1997Go), and indeed in recent years sulfotransferases which attach these anionic substituents to defined positions on the galactose (Kato and Spiro, 1989Go; Spiro and Bhoyroo, 1998Go; Torii et al., 2000Go), N-acetylgalactosamine (Skelton et al., 1991Go) and N-acetylglucosamine (Spiro et al., 1996Go) residues of these carbohydrate units have been described. Of particular interest has been the recognition that envelope glycoproteins including those from the vesicular stomatitis (Pinter and Compans, 1975Go; Karaivanova and Spiro, 1998Go), influenza (Compans and Pinter, 1975Go; Karaivanova and Spiro, 1998Go), and human immunodeficiency (Bernstein and Compans, 1992Go; Shilatifard et al., 1993Go) viruses as well as paramyxovirus SV5 (Prehm et al., 1979Go) contain N-linked sulfated substituents and it has been shown that the attachment of these groups is to a large extent dependent on the enzymatic machinery of the host (Karaivanova and Spiro, 1998Go).

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., 1985Go; Mir-Shekari et al., 1997Go) do not contain other anionic groups due to the intracellular action of the viral neuraminidase (Basak et al., 1985Go; Kaverin and Klenk, 1999Go). Furthermore, studies with influenza virus–infected 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, 1998Go).

Although it has been suggested from experiments employing brefeldin A that sulfation of N-linked oligosaccharides is a late biosynthetic event (Sampath et al., 1992Go; Karaivanova and Spiro, 1998Go), 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Subcellular distribution of influenza virus proteins after [35S]sulfate pulse-chase labeling of infected MDCK cells
SDS-PAGE indicated that after a 30 min incubation of infected MDCK cells with [35S]sulfate during a 30 min pulse, the influenza virus envelope proteins HA and neuraminidase become prominently radiolabeled in addition to host cell-derived high molecular weight proteoglycans (Figure 1); these components have previously been observed (Compans and Pinter, 1975Go; Karaivanova and Spiro, 1998Go). Examination of the subcellular fractions resolved by Nycodenz gradient centrifugation after a 90 min chase indicated that the radioactivity in these components moved from fractions of intermediate to low density.



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Fig. 1. Examination of subcellular fractions resolved by Nycodenz gradient centrifugation of [35S]sulfate-labeled influenza virus-infected MDCK cells. After an incubation of the cells with [35S]sulfate (450 µCi/plate) for 30 min without (Pulse) or with a subsequent 60 min Chase, the postnuclear supernants of the cell homogenates from 6 plates were applied to a Nycodenz (9–28%, w/v) gradient, as described in Materials and methods. The gradient was unloaded from the top in 1 ml fractions and equal aliquots were then submitted to SDS-PAGE. The components were visualized by fluorography and the position of the influenza HA and neuraminidase (NA) are indicated. The designated molecular weight markers were rabbit muscle phosphorylase (97,000), bovine serum albumin (66,000), and chicken egg ovalbumin (45,000). The electrophoresis of the Pulse and Chase samples were run for different lengths of time. The position of marker enzymes in this gradient are indicated in a subsequent section of the manuscript (i.e., Figure 5).

 
This intracellular movement is evident from plots of the radioactivity associated with the HA proteins during pulse-chase incubations of various lengths (Figure 2). It is apparent that during a 60–90 min chase, most of the radioactivity initially associated with the denser fractions (peak 2) has moved to the top of the gradient (peak 1), which is consistent with the reported half-time for the appearance of HA at the cell surface (Daniels and Edwardson, 1988Go). Since the exit site for influenza virus is believed to be the TGN (Griffiths and Simons, 1986Go; Gravotta et al., 1990Go; Wagner et al., 1994Go), it is likely that peak 1 contains this compartment.



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Fig. 2. Distribution of [35S]sulfate-labeled influenza virus HA on Nycodenz gradients after various times of incubation of MDCK cells. Infected cells were pulsed with [35S]sulfate for 10 or 30 min followed in the latter case by a 60 or 90 min chase. After Nycodenz gradient centrifugation, the fractions were electrophoresed as in Figure 1 and the amount of radioactivity in the HA band visualized by fluorography was determined by densitometry and plotted against the Nycodenz concentration. The fractions were pooled into low density (1) and intermediate density (2) peaks and the percent of total radioactivity in each peak is plotted as a function of total incubation time in the graph to the right of the elution diagrams. When virus-infected MDCK cells were radiolabeled with [2-3H]mannose for 30 min as described in Materials and methods the HA protein emerged in a peak indicated by the arrow (M)

 
After a 30-min pulse with [3H]mannose (arrow, Figure 2) the radiolabeled HA was found to be present at the bottom of the Nycodenz gradient consistent with the localization of newly synthesized N-glycosylated protein in the ER compartment. The separation of the [3H]mannose-(arrow) and [35S]sulfate-labeled HA (peak 2) suggests that the earliest sulfation takes place on already processed precursors in a post-ER (i.e., Golgi) location.

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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Fig. 3. Effect of PNGase and endo H on radiolabeled influenza virus HA obtained from MDCK cells by Nycodenz gradient centrifugation. After a 30 min pulse with [35S]sulfate, the HA in fraction 6–9 (Figure 2) was submitted to SDS-PAGE with (+) or without (-) prior PGNase or endo H as described in Materials and methods. The HA protein obtained in fractions 9 plus 10 after a 30 min incubation with [2-3H]mannose were also electrophoresed with (+) or without (-) a prior endo H digestion; the fast moving component which is split by the endo H represents oligosaccharide-lipids. The [35S]sulfate-labeled HA from fractions 1–3 (Figure 2) was also found to be resistant to endo H and susceptible to PNGase digestions (not shown).

 
Identification of the sulfated saccharides in HA from subcellular fractions separated by Nycodenz gradient centrifugation
Hydrazine/nitrous acid/NaBH4 treatment of the glycopeptides obtained from [35S]sulfate-labeled HA yielded two radioactive disaccharides which migrated on TLC to the position of Gal(3-SO4)ß1–4AnManH2 and Galß1–4AnManH2(6-SO4) (Figure 4). These disaccharides which are derived from Gal(3-SO4)ß1–4GlcNAc and Galß1–4GlcNAc(6-SO4) sequences, respectively, were present in fractions from both peaks 1 and 2, with the former disaccharide predominating. Furthermore, a slow migrating component obtained by cleavage of the di-N-acetylchitobiose sequence of the core and previously characterized as Man3AnManH2(6-S04) (Karaivanova and Spiro, 1998Go) was observed. Only small amounts of sulfated oligosaccharides were present in gradient fractions 8 to 9 which represent the tail of peak 2 and overlap with the ER region.



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Fig. 4. Identification by TLC of the [35S]sulfated saccharides derived from the HA protein by hydrazine/nitrous acid/NaBH4 treatment. Subsequent to a 30 min pulse of influenza virus-infected MDCK cells with [35S]sulfate, various fractions of a Nycodenz gradient (cf. Figure 2) were pooled and submitted to preparative electrophoresis. Glycopeptides from the HA bands prepared by Pronase digestion were submitted to hydrazine/nitrous acid/NaBH4 treatment as described in the Materials and methods and the products of this fragmentation procedure were then applied to a cellulose-coated plate which was developed for 24 h in pyridine/ethyl acetate/water/acetic acid (5:5:3:1); the radioactive components were visualized by fluorography. The positions of Gal(3-SO4)ß1–4AnManH2 [Gal(3S)aM] and Galß1–4AnManH2(6-SO4) [GalaM(6S)] were determined by chromatography of radiolabeled standards. Man3AnManH2(6-SO4) [Man3aM(6S)] was further identified by elution and rechromatography (Karaivanova and Spiro, 1998). The nature of the minor component migrating between Man3aM(6S) and GalaM(6S) is not known.

 
Characterization of subcellular fractions obtained by density gradient centrifugation
On the basis of marker enzymes it was apparent that the Nycodenz density gradient centrifugation of the MDCK cell postnuclear supernatant effectively separated the subcellular organelles into fractions relevant to the present investigation (Figure 5). Glucosidase II, an ER resident protein (Lucocq et al., 1986Go) was located in the densest part of the gradient which coincided with the region in which the endo H-sensitive HA was found subsequent to labeling with a [3H]mannose pulse (compare Figures 2 and 5). In contrast, sialyltransferase, which in MDCK cells (Brändli and Simons, 1989Go) and other cells (Chege and Pfeffer, 1990Go) has been localized to the TGN, was found in the least dense fractions of the gradient which coincided with the [35S]sulfate-labeled HA peak 1 (compare Figures 2 and 5).



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Fig. 5. Distribution of subcellular marker enzyme of MDCK cells after Nycodenz density gradient centrifugation. The postnuclear supernatants of unlabeled cells from six plates were centrifuged on a Nycodenz gradient as in Figures 1 and 2 and aliquots of each fraction were assayed for sialyltransferase (Sial-Tr), galactosyltransferase (Gal-Tr), endomannosidase (EnManase), and glucosidase II (Glcase) as described in Materials and methods. Results are plotted as percentages of maximum activity, which were as follows: sialyltransferase, 11.0 x 10–3 dpm; galactosyltransferase, 18 x 10–3 dpm; endomannosidase, 3.1 x 10–3 dpm; and glucosidase 7.0 x 10–3 dpm.

 
The UDP-Gal:N-acetylglucosamine galactosyltransferase was resolved into two peaks by the Nycodenz centrifugation, of which one overlapped with the sialyltransferase while the other emerged in the intermediate portion of the gradient (Figure 5). This is consistent with dual localization of this enzyme in the TGN and trans-Golgi cisternae as has been observed in a number of cells by immunoelectron microscopy (Taatjes et al., 1992Go; Nilsson et al., 1993Go; Rabouille et al., 1995Go) and moreover a dual localization has also been noted by sucrose density gradient centrifugation (Sugumaran et al., 1992Go). Endomannosidase which has been shown to be a Golgi enzyme by subcellular fractionation studies (Lubas and Spiro, 1987Go) and has more recently been localized to the cis/medial cisternae by immunoelectron microscopy (C.Zuber, M.J.Spiro, B.Guhl, R.G.Spiro, and J.Roth, unpublished observations) has a similar distribution to the more dense galactosyltransferase peak (Figure 5). This indicated that although the Nycodenz gradient achieved a clear separation of the TGN, ER, and Golgi fractions (Figure 5), resolution of the latter into its subcompartments was not effected, as previously noted (Gilbert et al., 1998Go). Nevertheless, the distribution of the enzyme markers clearly indicated that the denser [35S]sulfate-labeled HA fractions (peak 2) coincided with the Golgi region (compare Figures 2 and 5).

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, 1998Go) indicated no discernible activity throughout the gradient (data not shown).



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Fig. 6. Distribution of sulfotransferase activities in MDCK subcellular fractions. Nycodenz gradient centrifugation was carried out on unlabeled MDCK cells as described in Figure 5 and the activities of PAPS:Gal-3-O-sulfotransferase (Gal-3S-Tr) and PAPS:GlcNAc-6-O-sulfotransferase (GN-6S-Tr) were determined on aliquots of each fraction as described in Materials and methods. Results are plotted as percent of maximum activity which was: 6.3 x 10–5 dpm for the Gal-3-O-sulfotransferase and 6.7 x 10–3 dpm for the GlcNAc-6-O-sulfotransferase. The distribution of galactosyltransferase (Gal-Tr) is shown for comparison.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
From the results obtained in this study on the HA influenza virus envelope glycoprotein it became evident that sulfation of its N-linked oligosaccharides occurs only after they have been processed to an endo H-resistant state. This finding, which indicates that sulfate addition is limited to the medial/trans Golgi and the TGN, was supported by Nycodenz gradient centrifugation of the [35S]sulfate-labeled envelope glycoprotein during pulse-chase experiments.

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 ß1–4 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., 1985Go; Kaverin and Klenk, 1999Go), thereby providing additional galactose sites for 3-O-sulfation; in this instance, the sulfotransferase would most likely be in competition with the {alpha}1–3-galactosyltransferase, as galactose residues in {alpha}-linkage to the N-acetyllactosamine chains have been found in canine proteins (Spiro and Bhoyroo, 1984Go) and indeed terminal {alpha}-galactosyl groups have been reported to occur on HA complex oligosaccharides from influenza virus infected Madin-Darby bovine kidney cells (Mir-Shekari et al., 1997Go). 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 ß1–4 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 ß1–4 galactosyltransferase (Spiro et al., 1996Go).

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., 1985Go), where it has been located on the C-6 of this sugar residue (Karaivanova and Spiro, 1998Go), 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., 1985Go).

Studies employing brefeldin A in a bovine endothelial cell line (Sampath et al., 1992Go), as well as in LLC-PK1 cells (Karaivanova and Spiro, 1998Go) 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cell culture and viral infection
MDCK cells were grown on 100 mm plates in DMEM (Gibco) containing 4.5 g/l of glucose and 10% v/v FBS. The medium also contained streptomycin (100 µg/ml), penicillin (100 U/ml) and amphotericin (2.5 µg/ml). The incubations were carried out at 37°C in an atmosphere of 95% O2 and 5% CO2. When the cells were ~90% confluent, they were washed two times with serum-free medium and subsequently influenza virus (PR8/MS, a gift of Dr. Karl S.Matlin, Massachusetts General Hospital, Boston, MA) was added at a final concentration of ~50 pfu/cell and the infection was allowed to take place at room temperature for 4 h. At the end of this time, after aspiration of the virus-containing medium, 3 ml of growth medium containing 2% (v/v) FBS was added and the incubation was continued for 20 h at 37°C.

Radiolabeling of cells
Plates of the influenza virus–infected 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)Go. 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., 1982Go). 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., 1990Go) and to separate ER and Golgi fractions (Gilbert et al., 1998Go), was determined by the measurement of a number of enzymes. UDP-galactose:N-acetylglucosamine galactosyltransferase was assayed as previously described (Lubas and Spiro, 1987Go), while endomannosidase and glucosidase II activities were determined by the release of glucosylmannose or glucose, respectively, from 14C-labeled Glc1Man9GlcNAc substrate (Hiraizumi et al., 1993Go). To measure sialyltransferase, the incorporation of radioactivity from CMP-[3H]NeuAc (DuPont, New England Nuclear, 100,000 dpm, 3 nmol) into sialic acid–free 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)Go.

Assay of sulfotransferases
PAPS:galactose-3-O-sulfotransferase (Kato and Spiro, 1989Go) and PAPS: N-acetylglucosamine-6-O-sulfotransferase (Spiro et al., 1996Go) 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{alpha}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, 1989Go; Spiro et al., 1996Go). After thin layer chromatography of the material eluted from the columns on cellulose-coated plates, the sulfated saccharide products ([35S]Gal(3-SO4)ß1–4GlcNAc and [35S]GlcNAc(6-SO4)ß1–6Manß1-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., 1990Go). The glycopeptides were separated by filtration on Bio-Gel P-2 (Karaivanova and Spiro, 1998Go), dried and then treated with hydrazine for 28 h at 100°C followed by nitrous acid degradation and NaBH4 reduction (Edge and Spiro, 1985Go). 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, 1988Go).

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)Go. Radioactive components were visualized by fluorography at –80°C using X-Omatic AR film after treatment with ENHANCE (Du Pont–New 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, 1985Go), 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).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by Grant DK 17477 from the National Institutes of Health. We thank Vishnu Bhoyroo for help in some aspects of this investigation.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
HA, hemagglutinin; MDCK, Madin-Darby canine kidney; TGN, trans-Golgi network; ER, endoplasmic reticulum; PNGase, peptide:N-glycosidase; endo H, endo-ß-N-acetylglucosaminidase; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; Gal(3-SO4), sulfate group located on C-3 of galactose; GlcNAc(6-SO4), sulfate group located on C-6 of N-acetylglucosamine; AnManH2, 2,5-anhydromannitol; AnManH2(6-SO4), sulfate group located on C-6 of AnManH2; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; -O-Me, methylglycoside.


    Footnotes
 
1 To whom correspondence should be addressed at: Joslin Research Laboratory, One Joslin Place, Boston, MA 02215 Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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