Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, 9500 Gilman Drive, University of California, San Diego, La Jolla, CA 92093-0687, USA
Recevied on December 19, 2000; revised on April 5, 2001; accepted on April 5, 2001.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: sulfated N-linked oligosaccharides/sulfotransferase/CHO cell mutants/stable transfection/ MAA-lectin binding
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The various sulfation reactions that give rise to the sulfated glycans are catalyzed by distinct sulfotransferases located in the Golgi. Several of the glycoprotein-specific sulfotransferases have been purified and characterized (Kato and Spiro, 1989; Hooper et al., 1995
; Spiro et al., 1996
; Spiro and Bhoyroo, 1998
), and cDNAs for a Gal 6-O-sulfotransferases (Fukuta et al., 1997
), GlcNAc 6-sulfotransferases (Uchimura et al., 1998a
,b; Lee et al., 1999
; Bistrup et al., 1999
), and a GlcA 3-O-sulfotransferase (Ong et al., 1998
; Bakker et al., 1997
) have been cloned. Like other Golgi transferases, all of the enzymes appear to be type II transmembrane glycoproteins and show a high degree of specificity for their oligosaccharide substrates. By analogy to the sulfotransferases involved in glycosaminoglycan assembly, it seems likely that each enzyme is part of a multigene family whose members differ in distribution and developmental expression (Hashimoto et al., 1992
; Eriksson et al., 1994
; Orellana et al., 1994
; Bakker et al., 1997
; Honke et al., 1997
, Kobayashi et al., 1997
, 1999; Habuchi et al., 1998
; Ong et al., 1998
; Aikawa and Esko, 1999
; Shworak et al., 1999
; Aikawa et al., 2001
).
3-O-sulfation of Gal residues on glycoproteins was first described on thyroglobulins from various species (Spiro and Bhoyroo, 1988; de Waard et al., 1991
). The 3-O-sulfotransferase activity responsible for its assembly exhibits selectivity for the terminal disaccharide, Galß1-4GlcNAc-, independently of the underlying oligosaccharide or glycoprotein (Kato and Spiro, 1989
). Because
2-3 sialyltransferases,
1-2 fucosyltransferases, and
1-3 Gal transferase act on identical substrates, the four enzymes could potentially compete with each other and convey different chemical and possibly biological properties to the modified chains. Here, we report the activation of a glycoprotein Gal 3-O-sulfotransferase in two Chinese hamster ovary (CHO) cell lines by introduction of a wild-type cDNA library into a strain devoid of glycosaminoglycans (Esko et al., 1985
). As expected, sulfation results in decreased sialylation. Surprisingly, a heretofore unappreciated reactivity of Maackia amurensis lectin (MAL) for 3-O-sulfated Gal-terminated oligosaccharides was demonstrated.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analysis of the 35S-labeled macromolecules produced by these two lines yielded surprising results. As expected from the autoradiographic data, the amount of 35S-labeled macromolecules that precipitated with trichloroacetic acid was five- to sevenfold higher than in the parental line, pgsA-745 (Table I). Few of the 35S-counts were recovered in the glycolipid fraction in the clones and parental cells (Table I), although CHO cells are thought to make a small amount of sulfatide (Murphy-Ullrich et al., 1988). Treating clone 26 and 489 cells with trypsin released much of the 35S-counts, consistent with the idea that the enhanced incorporation had most likely occurred in glycoproteins or proteoglycans expressed on the cell surface (Table I). However, when the cells were analyzed by the glycan isolation procedure (GIP) (Norgard-Sumnicht et al., 2000
) the 35S-counts unexpectedly did not fractionate like glycosaminoglycans. This was confirmed using a slightly modified procedure for isolating the glycosaminoglycan chains (Table I). Instead, the radioactivity appeared in the glycopeptide fraction predicted to contain N- and O-linked glycans (GP1100 and GP3001000 in the GIP protocol). When samples were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE), multiple 35S-labeled proteins on the gels were observed in the transfectants. When samples from wild-type CHO cells were electrophoresed, the broad diffuse bands typical of proteoglycans obscured the gels. Only very faint bands were present in pgsA-745 cells (data not shown). Treatment with peptide:N-glycosidase F (PNGase F) caused the appearance of multiple low molecular mass sulfated oligosaccharide bands at the expense of the glycoprotein bands.
|
|
To locate the position of the sulfate groups, N-glycans biosynthetically labeled with 35SO4, [6-3H]Gal, and [6-3H]GlcN were isolated as above and treated with hydrazine and nitrous acid at high pH (Spiro et al., 1996). These conditions result in N-deacetylation of GlcNAc and GalNAc residues, cleavage of the hexosaminic bond, and deaminative ring contraction at the reducing end to form anhydromannose (aMan) or anhydrotalose, respectively. Reduction with NaBH4 then yields anhydromannitol (aManol) or anhydrotalitol, thus stabilizing the released oligosaccharides against further degradation. Based on the known structure of the chains in CHO cells, one would expect the labeled cleavage products to consist of sulfated, sialylated, or unmodified Gal-aManol units, and possibly sulfated anhydrotalitol if the cells contained terminal GalNAc residues. Gel filtration chromatography of the sample showed that
50% of the 35S-labeled material was cleaved under these conditions, yielding a major peak (Figure 2C, fractions 2631, 42% of counts) near the Vt of the column (fraction 35). Attempts to further improve the yield were unsuccessful, raising the possibility that some of the sulfate may have been located on the mannose rich core of the chains (e.g., Man4SO4; Yamashita et al., 1983
). However, the yield of [3H]Gal-labeled fragments from labeled glycans was also
50%, suggesting that the low yield of cleavage products was due to incomplete deacetylation, deamination, or cleavage of the chains. The [3H]Gal- and [3H]GlcN-containing products separated into three peaks, one of which coincided with the major 35S-labeled material (Figures 2A and 2B, 10% of counts). This suggested that
24% (10%/0.42) of the disaccharides contained sulfate groups. The elution position of the last peak was ahead of the location where standard aMan6SO4 eluted (Vt), suggesting that the transfectant did not produce terminal GalNAc4SO4 residues like those found on pituitary glycohormones (Green and Baenziger, 1988a
). The material in fractions 2224 was sensitive to ß-galactosidase, consistent with the structure Galß1-4aManol. The material in fractions 1315 was sensitive to neuraminidase, suggesting the structure Sia
2-3Galß1-4aManol.
|
|
|
|
|
|
|
Using these conditions as a guide, we established an assay for the sulfotransferase with synthetic Galß1-4GlcNAcß-O-naphthalenemethanol as acceptor. Using this analog facilitated product isolation because the hydrophobic aglycone bound strongly to C18 reversed-phase resins, whereas the [35S]PAPS donor did not. As shown in Figure 9, extracts from clone 26 and 489 transferred 35S-counts to products, whereas extracts from wild-type and pgsA-745 cells had very low to negligible activity. The products of the in vitro reaction and the oligosaccharides derived from the transfectant were subjected to acid hydrolysis, which yielded t1/2 values of 5580 min for the release of the sulfate groups. These values fall within the range expected for sulfate esters located at a secondary alcohol in an equatorial position (Rees, 1963), consistent with the idea that the sulfate group transferred in vitro was at the same position as that found in the oligosaccharides isolated from the cells.
|
|
Clones 26 and 489 should facilitate cloning of the cDNA encoding Gal 3-O-sulfotransferase. Initial attempts using the polymerase chain reaction (PCR) and probes that hybridize to the flanking sequence in the plasmid used to create the original cDNA library have not yielded a full-length clone with the expected properties of a sulfotransferase. This observation is consistent with the idea that the enhanced enzyme activity may be due to activation of an endogenous locus by juxtaposition of the strong immediate early CMV promoter in the plasmid. Nevertheless, the high level of expression of the enzyme activity suggests that it might be possible to isolate the cDNA by subtractive techniques or by hybridization to DNA arrays. A Gal 3-O-sulfotransferase involved in sulfatide synthesis was cloned (Honke et al., 1997), but this enzyme is selective for a glycolipid substrate (Gal-ceramide). As shown in Table I, the activated sulfotransferase in clone 26 does not result in enhanced synthesis of sulfatide in CHO cells, although the parental cells produce a small amount of sulfated glycolipid (Murphy-Ullrich et al., 1988
). Another 3-O-sulfotransferase that can act on both type I and type II Gal-GlcNAc repeats was recently reported (Honke et al., 2001
), but hybridization of mRNA and PCR analysis did not detect this transcript in the transfectants (data not shown). Thus, the Gal 3-O-sulfotransferase amplified in clones 26 and 489 appears to be unique and may act only on N-linked glycans versus other types of oligosaccharides (also see Chandrasekaran et al., 1997
, 1999).
Clones 26 and 489 also present interesting sulfated glycans at the cell surface that might act as ligands for mammalian lectins. Studies of synthetic analogs of carbohydrate ligands for selectins have shown that they can have either sialic acid at C3 of the terminal Gal residues or sulfate (Chandrasekaran et al., 1997; Koenig et al., 1997
; Ng and Weis, 1997
; Sanders et al., 1999
; Galustian et al., 1999
). Although the natural ligands are thought to be mucin-type oligosaccharides (i.e., O-linked glycans) containing sialylated or sulfated Lewis-type termini (Galß1-3/4(Fuc
1-4/3)GlcNAc-), N-glycans containing similar terminal structures may be active as well if present at sufficient density. The availability of clone 26 now makes it possible to test this and related hypotheses.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Screening technique
Mutant pgsA-745 cells were cotransfected with a wild-type CHO cell cDNA library prepared in pCDNA I (Invitrogen) and pMAMneo (Clontech) at a plasmid ratio of 5:1. Stable transfectants were selected with 0.4 mg/ml of geneticin (Gibco G418, corrected for activity). Resistant colonies were replica plated onto discs of polyester cloth as described previously (Esko, 1989
) and metabolically labeled with 10 µCi/ml of 35SO4 (2540 Ci/mg, DuPont NEN) in sulfate-free Hams F12 medium for 4 h. The colonies were fixed with 10% trichloroacetic acid (TCA), washed with 2% TCA, and exposed to X-ray film. Colonies that exhibited an enhanced level of incorporation of label by autoradiography were picked from the original master dishes and subcloned by a second round of replica plating. Two clones identified in this way (clones 26 and 489) had virtually identical properties and may have been siblings.
Metabolic labeling and lysis of cells
Cells were metabolically labeled for 1272 h with 10 µCi/ml of H235SO4 in sulfate-free Hams F12 medium or 10 µCi/ml of [6-3H]glucosamine HCl and 10 µCi/ml of [6-3H]galactose (2540 Ci/mg, DuPont NEN) in low-glucose medium for 12 h. After removing the spent medium, the cell layer was washed three times with phosphate buffered saline (PBS), scraped into 1 ml of PBS (Dulbecco and Vogt, 1954), and centrifuged at 1000 x g for 10 min. Cell pellets were resuspended in a lysis buffer of 50 mM TrisHCl, pH 7.5, 1% SDS, and 0.1 M 2-mercaptoethanol and boiled for 10 min (Roux et al., 1988
).
Quantitation of sulfated macromolecules
A portion of 35SO4-labeled cells was treated with 10% TCA, centrifuged, washed with 2% TCA, and counted by liquid scintillation spectrometry as a measure of sulfated macromolecules. Another portion of cells was treated with 0.125% trypsin for 5 min and centrifuged, and an aliquot of the supernatant was counted as a measure of trypsin-sensitive, cell surface glycoproteins and proteoglycans. A third portion of cells treated with protease and the glycosaminoglycan fraction was isolated by anion-exchange chromatography (Bame and Esko, 1989). A fourth portion of cells was extracted with chloroform:methanol:water (2:3:1, v/v/v), and the lipid fraction was analyzed by silicic acid chromatography to assess the amount of 35S-counts in glycolipids (Murphy-Ullrich et al., 1988
).
Samples were also analyzed by SDSPAGE before and after treatment for 24 h at 37°C with 10 mU Flavobacterium meningospecticum PNGase F (Boehringer Mannheim) in 20 mM TrisHCl buffer, pH 7.5, containing 0.1% SDS, 50 mM ethylenediamine tetraacetic acid (EDTA), and 20 mM 2-mecaptoethanol. Samples were boiled under reducing conditions and electrophoresed through a 10% gel. After electrophoresis, the gel was dried and exposed to X-ray film.
Isolation of Asn-linked N-glycans
Cells were radiolabeled with 35SO4 as described above, 100 µCi/ml of D-[6-3H]glucosamine HCl (40 Ci/nmol, DuPont NEN), or 100 µCi/ml of [6-3H]galactose in low-glucose medium. Radioactive N-glycans were isolated using a modification of the procedure described by Roux et al. (1988). Briefly, an aliquot of radiolabeled cells was dissolved in SDS and treated with PNGase F as described above, boiled for 10 min, and centrifuged to remove a small amount of precipitated protein. The supernatant containing the released glycans was applied to Sephacryl S-200 column (1.5 x 50 cm) in a buffer of 20 mM TrisHCl, pH 6.0, containing 0.2% SDS. The column was eluted at a flow rate of 6 ml/h, and 1-ml fractions were collected. Radioactive material eluting in the included volume was pooled and saturated KCl was added (1:100, v/v) to precipitate the SDS. After overnight incubation at 4°C, the sample was centrifuged at 5000 x g for 30 min, and the supernatant was lyophilized and desalted by passing through a PD-10 column (Pharmacia). The sample was lyophilized again and resuspended in appropriate buffer for further analysis.
To separate neutral and charged glycans, samples were dissolved in 2 mM Tris-base and applied to a 0.5-ml column of QAE-Sephadex (Sigma) prepared in a disposable pipette tip. The column was first washed with 2 mM Tris base (10 ml) and then eluted (2.5 ml each) sequentially with buffer containing 20 mM, 70 mM, 140 mM, 200 mM, 400 mM, and 1000 mM NaCl as described (Roux et al., 1988). The presence of labeled oligosaccharides was monitored by following the radioactivity in each fraction.
Enzymatic and chemical analysis
Purified N-glycans were treated for 24 h at 37°C with the following enzymes:
1. 20 mU of A. ureafaciens neuraminidase (Oxford Glycosystems) in 100 µl of 100 mM sodium acetate buffer, pH 6.0, containing 4 mM calcium acetate;
2. 20 mU of jack bean or bovine testicular ß-galactosidases (Oxford Glycosystems) in a buffer of 50 mM sodium acetate, pH 4, before and after treatment with neuraminidase or 10 mM HCl at 100°C for 30 min to chemically desialylated the chains;
3. 10 U human placental ß-hexosaminidase A (kindly provided by H. Freeze, Burnham Institute, La Jolla, CA). Samples were first chemically desialylated, lyophilized, resuspended in 100 µl of 50 mM sodium formate buffer, pH 4.5, and incubated with ß-galactosidase. An aliquot (15 µl) was mixed with 100 mM sodium formate buffer (185 µl), pH 3.5, and ß-hexosaminidase A was added. These conditions remove GlcNAc-6SO4 residues from the termini of N-glycans (Roux et al., 1988);
4. 10 mU of Flavobacterium heparinum heparin lyase II (Seikagaku) in 50 mM sodium phosphate buffer, pH 7.6, containing 0.1 M NaCl and 1 mM CaCl2;
5. 10 mU of Proteus vulgaris chondroitinase ABC (Seikagaku) in 50 mM TrisHCl buffer, pH 8.0, containing 50 mM sodium acetate;
6. 20 mU of Pseudomonas keratinase I (keratan sulfate endo-ß-galactosidase) in a buffer of 50 mM TrisHCl, pH 7.4; or
7. 1 mU of Bacillus keratanase II (keratan sulfate endo-ß-N-acetylglucosaminidase, Seikagaku) in a buffer of 10 mM sodium acetate, pH 6.5.
Enzymes were inactivated by heating the samples in a water bath at 100°C for 2 min. The material was applied to a Sephadex G-50 column (29 x 1 cm) and eluted with 0.5 M pyridinium acetate, pH 5.0 (6 ml/h), to separate intact chains from cleaved oligosaccharides. The eluate was collected (0.5-ml fractions) and counted by liquid scintillation. To remove the sulfate group, samples were solvolyzed at 100°C for 7 h in 0.3 ml of dimethyl sulfoxide reagent (90% dimethylsulfoxide/10% methanol, v/v) titrated with HCl to pH 4 (Nagasawa et al., 1977). In some experiments the material was analyzed by anion-exchange chromatography on a column of QAE-Sephadex as described above.
Hydrazine/nitrous acid treatment
Deamination of radiolabeled glycans to oligosaccharides was achieved by N-deacetylation of GlcNAc residues followed by nitrous acid cleavage at high pH (5). Briefly, 35S-, [3H]GlcN- and [3H]Gal-labeled N-glycans were deacetylated by treatment at 96°C for 16 h with 100% anhydrous hydrazine. Excess hydrazine was removed by repeated evaporation of the sample to dryness from toluene followed by desalting on a PD-10 column. After lyophilization, the sample was treated at room temperature for 2 h with nitrous acid at pH 4.0 prepared with 0.2 M NaNO2 in 0.4 N acetic acid. The cleaved oligosaccharides were reduced with 30 mM NaBH4 at room temperature for 2 h. After decomposition of the NaBH4 with acetic acid, the sample was repeatedly evaporated to dryness from methanol/acetic acid. Samples were dissolved in 0.5 ml of 0.5 M pyridinium acetate, pH 5.0, and chromatographed on a column of Bio-Gel P4 (Bio-Rad, 1.0 x 100 cm) in the same buffer (4 ml/h). Fractions (1 ml) were collected, and an aliquot was taken for liquid scintillation counting. Peak fractions were pooled as indicated in the figures, concentrated by evaporation, and desalted by chromatography on a 100-mg (3-ml) column of Hypersep PGC Pk30 (Hypersil, UK) as described (Packer et al., 1998). The column was prewashed with three bed volumes of 80% acetonitrile in 0.1% trifluoracetic acid (TFA) (v/v) followed by three volumes of water. After loading the sample, the column was washed with water to remove salts. The column was washed with 25% (v/v) acetonitrile to elute neutral oligosaccharides and then with 25% (v/v) acetonitrile containing 0.05% TFA (v/v) to elute charged oligosaccharides. These samples were dried under high vacuum.
In some experiments, the radiolabeled oligosaccharides liberated by hydrazine/nitrous acid treatment were fractionated by anion exchange chromatography (Spiro and Bhoyroo, 1988). Samples were chromatographed on a 0.8 x 5 cm column of AG-1 X2 (200400 mesh, acetate) by step elution with different buffers. Neutral products were eluted with 35 ml of water (fraction 1); sialic acidcontaining products were eluted with 45 ml of 1 M formic acid (fraction 2); monosulfated components with 35 ml of 0.7 M pyridine acetate, pH 5 (fraction 3); and disulfated saccharides with 35 ml of 2 M pyridine acetate, pH 5. The samples were lyophilized for further analysis.
TLC
The 35S- and 3H-labeled oligosaccharides in fraction 3 described above (monosulfated components) and disaccharides separated by Bio-gel P4 chromatography were analyzed by cellulose TLC (0.1 mm thickness, plastic sheets, Merck) (Spiro and Bhoyroo, 1988). Standards of [3H]Galß1-4aManol, [3H]Gal3Sß1-4aManol, [3H]Galß1-4aManol6S were kindly provided by R.G. Spiro (Harvard University, Cambridge, MA). Ascending chromatography was performed in a solvent of pyridine/ethyl acetate/water/acetic acid (5:5:3:1, v/v/v/v) using a wick of Whatman No. 3MM paper clamped to the top of the plate during the chromatography. After 22 h, the plate was dried and cut into 1/4-inch strips. The radiolabeled material was extracted with water and counted by liquid scintillation.
Periodate oxidation
Samples were oxidized at 4°C for 16 h in the dark in 100 µl of 0.1 M sodium metaperiodate in 0.04 M sodium acetate buffer, pH 4.5 (Spiro and Bhoyroo, 1988). The reaction was stopped by the addition of 100 µl of 0.2 M glycerol at room temperature. After 1 h, the samples were adjusted to pH 10 with sodium borate buffer and reduced with 30 mM NaBH4 at room temperature for 2 h. After removal of excess borohydride, samples were analyzed by descending paper chromatography on Whatman No. 3MM as described above. A standard sample of [6-3H]galactose showed >95% loss of counts, indicating that the reaction was nearly quantitative. Some samples were subjected to methanolysis prior to periodate oxidation to remove the sulfate ester (Wing et al., 1992
). Briefly, desalted glycans were resuspended in 50 mM HCl in methanol and incubated in a sealed tube at room temperature for 15 h. The sample was then dried under vacuum. The extent of desulfation was checked by QAE-Sephadex chromatography as described above.
MAL affinity chromatography.
MAL agarose affinity chromatography was performed as described (Wang and Cummings, 1988). MAL lectin (a mixture of MAL-I and -II) immobilized on agarose beads (EY Laboratories) was poured into pipette tips to make small columns (
0.2 ml), and pre-equilibrated with a buffer of 20 mm KH2PO4, pH 7.4, 150 mM NaCl, and 0.02% sodium azide. The samples were loaded on the column and eluted with 10 bed volumes of the equilibration buffer, followed by 10 bed volumes of buffer containing of 100 mM lactose. Fractions (1.5 bed volumes) were collected and monitored by liquid scintillation counting.
Galactose:O-sulfotransferase assay.
The activity of PAPS:galactose O-sulfotransferase was measured in CHO cell homogenates essentially as described by Kato and Spiro (1989). Cells were grown to confluence, rinsed three times with cold PBS, and detached with a rubber policeman in 50 µl of buffer containing 0.25 M sucrose, 50 mM Tris acetate buffer (pH 7.4), 1% (w/v) Triton X-100, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride. Total cell protein was estimated by the Bio-Rad protein assay kit using bovine serum albumin as standard. The cells were homogenized by brief sonication and the solution was clarified by centrifugation at 12,000 x g for 15 min. The sulfate donor, [35S]PAPS, was prepared using yeast homogenates as described by Renosto and Segel (1977)
and Robbins (1962)
.
The standard reaction mixture contained 50 mM Tris acetate, pH 7.0, 0.1% (v/v) Triton X-100, 20 mM manganese acetate, 100 mM NaF, 10 mM EDTA, 2 mM ATP, 50 µM [35S]PAPS (0.2 Ci/mmol), 7.5 mM of Galß1-4GlcNAcß-O-naphthalenemethanol or Galß1-3GlcNAcß-O-naphthalenemethanol (Sarkar et al., 2000
), and 50100 µg of cell protein in a final volume of 25 µl. The mixture was incubated for 60 min at 37°C, and the reaction was stopped by adding 1 ml of 0.5 M NaCl. Next, the samples were applied to a 100-mg Sep-Pak Vac RC C18 Cartridges (Waters), which were prewashed with 100% methanol, water, and 0.5 M NaCl. After sample application, unincorporated radioactive material were removed by washing the column with 0.5 M NaCl (2.5 ml) and water (25 ml). Product was eluted with 40% methanol in water (2.5 ml) and counted by liquid scintillation spectrometry.
The in vitro product was further characterized by acid hydrolysis and periodate oxidation. The samples was first separated from unreacted substrate by sequential chromatography on QAE and C18 resins. One portion was subjected to acid hydrolysis to determine the rate of sulfate loss (Rees, 1963). Another portion was subjected to periodate oxidation, followed by TLC on aluminum-backed silica gel 60 high-performance TLC plates (Merck) in a solvent of ethyl-acetate: glacial acetic acid:methanol:H2O (10:3:3:2, v/v/v/v). The plate was dried and subjected to autoradiography. The disaccharide standard, [3H]Galß1,4GlcNAc-naphthalenemethanol, was synthesized enzymatically by galactosylation of GlcNAc-naphthalenemethanol using UDP-[3H]Glc (8.4 Ci/mmol, NEN Life Sciences Products) together with crude bacterial lysate containing a GalT/Epimerase fusion protein (pcw:galE-ltgB) as the enzyme source (Blixt et al., 2001
).
Lectin binding
PgsA-745 and clone 26 cells were grown in sulfate-free medium with and without 20 mM sodium chlorate. One set of plates contained both sulfate and chlorate. After 3 days, cells were detached in buffer containing 5 mM EDTA, 20 mM NaH2PO4, pH 7.4, and 150 mM NaCl. The cells were washed three times with phosphate buffer, and 15 x 106 cells were digested with 20 mU of A. ureafaciens neuraminidase for 1 h at room temperature in 50 mM HEPES buffer, pH 6.9, containing 2 mM CaCl2 and 0.15 M NaCl. The cells were washed three times with PBS and incubated for 30 min at 4°C with 100 µl of 10 µg/ml biotinylated MAL-I or -II (Vector Laboratory) in PBS. After three washes, the cells were resuspended in 0.2 ml of buffer containing 5 mg/ml of fluorescein-avidin-DCS (Vector Laboratory). The cells were incubated for 20 min at 4°C in the dark, washed, and resuspended in cold PBS. Flow cytometry analysis was done on a FACS Star (Becton-Dickinson).
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aikawa, J., Grobe, K., Tsujimoto, M., and Esko, J.D. (2001) Multiple isozymes of heparan sulfate/heparin GlcNAc N-deacetylase/N-sulfotransferase: structure and activity of the fourth member, NDST4. J. Biol. Chem., 276, 58765882.
Amado, M., Almeida, R., Carneiro, F., Levery, S.B., Holmes, E.H., Nomoto, M., Hollingsworth, M.A., Hassan, H., Schwientek, T., Nielsen, P.A., and others (1998) A family of human beta3-galactosyltransferases. Characterization of four members of a UDP-galactose:beta-N-acetyl-glucosamine/beta-nacetyl- galactosamine beta-1, 3-galactosyltransferase family. J. Biol. Chem., 273, 1277012778.
Bakker, H., Friedmann, I., Oka, S., Kawasaki, T., Nifantev, N., Schachner, M. and Mantei, N. (1997) Expression cloning of a cDNA encoding a sulfotransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope. J. Biol. Chem., 272, 2994229946.
Bame, K.J., and Esko, J.D. (1989) Undersulfated heparan sulfate in a Chinese hamster ovary cell mutant defective in heparan sulfate N-sulfotransferase. J. Biol. Chem., 264, 80598065.
Baumeister, F.A., and Herzog, V. (1988) Sulfation of thyroglobulin: a ubiquitous modification in vertebrates. Cell Tissue Res., 252, 349358.[ISI][Medline]
Bergwerff, A.A., Van Oostrum, J., Kamerling, J.P., and Vliegenthart, J.F.G. (1995) The major N-linked carbohydrate chains from human urokinasethe occurrence of 4-O-sulfated, (2-6)-sialylated or (
1-3)-fucosylated N-acetylgalactosamine(ß1-4)-N-acetylglucosamine elements. Eur. J. Biochem., 228, 10091019.[Abstract]
Bistrup, A., Bhakta, S., Lee, J.K., Belov, Y.Y., Gunn, M.D., Zuo, F.R., Huang, C.C., Kannagi, R., Rosen, S.D., and Hemmerich, S. (1999) Sulfotransferases of two specificities function in the reconstitution of high endothelial cell ligands for L-selectin. J. Cell Biol., 145, 899910.
Blixt, O., Brown, J., Schur, M.J., Wakarchuk, W., and Paulson, J.C. (2001) Efficient preparation of natural and synthetic galactosides with a recombinant beta-1, 4-galactosyltransferase-/UDP-4'-Gal epimerase fusion protein. J. Org. Chem., 66, 24422448.[ISI][Medline]
Brown, G.M., Huckerby, T.N., Morris, H.G., Abram, B.L., and Nieduszynski, I.A. (1994) Oligosaccharides derived from bovine articular cartilage keratan sulfates after keratanase II digestion: implications for keratan sulfate structural fingerprinting. Biochemistry, 33, 48364846.[ISI][Medline]
Chandrasekaran, E.V., Jain, R.K., Rhodes, J.M., Chawda, R., Piskorz, C., and Matta, K.L. (1999) Characterization of distinct Gal:3-O-sulfotransferase activities in human tumor epithelial cell lines and of calf lymph node GlcNAc:6-O-sulfotransferase activity. Glycoconj. J., 16, 523536.[ISI][Medline]
Chandrasekaran, E.V., Jain, R.K., Vig, R., and Matta, K.L. (1997) The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal. Glycobiology, 7, 753768.[Abstract]
Chou, D.K., Ilyas, A.A., Evans, J.E., Costello, C., Quarles, R.H., and Jungalwala, F.B. (1986) Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK-1 antibody and some IgM paraproteins in neuropathy. J. Biol. Chem., 261, 1171711725.
Chou, D.K., Schwarting, G.A., Evans, J.E., and Jungalwala, F.B. (1987) Sulfoglucuronyl-neolacto series of glycolipids in peripheral nerves reacting with HNK-1 antibody. J. Neurochem., 49, 865873.[ISI][Medline]
Crommie, D., and Rosen, S.D. (1995) Biosynthesis of GlyCAM-1, a mucin-like ligand for L-selectin. J. Biol. Chem., 270, 2261422624.
de Waard, P., Koorevaar, A., Kamerling, J.P., and Vliegenthart, J.F. (1991) Structure determination by 1H NMR spectroscopy of (sulfated) sialylated N-linked carbohydrate chains released from porcine thyroglobulin by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F. J. Biol. Chem., 266, 42374243.
Dulbecco, R. and Vogt, M. (1954) Plaque formation and isolation of pure cell lines with poliomyelitis viruses. J. Exp. Med., 99, 167182.[ISI][Medline]
Edge, A.S., and Spiro, R.G. (1984) Presence of sulfate in N-glycosidically linked carbohydrate units of calf thyroid plasma membrane glycoproteins. J. Biol. Chem., 259, 47104713.
Eriksson, I., Sandbäck, D., Ek, B., Lindahl, U., and Kjellén, L. (1994) cDNA cloning and sequencing of mouse mastocytoma glucosaminyl N-deacetylase/N-sulfotransferase, an enzyme involved in the biosynthesis of heparin. J. Biol. Chem., 269, 1043810443.
Esko, J.D. (1989) Replica plating of animal cells. Meth. Cell Biol., 32, 387422.[ISI][Medline]
Esko, J.D., Stewart, T.E., and Taylor, W.H. (1985) Animal cell mutants defective in glycosaminoglycan biosynthesis. Proc. Natl Acad. Sci. USA, 82, 31973201.[Abstract]
Esko, J.D., Weinke, J.L., Taylor, W.H., Ekborg, G., Rodén, L., Anantharamaiah, G., and Gawish, A. (1987) Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I. J. Biol. Chem., 262, 1218912195.
Fiete, D., Srivastava, V., Hindsgaul, O., and Baenziger, J.U. (1991) A hepatic reticuloendothelial cell receptor specific for SO4-4GalNAc beta 1, 4GlcNAc beta 1, 2Man alpha that mediates rapid clearance of lutropin [see comments]. Cell, 67, 11031110.[ISI][Medline]
Freeze, H.H., and Wolgast, D. (1986) Structural analysis of N-linked oligosaccharides from glycoproteins secreted by Dictostelium discoideum. Identification of mannose 6-sulfate. J. Biol. Chem., 261, 127134.
Fukuta, M., Inazawa, J., Torii, T., Tsuzuki, K., Shimada, E., and Habuchi, O. (1997) Molecular cloning and characterization of human keratan sulfate Gal-6-sulfotransferase. J. Biol. Chem., 272, 3232132328.
Galustian, C., Lubineau, A., le Narvor, C., Kiso, M., Brown, G., and Feizi, T. (1999) L-selectin interactions with novel mono- and multisulfated Lewisx sequences in comparison with the potent ligand 3'-sulfated Lewisa. J. Biol. Chem., 274, 1821318217.
Green, E.D., and Baenziger, J.U. (1988a) Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. I. Structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J. Biol. Chem., 263, 2535.
Green, E.D., and Baenziger, J.U. (1988b) Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J. Biol. Chem., 263, 3644.
Habuchi, H., Kobayashi, M., and Kimata, K. (1998) Molecular characterization and expression of heparan-sulfate 6-sulfotransferasecomplete cDNA cloning in human and partial cloning in Chinese hamster ovary cells. J. Biol. Chem., 273, 92089213.
Ham, R.G. (1965) Clonal growth of mammalian cells in a chemically defined, synthetic medium. Proc. Natl Acad. Sci. USA, 53, 288293.[ISI][Medline]
Hard, K., Van Zadelhoff, G., Moonen, P., Kamerling, J.P., and Vliegenthart, F.G. (1992) The Asn-linked carbohydrate chains of human Tamm-Horsfall glycoprotein of one male. Novel sulfated and novel N-acetylgalactosamine-containing N-linked carbohydrate chains. Eur. J. Biochem., 209, 895915.[Abstract]
Hashimoto, Y., Orellana, A., Gil, G., and Hirschberg, C.B. (1992) Molecular cloning and expression of rat liver N-heparan sulfate sulfotransferase. J. Biol. Chem., 267, 1574415750.
Hemmerich, S., and Rosen, S.D. (1994) 6'-sulfated sialyl Lewis x is a major capping group of GlyCAM-1. Biochemistry, 33, 48304835.[ISI][Medline]
Hemmerich, S., Bertozzi, C.R., Leffler, H., and Rosen, S.D. (1994) Identification of the sulfated monosaccharides of GlyCAM-1, an endothelial derived ligand for L-selectin. Biochemistry, 33, 48204829.[ISI][Medline]
Hemmerich, S., Leffler, H., and Rosen, S.D. (1995) Structure of the O-glycans in GlyCAM-1, an endothelial-derived ligand for L-selectin. J. Biol. Chem., 270, 1203512047.
Hennet, T., Dinter, A., Kuhnert, P., Mattu, T.S., Rudd, P.M., and Berger, E.G. (1998) Genomic cloning and expression of three murine UDP-galactose: beta-N- acetylglucosamine beta1, 3-galactosyltransferase genes. J. Biol. Chem., 273, 5865.
Hokke, C.H., Damm, J.B., Kamerling, J.P., and Vliegenthart, J.F. (1993) Structure of three acidic O-linked carbohydrate chains of porcine zona pellucida glycoproteins. FEBS Lett., 329, 2934.[ISI][Medline]
Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A., and Wada, Y. (1997) Molecular cloning and expression of cDNA encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase. J. Biol. Chem., 272, 48644868.
Honke, K., Tsuda, M., Koyota, S., Wada, Y., Iida-Tanaka, N., Ishizuka, I., Nakayama, J., and Taniguchi, N. (2001) Molecular cloning and characterization of a human ß-gal-3'-sulfotransferase that acts on both type 1 and type 2 (Galß1-3/1-4GlcNAc-R) oligosaccharides. J. Biol. Chem., 276, 267274.
Hooper, L.V., Hindsgaul, O., and Baenziger, J.U. (1995) Purification and characterization of the GalNAc-4-sulfotransferase responsible for sulfation of GalNAcß1, 4GlcNAc-bearing oligosaccharides. J. Biol. Chem., 270, 1632716332.
Imberty, A., Gautier, C., Lescar, J., Perez, S., Wyns, L., and Loris, R. (2000) An unusual carbohydrate binding site revealed by the structures of two Maackia amurensis lectins complexed with sialic acid-containing oligosaccharides. J. Biol. Chem., 275, 1754117548.
Isshiki, S., Togayachi, A., Kudo, T., Nishihara, S., Watanabe, M., Kubota, T., Kitajima, M., Shiraishi, N., Sasaki, K., Andoh, T., and Narimatsu, H. (1999) Cloning, expression, and characterization of a novel UDP-galactose:beta- N-acetylglucosamine beta1, 3-galactosyltransferase (beta3Gal-T5) responsible for synthesis of type 1 chain in colorectal and pancreatic epithelia and tumor cells derived therefrom. J. Biol. Chem., 274, 1249912507.
Kaku, H., Mori, Y., Goldstein, I.J., and Shibuya, N. (1993) Monomeric, monovalent derivative of Maackia amurensis leukoagglutinin. Preparation and application to the study of cell surface glycoconjugates by flow cytometry. J. Biol. Chem., 268, 1323713241.
Kamerling, J.P., Rijkse, I., Maas, A.A., van Kuik, J.A., and Vliegenthart, J.F. (1988) Sulfated N-linked carbohydrate chains in porcine thyroglobulin. FEBS Lett., 241, 246250.[ISI][Medline]
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.
Knibbs, R.N., Goldstein, I.J., Ratcliffe, R.M., and Shibuya, N. (1991) Characterization of the carbohydrate binding specificity of the leukoagglutinating lectin from Maackia amurensis. Comparison with other sialic acid-specific lectins. J. Biol. Chem., 266, 8388.
Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) Molecular cloning and expression of Chinese hamster ovary cell heparan-sulfate 2-sulfotransferase. J. Biol. Chem., 272, 1398013985.
Kobayashi, M., Sugumaran, G., Liu, J.A., Shworak, N.W., Silbert, J.E., and Rosenberg, R.D. (1999) Molecular cloning and characterization of a human uronyl 2-sulfotransferase that sulfates iduronyl and glucuronyl residues in dermatan chondroitin sulfate. J. Biol. Chem., 274, 1047410480.
Koenig, A., Jain, R., Vig, R., Norgard-Sumnicht, K.E., Matta, K.L., and Varki, A. (1997) Selectin inhibition: synthesis and evaluation of novel sialylated, sulfated and fucosylated oligosaccharides, including the major capping group of GlyCAM-1. Glycobiology, 7, 7993.[Abstract]
Konami, Y., Yamamoto, K., Osawa, T., and Irimura, T. (1994) Strong affinity of Maackia amurensis hemagglutinin (MAH) for sialic acid-containing Ser/Thr-linked carbohydrate chains of N-terminal octapeptides from human glycophorin A. FEBS Lett., 342, 334338.[ISI][Medline]
Lee, E.U., Roth, J., and Paulson, J.C. (1989) Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of beta-galactoside alpha 2, 6-sialyltransferase. J. Biol. Chem., 264, 1384813855.
Lee, J.K., Bhakta, S., Rosen, S.D., and Hemmerich, S. (1999) Cloning and characterization of a mammalian N-acetylglucosamine-6-sulfotransferase that is highly restricted to intestinal tissue. Biochem. Biophys. Res. Commun., 263, 543549.[ISI][Medline]
Lo-Guidice, J.M., Périni, J.M., Lafitte, J.J., Ducourouble, M.P., Roussel, P., and Lamblin, G. (1995) Characterization of a sulfotransferase from human airways responsible for the 3-O-sulfation of terminal galactose in N-acetyllactosamine-containing mucin carbohydrate chains. J. Biol. Chem., 270, 2754427550.
Lo-Guidice, J.M., Wieruszeski, J.-M., Lemoine, J., Verbert, A., Roussel, P., and Lamblin, G. (1994) Sialylation and sulfation of the carbohydrate chains in respiratory mucins from a patient with cystic fibrosis. J. Biol. Chem., 269, 1879418813.
Margolis, R.K., and Margolis, R.U. (1993) Nervous tissue proteoglycans. Experientia, 49, 429446.[ISI][Medline]
Murphy-Ullrich, J.E., Westrick, L.G., Esko, J.D., and Mosher, D.F. (1988) Altered metabolism of thrombospondin by Chinese hamster ovary cells defective in glycosaminoglycan synthesis. J. Biol. Chem., 263, 64006406.
Nagasawa, K., Inoue, Y., and Kamata, T. (1977) Solvolytic desulfation of glycosaminoglycuronan sulfates with dimethyl sulfoxide containing water or methanol. Carbohydr. Res., 58, 4755.[ISI][Medline]
Ng, K.K., and Weis, W.I. (1997) Structure of a selectin-like mutant of mannose-binding protein complexed with sialylated and sulfated Lewis(x) oligosaccharides. Biochemistry, 36, 979988.[ISI][Medline]
Noguchi, S., Hatanaka, Y., Tobita, T., and Nakano, M. (1992) Structural analysis of the N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur. J. Biochem., 207, 1130.[ISI][Medline]
Norgard-Sumnicht, K., Bai, X., Esko, J.D., Varki, A., and Manzi, A.E. (2000) Exploring the outcome of genetic modifications of glycosylation in cultured cell lines by concurrent isolation of the major classes of vertebrate glycans. Glycobiology, in press.
Ong, E., Yeh, J.C., Ding, Y.L., Hindsgaul, O., and Fukuda, M. (1998) Expression cloning of a human sulfotransferase that directs the synthesis of the HNK-1 glycan on the neural cell adhesion molecule and glycolipids. J. Biol. Chem., 273, 51905195.
Orellana, A., Hirschberg, C.B., Wei, Z., Swiedler, S.J., and Ishihara, M. (1994) Molecular cloning and expression of a glycosaminoglycan N-acetylglucosaminyl N-deacetylase/N-sulfotransferase from a heparin-producing cell line. J. Biol. Chem., 269, 22702276.
Packer, N.H., Lawson, M.A., Jardine, D.R., and Redmond, J.W. (1998) A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj. J., 15, 737747.[ISI][Medline]
Rees, D.A. (1963) A note on the characterization of carbohydrate sulfates by acid hydrolysis. Biochem. J., 88, 343345.[ISI][Medline]
Renosto, F., and Segel, I.H. (1977) Choline sulfokinase of Penicillium chrysogenum: partial purification and kinetic mechanism. Arch. Biochem. Biophys., 180, 416428.[ISI][Medline]
Robbins, P.W. (1962) Sulfate-activating enzymes. Meth. Enzymol., 5, 964977.[ISI]
Roux, L., Holojda, S., Sundblad, G., Freeze, H.H., and Varki, A. (1988) Sulfated N-linked oligosaccharides in mammalian cells. I. Complex-type chains with sialic acids and O-sulfate esters. J. Biol. Chem., 263, 88798889.
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.
Sanders, W.J., Gordon, E.J., Dwir, O., Beck, P.J., Alon, R., and Kiessling, L.L. (1999) Inhibition of L-selectin-mediated leukocyte rolling by synthetic glycoprotein mimics. J. Biol. Chem., 274, 52715278.
Sarkar, A.K., Brown, J.R., and Esko, J.D. (2000) Synthesis and glycan priming activity of acetylated disaccharides. Carbohydr. Res., 329, 287300.[ISI][Medline]
Sata, T., Lackie, P.M., Taatjes, D.J., Peumans, W., and Roth, J. (1989) Detection of the Neu5 Ac (alpha 2, 3) Gal (beta 1, 4) GlcNAc sequence with the leukoagglutinin from Maackia amurensis: light and electron microscopic demonstration of differential tissue expression of terminal sialic acid in alpha 2, 3- and alpha 2, 6-linkage. J. Histochem. Cytochem., 37, 15771588.[Abstract]
Schachner, M., and Martini, R. (1995) Glycans and the modulation of neural-recognition molecule function. Trends Neurosci., 18, 183191.[ISI][Medline]
Shailubhai, K., Huynh, Q.K., Boddupalli, H., Yu, H.H., and Jacob, G.S. (1999) Purification and characterization of a lymph node sulfotransferase responsible for 6-O-sulfation of the galactose residues in 2'-fucosyllactose and other sialyl LewisX-related sugars. Biochem. Biophys. Res. Commun., 256, 170176.[ISI][Medline]
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]
Shworak, N.W., Liu, J.A., Petros, L.M., Zhang, L.J., Kobayashi, M., Copeland, N.G., Jenkins, N.A., and Rosenberg, R.D. (1999) Multiple isoforms of heparan sulfate D-glucosaminyl 3-O-sulfotransferaseisolation, characterization, and expression of human cDNAs and identification of distinct genomic loci. J. Biol. Chem., 274, 51705184.
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-galactosyl-N-acetylglucosamine 6-sulphate sequence of N-linked oligosaccharides. Biochem. J., 319, 209216.[ISI][Medline]
Toma, L., Pinhal, M.A.S., Dietrich, C.P., Nader, H.B., and Hirschberg, C.B. (1996) Transport of UDP-galactose into the Golgi lumen regulates the biosynthesis of proteoglycans. J. Biol. Chem., 271, 38973901.
Uchimura, K., Muramatsu, H., Kadomatsu, K., Fan, Q.W., Kurosawa, N., Mitsuoka, C., Kannagi, R., Habuchi, O., and Muramatsu, T. (1998a) Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase. J. Biol. Chem., 273, 2257722583.
Uchimura, K., Muramatsu, H., Kaname, T., Ogawa, H., Yamakawa, T., Fan, Q.W., Mitsuoka, C., Kannagi, R., Habuchi, O., Yokoyama, I., and others (1998b) Human N-acetylglucosamine-6-O-sulfotransferase involved in the biosynthesis of 6-sulfo sialyl Lewis X: molecular cloning, chromosomal mapping, and expression in various organs and tumor cells. J. Biochem. (Tokyo), 124, 670678.[Abstract]
Vestweber, D., and Blanks, J.E. (1999) Mechanisms that regulate the function of the selectins and their ligands. Physiol. Rev., 79, 181213.
Wang, W.-C., and Cummings, R.D. (1988) The immobilized leukoagglutinin from the seeds of Maackia amurensis binds with high affinity to complex-type Asn-linked oligosaccharides containing terminal sialic acid-linked alpha-2, 3 to penultimate galactose residues. J. Biol. Chem., 263, 45764585.
Wing, D.R., Rademacher, T.W., Field, M.C., Dwek, R.A., Schmitz, B., Thor, G., and Schachner, M. (1992) Use of large-scale hydrazinolysis in the preparation of N-linked oligosaccharide libraries: application to brain tissue. Glycoconj. J., 9, 293301.[ISI][Medline]
Yamashita, K., Ueda, I., and Kobata, A. (1983) Sulfated asparagine-linked sugar chains of hen egg albumin. J. Biol. Chem., 258, 1414414147.