Department of Animal Physiology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30060 Kraków, Poland, 2Institute of Medical Biochemistry, Collegium Medicum, Jagiellonian University, Kopernika 7, 31034 Kraków, Poland, and 3Department of Psychiatry and Neurochemistry, Institute of Clinical Neuroscience, Göteborg University, Sahlgrenska University Hospital/Mölndal, S431 80 Mölndal, Sweden
Received on June 25, 1999; revised on December 22, 1999; accepted on December 29, 1999.
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Abstract |
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Key words: arylsulfatase A/core fucosylation/high mannose type glycans/MALDI MS
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Introduction |
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Despite numerous studies on human ASA, the structure of its carbohydrate moieties has not been fully elucidated. ASA is glycosylated in vivo at each of its three glycosylation sites and carries high mannose or hybrid type oligosaccharides (Sommerlade et al., 1994). The presence of minor endo-ß-N-acetylglucosaminidase H resistant glycans (Laidler et al., 1988
; Fujii et al., 1992
) as well as complex type glycans (Gieselmann et al., 1992
) in human enzymes in addition to the expected high mannose glycans was also reported. Recently Poretz et al. (1992)
and Laidler et al. (1994)
observed that human platelet and placental ASA contains high mannose and/or hybrid glycan with 6-O-L-fucose bound to the innermost N-acetylglucosamine residue. This has been established by lectin affinity chromatography on LCA-agarose column or by reaction with Aleuria aurantia agglutinin. These observations prompted us to definitely resolve the problem of core fucosylation and answer the question of which type of carbohydrate residue is present in human ASA.
Structural elucidation of the carbohydrate moieties typically involves either chemical (e.g., hydrazinolysis) or enzymatic (e.g., PNGase F) release of the glycan moiety from the glycoprotein followed by separation of the mixture into its constituents and detailed analyses of the resulting glycans. Oligosaccharides released by chemical or enzymatic methods can be separated by high-performance liquid chromatography (HPLC), high pH anion-exchange chromatography (HPAEC), gas-liquid chromatography (GC), serial lectin affinity chromatography (SLAC) (Lee et al., 1990; Hounsell, 1998
), and high-resolution polyacrylamide gel electrophoresis (Westfall, 1998
). The structures of the obtained glycans may then be solved by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy (Lee et al., 1990
). Integrated methods are available by which the sequence of oligosaccharides can be determined (Tomiya et al., 1988
; Webb et al., 1988
; Hoja-£ukowicz et al., 1999). Küster et al. (1997)
reported a mass-spectrometric-based or normal-phase high-performance liquid chromatography-based strategy for determining the detailed structural features of N-linked oligosaccharides from protein gels in conjunction with exoglycosidase digestion. This comprehensive strategy include: sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) for protein separation and purification, in-gel deglycosylation using PNGase F for glycan release, and structural characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) or by normal-phase high-performance liquid chromatography following fluorescent labeling. Then oligosaccharides were sequenced using specific exoglycosidases, and digestion products were analyzed by MALDI MS.
In this study, we demonstrate by a combination of SDSPAGE, PNGase F on-blot deglycosylation, and structural characterization of released glycans by MALDI MS in conjunction with exoglycosidase digestion (Hoja-£ukowicz et al., 1999) that human ASA from placenta possesses core fucosylated high-mannose-type oligosaccharides.
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Results |
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Discussion |
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The previous studies indicating the presence of complex type glycans, were carried out mainly on the enzyme synthesized in transfected BHO cell with cDNA of placental ASA (Braulke et al., 1987; Gieselmann et al., 1992
). However, these mutants have different glycosylation, phosphorylation, and intracellular sorting of ASA. It is noteworthy that the higher secretion of these mutants was associated with an increased processing of its oligosaccharide to an Endo Hresistant form. Gieselmann et al. (1992)
assumed a priori that Endo Hresistant glycans were complex type oligosaccharides. However, it is well established that Endo H can effectively hydrolyze the chitobiose unit in mannose-containing N-linked oligosaccharides possessing at least three mannose residues, providing the
1,6-mannose arm with at least another mannose attached to it (Maley et al., 1989
). This specificity of Endo H is in agreement with the presence of glycans: Man4GlcNAc2 and Man5GlcNAc2 (1095.4, 1111.4, 1257.5, 1273.5; Figure 2A). Moreover, the presence of fucose in the core region of Man3GlcNAc2 compound is an additional limiting factor toward hydrolysis by Endo H (compare compound Man3GlcNAc(Fuc)GlcNAc, m/z 1079.4, Figure 2(A)) (Maley et al., 1989
). In any event, when the
1,6-mannosyl residue, linked to the core ß-mannose, is unsubstituted in mannose-containing oligosaccharides, resistance to Endo H hydrolysis is encountered. A more detailed analysis of arylsulfatase A glycans based on PAGE, SDSPAGE, and Western blots did not confirm the presence of complex type glycans. Moreover, subunits of arylsulfatase A did not react with either Datura stramonium agglutinin (DSA) or Ricinus communis I agglutinin (RCA), and native placental enzyme did not bind to RCA (I)agarose even though it contained core fucose (Laidler et al., 1994
).
Another unresolved question so far has been whether the glycans present on each subunit were of the high-mannose and/or hybrid type. This uncertainty arose mainly from the fact that the enzyme showed a positive reaction with Aleuria aurantia lectin (AAL) and Lens culinaris lectin (LCA), the probes for core fucose (Poretz et al., 1992; Laidler and Lityñska, 1997). It has been established that 6-O-L fucosylation of the innermost GlcNAc is one of the later events during the oligosaccharide processing, which occurs almost exclusively in complex- and hybrid-type structures. The enzyme responsible for the addition of the core fucose (
1,6-fucosyltransferase) requires at least the presence of a ß1,2GlcNAc unit on the core (
13)Man (Schwarz and Elbein, 1985
; Voynow et al., 1991
). As the core fucose was not expected, since high-mannose-type oligosaccharides have been reported not to be substrates for the
1,6-fucosyltransferase, Laidler and Lityñska (1997) did not exclude the presence of hybrid-type glycans. The early view of posttranslational glycosylation suggested that
-mannosidase I and II were found in the cis and medial Golgi, and GlcNAc-transferase I and
-1,6-fucosyltransferase in the medial Golgi. Further investigation demonstrated cell-type-specific Golgi subcompartmentation and showed a different overlapping in the localization of some glycosylation enzymes (Velasco et al., 1993
; Rabouille et al., 1995
) and the different sequence requirements for the Golgi retention of the same enzyme in various cell types (Colley, 1997
). Although no rigorous localization studies on the
1,6-fucosyltransferase have been performed, Magner et al. (1986)
, 1992) demonstrated that active mouse thyrotrophs appeared to shift the subcellular site of fucosylation partially from Golgi to RER. In addition, the investigation of the Golgi to RER recycling pathway could explain the presence of fucosyltransferase in the RER in some cell types (Doms et al., 1989
; Ulmer and Palade, 1989
; Lippincott-Schwartz et al., 1990
). In fact, a few naturally occurring lysosomal glycoproteins (cathepsin B, cathepsin D, ß-glucuronidase, and
-mannosidase) have been reported to bear core-fucosylated small oligomannose-type (Man5GlcNAc2 and smaller) N- glycans (Howard et al., 1982
; Takahashi et al., 1983
, 1984; Taniguchi et al., 1985
; Kozutsumi et al., 1986
; Maley et al., 1989
). All of these enzymes possess a high content of oligomannose structures and bear a high percentage of large oligomannose chains (e.g., Man89GlcNAc2), but only Man5GlcNAc2 and smaller chains were found to be fucosylated.
Our experiment has led us to somewhat confusing results, because so far nobody has detected Man6GlcNAc(Fuc)GlcNAc oligosaccharide on glycoprotein. In the literature it has been debated whether a biosynthetic or degradative pathway is responsible for the formation of these structures (Lin et al., 1994). Taking together all of these investigations we postulate two possibilities for the processing pathway of glycan residues of human placental ASA. (1) Since trimming of Man8GlcNAc2 to Man5GlcNAc2 occurs in cis Golgi and neither Man5GlcNAc2 nor shorter glycans serve as an acceptor substrate for
1,6-fucosyltransferase, core fucosylation of Man8GlcNAc2 or Man9GlcNAc2 (if (ER)
1,2-mannosidase and
1,6-fucosyltransferase compete for the same substrate) takes place in ER. Then core fucosylated high-mannose residues acquire Man 6-P marker and ASA is directed to lysosomes. (2) Or, if activity of (Golgi)-
1,2-mannosidase I is shifted to medial Golgi and
1,6 fucosyltransferase is localized in cis Golgi, Man8GlcNAc2 undergoes core fucosylation and then phosphorylation, and ASA is directed to lysosomes, thus escaping from the subsequent modification by processing enzymes.
The presence of Man6GlcNAc(Fuc)GlcNAc structure clearly indicated that the smaller structures with fucose are not the product of degradation of fucosylated complex- or hybrid-type oligosaccharides. In the environment of lysosomal mannosidases, part of the sugar chains of ASA may undergo degradation to shorter chains. These hypotheses are in agreement with our results (Figure 2A; Man35GlcNAc(Fuc)GlcNAc, m/z 1079.4, 1242.5, 1403.5).
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Materials and methods |
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Gel electrophoresis
The gel (85 x 70 x 1 mm) was prepared in SDS conditions and using a discontinuous buffer system according to Laemmli (1970). Purified ASA was separated on 4.5% stacking gel and on 10% separation gel. Prior to electrophoresis, protein sample (6 µg of ASA) and control sample (1 µg of ASA) were boiled at 100°C for 10 min. The HMW protein standards (8 µl of the color markers) were boiled at 100°C for 3 min. Electrophoresis was run using a Mini-PROTEAN II cell (Bio-Rad) for ~3 h.
Western blotting
Electroblotting was run using a Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) at 40 V constant voltage in 25 mM Tris/192 mM glycine/20% methanol, pH 8.4. Proteins (sample, 6 µg of ASA; control, 1 µg of ASA) were transferred onto an Immobilon P sheet overnight. Prior to electrophoretic transfer, the gel was preequilibrated in a transfer buffer for 5 min.
Protein alkylation
The procedure essentially follows the method described by Küster et al. (1998). The excised Immobilon P pieces (8 x 2 mm) were placed into Eppendorf tubes and washed twice with 20 mM NaHCO3, pH 7.0, 15 min for each. The wash was discarded and replaced by 300 µl of fresh 20 mM NaHCO3, pH 7.0. To this solution 20 µl of 45 mM dithiothreitol (DDT) was added and the protein reduced at 60°C for 30 min. After cooling to room temperature (RT), 20 µl of 100 mM iodoacetamide (IAA) was added and the protein alkylated for 30 min at RT in the dark. The reducing and alkylation reagents, as well as residual SDS, were then removed by incubation in 1/1 acetonitrile/fresh 20 mM NaHCO3, pH 7.0, for 60 min. Subsequently, the membrane pieces were incubated in the blocking solution (Boehringer Mannheim Biochimica).
In situ digestion (Weitzhändler et al., 1993)
Prior to deglycosylation, Immobilon P pieces were washed with 20 mM NaHCO3, pH 7.0, for 3 x 15 min. The washings were discarded and replaced by 3 U of PNGase F in 50 µl of 20 mM NaHCO3, pH 7.0, and incubated at 37°C for 1216 h (Tarentino et al., 1985). Simultaneously, the control was incubated with buffer alone. The removal of oligosaccharides from the protein was confirmed by the negative reaction of deglycosylated ASA with digoxigenin-labeled GNA (Laidler et al., 1994
).
Sugar extraction
The incubation buffer, after deglycosylation, was completely dried in a SpeedVac (JW Electronic, Poland), and then dissolved in 10 µl of ultrapure water (Milli-Q Plus, Millipore, Bedford, CA, USA), and applied to a microcolumn clean-up.
Microcolumn clean-up of sugars
Prior to MALDI MS, a microcolumn consisting of an Eppendorf GELoader pipette tip (Küster et al., 1997) packed with ~5 µl each of (AG-3 OH- form, bottom) and AG-50 (H+ form, top) was used. The column was washed with 100 µl water and an aliquot of the sugar sample was applied. Glycans were eluted with 100 µl water and dried in a SpeedVac.
Exoglycosidase digestion
Oligosaccharides, obtained by on-blot deglycosylation from 6 µg (50 pmol) human ASA, were split into aliquots, each containing about 20% of total glycans, and incubated overnight at 37°C in 20 mM sodium acetate at pH 5.5 in parallel with the following exoglycosidase arrays: (1) 1 mU of bovine epididymis -fucosidase in 3 µl of 20 mM sodium acetate at pH 5.5; (2) 0.5 mU of fucosidase and 15 mU of jack bean ß-N-acetylhexosaminidase in 3 µl of 20 mM sodium acetate at pH 5.5; (3) 0.33 mU of fucosidase, 10 mU of ß-N-acetylhexosaminidase and 33 mU of Jack bean
-mannosidase in 3 µl of 20 mM sodium acetate at pH 5.5 (Küster et al., 1997
). Prior to MALDI MS, each reaction mixture was desalted using the microcolumn clean-up described above.
MALDI mass spectrometry
Angiotensin II (1045.5 u), ACTH clip 1839 (2464.2 u), bovine insulin (5733.5 u) and equine cytochrome c (12360.1 u) were used for external calibration of the mass spectrometer. The following glycans were used as oligosaccharide standards: high-mannose MAN-9; hybrid HYBR; asialo-, galactosylated tetraantennary NA4; di-sialylated, galactosylated biantennary, core substituted with fucose A2F. All calibrants (15 pmol/µl) were dissolved in 0.1% trifluoroacetic acid (TFA) in ultrapure Milli-Q Plus water. 2,5-dihydroxy benzoic acid (DHB) was used as MALDI matrix. The matrix was dissolved in HPLC-grade acetronitrile (1 g/l for the seed layer) (Westman et al., 1998) or in 0.1% TFA in acetonitrile/ultrapure water [1:1 v/v] (10 g/l, saturated for the sample/matrix mixture).
All samples were prepared with the seed layer method (Westman et al., 1998). First, a matrix seed layer was created by depositing a droplet (1 µl) of a 1 g/l solution of matrix dissolved in acetonitrile on a highly polished, stainless steel sample probe. Thereafter, the 10 g/l matrix and sample solutions were mixed in a test tube 1:1, and a droplet (1 µl) of sample/matrix was deposited on the matrix seed layer. Samples were then left to dry totally in air.
All MALDI analyses were performed with an upgraded Reflex II MALDI-TOF mass spectrometer (Bruker-Franzen Analytic Gmbh, Bremen, Germany). Samples were irradiated with a 337 nm nitrogen and a laserspot ~1020 µm in diameter. All spectra were acquired in the reflectron mode at an accelerating voltage of 20 kV. Mass spectra were analyzed using the software provided by Bruker. All spectra shown were calibrated using external calibration with a mass deviation of within 0.08%. Sample-potential/first-electrode potential ratio was optimized to achieve optimal resolution for the molecules studied. The sample probe was made of highly polished stainless steel. Since the spectrometer was equipped with a computer-controlled XY sample stage and high-definition observation optics connected to a video camera, visual inspection of the sample inside the MALDI-TOF MS was possible. This made it possible to aim the laser beam at specific sample spots and take full advantage of the small deposits on the target.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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