2 Proteome Analysis Team, Korea Basic Science Institute, Daejeon 305-806, Korea
3 Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK
4 Magnetic Resonance Team, Korea Basic Science Institute, Daejeon 305-806, Korea
5 Department of Sericultural and Entomological Biology, Miryang National University, Miryang 627-702, Korea
Received on May 2, 2002; revised on October 8, 2002; accepted on October 8, 2002
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Abstract |
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Key words: Antheraea pernyi storage protein / arylphorin / glycosylation / monoglucosylated oligomannose
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Introduction |
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Unusual GlcNAc-terminated glycans with carbohydrate-linked 2-aminoethylphosphonate (Hard et al., 1993),
1-3-monofucosylated or
1-3-(
1-6)difucosylated oligosaccharides, and terminal trisaccharide GalNAcß1-4[Fuc
1-3]GlcNAcß1- on the core
1-3 mannosyl residue (Kubelka et al., 1993
, 1995
) have been also described in insects. Recent studies with nonengineered insect cells Trichoplusia ni (Rudd et al., 2000
; Ailor et al., 2000
) or Spodoptera frugiperda (Davidson et al., 1990
) have revealed hybrid and complex-type oligosaccharide structures, albeit with limited amounts. These may indicate that insects have the potential to synthesize diverse glycan structures, although the majority of the N-linked oligosaccharides of insects found to date are of the oligomannose type.
Storage proteins, such as arylophorin, are synthesized from the fat body of a wide range of lepidopteran and dipteran larvae and also in other insect orders. They are the major proteins in the hemolymph. Storage proteins are taken up by the fat body shortly before pupation and stored in protein granules. The central function of the proteins seems to be a storage pool of the amino acid resources for complete development of the adult. There are at least two kinds of storage proteins in lepidoptera. They form loose clusters on a bivariate plot of the proportion of aromatic amino acid (tyrosine plus phenylalanine) versus methionine (Telfer and Kunkel, 1991). Arylphorin is rich in aromatic amino acids, and the second protein is referred to as methionine-rich storage protein. All have molecular masses of nearly 500 kDa and are hexamers composed of approximately 80 kDa subunits.
Antheraea pernyi is a lepidopteran and an economically important wild silk moth found in Europe and Far Eastern Asia. There is no report to describe oligosaccharide structure or function in A. pernyi. The work described here is the most detailed study of oligosaccharides in the lepidopteran storage protein arylphorin of A. pernyi, so far. Unexpectedly, arylphorin had monoglucosylated Glc1Man9GlcNAc2 in the oligosaccharide pool of A. pernyi as well as in that of Bombyx mori. This sugar may play a role in the folding of arylphorin or in the assembly of hexamer.
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Results |
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Oligosaccharide profile
As a final step, A. pernyi arylphorin used for oligosaccharide analysis was purified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) to remove other possible contaminating glycoproteins or glycans, even though it was estimated to be already more than 95% pure. An aliquot of the fluorescent-labeled glycans released by in-gel digestion with peptide-N-glycosidase F (PNGase F) was analyzed by normal-phase (NP) high-performance liquid chromatography (HPLC). The profile showed a clearly separated set of peaks, suggesting a series of oligosaccharides each differing by one monosaccharide unit (Figure 1A). Individual peaks were assigned glucose unit (GU) values by comparison with the elution positions of a standard 2-aminobenzamide (AB)-labeled dextran hydrolysate ladder. Then, each peak was assigned a preliminary structure using the elution positions and predetermined incremental values for monosaccharide residues (Guile et al., 1996). These data indicated that the major glycans were probably of the nonfucosylated oligomannose type; however, the predominant species was contained in the peak 11 (10.20 GU) with higher retention time than Man9GlcNAc2 (Figure 1A). Arylphorin of A. pernyi was well separated from other hemolymph proteins by gel-filtration chromatography, and the protein recovered in this way had the same oligosaccharide profile as glycans released from arylphorin in SDSPAGE gel bands (data not shown). Other experiments, including the hydrazinolysis described later, were carried out with arylphorin purified by gel filtration.
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Exoglycosidase sequencing of the oligosaccharides
The results strongly suggested that A. pernyi arylphorin contained mainly oligomannose-type oligosaccharides. To confirm this, aliquots of the glycan pool were digested with exoglycosidases alone or in combination. The specific activity of each enzyme was confirmed by digestion of authentic standards. The digestion mixtures were resolved by NP HPLC (Figure 1BF). The assignment of each peak in the glycan pool was checked by following its predicted elution position through each of the enzyme digests and by comparison with authentic standards. As expected, with the exception of three minor peaks, all of the structures collapsed into Man1GlcNAc2 (peak 12) following incubation with jack bean -mannosidase (Figure 1B), which removes
-mannose (Man
1-2, 3, 6Man). The minor peaks were not digested with a combination of
-mannosidase and almond meal
-fucosidase (Figure 1C). However, simultaneous treatment of the glycan with the
-mannosidase and bovine kidney
-fucosidase removed peak 13 from the total glycan pool, suggesting that Fuc was attached to proximal GlcNAc via an
1-6 linkage (Figure 1D).
Two undigested peaks (14 and 15) remained after jack bean -mannosidase and bovine kidney
-fucosidase treatment (Figure 1D). These structures were resistant to a range of exoglycosidases either alone or in combinations (A. ureafaciens sialidase, bovine testes ß-galactosidase, jack bean ß-N-acetylhexosaminidase, bovine kidney
-fucosidase, almond meal
-fucosidase, or bovine testes ß-galactosidase and jack bean ß-N-acetylhexosaminidase with jack bean
-mannosidase) (data not shown). There was no peak corresponding to peak 14 (6.00 GU) in the total glycan pools, which indicated that the peak was generated by the
-mannosidase digestion. Peak 15 (6.89 GU) eluted close to peak 7 (6.95 GU), corresponding to Man6GlcNAc2. Peak 7 decreased significantly after digestion with jack bean
-mannosidase, however peak 15 remained, suggesting that the two glycans are of the same size but of different composition.
When the glycan pool was subjected under A. saitoi -mannosidase treatment that is specific for Man
1-2Man, positions and patterns of peaks 14 were not changed, indicating no terminal Man linked via
1-2 to these glycans (Figure 1E). Peak 5 that seemed to be Man5GlcNAc2 compared to standard oligosaccharide was increased, whereas peaks 611 disappeared with emerging peak 16 (8.60 GU) around peak 8 (8.67 GU). It was likely that peak 16 was moved from peak 11, because the amount of peak 16 fraction (25.5%) was only comparable to that of peak 11 (28.3%). Furthermore, an extra aliquot of the exoglycosidase did not change the chromatogram, suggesting that peak 16 was the final product of A. saitoi
-mannosidase digestion. The jack bean and A. saitoi
-mannosidase digestion results together with the profiling of the total oligosaccharide pool demonstrated that, with the exception of peak 11, the major glycan pool of arylphorin might consist of typical oligomannose-type structures. Xanthomonas manihotis
-mannosidase that is specific for unbranched Man
1-6Man was used to determine if there was a Man with other than
1-2 linkage. Definitely, peaks 1 and 2 were moved to peaks 12 and 13, respectively, but other glycans were not changed at all on the chromatogram (Figure 1F). It demonstrated that peaks 1 and 2 had a terminal
1-6-linked Man on ß-mannosyl chitobiose and core-fucosylated one, respectively.
To confirm or determine the structures of all the glycans, each major glycan peak larger than 6.0 GU was fractionated from NP HPLC runs and digested with jack bean -mannosidase, individually. Purified peak 11 split into two peaks (14 and 15) (Figure 2), and other oligosaccharide fractions were fully digested to Man1GlcNAc2 (data not shown). The proportion of peak 14 increased; although peak 15 decreased it was still detectable even when incubation was carried out with a second aliquot or higher enzyme concentration (Figure 2B, C). This suggested that peak 14 was the final digestion product of the peak 11 by jack bean
-mannosidase but that peak 15 was very resistant to the enzyme digestion. Moreover, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) showed clearly that the two peaks were composed of 2GlcNAc and 4 or 5 hexoses (Figure 2D). We postulated that peaks 11, 14, and 15 corresponded to Hex1Man9GlcNAc2, Hex1Man4GlcNAc2, and Hex1Man5 GlcNAc2, respectively (Figure 2E), and peak 11 produced two peaks by jack bean
-mannosidase treatment. These data are consistent with the presence of Glc1Man9 GlcNAc2. In structure 16 (Figure 2E), the
1,6-linked Man is particularly resistance to digestion with jackbean
-mannosidase when the
1,3 arm is substituted (Cacan et al., 2001
).
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Discussion |
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Here the calculated numbers of each monosaccharide from oligosaccharide sequencing (Glc:Man:GlcNAc:Fuc=1:12:4:0.4) were quite well matched with the result of sugar composition analysis. Four moles of GlcNAc in oligomannose oligosaccharides suggests two N-glycosylation sites in arylphorin, and this is consistent with two conserved N-glycosylation sites in the N-terminal region of arylphorins of A. pernyi, B. mori, M. sexta, and Spodoptera litura (Fujii et al., 1989; Wu et al., 1996
; Zheng et al., 2000
).
The presence of Man3GlcNAc2 may indicate that the A. pernyi glycan processing pathway is, in this respect, the same as that of higher animals in that complete mannose trimming is achieved (März et al., 1995). Recently, ß(1,2)-N-acetylglucosaminyltransferase I (Velardo et al., 1993
), ß(1,2)-N-acetylglucosaminyltransferase II (Altmann et al., 1993
), and ß(1,4)-galactosaminyltransferase (Van Die et al., 1996
) activities have been detected in insect cells. In addition, D. melanogaster UDP-GlcNAc:
-3-D-mannoside ß1,2-N-acetylglucosaminyltransferase I (Sakar and Schachter, 2001
) was cloned and expressed, which suggested that insects have the potential to make glycans other than oligomannose type. However, in the storage proteins we found only oligomannose-type oligosaccharides. There is no report to date describing glycans carrying Gal or GlcNAc residues from an insect storage protein. Fucosylation and processing of Man5GlcNAc2 to Man3 GlcNAc2 require prior addition of an outer-arm GlcNAc by GlcNAc transferase I and subsequent removal of the GlcNAc (Marchal et al., 1999
). Although we found no evidence of GlcNAc-terminated chains, the fucosylation on the truncated oligosaccharides suggested GlcNAc transferase I activity in this silk moth, A. pernyi. No difucosylated glycans, such as those on honeybee venom phospholipase A2 (Staudacher et al., 1991
), were detected in this study. All glycans studied were neutral.
The most abundant peak was monoglucosylated Man9GlcNAc2, and this assignment was consistent with all the results described in this study. The last Glc was linked via 1-3 to Man9GlcNAc2, which suggested that it was partially processed in the endoplasmic reticulum (ER). Monoglucosylated oligomannosides on newly synthesized glycoproteins can participate in the protein folding pathway that involves the lectin chaperones calnexin and calreticulin and deglucosylation by glucosidase I and II (Bergeron et al., 1994
; Helenius, 1994
) and reglucosylation by UDP-Glc:glycoprotein glucosyltransferase in the ER (Parodi, 2000
). The reaction is, however, transient, and the oligosaccharides on protein are finally deglucosylated and further processed after acquisition of their native tertiary and quaternary structures. Monoglucosylated oligosaccharide on mature glycoproteins was found in Leishmania spp. promastigote (Funk et al., 1997
). The glycan was one of two predominant species and a common feature of the cell surface glycoproteins. The authors concluded that the Glc1Man6GlcNAc2 structure was present on mature glycoproteins when organisms either lack glucosidase II enzymes or have a low level of glucosidase II activity. This may explain the presence of glucosylated glycans; however, the oligosaccharide is presented only on arylphorins, not on overall hemolymph proteins of A. pernyi (Figure 6), suggesting that if the explanation given by Funk et al. (1997)
is correct, the monoglucosylated glycans on arylphorin are somewhat inaccessible. Characterization of putative ER and Golgi processing enzymes from A. pernyi's fat body, which is a biosynthetic place of arylphorin, would be necessary to address how this oligosaccharide comes to remain on arylphorins.
Even though it is very rare to find Glc1Man9GlcNAc2 in native mature glycoproteins, it has been described that normal storage proteins or tissues have the oligosaccharide as a major N-glycan. First of all, the standard Glc1Man9GlcNAc2 used in this study was purified from hen albumen, in which the structure was determined from immunoglobulins of egg yolk of hen or Japanese quail (Ohta et al., 1991; Matsuura et al., 1993
). A major glycoprotein present in the egg jelly coat of a starfish, Asterias amurensis, and vitellogenic substances from the ovary of A. rubens (L.) have the same glycan structure (Endo et al., 1987
; De Waard et al., 1987
). Moreover, Glc1Man9GlcNAc2 makes up some 2040% of the total glycans in
-mannosidase extracted from jackbean that is a plant storage tissue (Kimura et al., 1999
). Glc1Man9GlcNAc2 was not present in other hemolymph proteins (Figure 6A) but was present in arylphorin (SP2) of another silkworm, B. mori (Figure 6B). These data strongly suggest that Glc1Man9GlcNAc2 might play a specific role in storage proteins.
We could not describe the exact function of the oligosaccharides on storage protein, arylphorin, of A. pernyi. It is, however, worth pointing out that the Glc1Man9GlcNAc2 structure was not released efficiently by PNGase F (Figure 7A), although a significant amount of the glycan was reproducibly released from denatured arylphorin. If the oligosaccharide is exposed on the surface of arylphorin, we may expect to find Glc1Man5-8GlcNAc2, since the lepidopteran insect cell line has class I and II -mannosidase, which can convert Man9GlcNAc2 to Man3GlcNAc2 in the Golgi apparatus (Kawar et al., 2000
, 2001
). Considering that there are no detectable intermediate glucosylated oligomannosides, arylphorin, which is a hexameric protein, may be assembled with the Glc1Man9GlcNAc2 oligosaccharide in a location that restricts further processing of the glycan in the ER and Golgi. This speculation is supported by the evidence that the carbohydrate moiety of housefly, Musca domestica, arylphorin was not involved in its uptake by the fat bodies and integument (Marinotti et al., 1988
). In addition, removal of glucose-containing oligomannose-type oligosaccharides resulted in difficulty with the assembly of enzyme subunits to form the active quaternary structure for jackbean
-mannosidase. The authors concluded that the oligomannose type glycan of
-mannosidase might be necessary for the assembly of protein subunits rather than folding of the protein molecule (Kimura et al., 1999
). Taken together with these observations, it suggests that A. pernyi arylphorin requires Glc1Man9GlcNAc2 oligosaccharide for assembly of the hexamer as well as protein folding.
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Materials and methods |
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Animals
The Chinese oak silkworms, A. pernyi, were raised throughout all the stages in the oak tree field established in the National Institute of Agricultural Science and Technology, RDA, Korea. The A. pernyi and B. mori were reared on fresh mulberry leaves.
Hemolymph collection
The abdominal legs of the corresponding silkmoth larvae were injured with sharpened scissors and hemolymph bled from the wound. It was directly collected into a precooled test tube with a few crystals of 1-phenyl-2-thiourea to inhibit phenol oxidase. The hemolymph was centrifuged at 5000xg for 15 min to remove hemocytes and other debris. The hemolymph prepared was kept at -70°C until analysis.
Purification of storage proteins
Storage proteins were separated by simple native-gel electrophoresis described previously (Shimada et al., 1987) or gel filtration with Superdex 200 pg (16x60 cm, Pharmacia, Upsala, Sweden). The solvent was 50 mM sodium phosphate, pH 7.5, containing 0.1 M sodium chloride. Flow rate was 1.0 ml/min, and the eluting proteins were monitored at 280 nm. Storage proteins were eluted just after the void volume of the column.
Monosaccharide composition analysis
Each 0.1 mg of the protein was subjected to different conditions of acid hydrolysis. For analysis of amino or neutral sugars, the protein was hydrolyzed in 6 N HCl or 2 M trifluoroacetic acid at 100°C for 4 h, respectively. The hydrolyzate was evaporated to dryness using a SpeedVac (Savant Instruments, Holbrook, NY), resuspended in distilled water, and injected into a Bio-LC DX-300 (Dionex, Sunnyvale, CA) HPAEC with pulsed amperometric detection. Sixteen millimolar NaOH at a flow rate of 1 ml/min was used to separate monosaccharides on a CarboPac PA-1 column (Dionex, 4x250 mm) and a guard column (4x50 mm).
Extraction and fluorescence labeling of oligosaccharides
Approximately 5 µg of a protein was separated on a 10% SDSPAGE. Protein bands visualized by Coomassie Brilliant Blue staining were excised from the gel. Proteins were alkylated, digested with PNGase F, and extracted as described previously (Küster et al., 1997). All extracts were combined and dried in a SpeedVac. Automated glycan preparation by hydrazinolysis with arylphorin that was isolated by gel-filtration chromatography was performed using a GlycoPrep1000 (Oxford GlycoSciences). The dried samples were labeled with 2-AB using the Glyko Signal labeling kit (Bigge et al., 1995
).
Exoglycosidase sequencing with NP HPLC
The 2-AB-labeled glycans were dried and incubated with several exoglycosidases at 37°C for 1624 h in 100 mM sodium acetate buffer, pH 5.0, containing 2 mM Zn2+. Conditions for individual enzymes in the array were as follows: jack bean -mannosidase, 100 U/ml; A. saitoi
-mannosidase, 2 mU/ml; X. manihotis
-mannosidase, 0.2 U/ml; bovine kidney
-fucosidase, 0.2 U/ml; almond meal
-fucosidase, 0.2 mU/ml; A. ureafaciens sialidase, 1 U/ml; jack bean ß-N-acetylhexosaminidase, 10 U/ml; bovine testes ß-galactosidase, 2 U/ml. Each reaction mixture was cleaned up by filtration through a cellulose nitrate centrifugal filter with 0.45 µm pore size. The filter was preequilibrated with each 20 µl water for 30 min and 5% (v/v) acetonitrile for 5 min. After brief centrifugation, the mixture was applied on the top of the filter followed by incubation for 30 min. The filter was washed twice with 5% acetonitrile. The filtrates were combined, dried, and analyzed by HPLC.
The 2-AB-labeled sugars were separated on a 4.6x250 mm GlycoSep N column (Glyko, Milford, MA) using two Waters 510 pumps, 717 autosampler, and 474 fluorescence detector. The initial conditions were 20% solvent A, 50 mM ammonium formate, pH 4.4, and 80% solvent B, acetonitrile at a flow rate of 0.4 ml/min, followed by a linear gradient of 2058% A over 152 min, followed by 58100% A over the next 3 min. The total run time was 180 min, and column temperature was 30°C.
MALDI MS
All mass spectra were acquired on Micromass (Altrincham, Cheshire, UK) AutoSpec-FPDQ magnetic sector instrument fitted with a pulsed nitrogen laser (337 nm) and an array detector. Oligosaccharide pool or each peak of total 2-AB-labeled glycan on the HPLC chromatogram was collected to determine mass. Prior to MALDI MS, collections were dried in a SpeedVac and resuspended in 20 µl distilled water. Samples were applied to Ziptip C18 (Millipore, Bedford, MA) equilibrated with acetonitrile. The Ziptip was washed with water, and labeled glycans were eluted with twice 20 µl of 50% (v/v) acetonitrile. The eluate was dried in vacuo. Sugar samples were loaded onto the mass spectrometer target in 1 µl water, mixed with 1 µl dihydroxybenzoic acid (10 mg/ml in acetonitrile), and allowed to dry. The array detector was set to the high-resolution position, and the mass range was set to be appropriate to the sample being examined. For data acquisition, the laser was operated at full power, and the laser beam was moved manually over the sample to compensate for sample depletion under the laser beam.
Glycan preparation for 1H NMR analysis
N-linked oligosaccharides were released from hemolymph proteins (1020 mg) by treatment with PNGase F according to the standard deglycosylation protocol (Tarentino et al., 1989). Oligosaccharides were extracted by selective organic procedures (Verostek et al., 2000
). Briefly, the pH of the mixture was adjusted to around pH 5.5 using 1 N phosphoric acid after the deglycosylation. Proteins and oligosaccharides were precipitated by adding 4 volumes of ice-cold acetone followed by incubation at -20°C for 30 min. The mixtures were centrifuged at 14,000xg at 4°C for 10 min, and the salt- and SDS-containing supernatant was discarded. The pellet was extracted with 0.25 ml of ice-cold 60% (v/v) aqueous methanol by sonication and 1 h incubation at -20°C. A second 60% methanol extract of the pellet was combined with the first and dried. The dried material was dissolved in a minimal volume of water. Any residual SDS and peptides were removed from the oligosaccharide pools by passage through a Sep-Pak C18 cartridge (Waters). The run-through fraction containing oligosaccharides was concentrated with a SpeedVac or injected directly into HPAEC with CarboPac PA1 (9x250 mm, Dionex). The oligosaccharides were eluted with a linear gradient of 00.2 M sodium acetate in 0.25 M NaOH in 30 min at a flow rate of 4.2 ml/min, and the eluant was monitored with pulsed amperometric detection. Oligosaccharide fractions were collected and desalted by passage through cation-exchange resin, AG50X-12 (H+ form, Bio-Rad). The glycan pools were dried in vacuo.
1H NMR spectrometry
Oligosaccharide was exchanged several times by rotary evaporation from 99.9% D2O then dissolved in 0.25 ml 99.996% D2O (Aldrich, Milwaukee, WI). A microtube (Shigemi, Allison Park, PA) was used to decrease the sample volume. 1H NMR spectrum was obtained with 600 MHz Bruker DMX 600 Avance instrument (Bruker Biospin GmbH, Karlsruhe, Germany) at 25°C. One-dimensional 1H NMR spectrum was acquired by suppressing the water peak using the presaturation method. Four hundred scans were averaged of 32K complex data points using spectral widths of 600 MHz. Chemical shifts () were given relative to the residual HDO signal at 4.8 ppm.
N-terminal amino acid sequencing
Amino acid sequences were determined using a 491A protein sequencer (Perkin-Elmer, Foster City, CA). The N-terminal sequence obtained was used for protein identification by a BLAST search of NCBI.
PNGase F digestion of native protein
Ten micrograms each of purified arylphorin was subjected under nondenaturing digestion for 18 h with 10 mU PNGase F in 10 µl 50 mM sodium phosphate, pH 7.5. An extra aliquot of PNGase F was added to complete the reaction. Released oligosaccharides were recovered, 2-AB-labeled, and separated on NP HPLC as described. Oligosaccharides from denatured arylphorin by PNGase F were used as control.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail:shkim{at}kbsi.re.kr
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Abbreviations |
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References |
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