2Department of Molecular Biology, University of Wyoming, Laramie, WY 820713944, USA and 3McGill Cancer Centre, McGill University, Montréal, Québec H3G 1Y6, Canada
Received on February 4, 1999; revised on October 28, 1999; accepted on October 28, 1999.
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Abstract |
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Key words: 1,2-mannosidase/substrate specificity/insect/GST/N-glycosylation
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Introduction |
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Sf9 is a lepidopteran insect cell line which is frequently used as a host for baculovirus-mediated glycoprotein production. As part of our efforts to more definitively elucidate the N-glycan processing capabilities of insect cells, we previously isolated an 1,2-mannosidase cDNA from Sf9 cells and demonstrated that it encodes an enzyme, SfManI, which releases [3H]mannose from [3H]Man9GlcNAc, requires calcium, and is inhibited by 1-deoxymannojirimycin (Kawar et al., 1997
). SfManI is a member of the class I
-mannosidase family, which includes processing enzymes that specifically cleave
1,2-linked mannose residues from N-linked oligosaccharides (reviewed in Moremen et al., 1994
; Herscovics, 1999a
). The precise number of enzymes within this family and their inter-relationships are being investigated, and the relationship between SfManI and other
1,2-mannosidases is unknown.
In Saccharomyces cerevisiae there is only one class I -mannosidase and this enzyme specifically cleaves only the
1,2-mannose on the middle arm of Man9GlcNAc2 (M10 in Figure 1; Byrd et al., 1982
; Jelinek-Kelly and Herscovics, 1988
). In contrast, mammalian cells have several
1,2-mannosidases with different tissue expression patterns, subcellular localizations, and substrate specificities (reviewed in Moremen et al., 1994
; Herscovics, 1999b
). Among these is a human ortholog with the same specificity as the yeast processing
1,2-mannosidase (Gonzalez et al., 1999
; Tremblay and Herscovics, 1999
) and a separate group of enzymes, including two
1,2-mannosidases (IA and IB) from mouse (Herscovics et al., 1994
; Lal et al., 1994
, 1998) and human tissues (Bieberich and Bause, 1995
; Tremblay et al., 1998
), and a pig
1,2-mannosidase (Bause et al., 1992
; Bieberich et al., 1997
). The latter enzymes have similar substrate specificities, readily cleaving three of the four
1,2-mannose residues from Man9GlcNAc2 (M8, M11, and M9 in Figure 1), but cleaving the fourth (M10) at a much slower rate. There are also additional
1,2-mannosidases in the ER of mammalian cells, but these have not been cloned (Bischoff and Kornfeld, 1983
; Bischoff et al., 1986
; Rizzolo and Kornfeld, 1988
; Weng and Spiro, 1993
, 1996). Mammalian cells also contain an endo-
1,2-mannosidase (Lubas and Spiro, 1987
; Spiro et al., 1997
) which specifically cleaves M11 (Figure 1) if one or more glucose residues remain attached to this mannose residue.
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The goal of the present study was to establish the substrate specificity of SfManI and its relationship to known 1,2-mannosidases. To accomplish this, we produced a GST-tagged, secreted form of SfManI, purified it by affinity chromatography, and used this recombinant protein for substrate specificity assays.
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Results |
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Substrate specificity of GST-SfManI
The substrate specificity of the affinity purified GST-SfManI was examined in vitro with [3H]Man9GlcNAc as the substrate. To determine how many 1,2-linked mannose residues were removed from this oligosaccharide, samples were taken from the reaction mixture after various incubation times and analyzed by HPLC (Figure 5A). Although Man9GlcNAc was ultimately converted to Man5GlcNAc, there was significant accumulation of Man6GlcNAc during the reaction (Figure 5A, graphs 25), and a substantial amount of this intermediate remained even after 8 h of incubation with the enzyme (Figure 5A, graph 5). However, when [3H]Man8GlcNAc isomer B, which lacks M10 (structure shown in Figure 5B) was used as the substrate, it was quantitatively converted to Man5GlcNAc after only 3 h of incubation with GST-SfManI, with no accumulation of any intermediates (Figure 5B, graphs 25). Control reactions demonstrated that GST-SfManI that had been boiled for 3 min before being assayed had no activity against Man9GlcNAc (Figure 5A, graph 1) or Man8GlcNAc isomer B (Figure 5B, graph 1). These results indicate that cleavage of M10 is the rate-limiting step in the conversion of Man9GlcNAc to Man5GlcNAc by GST-SfManI.
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Discussion |
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The precise relationship between SfManI and the 1,2-mannosidase which was purified from Sf9 cells by Ren and coworkers (Ren et al., 1995
) remains unclear. When the latter enzyme was incubated with Man9GlcNAc2, some of this substrate was converted to Man8GlcNAc2 and Man7GlcNAc2. In contrast, SfManI converted Man9GlcNAc to Man6GlcNAc and Man5GlcNAc. These results might indicate that SfManI and the purified mannosidase are different enzymes with different substrate specificities. Alternatively, these results could simply reflect differences in the amount of enzyme added to the assays in these two different studies. Interestingly, the enzyme purified by Ren and co-workers preferred an oligosaccharide substrate with M8 as the only
1,2-linked mannose residue over substrates containing other
1,2-linked mannose residues. Therefore, it is possible that the purified enzyme would remove M11 then M8 from a Man9GlcNAc2 substrate to produce Man7GlcNAc2 isomer C, as was demonstrated for SfManI in the present study.
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Materials and methods |
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Construction of a recombinant baculovirus expressing a GST-tagged, secreted form of SfManI
A DraI (New England Biolabs, Beverly, MA) fragment of the SfManI cDNA (Kawar et al., 1997) encoding the C-terminal stem and catalytic domains of the enzyme, but not its N-terminal cytoplasmic tail or membrane spanning region, was subcloned into the baculovirus transfer plasmid pAcSecG2T (Pharmingen, San Diego, CA). The resulting plasmid, pAcGST-SfManI, encoded a hybrid protein consisting of a cleavable signal peptide, the GST protein, and the SfManI protein fragment under the transcriptional control of the polyhedrin promoter (Figure 2). This plasmid was used to produce a recombinant baculovirus vector, designated AcGST-SfManI, which was capable of expressing a GST-tagged, secreted form of SfManI during the very late phase of infection. The recombinant virus was produced by using a standard allelic transplacement method (Summers and Smith, 1987
; O'Reilly et al., 1992
) with Bsu36I-digested BacPAK6 viral DNA (Kitts and Possee, 1993
) as the target for homologous recombination. The recombinant virus was plaque purified twice, amplified in Sf9 cells, and titered by plaque assay.
Baculovirus-mediated production of GST-SfManI
Sf9 cells were seeded into 75 cm2 tissue culture flasks (Corning Glass Works, Corning, NY) at a density of 10 million cells per flask and infected at a multiplicity of infection of 2 plaque forming units per cell with either AcGST-SfManI or wild type baculovirus (strain E2) as a negative control. The cells were cultured in TNM-FH medium containing 10% (v/v) heat-inactivated fetal bovine serum for 20 h, and then the medium was removed and the cells were washed twice with Graces medium (Grace, 1966) containing 0.5% (v/v) heat-inactivated fetal bovine serum and fed with 12 ml of the same medium. At selected times post infection, the extracellular medium was harvested, clarified by centrifugation at 1000 x g for 5 min, and a sample from the supernatant was mixed with an equal volume of 2x protein sample buffer containing 30 µg/ml of the protease inhibitor E-64 (Sigma, St. Louis, MO; Hom and Volkman, 1998
). In parallel, the cells in the culture flask were rinsed twice with TBS and lysed with protein sample buffer containing 15 µg/ml of E-64. Samples of these extra- and intracellular total protein extracts were then analyzed by discontinuous SDSPAGE (Laemmli, 1970
) followed by Coomassie blue staining to monitor production of the GST-SfManI protein. Alternatively, the proteins were electrophoretically transferred from the polyacrylamide gel to a Westran® membrane (Schleicher & Schuell, Keene, NH; Towbin et al., 1979
) and the membrane was probed with a rabbit antibody raised against E.coli-expressed SfManI (Z.Kawar and D.L.Jarvis, unpublished observations). The primary antibody reaction was followed by a secondary reaction with an alkaline phosphatase-conjugated antibody against rabbit IgG (Promega, Inc., Madison, WI), which was detected using a standard color reaction (Blake et al., 1984
).
Affinity purification of the baculovirus-produced GST-SfManI
Extracellular medium was harvested from Sf9 cells at 72 h after infection with AcGST-SfManI, centrifuged at 20,000 x g for 30 min to remove virus particles and other particulates, adjusted to 20 mM TrisHCl (pH 8.0), 250 mM NaCl, and 1.25% Triton X-100, and passed through a 0.22 µm Costar cellulose acetate filter (Corning). A volume of 3060 ml of this medium was then applied at room temperature to an immobilized glutathione-agarose column (Pierce, Rockford, IL) equilibrated with binding buffer (20 mM TrisHCl, pH 8.0; 250 mM NaCl; and 1.25% Triton X-100). The medium was passed through the column twice and the medium remaining in the column was displaced with 1 ml of binding buffer. The column was then washed with 10 ml of mannosidase reaction buffer (100 mM Na+MES, pH 6.0; 10 mM CaCl2; 10 mM MgCl2; and 0.5% Triton X-100) and the GST-SfManI was eluted with 3 ml of the same buffer supplemented with 10 mM reduced glutathione. Samples of the starting material, flow-through, wash, and eluant were analyzed by SDSPAGE and Coomassie blue staining or Western blotting as described above. PNGase F and recombinant Endoglycosidase Hf (New England Biolabs) treatments of the affinity purified GST-SfManI were carried out at 37°C for 2 h under the conditions recommended by the supplier. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) according to the manufacturers instructions, using the microassay procedure with IgG as the standard.
HPLC analysis of -mannosidase reaction products
1,2-Mannosidase substrate specificity assays were performed at 37°C using 2.5 µg of affinity-purified GST-SfManI in 30 µl reactions containing 50 mM Na+MES (pH 6.0), 5 mM CaCl2, 5 mM MgCl2, 0.25% Triton X-100, 180 µg of Man9GlcNAc, and 30,000 d.p.m. of [3H]Man9GlcNAc. Equivalent assays were done with Man8GlcNAc isomer B as the substrate, which was produced by digestion of Man9GlcNAc with the yeast specific
1,2-mannosidase (Lipari and Herscovics, 1994
). Enzyme that had been inactivated by boiling for 3 min was used as a negative control with both substrates. After various incubation times, 5 µl samples were removed and added to 100 µl of water. The diluted samples were then boiled for 3 min and mixed with 1000 d.p.m. of [14C]Glc(13)Man9GlcNAc standards in a final volume of 250 µl containing 40% acetonitrile. Finally, these samples were chromatographed on a Spherisorb S5 NH2 Column (Waters Corp., Milford, MA) with a Varian model 5000 liquid chromatograph, as described previously (Romero et al., 1985
). The column was equilibrated with 40% acetonitrile containing 15 mM KH2PO4 and 5 mM NaN3 (pH 5.0). Samples were applied and eluted isocratically with this same solvent at a flow rate of 1 ml per min for 20 min. This was followed by elution with a linear gradient of 40% to 58% acetonitrile in 15 mM KH2PO4 and 5 mM NaN3 (pH 5.0) at the same flow rate for 95 min. Fractions were collected every min, each was added to 4 ml of Universol scintillation cocktail (ICN, Costa Mesa, CA), and radioactivity was measured using an LKB model 1218 Rackbeta liquid-scintillation counter equipped with a d.p.m. conversion program.
To characterize the isomeric configurations of the Man(68)GlcNAc2 intermediates, an -mannosidase assay was carried out as described above using 200 pmol of Man9GlcNAc2-PA (Takara Shuzo Co., LTD., Otsu, Shiga, Japan) as substrate with 1.5 µg of affinity-purified GST-SfManI in a 40 µl reaction. The reaction was stopped after 1 h at 37°C and fractionated by HPLC using a TSKgel Amide 80 column (Toso Haas, Montgomeryville, PA) with a Varian model 360 fluorescence detector (Varian Canada Inc., Mississauga, Ontario, Canada) to separate the Man(68)GlcNAc2-PA intermediates. An aliquot of each intermediate was then resolved by analytical reverse phase HPLC using a Micro Pak C18 column (C18Ip-40, Varian), as described previously (Yoshida et al., 1998
). PA-tagged oligosaccharide standards were purchased from Takara.
1H-NMR analysis of the Man7GlcNAc intermediate
For 1H-NMR analysis, an -mannosidase assay was carried out as described above using 3 mg of Man9GlcNAc mixed with 500,000 c.p.m. of [3H]Man9GlcNAc as substrate with 42 µg of affinity-purified GST-SfManI in a 500 µl reaction. The reaction was stopped after 105 min at 37°C, and the products were applied to a Sep-Pak C18 cartridge (Waters Corp.) to remove Triton X-100, as described previously (Romero and Herscovics, 1989
). The material that passed through the cartridge was lyophilized, resuspended in water-acetonitrile and fractionated by HPLC on a Spherisorb S5 NH2 Column, as described above. The fractions containing Man7GlcNAc were pooled, lyophilized, and resuspended in water. This material was then applied to a Bio-Gel P-6 column equilibrated with water, and the oligosaccharide-containing fractions were pooled, freeze-dried, dissolved in 99.9% D2O, lyophilized three times, and finally stored overnight in a dessicator over P2O5 in vacuo. It was then redissolved in 99.996% D2O and analyzed by 1H-NMR at the Université de Montréal NMR Facility using a 600 MHz spectrometer (Bruker) at 30°C. Acetone, with a chemical shift of 2.225 p.p.m. compared to 4.4-dimethyl-4-silapentane sulfonate, was used as an internal standard.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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