N-Glycan processing by a lepidopteran insect {alpha}1,2-mannosidase

Ziad Kawar2, Pedro A. Romero3, Annette Herscovics3 and Donald L. Jarvis1,2

2Department of Molecular Biology, University of Wyoming, Laramie, WY 82071–3944, 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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Protein glycosylation pathways are relatively poorly characterized in insect cells. As part of an overall effort to address this problem, we previously isolated a cDNA from Sf9 cells that encodes an insect {alpha}1,2-mannosidase (SfManI) which requires calcium and is inhibited by 1-deoxymanno­jirimycin. In the present study, we have characterized the substrate specificity of SfManI. A recombinant baculovirus was used to express a GST-tagged secreted form of SfManI which was purified from the medium using an immobilized glutathione column. The purified SfManI was then incubated with oligosaccharide substrates and the resulting products were analyzed by HPLC. These analyses showed that SfManI rapidly converts Man9GlcNAc2 to Man6Glc­NAc2 isomer C, then more slowly converts Man6GlcNAc2 isomer C to Man5GlcNAc2. The slow step in the processing of Man9GlcNAc2 to Man5GlcNAc2 by SfManI is removal of the {alpha}1,2-linked mannose on the middle arm of Man9GlcNAc2. In this respect, SfManI is similar to mammalian {alpha}1,2-manno­sidases IA and IB. However, additional HPLC and 1H-NMR analyses demonstrated that SfManI converts Man9GlcNAc2 to Man5GlcNAc2 primarily through Man7GlcNAc2 isomer C, the archetypal Man9GlcNAc2 missing the lower arm {alpha}1,2-linked mannose residues. In this respect, SfManI differs from mammalian {alpha}1,2-mannosidases IA and IB, and is the first {alpha}1,2-mannosidase directly shown to produce Man7GlcNAc2 isomer C as a major processing intermediate.

Key words: {alpha}1,2-mannosidase/substrate specificity/insect/GST/N-glycosylation


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The ability to modify proteins by N-glycosylation is conserved among all eukaryotic organisms, indicating its importance in modulating protein function. N-glycan biosynthesis usually begins with the transfer of a preassembled Glc3Man9GlcNAc2 oligosaccharide to newly-formed proteins, followed by removal of the terminal glucose residues by two {alpha}-gluco­sidases. The oligosaccharide then undergoes further trimming and extension reactions that vary according to local protein structure and the nature of the cellular processing machinery. Mammalian cells have several {alpha}-mannosidases that can remove up to six mannose residues (reviewed in Moremen et al., 1994Go; Herscovics, 1999aGo,b) and a variety of glycosyltransferases that can extend the trimmed oligosaccharides by adding N-acetylglucosamine, fucose, galactose, and sialic acid residues (reviewed in Kornfeld and Kornfeld, 1985Go). In contrast, Saccharomyces cerevisiae has a single processing {alpha}-mannosidase and several mannosyltransferases that produce characteristic polymannose N-glycans (reviewed in Tanner and Lehle, 1987Go; Ballou, 1990Go; Herscovics, 1999cGo; Lussier et al., 1999Go). The N-glycan processing pathways of insects are not as well-characterized as those of yeast and mammals. However, it is known that N-linked oligosaccharides produced by insect cells usually range from Man9GlcNAc2 to Man3GlcNAc2, and there is evidence to suggest that insects can also produce more highly extended mammalian-like structures (reviewed in Marz et al., 1995Go; Jarvis et al., 1998Go). A clearer understanding of the insect cell N-glycosylation pathway is needed because these cells serve as hosts for baculovirus expression vectors which are widely used to produce recombinant glycoproteins for basic research and biomedical applications (Summers and Smith, 1987Go; O'Reilly et al., 1992Go).

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 {alpha}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., 1997Go). SfManI is a member of the class I {alpha}-mannosidase family, which includes processing enzymes that specifically cleave {alpha}1,2-linked mannose residues from N-linked oligosaccharides (reviewed in Moremen et al., 1994Go; Herscovics, 1999aGo). The precise number of enzymes within this family and their inter-relationships are being investigated, and the relationship between SfManI and other {alpha}1,2-mannosidases is unknown.

In Saccharomyces cerevisiae there is only one class I {alpha}-mannosidase and this enzyme specifically cleaves only the {alpha}1,2-mannose on the middle arm of Man9GlcNAc2 (M10 in Figure 1; Byrd et al., 1982Go; Jelinek-Kelly and Herscovics, 1988Go). In contrast, mammalian cells have several {alpha}1,2-mannosidases with different tissue expression patterns, subcellular localizations, and substrate specificities (reviewed in Moremen et al., 1994Go; Herscovics, 1999bGo). Among these is a human ortholog with the same specificity as the yeast processing {alpha}1,2-mannosidase (Gonzalez et al., 1999Go; Tremblay and Herscovics, 1999Go) and a separate group of enzymes, including two {alpha}1,2-mannosidases (IA and IB) from mouse (Herscovics et al., 1994Go; Lal et al., 1994Go, 1998) and human tissues (Bieberich and Bause, 1995Go; Tremblay et al., 1998Go), and a pig {alpha}1,2-mannosidase (Bause et al., 1992Go; Bieberich et al., 1997Go). The latter enzymes have similar substrate specificities, readily cleaving three of the four {alpha}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 {alpha}1,2-mannosidases in the ER of mammalian cells, but these have not been cloned (Bischoff and Kornfeld, 1983Go; Bischoff et al., 1986Go; Rizzolo and Kornfeld, 1988Go; Weng and Spiro, 1993Go, 1996). Mammalian cells also contain an endo-{alpha}1,2-mannosidase (Lubas and Spiro, 1987Go; Spiro et al., 1997Go) which specifically cleaves M11 (Figure 1) if one or more glucose residues remain attached to this mannose residue.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1. Man9GlcNAc2 structure. M, Mannose; G, N-acetylglucosamine; Asn, asparagine. Linkages are indicated in parentheses, and mannose residues are labeled 3–11.

 
The presence of at least one {alpha}1,2-mannosidase in insect cells is clearly indicated by their ability to produce glycoproteins with trimmed N-linked glycans and by the fact that 1-deoxymanno­jirimycin can block these processing reactions (Naim and Koblet, 1988Go; Jarvis and Summers, 1989Go; Jarvis and Garcia, 1994Go). An {alpha}1,2-mannosidase was purified from Sf9 cells and was shown to cleave M8 (Figure 1) at a faster rate than the other {alpha}1,2-linked mannose residues (Ren et al., 1995Go). However, the relationship between this enzyme and SfManI has not been determined. A putative {alpha}1,2-mannosidase cDNA was isolated from Drosophila (Kerscher et al., 1995Go), but the protein encoded by this cDNA has not been characterized. Finally, it was reported that, unlike mammalian cells, Sf9 and other insect cell lines lack endo-mannosidase activity (Dairaku and Spiro, 1997Go).

The goal of the present study was to establish the substrate specificity of SfManI and its relationship to known {alpha}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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Baculovirus-mediated production of a GST-tagged, secreted form of SfManI
A fragment of the SfManI cDNA encoding the C-terminal catalytic domain of the protein was inserted into a baculovirus transfer plasmid downstream of the polyhedrin promoter, a translational initiation site, a cleavable signal peptide sequence, and a GST coding sequence (Figure 2). The resulting plasmid, which encodes a secreted GST-SfManI hybrid protein, was used to produce a recombinant baculovirus vector, designated AcGST-SfManI. Expression of the GST-SfManI protein was assessed by comparing the total intracellular and extracellular protein profiles of Sf9 cells at various times after infection with either wild type baculovirus or AcGST-SfManI (data not shown). Discontinuous SDS–PAGE followed by Coomassie blue staining showed that cells infected with the recombinant virus contained a protein with an apparent molecular weight of about 96 kDa, which is the size expected for GST-SfManI. This protein was also secreted into the medium, was not observed in mock-infected or wild type baculovirus-infected cells, and was specifically recognized in Western blots probed with a rabbit antibody raised against E.coli-expressed SfManI (Z.Kawar and D.L.Jarvis, unpublished). The timing of expression was consistent with polyhedrin-mediated transcription of the chimeric gene by the recombinant baculovirus, as the hybrid protein was first detected at 24 h post infection by Western blotting and at 48 h postinfection by Coomassie blue staining. These results demonstrate that the recombinant baculovirus, AcGST-SfManI, can produce large amounts of GST-tagged SfManI, a significant proportion of which is secreted into the medium.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2. Genetic map of pAcGST-SfManI. A fragment of the SfManI cDNA encoding the C-terminal domain of the protein was inserted into a baculovirus transfer plasmid downstream of the polyhedrin promoter (Polh), the signal sequence of the baculovirus glycoprotein gp64 (gp64 SS), and a GST coding sequence. This expression cassette is flanked by homologous recombination sequences (HRS) from the polyhedrin region of the baculovirus genome. The diagram is not drawn to scale.

 
Affinity purification of GST-SfManI
For affinity purification of GST-SfManI, the medium was harvested from Sf9 cells at 72 h after infection with AcGST-SfManI and applied to an immobilized glutathione agarose column. The GST-SfManI protein was then eluted from the affinity column with mannosidase reaction buffer containing 10 mM reduced glutathione. Initially, we attempted to purify GST-SfManI from infected cells that had been cultured in TNM-FH medium or serum-free media (HyQ®SFX-Insect and EX-CELL{spadesuit} 420) from two different commercial sources. However, almost none of the GST-SfManI secreted into these media bound to the immobilized glutathione column (data not shown). We subsequently determined that the pluronic F68, yeast extract, and/or lactalbumin hydrolysate in these media interfered with binding of the hybrid protein to the column (data not shown). In contrast, the hybrid protein bound efficiently to the column when infected cells were cultured in Grace’s medium with 0.5% (v/v) heat-inactivated fetal bovine serum, but without pluronic F68, yeast extract, or lactalbumin hydrolysate. This was demonstrated by SDS–PAGE and Coomassie blue staining (Figure 3A). Most of the GST-SfManI bound to the column under these conditions and could be eluted with reduced glutathione. The column eluant contained a single major protein detectable by Coomassie blue staining. This protein was identified as GST-SfManI by its size and immunoreactivity with the anti-SfManI antibody in a Western blot (Figure 3B). Coomassie blue staining also revealed that there were very minor amounts of several smaller proteins in the eluant. Since all of these proteins reacted with the polyclonal anti-SfManI antiserum, they were probably degradation products of GST-SfManI. This purification procedure yielded about 0.5 mg of GST-SfManI from 50 ml of medium, and the enzyme was stable at –80°C for at least 9 months.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 3. Affinity purification of GST-SfManI. Extracellular medium was harvested from Sf9 cells 72 h after infection with AcGST-SfManI, adjusted to the appropriate buffer conditions, and applied to an immobilized glutathione column. The column was washed with mannosidase reaction buffer and eluted with the same buffer containing 10 mM reduced glutathione. Equivalent samples were taken from the medium before (lanes 1) and after (lanes 2) it was applied to the column as well as from the column wash (lanes 3) and eluant (lanes 4). These normalized samples were analyzed by SDS–PAGE followed by Coomassie blue staining (A) or by Western blotting with a polyclonal antiserum against SfManI (B). Lane 5 of (A) shows a 5-fold excess of the column eluant stained with Coomassie blue. The positions of protein markers are shown in kDa on the left-hand side of the figure and the positions of GST-SfManI and BSA are shown on the right.

 
Since the major GST-SfManI band was a doublet, we investigated whether the two forms of this protein were due to partial N-glycosylation at a sequon present in the SfManI portion of GST-SfManI. Treatment of the affinity purified GST-SfManI with PNGase F converted the larger form to the smaller one (Figure 4), indicating that it was N-glycosylated. However, the larger form of SfManI was resistant to treatment by endoglycosidase H, showing that the N-glycan was processed beyond the {alpha}-mannosidase II step of the N-linked oligosaccharide processing pathway.



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 4. Glycosylation of GST-SfManI. Affinity-purified GST-SfManI was treated for 2 h at 37°C with buffer alone (lane 1), endoglycosidase Hf (lane 2), or PNGase F (lane 3). The reaction products were analyzed by SDS–PAGE followed by Coomassie blue staining. The positions of protein markers are shown in kDa on the left-hand side of the figure and the positions of GST-SfManI, endoglycosidase Hf (endo Hf), and PNGase F are shown on the right. Endo Hf is a recombinant fusion protein of endoglycosidase H and maltose binding protein, with an apparent molecular weight of about 70 kDa.

 
The purified GST-SfManI retained its enzymatic activity as evidenced by its ability to release [3H]Mannose from [3H]Man9GlcNAc, using a previously described assay (Kawar et al., 1997Go). Removal of the GST tag by treatment with thrombin had no effect on this activity (data not shown).

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 {alpha}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 2–5), 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 2–5). 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.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5. Time course of digestion of Man9GlcNAc and Man8GlcNAc isomer B with GST-SfManI. Affinity-purified GST-SfManI was incubated with either [3H]Man9GlcNAc (A) or [3H]Man8GlcNAc isomer B (B) for the indicated times, and the oligosaccharide products were analyzed by HPLC on a Spherisorb S5 NH2 Column, as described in Materials and methods. Fractions were collected every min, and radioactivity was measured. Graph 1 in (A) and (B) shows the result of assays done for 8 h with GST-SfManI that had been inactivated by boiling. The small amount of Man7GlcNAc seen in (B) graph 1 was a contaminant present in the substrate. G1, G2, and G3 refer to [14C]Glc(1–3)Man9GlcNAc internal standards; M9 to M5 refer to [3H]Man(9–5)GlcNAc, and M refers to mannose.

 
To establish the order in which {alpha}1,2-linked mannose residues are removed, each processing intermediate produced by incubating GST-SfManI with Man9GlcNAc2-PA was isolated and subsequently fractionated by reverse phase HPLC to resolve the different isomers. The potential isomers of each oligosaccharide intermediate are shown in figure 6A, while the reverse phase HPLC profiles of the intermediates produced by GST-SfManI are shown in Figure 6B–D. Man8GlcNAc2-PA was mainly isomer A (Figure 6B) and Man6GlcNAc2-PA was mainly isomer C (Figure 6D), while the major peak produced during reverse phase HPLC analysis of Man7GlcNAc2-PA migrated at the position expected for isomer C (Figure 6C). Since there is no commercially available Man7GlcNAc2-PA isomer C standard, we analyzed this intermediate using 1H-NMR. The results showed that 80% of this intermediate was isomer C, while the other 20% was isomer D (Figure 7), which is barely detectable in the reverse phase HPLC analysis (Figure 6C).




View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6. Isomeric configurations of intermediates. Man9GlcNAc2-PA was treated with GST-SfManI for 1 h. The oligosaccharide intermediates were first size-fractionated by HPLC on a TSKgel Amide 80 column, and each intermediate was then analyzed by reverse phase HPLC. (A) shows the potential isomeric configurations of each intermediate. (B–D) show the reverse phase HPLC profiles obtained with Man8GlcNAc2-PA, Man7GlcNAc2-PA, and Man6GlcNAc2-PA respectively. Peaks were identified by comparison to PA-labeled standards of known isomeric configurations. M, mannose; G, N-acetylglucosamine; PA, pyridylamino.

 


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7. 1H-NMR analysis of the Man7GlcNAc intermediate. Man9GlcNAc was treated with GST-SfManI for 105 min and the Man7GlcNAc intermediate was isolated by HPLC on a Spherisorb S5 NH2 Column and analyzed by 1H-NMR. The numbers above each peak indicate which mannose (M) or N-acetylglucosamine (G) residue generated that signal, according to the numbering scheme shown above the NMR profile. The isomer structures and the percent representation of each in the sample are also indicated.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This study reveals the sequence of Man9GlcNAc2 processing by SfManI in vitro, as summarized in Figure 8. SfManI initially converts the Man9GlcNAc2 precursor to Man8GlcNAc2 isomer A by cleaving M11. Subsequently, this enzyme cleaves M8 and, to a lesser extent, M9 to produce Man7GlcNAc2 isomer C or D, respectively. SfManI is the first {alpha}1,2-mannosidase directly shown to produce Man7GlcNAc2 isomer C as a major intermediate. Mammalian {alpha}1,2-mannosidases IA and IB do not produce Man7GlcNAc2 isomer C (Lal et al., 1998Go; Tabas and Kornfeld, 1979Go), and studies on {alpha}1,2-mannosidases from Aspergillus (Ichishima et al., 1999Go) and pig liver (Bause et al., 1992Go) suggest that they produce only small amounts of this intermediate. SfManI then converts Man7GlcNAc2 to Man6GlcNAc2 isomer C by cleaving M9 or M8 from Man7GlcNAc2 isomer C or D respectively. Finally, SfManI cleaves M10 to produce the Man5GlcNAc2 end product, but this occurs at a significantly slower rate than cleavage of the first three mannose residues. In this respect, SfManI behaves like the mammalian {alpha}1,2-mannosidases IA and IB, which also cleave M10 more slowly than the other {alpha}1,2-linked mannose residues (Tabas and Kornfeld, 1979Go; Bause et al., 1992Go; Herscovics et al., 1994Go; Lal et al., 1994Go; Bieberich et al., 1997Go; Lal et al., 1998Go; Tremblay et al., 1998Go).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 8. Conversion of Man9GlcNAc2 to Man5GlcNAc2 by GST-SfManI. Nomenclature is the same as in Figure 6A.

 
It is important to recognize that the model shown in Figure 8 depicts oligosaccharide processing by SfManI in vitro. However, SfManI is probably not the only {alpha}1,2-mannosidase involved in N-glycan trimming in Sf9 cells. In other systems, there are complementary {alpha}1,2-mannosidases that can more effectively cleave M10. Among these are the single yeast {alpha}1,2-mannosidase (Byrd et al., 1982Go; Jelinek-Kelly and Herscovics, 1988Go) and its human ortholog (Gonzalez et al., 1999Go; Tremblay and Herscovics, 1999Go) which only cleave M10, an {alpha}1,2-mannosidase from Trypanosoma cruzi which cleaves M10 and M9 (Bonay and Fresno, 1999Go), and various mammalian {alpha}1,2-mannosidases which cleave all {alpha}1,2-linked mannose residues (reviewed in Herscovics, 1999aGo,b). Insect cells are also likely to have multiple {alpha}1,2-mannosidases since a mutant strain of Drosophila which has a defective copy of an uncharacterized {alpha}1,2-mannosidase gene can still trim {alpha}1,2-linked mannose residues (Roberts et al., 1998Go). Additionally, Southern blots of Sf9 genomic DNA probed with a fragment of the SfManI gene revealed that there is at least one other cross-hybridizing {alpha}1,2-mannosidase gene in these cells (Kawar et al., 1997Go).

The precise relationship between SfManI and the {alpha}1,2-mannosidase which was purified from Sf9 cells by Ren and coworkers (Ren et al., 1995Go) 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 {alpha}1,2-linked mannose residue over substrates containing other {alpha}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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cells and cell culture
Sf9 cells are derived from the IPLB-Sf21-AE cell line, which was originally isolated from Spodoptera frugiperda (fall armyworm) ovaries (Vaughn et al., 1977Go). Sf9 cells were maintained as a suspension culture at densities between 0.3 and 3.0 x 106 cells per ml in TNM-FH medium (Summers and Smith, 1987Go) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Summit Biotechnology, Fort Collins, CO) and 0.1% (w/v) pluronic F68 (BASF Wynandotte Corp., Parsippany, NJ; Murhammer and Goochee, 1988Go). For some experiments, Sf9 cells were cultured in HyQ®SFX-Insect serum-free medium (HyClone, Logan, UT) or in EX-CELLTM 420 serum-free medium (JRH Biosciences, Lenexa, KS).

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., 1997Go) 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, 1987Go; O'Reilly et al., 1992Go) with Bsu36I-digested BacPAK6 viral DNA (Kitts and Possee, 1993Go) 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 Grace’s medium (Grace, 1966Go) 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, 1998Go). 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 SDS–PAGE (Laemmli, 1970Go) 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., 1979Go) 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., 1984Go).

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 Tris–HCl (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 30–60 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 Tris–HCl, 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 SDS–PAGE 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 manufacturer’s instructions, using the microassay procedure with IgG as the standard.

HPLC analysis of {alpha}-mannosidase reaction products
{alpha}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 {alpha}1,2-mannosidase (Lipari and Herscovics, 1994Go). 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(1–3)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., 1985Go). 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(6–8)GlcNAc2 intermediates, an {alpha}-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(6–8)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., 1998Go). PA-tagged oligosaccharide standards were purchased from Takara.

1H-NMR analysis of the Man7GlcNAc intermediate
For 1H-NMR analysis, an {alpha}-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, 1989Go). 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.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Barry Sleno for technical assistance. This work was supported by grants from the National Institutes of Health (GM 49734 to D.L.J. and GM 31265 to A.H.) and from the Medical Research Council of Canada (to A.H.).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
BSA, bovine serum albumin; GST, glutathione S-transferase; Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electro­phoresis; PA, pyridylamino.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Ballou,C.E. (1990) Isolation, characterization and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol., 185, 440–470.[Medline]

Bause,E., Breuer,W., Schweden,J., Roeser,R. and Geyer,R. (1992) Effect of substrate structure on the activity of Man9-mannosidase from pig liver involved in N-linked oligosaccharide processing. Eur. J. Biochem., 208, 451–457.[Abstract]

Bieberich,E. and Bause,E. (1995) Man9-mannosidase from human kidney is expressed in COS cells as a Golgi- resident type II transmembrane N-glycoprotein. Eur. J. Biochem., 233, 644–649.[Abstract]

Bieberich,E., Treml,K., Volker,C., Rolfs,A., Kalz-Fuller,B. and Bause,E. (1997) Man9-mannosidase from pig liver is a type-II membrane protein that resides in the endoplasmic reticulum. cDNA cloning and expression of the enzyme in COS 1 cells. Eur. J. Biochem., 246, 681–689.[Abstract]

Bischoff,J. and Kornfeld,R. (1983) Evidence for an {alpha}-mannosidase in endoplasmic reticulum of rat liver. J. Biol. Chem., 258, 7907–7910.[Abstract/Free Full Text]

Bischoff,J., Liscum,L. and Kornfeld,R. (1986) The use of 1-deoxymannojirimycin to evaluate the role of various {alpha}-mannosidases in oligosaccharide processing in intact cells. J. Biol. Chem., 261, 4766–4774.[Abstract/Free Full Text]

Blake,M.S., Johnston,K.H., Russell-Jones,G.J. and Gotschlich,E.C. (1984) A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal. Biochem., 136, 175–179.[ISI][Medline]

Bonay,P. and Fresno,M. (1999) Isolation and purification of a neutral {alpha} (1,2)-mannosidase from Trypanosoma cruzi. Glycobiology, 9, 423–433.[Abstract/Free Full Text]

Byrd,J.C., Tarentino,A.L., Maley,F., Atkinson,P.H. and Trimble,R.B. (1982) Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing. J. Biol. Chem., 257, 14657–14666.[Abstract/Free Full Text]

Dairaku,K. and Spiro,R.G. (1997) Phylogenetic survey of endomannosidase indicates late evolutionary appearance of this N-linked oligosaccharide processing enzyme. Glycobiology, 7, 579–586.[Abstract]

Gonzalez,D.S., Karaveg,K., Vandersall-Nairn,A.S., Lal,A. and Moremen,K.W. (1999) Identification, expression and characterization of a cDNA encoding human endoplasmic reticulum mannosidase I, the enzyme that catalyzes the first mannose trimming step in mammalian Asn-linked oligosaccharide biosynthesis. J. Biol. Chem., 274, 21375–21386.[Abstract/Free Full Text]

Grace,T.D. (1966) Establishment of a line of mosquito (Aedes aegypti L.) cells grown in vitro. Nature, 211, 366–367.[ISI][Medline]

Herscovics,A. (1999a) Glycosidases of the asparagine-linked oligosaccharide processing pathway. In Pinto,B.M. (ed.), Comprehensive Natural Products Chemistry. Elsevier, Amsterdam, Vol. 3, pp. 13–35.

Herscovics,A. (1999b) Importance of glycosidases in mammalian glycoprotein biosynthesis. Biochim. Biophys. Acta., in press.

Herscovics,A. (1999c) Processing glycosidases of Saccharomyces cerevisiae. Biochim. Biophys. Acta, 1426, 275–285.[ISI][Medline]

Herscovics,A., Schneikert,J., Athanassiadis,A. and Moremen,K.W. (1994) Isolation of a mouse Golgi mannosidase cDNA, a member of a gene family conserved from yeast to mammals. J. Biol. Chem., 269, 9864–9871.[Abstract/Free Full Text]

Hom,L.G. and Volkman,L.E. (1998) Preventing proteolytic artifacts in the baculovirus expression system. Biotechniques, 25, 18–20.[ISI][Medline]

Ichishima,E., Taya,N., Ikeguchi,M., Chiba,Y., Nakamura,M., Kawabata,C., Inoue,T., Takahashi,K., Minetoki,T., Ozeki,K., Kumagai,C., Gomi,K., Yoshida,T. and Nakajima,T. (1999) Molecular and enzymic properties of recombinant 1,2-{alpha}-mannosidase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells. Biochem. J., 339, 589–597.[ISI][Medline]

Jarvis,D.L. and Garcia,A., Jr. (1994) Biosynthesis and processing of the Autographa californica nuclear polyhedrosis virus gp64 protein. Virology, 205, 300–313.[ISI][Medline]

Jarvis,D.L. and Summers,M.D. (1989) Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells. Mol. Cell Biol., 9, 214–223.

Jarvis,D.L., Kawar,Z.S. and Hollister,J.R. (1998) Engineering N-glycosylation pathways in the baculovirus-insect cell system. Curr. Opin. Biotechnol., 9, 528–533.[ISI][Medline]

Jelinek-Kelly,S. and Herscovics,A. (1988) Glycoprotein biosynthesis in Saccharomyces cerevisiae. Purification of the {alpha}-mannosidase which removes one specific mannose residue from Man9GlcNAc. J. Biol. Chem., 263, 14757–14763.[Abstract/Free Full Text]

Kawar,Z., Herscovics,A. and Jarvis,D.L. (1997) Isolation and characterization of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9. Glycobiology, 7, 433–443.[Abstract]

Kerscher,S., Albert,S., Wucherpfennig,D., Heisenberg,M. and Schneuwly,S. (1995) Molecular and genetic analysis of the Drosophila mas-1 (mannosidase-1) gene which encodes a glycoprotein processing {alpha} 1,2-mannosidase. Dev. Biol., 168, 613–626.[ISI][Medline]

Kitts,P.A. and Possee,R.D. (1993) A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques, 14, 810–817.[ISI][Medline]

Kornfeld,R. and Kornfeld,S. (1985) Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem., 54, 631–664.[ISI][Medline]

Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[ISI][Medline]

Lal,A., Pang,P., Kalelkar,S., Romero,P.A., Herscovics,A. and Moremen,K.W. (1998) Substrate specificities of recombinant murine golgi {alpha}1,2-mannosidases IA and IB and comparison with endoplasmic reticulum and golgi processing {alpha}1,2-mannosidases. Glycobiology, 8, 981–995.[Abstract/Free Full Text]

Lal,A., Schutzbach,J.S., Forsee,W.T., Neame,P.J. and Moremen,K.W. (1994) Isolation and expression of murine and rabbit cDNAs encoding an {alpha}1,2-mannosidase involved in the processing of asparagine-linked oligosaccharides. J. Biol. Chem., 269, 9872–9881.[Abstract/Free Full Text]

Lipari,F. and Herscovics,A. (1994) Production, purification and characterization of recombinant yeast processing {alpha}1,2-mannosidase. Glycobiology, 4, 697–702.[Abstract]

Lubas,W.A. and Spiro,R.G. (1987) Golgi endo-{alpha}-D-mannosidase from rat liver, a novel N-linked carbohydrate unit processing enzyme. J. Biol. Chem., 262, 3775–3781.[Abstract/Free Full Text]

Lussier,M., Sdicu,A.M. and Bussey,H. (1999) The KTR and MNN1 mannosyltransferase families of Saccharomyces cerevisiae. Biochim. Biophys. Acta, 1426, 323–334.[ISI][Medline]

Marz,L., Altmann,F., Staudacher,E. and Kubelka,V. (1995) Protein glycosylation in insects. In Montreuil,J., Vliegenthart,J.F.G. and Schachter,H. (eds.), Glycoproteins. Elsevier, Amsterdam, Vol. 29a, pp. 543–563.

Moremen,K.W., Trimble,R.G. and Herscovics,A. (1994) Glycosidases of the asparagine-linked oligosaccharide processing pathway. Glycobiology, 4, 113–125.[ISI][Medline]

Murhammer,D.W. and Goochee,C.F. (1988) Scaleup of insect cell cultures: protective effects of pluronic F-68. Bio/Technology, 6, 1411–1418.[ISI]

Naim,H.Y. and Koblet,H. (1988) Investigation of the role of glycans for the biological activity of Semliki Forest virus grown in Aedes albopictus cells using inhibitors of asparagine-linked oligosaccharides trimming. Arch. Virol., 102, 73–89.[ISI][Medline]

O’Reilly,D.R., Miller,L.K. and Luckow,V.A. (1992) Baculovirus expression vectors. W.H. Freeman, New York.

Ren,J., Bretthauer,R.K. and Castellino,F.J. (1995) Purification and properties of a Golgi-derived ({alpha} 1,2)-mannosidase-I from baculovirus-infected lepidopteran insect cells (IPLB-SF21AE) with preferential activity toward mannose6-N-acetylglucosamine2. Biochemistry, 34, 2489–2495.[ISI][Medline]

Rizzolo,L.J. and Kornfeld,R. (1988) Post-translational protein modification in the endoplasmic reticulum. Demonstration of fatty acylase and deoxymannojirimycin-sensitive {alpha}-mannosidase activities. J. Biol. Chem., 263, 9520–9525.[Abstract/Free Full Text]

Roberts,D.B., Mulvany,W.J., Dwek,R.A. and Rudd,P.M. (1998) Mutant analysis reveals an alternative pathway for N-linked glycosylation in Drosophila melanogaster. Eur. J. Biochem., 253, 494–498.[Abstract]

Romero,P.A. and Herscovics,A. (1989) Glycoprotein biosynthesis in Saccharomyces cerevisiae. Characterization of {alpha}-1,6-mannosyltransferase which initiates outer chain formation. J. Biol. Chem., 264, 1946–1950.[Abstract/Free Full Text]

Romero,P.A., Saunier,B. and Herscovics,A. (1985) Comparison between 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin as inhibitors of oligosaccharide processing in intestinal epithelial cells. Biochem. J., 226, 733–740.[ISI][Medline]

Spiro,M.J., Bhoyroo,V.D. and Spiro,R.G. (1997) Molecular cloning and expression of rat liver endo-{alpha}-mannosidase, an N-linked oligosaccharide processing enzyme. J. Biol. Chem., 272, 29356–29363.[Abstract/Free Full Text]

Summers,M.D. and Smith,G.E. (1987). A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agric. Exp. Station Bull., 1555.

Tabas,I. and Kornfeld,S. (1979) Purification and characterization of a rat liver Golgi {alpha}- mannosidase capable of processing asparagine-linked oligosaccharides. J. Biol. Chem., 254, 11655–11663.[Abstract]

Tanner,W. and Lehle,L. (1987) Protein glycosylation in yeast. Biochim. Biophys. Acta., 906, 81–99.[ISI][Medline]

Towbin,H., Staehelin,T. and Gordon,J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76, 4350–4354.[Abstract]

Tremblay,L.O., Campbell Dyke,N. and Herscovics,A. (1998) Molecular cloning, chromosomal mapping and tissue-specific expression of a novel human {alpha}1,2-mannosidase gene involved in N-glycan maturation. Glycobiology, 8, 585–595.[Abstract/Free Full Text]

Tremblay,L.O. and Herscovics,A. (1999) Cloning and expression of a specific human {alpha}1,2-mannosidase that trims Man9GlcNAc2 to Man8GlcNAc2 isomer B during N-glycan biosynthesis. Glycobiology, 10, 1073–1078.

Vaughn,J.L., Goodwin,R.H., Thompkins,G.J. and McCawley,P. (1977) The establishment of two insect cell lines from the insect Spodoptera frugiperda (Lepidoptera:Noctuidae). In Vitro, 13, 213–217.

Weng,S. and Spiro,R.G. (1993) Demonstration that a kifunensine-resistant alpha-mannosidase with a unique processing action on N-linked oligosaccharides occurs in rat liver endoplasmic reticulum and various cultured cells. J. Biol. Chem., 268, 25656–25663.[Abstract/Free Full Text]

Weng,S. and Spiro,R.G. (1996) Endoplasmic reticulum kifunensine-resistant {alpha}-mannosidase is enzymatically and immunologically related to the cytosolic {alpha}-mannosidase. Arch. Biochem. Biophys., 325, 113–123.[ISI][Medline]

Yoshida,T., Nakajima,T. and Ichishima,E. (1998) Overproduction of 1,2-{alpha}-mannosidase, a glycochain processing enzyme, by Aspergillus oryzae. Biosci. Biotechnol. Biochem., 62, 309–315. [ISI][Medline]