Institut für Physiologische Chemie, Nussallee 11, 53115 Bonn, Germany
Received on September 7, 1999; revised on October 27, 1999; accepted on November 16, 1999.
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Abstract |
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Key words: glucosidase II complex/- and ß-subunit/cDNA cloning/alternative splicing/expression of catalytic activity/subcellular localization
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Introduction |
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The biological significance of the three transient glucoses, as well as the specific role of -glucosidases I and II, are still not fully understood. Possible functions include protection of the lipid-linked Glc3-Man9-GlcNAc2 precursor from degradation by phosphodiesterases (Hoflack et al., 1981
), control of protein glycosylation (Spiro et al., 1979
; Breuer and Bause, 1995
) and regulation of N-glycoprotein transport (Arakaki et al., 1987
; Edwards et al., 1989
), as well as protein degradation (Moore and Spiro, 1993
) and folding (Helenius, 1994
). A "quality control mechanism" for N-glycoprotein folding has been proposed, in which mono- and de-glucosylated Man9-GlcNAc2 oligosaccharide intermediates play a central role (Hammond and Helenius, 1995
; Helenius et al., 1997
; Parodi, 1998
). This control mechanism, which operates in the ER, involves binding of Glc1-Man9-GlcNAc2 to and dissociation of Man9-GlcNAc2 from lectin-like chaperones, such as calnexin and/or calreticulin, during different stages of protein folding. The obviously repetitive process is driven by glucosidase II-mediated deglucosylation and UDP-glucose:glycoprotein glucosyltransferase-catalyzed reglucosylation (Ganan et al., 1991
; Sousa et al., 1992
; Sousa and Parodi, 1995
).
Glucosidases I and II have already been purified from a variety of cell types and their molecular and catalytic properties characterized in detail (Moremen et al., 1994; Herscovics, 1999
). Among other features, both enzymes differ substantially in their substrate specificity. Thus glucosidase I which initiates the N-linked processing pathway, removes the distal
1,2-linked glucose residue from Glc3-Man9-GlcNAc2, while glucosidase II cleaves the two inner
1,3-glucose residues in Glc2,1-intermediate(s). Studies by Trombetta et al. (1996)
have identified glucosidase II from rat liver as existing as a hetero-dimeric complex, consisting of an
-subunit (~110 kDa) and a ß-subunit (~80 kDa). The larger
-subunit, which is assumed to house the catalytic activity, appears to be soluble and retained in the ER lumen by contact with the ß-subunit, which itself contains a HDEL tetrapeptide sequence known to act as an ER-retrieval signal (Robbi and Beaufay, 1991
; Ozawa and Muramatsu, 1993
). During affinity purification of glucosidase II from pig liver we occasionally observed a polypeptide of similar size to the ß-subunit. We finally, however, isolated a catalytically active enzyme consisting of a ~107/112 kDa protein doublet, with the smaller component being the major species (Hentges and Bause, 1997
). The molecular masses of these two proteins are close to that for the polypeptide encoded by the glucosidase II-specific cDNA, as recently cloned from a pig liver cDNA library (Flura et al., 1997
).
In this paper we report on the cDNA cloning and expression in COS-1 cells of the glucosidase II - and ß-subunits from human liver, thus extending our studies on the
-glycosidases of the "early" processing pathway. The full-length cDNA, reconstructed for either subunit from independent lambda clones, encodes a polypeptide of 944 (
-subunit) and 534 amino acids (ß-subunit), respectively. Evidence is presented showing that alternative splicing variants are likely to exist for both subunit transcripts and that increased levels of glucosidase II activity require simultaneous coexpression of both subunit proteins in COS-1 cells, indicating that the ß-subunit directly affects expression and formation of an active glucosidase II complex during biosynthesis.
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Results and discussion |
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The hydrophobicity profile (Kyte and Doolittle, 1982) established for the human liver
-subunit reveals two stretches of hydrophobic amino acids, one being located close to the N-terminus (residues 1533) and a second one between amino acid 628646 (Figure 1). The hydrophobic sequence within the N-terminal domain is framed upstream by four consecutive arginines and downstream by Asp-Arg. This apolar region could, therefore, act in principle either as part of a cleavable signal sequence, involved in the conversion of the
-subunit to a soluble protein, or as a membrane anchor, resulting in a type II transmembrane topology. Membrane anchoring of the
-subunit via the hydrophobic sequence located between amino acids 628646 of the coding region can be excluded because we were unable to detect degradation products of the
-subunit on treatment of pig or human liver microsomes with trypsin in the absence of detergents (not shown).
In order to study the function of the ß-subunit in the hetero-dimeric glucosidase II complex, we have cloned the ß-subunit cDNA taking advantage of published sequence data (Sakai et al., 1989). Two independent
-clones were isolated from the mixed-primed human liver cDNA library, allowing the reconstruction of a 2095 bp full-length cDNA. Its nucleotide sequence was identical with that of the human liver ß-subunit previously published (Sakai et al., 1989
), except that we found an additional 21 bp fragment inserted downstream at bp 1140 of the 2095 bp construct. The ORF in the ß-subunit cDNA, starting at bp 137 and terminating at bp 1741, encodes 534 amino acids corresponding to a ~60 kDa polypeptide. The ~60 kDa protein contains a typical signal sequence at the N-terminus, two potential N-glycosylation sites of the Asn-Xaa-Ser type and a C-terminal HDEL ER-retrieval signal. In vitro translation and glycosylation experiments, using ß-subunit specific mRNA to program a particulate reticulocyte lysate/dog pancreas glycosylation system, revealed that the signal sequence is cleaved in the presence of dog pancreatic microsomes, whereas the two Asn-Xaa-Ser acceptor sites are not N-glycosylated (data not shown). A strikingly long hydrophilic stretch of ~20 consecutive glutamic acid residues is located in the central region of the ß-subunit polypeptide, the function of which is unknown at present.
The size of the poly(A)+mRNA specific for the - and ß-subunits was determined for different human tissues by Northern blotting using PCR amplified probes as analytical tools. The
-subunit-specific cDNA was found to hybridize strongly with a 4.4 kb transcript, the size of which was in good agreement with that of the 4106 bp construct. On the other hand the cDNA probe specific for the ß-subunit hybridized with one major transcript of 2.4 kb, corresponding with the 2095 bp cDNA (not shown).
The pre-mRNA for the - and ß-subunits is subject to alternative splicing
During library screening, several independent -clones were isolated which differed from the
-clones used for reconstruction of the
- and ß-subunit-specific cDNAs by short nucleotide fragments inserted or deleted in the coding region of either cDNA. In neither case did these sequence alterations cause a frame-shift, strongly suggesting that they result from alternative splicing of the primary transcripts rather than being artefacts introduced during cDNA library preparation. In case of the
-subunit, one independent
-clone was isolated which contained a 66 bp insertion at bp 773 of the 4106 bp cDNA sequence. As outlined schematically in Figure 2, the resulting two sequence variants are likely to be generated by alternative splicing, involving differential utilization of the splice sites in the two introns (445 bp and 793 bp), which we have previously identified in the genomic DNA (unpublished observations). Both splice variants have also been described for the glucosidase II
-subunit from mouse T-cells by Arendt et al. (1999)
, supporting our interpretation.
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As expected, the cDNA sequence of the ß-subunit which we had cloned in order to carry out co-expression studies, turned out to be identical with the human sequence previously published (Sakai et al., 1989). The amino acid sequence displayed 80% and 78% identity with the sequence of the ß-subunit polypeptide from cattle and mouse, respectively (Arendt and Ostergaard, 1997
; GenBank accession number U49178). No significant homology was detectable compared to other proteins.
Expression of glucosidase II activity in COS-1 cells requires the presence of both the - and ß-subunit
In order to study the subunit function of the hetero-dimeric glucosidase II complex, cDNA constructs encoding either the - or the ß-subunit were subcloned into the EcoRI/NotI sites of the expression vector pcDNA3 which contains the CMV promotor. COS-1 cells were transfected with one or both of the recombinant vector constructs (HL-GII
and HL-GIIß) using the FuGENE 6 reagent for transfection. The overexpressed proteins were then analyzed by SDSPAGE/immunoblotting (
-subunit) and their catalytic activity was determined with [14C]Glc2,1-Man9-GlcNAc2. Immunodetection of the
-subunit was carried out using a polyclonal antibody raised against the ~107 kDa glucosidase II protein from pig liver (Hentges and Bause, 1997
).
The results in Figure 4A show that after transfection of COS-1 cells with HL-GII, the antibody raised against the pig liver ~107 kDa-protein reacted strongly with a protein band of similar molecular mass (lane 2), whereas the amount of cross-reacting material in control cells transfected with pcDNA-3 was rather low (lane 1). The molecular mass of the overexpressed
-subunit was close to that calculated from the ORF (~107 kDa), taking into account cleavage of the N-terminal signal sequence (aa 132) and that the resulting polypeptide acquires one N-linked sugar chain at Asn97. N-glycosylation at this site is consistent with the observation that Endo H reduced its molecular mass by ~1.5 kDa (Figure 4B). In order to determine the catalytic activity of the over-expressed
-subunit protein a detergent extract, prepared from HL-GII
transfected COS-1 cells, was incubated in the presence of [14C]Glc2,1-Man9-GlcNAc2. In several experiments no significant increase of substrate hydrolysis was detectable compared to control cells, indicating that the overexpressed polypeptide is catalytically inactive. It should be noted, however, that the background activity resulting from nonspecific enzyme cleavage of [14C]Glc2,1-Man9-GlcNAc2 was found to be rather high in COS-1 cells, thus rendering it difficult to assign moderate increases of catalytic activity contributed by the over-expressed
-subunit reliably.
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The antibody raised against the ~107 kDa -subunit from pig liver recognized a ~107/112 kDa protein doublet on immunoblots prepared from intact pig liver and different human tissues as well as from COS-1 cells, as previously shown by Hentges and Bause (1997)
. The relative abundances of the components of this protein doublet were similar in all cell types. A reasonable explanation for this observation is that the 107 kDa and the 112 kDa protein species are iso-forms of the
-subunit resulting from translation of alternatively spliced transcripts. This possibility is supported by the isolation of a cDNA clone which was found to contain an additional in-frame 66 bp sequence in the coding region, corresponding to 22 amino acids (Figure 2). Thus, the two iso-forms of the
-subunit could be encoded by differently spliced transcripts, with only the ~112 kDa protein containing this additional 22 amino acid peptide. This interpretation is consistent with the observation that transfection of COS-1 cells with
-subunit-specific cDNA lacking the 66 bp insert (HL-GII
) gave rise to a single polypeptide, similar in size to that of the ~107 kDa component of purified glucosidase II from pig liver. The function of the two
-subunit sequence variants, as well as those for the ß-subunit, remains unknown at present.
The immunofluorescence data show that the glucosidase II -subunit is expressed in COS-1 cells as an ER-resident protein. Location within the ER is apparently not dependent on whether COS-1 cells are transfected just with the
-subunit-specific cDNA, resulting in the synthesis of a catalytically inactive protein, or on the simultaneous presence of the ß-subunit essential for expression of enzymatically active glucosidase II. Given the cleavage of the N-terminal signal peptide in the
-subunit, the remaining polypeptide lacks any typical signal sequence for ER-retention or retrieval, whereas the ß-subunit contains a HDEL retrieval signal at its C-terminus. It is reasonable to assume, therefore, that the
-subunit in the glucosidase II complex is retained in the ER through its association with the ß-subunit. The ER-location of the catalytically inactive form of the
-subunit suggests, on the other hand, that due to incomplete maturation the inactive protein may be retained in the ER by an as yet unknown mechanism, which could involve the "quality control machinery" suggested by Helenius (1994)
. This interpretation is, at least, not inconsistent with the observation that the
-subunit contains one N-linked sugar chain at Asn97.
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Materials and methods |
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Amino acid sequencing of the ~107 kDa glucosidase II protein from pig liver
The ~107/112 kDa protein doublet in the glucosidase II preparation, isolated from pig liver microsomes, was separated by preparative SDSPAGE, using the continuous flow Bio-Rad PrepCell system. Eluate fractions containing the ~107 kDa enzyme were dialyzed against water and the protein precipitated with acetone under acidic conditions (Henderson et al., 1979). Sixty micrograms of the homogenous ~107 kDa protein were dissolved in 0.1% trifluoroacetic acid and the NH2-terminal amino acid sequence determined by automatic Edman degradation on an Applied Biosystem gas-phase sequencer, using an on-line phenylthiohydantoin-Xaa analyzer for detection.
Chemical cleavage was carried out by treating ~100 µg of the SDSPAGE-purified ~107 kDa protein in 50 µl 90% formic acid/water with 2.0 mg cyanogen bromide (CNBr) for 24 h at room temperature under nitrogen, followed by a second 24 h incubation with an additional 2.0 mg CNBr (Gross, 1967). The sample was diluted with water and lyophilized. The peptide mixture was then dissolved in sample buffer and separated by SDSPAGE using the high resolution gel-system previously described by Schägger and von Jagow (1987)
. After electrophoretic transfer onto poly(vinylidenefluoride) membranes, peptides were localized by Coomassie blue staining, the corresponding regions excised and subjected directly to automatic Edman degradation.
For tryptic digestion ~200 µg of affinity-purified glucosidase II from pig liver were subjected to SDSPAGE under reducing conditions, followed by electrophoretic transfer of the proteins onto nitrocellulose. Nitrocellulose areas containing the ~107 kDa polypeptide were excised and free binding sites saturated by treatment with 0.5% polyvinylpyrrolidone in 100 mM acetic acid. The nitrocellulose fragments were then washed with water and incubated for 12 h at 37 °C with 2 µg trypsin in 0.5 ml 100 mM Tris/HCl, pH 8.5, containing 5% acetonitrile (v/v) in order to limit binding of cleavage products to the nitrocellulose. The nitrocellulose fragments were removed by low-speed centrifugation and the tryptic peptides in the supernatant separated by reversed phase HPLC on RP18 columns, using an acetonitrile gradient (070%) in either 0.1% ammonium acetate, pH 6.0 or 0.1% trifluoroacetic acid/water. Fractions containing homogenous peptides were collected and subjected to Edman degradation.
Synthesis of pig liver first strand cDNA and preparation of - and ß-subunit-specific screening probes
Total mRNA was isolated from pig liver tissue using cesium chloride density centrifugation. The poly(A)+ RNA fraction, separated by affinity chromatography on oligo(dT)-cellulose, was incubated with reverse transcriptase in the presence of oligo(dT) and/or "random" hexanucleotide primers. The first strand cDNA was then used as the template in a PCR reaction (MOPAC). For amplification of an -subunit-specific screening probe, oligonucleotides were designed based on amino acid sequence data derived from the pig liver ~107 kDa protein. The ß-subunit-specific screening probe was synthesized by PCR, taking advantage of published sequence data for primer design (Sakai et al., 1989
). The PCR products were then purified by agarose gel electrophoresis and characterized by sequencing using the dideoxynucleotide chain termination method (Sanger et al., 1977
).
Isolation of cDNA clones from the human liver cDNA library
The purified PCR-products were radio-labeled with [-32P]dATP by primer extension (Feinberg and Vogelstein, 1983
) and used to screen an oligo-(dT)/random-primed human liver cDNA library constructed in
gt10 (HL3006a, Clontec), by applying standard plaque hybridization methods. The cDNA inserts of the lambda clones were excised and subcloned into the EcoRI restriction site of the pUC BM20 vector for sequencing.
Construction of recombinant expression vectors and transfection of COS-1 cells
The cDNA of the -subunit was reconstructed from overlapping
-clones by restriction and ligation at a common KpnI-site. The construct was then subcloned into the EcoRI- and NotI-site of the mammalian expression vector pcDNA3. The recombinant vector (HL-GII
) contained a 2845 bp insert covering the complete 2835 bp coding region and including 10 bp of the 5'-non-coding sequence. Transcription promotor and polyadenylation signal were contributed by the pcDNA3 vector. The cDNA containing the coding region of the ß-subunit, was reconstructed from two independent
-clones taking advantage of a common HindIII site, followed by subcloning of the construct into the EcoRI site of pcDNA3 (HL-GIIß). For construction of an expression vector containing the cDNA of both the
- and the ß-subunit (HL-GII
ß), HL-GII
and HL-GIIß were cleaved with BspDI and ScaI, respectively. Sticky ends in HL-GII
were filled in, followed by blunt-end ligation of the two plasmid fragments.
COS-1 cells propagated in DMEM containing 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin, were subcultured for 24 h prior to transfection. At ~60% confluency cells were transfected with 4.5 µg of either cDNA3 (control), HL-GII, HL-GIIß, HL-GII
ß or HL-GII
plus HL-GIIß, using the FuGENE 6 transfection reagent as described in the suppliers manual. After 48 h cells were washed with NaCl/Pi (140 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) and solubilized in 50 mM phosphate buffer, pH 6.5, containing 1% Thesit, 10 µM phenylmethansulfonylfluoride, followed by centrifugation for 5 min at 10,000 x g. The resulting supernatant was used for further studies.
Determination of glucosidase II activity
The glucosidase II activity in transfected COS-1 cells was measured by incubating 30 µl aliquots of detergent extracts (containing ~6 µg of protein) with 5 µl of a substrate solution containing 500 c.p.m. [14C]Glc2,1-Man9-GlcNAc2. After given times the reactions were stopped by addition of 35 µl of acetic acid and cleavage products separated by paper chromatography, using iso-propanol/acetic acid/water (29/8/15, v/v). Released [14C]glucose was then determined by scintillation counting (Hettkamp et al., 1984). Inhibition studies were carried out by preincubating aliquots of the detergent extracts with dNM, N-methyl-dNM and N,N-dimethyl-dNM for 5 min, followed by measuring the residual glucosidase II activity as described.
General methods
SDS-PAGE, Western and immunoblotting as well as library screening and PCR amplification were all carried out as described previously (Laemmli, 1970; Saiki et al., 1988
; Schweden and Bause, 1989
; White, 1993
). Immunofluorescence microscopy, in vitro translation/glycosylation and Endo H cleavage were done according to the procedures described by Bieberich and Bause (1995)
. Nucleotide sequencing was performed according to Sanger using the Pharmacia T7Sequencing Kit and [35S]dATP-
-S as tracer (Sanger et al., 1977
). [14C]Glc2,1Man9GlcNAc2 was synthesized as detailed in Hettkamp et al. (1984)
. The hydrophobicity profile was calculated by the Kyte and Doolittle method (1982) using a window size of seven residues. Homology searches were carried out using the GenBank database (release date 20.02.97) and BLASTN and BLASTP programs. All other procedures were carried out as described by Ausubel et al. (1987)
and Sambrook et al. (1989)
.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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