The {alpha}- and ß-subunits are required for expression of catalytic activity in the hetero-dimeric glucosidase II complex from human liver

Kornelia Treml, Dido Meimaroglou, Andrea Hentges and Ernst Bause1

Institut für Physiologische Chemie, Nussallee 11, 53115 Bonn, Germany

Received on September 7, 1999; revised on October 27, 1999; accepted on November 16, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The {alpha}- and ß-subunits of the hetero-dimeric glucosidase II complex from human liver were cloned and expressed in COS-1 cells. The 4106 bp full-length cDNA for the {alpha}-subunit contained a 2835 bp ORF encoding a 107 kDa polypeptide. The 2095 bp cDNA for the ß-subunit encodes a ~60 kDa protein in a continuous 1605 bp ORF. The {alpha}- and ß-subunits each contain two potential Asn-Xaa-Thr/Ser acceptor sites, with only one site in the {alpha}-subunit (Asn97) being glycosylated. Additional {lambda}-clones were isolated for each subunit containing in-frame insertions/deletions within the coding region, indicating alternative splicing. Analysis of different human tissues revealed ~4.4 kb and ~2.4 kb transcripts for {alpha}- and ß-subunit, respectively, consistent with their full-length cDNA. Coexpression of the {alpha}- and ß-subunits in COS-1 cells resulted in >4-fold increase of glucosidase II activity. An inactive protein was obtained, however, after transfection with the {alpha}-subunit alone, showing that both subunits are essential for expression of active glucosidase II. The observation that the enzyme, previously purified from pig liver and lacking the ß-subunit, was catalytically active indicates that the ß-subunit is involved in {alpha}-subunit maturation rather than being required for enzymatic activity once the {alpha}-subunit has acquired its mature form. The {alpha}-subunit is expressed in COS-1 cells as an ER-located protein, whether inactive or part of a catalytically active complex. This suggests that ER-localization of the {alpha}-subunit, when associated with the dimeric enzyme complex, is mediated by the C-terminal HDEL-signal in the ß-subunit, whereas the apparently incompletely folded form of the inactive {alpha}-subunit could be retained in the ER by the putative "glycoprotein-specific quality control machinery."

Key words: glucosidase II complex/{alpha}- and ß-subunit/cDNA cloning/alternative splicing/expression of catalytic activity/subcellular localization


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Remodeling of the asparagine-linked Glc3-Man9-GlcNAc2 precursor to give the mature glycan structure occurs through a complex pathway involving a large number of ER- and Golgi-resident {alpha}-glycosidases and glycosyltransferases (Kornfeld and Kornfeld, 1985Go). The cleavage of three glucose residues from the peptide-bound Glc3-Man9-GlcNAc2 oligosaccharide, catalyzed by glucosidases I and II, is a key event in this pathway. The resulting Man9-GlcNAc2 is then acted upon by different {alpha}1,2-mannosidases, which remove up to four of the {alpha}1,2-linked mannose residues generating distinct "high-mannose" structures (Moremen et al., 1994Go; Herscovics, 1999Go). After transfer of a GlcNAc residue onto Man5-GlcNAc2, the outer {alpha}1,3/{alpha}1,6-mannose branch is degraded by mannosidase II with complex type oligosaccharides then being generated by reglycosylation.

The biological significance of the three transient glucoses, as well as the specific role of {alpha}-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., 1981Go), control of protein glycosylation (Spiro et al., 1979Go; Breuer and Bause, 1995Go) and regulation of N-glycoprotein transport (Arakaki et al., 1987Go; Edwards et al., 1989Go), as well as protein degradation (Moore and Spiro, 1993Go) and folding (Helenius, 1994Go). 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, 1995Go; Helenius et al., 1997Go; Parodi, 1998Go). 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., 1991Go; Sousa et al., 1992Go; Sousa and Parodi, 1995Go).

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., 1994Go; Herscovics, 1999Go). Among other features, both enzymes differ substantially in their substrate specificity. Thus glucosidase I which initiates the N-linked processing pathway, removes the distal {alpha}1,2-linked glucose residue from Glc3-Man9-GlcNAc2, while glucosidase II cleaves the two inner {alpha}1,3-glucose residues in Glc2,1-intermediate(s). Studies by Trombetta et al. (1996)Go have identified glucosidase II from rat liver as existing as a hetero-dimeric complex, consisting of an {alpha}-subunit (~110 kDa) and a ß-subunit (~80 kDa). The larger {alpha}-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, 1991Go; Ozawa and Muramatsu, 1993Go). 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, 1997Go). 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., 1997Go).

In this paper we report on the cDNA cloning and expression in COS-1 cells of the glucosidase II {alpha}- and ß-subunits from human liver, thus extending our studies on the {alpha}-glycosidases of the "early" processing pathway. The full-length cDNA, reconstructed for either subunit from independent lambda clones, encodes a polypeptide of 944 ({alpha}-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.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
cDNA cloning and structural properties of the {alpha}- and ß-subunits of glucosidase II from human liver
Recently Trombetta et al. (1996)Go isolated a hetero-dimeric glucosidase II complex from rat liver consisting of a ~110 kDa protein ({alpha}-subunit) which was associated with a smaller polypeptide (ß-subunit) that migrated on SDS–PAGE as a ~80 kDa protein. During purification of glucosidase II from pig liver we observed proteins of similar size, although the catalytically active glucosidase II preparation which we have finally isolated, contained two polypeptides with molecular masses of ~107 kDa and ~112 kDa, respectively (Hentges and Bause, 1997Go). Immunological studies using antibodies against the ~107 kDa protein showed that these two proteins were structurally related, suggesting that they represent differently processed forms of the same {alpha}-subunit. Based on sequence information derived from the pig liver ~107 kDa polypeptide, we have cloned the glucosidase II {alpha}-subunit using a mixed-primed human liver cDNA library for screening. Three independent {lambda}-clones were isolated which allowed reconstruction of a 4106 bp full-length cDNA (Figure 1). The 4106 bp cDNA sequence was found to be homologous with a previously unidentified human gene (accession number D42041). This sequence, shown to encode the {alpha}-subunit of glucosidase II (Trombetta et al., 1996Go), has a 5'-base which aligns with bp 216 of the 4106 bp cDNA construct, indicating that this database sequence lacks the first two bases of the initiator ATG and the 5' untranslated region. The 4106 bp cDNA-construct contained a continuous 2835 bp open reading frame, starting at bp 214 with ATG and terminating with a TAA stop codon at bp 3046. The amino acid sequence of all tryptic and CNBr peptides previously derived from the pig liver ~107 kDa protein were recovered within this ORF revealing a high degree of sequence similarity between the {alpha}-subunit of the pig and human liver enzyme. An additional in-frame ATG at bp 79 in the 5'-untranslated region is followed downstream by several in-frame stop codons, supporting the view that ATG-214 does indeed code for the first amino acid of the {alpha}-subunit polypeptide. The 1058 bp 3'-untranslated sequence, terminated by a 75 bp poly(A)tail, contains a classical AATAAA consensus sequence 18 bp upstream from the poly(A)tract. This is likely to be used as signal for pre-mRNA cleavage and polyadenylation (Wahle and Keller, 1992Go).




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Fig. 1. cDNA and derived amino acid sequence of the glucosidase II {alpha}-subunit from human liver. Numbers on the right designate amino acids 1–944 in the ORF, numbers on the left the nucleotide sequence with 214 representing the first base of the initiator methionine. The peptide sequences derived from the pig liver ~107 kDa protein are shown in heavy type. The two hydrophobic domains in the coding region are underlined; potential N-glycosylation sites (Asn97 and Asn514) are shown in boxes. The polyadenylation signal is shown as reversed. The site of the variably expressed 66 bp-insert is indicated by a black triangle (see Figure 2).

 
The 2835 bp ORF within the 4106 bp cDNA construct codes for 944 amino acids corresponding to a ~107 kDa polypeptide (Figure 1). The coding sequence contains two potential acceptor sites of the Asn-Xaa-Thr/Ser type (Asn97 and Asn514), of which only the Asn97-site can be glycosylated because proline in the Xaa position of the Asn514 triplet is known to impair N-glycosylation (Bause, 1983Go). This suggests that the {alpha}-subunit from human liver as for the pig liver enzyme (Hentges and Bause, 1997Go), is a glycoprotein with one N-linked sugar chain. The N-terminal amino acid of the pig liver ~107 kDa protein (from which sequence data for primer design were obtained) aligns with Val33 in the human liver ~107 kDa polypeptide, indicating cleavage of a 4–5 kDa fragment from the N-terminus of the pig liver enzyme.

The hydrophobicity profile (Kyte and Doolittle, 1982Go) established for the human liver {alpha}-subunit reveals two stretches of hydrophobic amino acids, one being located close to the N-terminus (residues 15–33) and a second one between amino acid 628–646 (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 {alpha}-subunit to a soluble protein, or as a membrane anchor, resulting in a type II transmembrane topology. Membrane anchoring of the {alpha}-subunit via the hydrophobic sequence located between amino acids 628–646 of the coding region can be excluded because we were unable to detect degradation products of the {alpha}-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., 1989Go). Two independent {lambda}-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., 1989Go), 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 {alpha}- and ß-subunits was determined for different human tissues by Northern blotting using PCR amplified probes as analytical tools. The {alpha}-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 {alpha}- and ß-subunits is subject to alternative splicing
During library screening, several independent {lambda}-clones were isolated which differed from the {lambda}-clones used for reconstruction of the {alpha}- 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 {alpha}-subunit, one independent {lambda}-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 {alpha}-subunit from mouse T-cells by Arendt et al. (1999)Go, supporting our interpretation.



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Fig. 2. Alternative splicing of the {alpha}-subunit of glucosidase II from human liver. The diagram shows part of the genomic sequence. Exons are shown in boxes together with eight nucleotide residues at the 5'- and 3'-end of the two introns (445 bp and 793 bp). Potential splice sites (1–4) are shown as reversed. Cleavage at sites 1 and 4 yields the nucleotide sequence lacking the 66 bp-insert. Utilization of splice sites 1 and 2, as well as 3 and 4, removes the two introns only, resulting in the sequence variant containing the 66 bp insert.

 
As for the {alpha}-subunit, library screening with ß-subunit-specific cDNA probes yielded several independent {lambda}-clones, whose nucleotide sequences differed from the sequence of the 2095 bp cDNA construct by two distinct in-frame deletions. These deletions were found immediately downstream of the nucleotide sequence, encoding the consecutive glutamic acid residues (Figure 3A). Since the 5'-GT of Sß-1 and the 3'-AG in Sß-2 are potential splice sites, formation of the three observed variants is best explained by postulating an intron sequence between Sß-1 and Sß-2 in the human liver genomic DNA analogous to that previously identified in the ß-subunit genomic DNA from mouse T-cells (Figure 3B; Arendt et al., 1999Go). Although in mouse T-cells Sß-2 was not identified as a variably spliced sequence, as we found in the human liver ß-subunit, the high degree of similarity in the structural organization of the glucosidase II ß-subunit gene in mouse and man is obvious.



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Fig. 3. Alternative splicing of the ß-subunit of glucosidase II from human liver. (A) shows the amino acid sequence of the ß-subunit, as well as the region in which the nucleotide sequence of independent {lambda}-clones isolated from the human liver cDNA library were found to differ. Variably expressed regions are given in boxes. (B) Diagram explaining the formation of the three sequence variants by alternative splicing, assuming the occurrence of an intron sequence between Sß-1 and Sß-2 in the human genomic DNA, analogous to that recently observed by Arendt et al. (1999)Go for mouse T-cells. Possible splice sites (1–4) are shown as reversed. Excision of the postulated intron would result in the sequence variant containing both Sß-1 and Sß-2, whereas cleavage at sites 1/3 and 2/4, respectively, would explain the formation of splicing variants containing either Sß-1 or Sß-2.

 
Sequence similarities to other {alpha}-glycosidases
The nucleotide sequence of the glucosidase II {alpha}- and ß-subunits from human liver was compared with other enzymes in the GenBank database in order to determine regions of similarity. The coding region in the 4106 bp cDNA exhibits ~90%, ~85%, and ~39% identity with the glucosidase II {alpha}-subunit from pig liver, mouse, and yeast, respectively (Feldmann et al., 1994Go; Arendt and Ostergaard, 1997Go; Flura et al., 1997Go). Comparison with other processing {alpha}-glycosidases, including glucosidase I from human hippocampus (Kalz-Füller et al., 1995Go), Man9-mannosidase from pig liver and human kidney (Bause et al., 1993Go; Bieberich et al., 1997Go), as well as mannosidase II (Moremen and Robbins, 1991Go) revealed no striking similarities in the sequence, either at nucleotide or protein levels. These enzymes must, therefore, be derived from different ancestral genes. The degree of sequence homology was rather low (25–30%) compared to {alpha}-glucosidases belonging to the glycosyl hydrolase family 31 (Henrissat, 1991Go; Henrissat and Bairoch, 1993Go; Frandsen and Svensson, 1998Go). These {alpha}-glucosidases contain a short peptide segment of conserved amino acids (D-G-X-W-I-D-M-N-E-X-S-X-F) including an aspartic acid residue assumed to be involved in catalysis (Hermans et al., 1991Go; Kimura et al., 1992Go; Iwanami et al., 1995Go; Hülseweh et al., 1997Go). A similar peptide sequence (L-F-V-W-N-D-M-N-E-P-S-V-F) appears in the glucosidase II {alpha}-subunit from human liver (aa 538–549), indicating that the catalytically active {alpha}-subunit of the glucosidase II complex may also belong to this group of glycosyl hydrolases (family 31) despite its rather low overall sequence homology.

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., 1989Go). 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, 1997Go; 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 {alpha}- and ß-subunit
In order to study the subunit function of the hetero-dimeric glucosidase II complex, cDNA constructs encoding either the {alpha}- 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{alpha} and HL-GIIß) using the FuGENE 6 reagent for transfection. The overexpressed proteins were then analyzed by SDS–PAGE/immunoblotting ({alpha}-subunit) and their catalytic activity was determined with [14C]Glc2,1-Man9-GlcNAc2. Immunodetection of the {alpha}-subunit was carried out using a polyclonal antibody raised against the ~107 kDa glucosidase II protein from pig liver (Hentges and Bause, 1997Go).

The results in Figure 4A show that after transfection of COS-1 cells with HL-GII{alpha}, 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 {alpha}-subunit was close to that calculated from the ORF (~107 kDa), taking into account cleavage of the N-terminal signal sequence (aa 1–32) 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 {alpha}-subunit protein a detergent extract, prepared from HL-GII{alpha} 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 {alpha}-subunit reliably.



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Fig. 4. Immunoblot and structural analysis of the glucosidase II {alpha}-subunit from human liver. (A) COS-1 cells transfected with either pcDNA-3 (lane 1, control), HL-GII{alpha} (lane 2), HL-GIIß (lane 3), or HL-GII{alpha} plus HL-GIIß (lanes 4 and 5), were solubilized in 50 mM phosphate buffer, pH 6.5, containing 1% Triton X-100. Aliquots of the detergent extracts were separated by SDS–PAGE and proteins transferred electrophoretically onto nitrocellulose, followed by immunostaining using a polyclonal antibody raised against the pig liver ~107 kDa protein (Hentges and Bause, 1997Go). (B) COS-1 cells transfected with HL-GII{alpha} were solubilized in 50 mM phosphate buffer, pH 6.5, containing 1% Triton X-100. Aliquots of the detergent extract were incubated with 500 units of Endo H for 3 h at 37°C, followed by SDS–PAGE and immunoblotting. Lane 1, control minus Endo H; lane 2, as in lane 1 plus Endo H-treated {alpha}-subunit; lane 3, {alpha}-subunit plus Endo H.

 
In order to analyze whether the ß-subunit affects the expression of a catalytically active {alpha}-subunit, COS-1 cells were cotransfected simultaneously with both the HL-GII{alpha} and the HL-GIIß vector. The immunoblot results, shown in Figure 4A, indicate that the level of over-expression of the {alpha}-subunit protein is comparable in cells transfected either with HL-GII{alpha} alone (lane 2) or with both HL-GII{alpha} and HL-GIIß (lanes 4 and 5). A typical time course for [14C]Glc2,1-Man9-GlcNAc2 hydrolysis by detergent extracts of these (co-)transfected cells is outlined in Figure 5. As can be seen, the relative rate of substrate degradation in HL-GII{alpha}-transfected cells is similar to that of control cells, whereas after cotransfection with both vectors glucosidase II activity is increased more than 4-fold compared with initial cleavage rates. A similar increase in catalytic activity was observed using a vector plasmid for transfection which contained the cDNA for both the {alpha}- and ß-subunit (HL-GII{alpha}ß). This HL-GII{alpha}ß vector was constructed to ensure coexpression of comparable levels of both subunits in the same cell. These data show that (1) the ß-subunit, for which antibodies were not available, is efficiently expressed in COS-1 cells and (2) the expression of a catalytically active form of the {alpha}-subunit is intimately linked to the simultaneous presence of the ß-subunit. The overexpressed glucosidase II activity was strongly inhibited by 1-deoxynojirimycin (dNM) and N-methyl-dNM with 50% inhibition being observed at inhibitor concentrations of 4 µM and 10 µM, respectively, whereas N,N-dimethyl-dNM was not inhibitory at 1 mM (not shown). The relative susceptibility of the overexpressed activity against dNM and its N-methylated derivatives resembles that observed for glucosidase II previously purified from pig liver (Hentges and Bause, 1997Go). Thus the catalytic properties of the enzyme from pig and human liver are highly similar.



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Fig. 5. Hydrolysis of [14C]Glc2,1-Man9-GlcNAc2 by glucosidase II expressed in COS-1 cells. COS-1 cells were transfected with either pcDNA-3 (open circles, control), HL-GII{alpha} (triangles), or simultaneously with HL-GII{alpha} and HL-GIIß (solid circles). Cell pellets were solubilized in 50 mM phosphate buffer, pH 6.5, containing 1% Triton X-100 and aliquots of the detergent extract (containing 6 µg protein) incubated with 500 c.p.m. [14C]Glc2,1-Man9-GlcNAc2. After given times, released [14C]glucose was analyzed as described by Hentges and Bause (1997)Go.

 
The glucosidase II {alpha}-subunit is expressed in COS-1 cells as an ER-resident protein
The subcellular location of the glucosidase II {alpha}-subunit was determined by immunfluorescence microscopy using a polyclonal antibody raised against the ~107 kDa enzyme protein from pig liver as analytical probe. The results summarized in Figure 6 show that, after transfection of COS-1 cells with HL-GII{alpha}, the antibody reacted strongly with a diffuse reticular network throughout the cells typical of endoplasmic reticulum structures whereas no immunostaining was detectable in control cells (Figure 6A). Except for less intense labeling of the nuclear envelope membrane, this staining pattern is similar to that previously found for ER-glucosidase I (Figure 6C) (Kalz-Füller et al., 1995Go) but differs clearly from that observed for Golgi-located Man9-mannosidase from human kidney (Figure 6D) (Bieberich and Bause, 1995Go). Immunofluorescence labeling seen for the {alpha}-subunit was not altered when COS-1 cells were cotransfected with either HL-GII{alpha} and HL-GIIß (Figure 6B), or transfected with the vector construct HL-GII{alpha}ß containing the cDNA for both subunits (not shown). Transfection of COS-1 cells with HL-GII{alpha}ß resulted in an increase of glucosidase II activity similar to that observed after cotransfection with HL-GII{alpha} and HL-GIIß as described above, indicating specificity and efficiency of both experimental approaches. The various data show that the simultaneous expression of the two subunits has no influence on the subcellular location of the {alpha}-subunit, although the molecular cause for the ER-retention of the catalytically inactive and active form of the {alpha}-subunit are most likely to be different (see Conclusions).



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Fig. 6. Immunofluorescence microscopy of COS-1 cells expressing glucosidase II. COS-1 cells were transfected with either HL-GII{alpha} (A) or with HL-GII{alpha} plus HL-GIIß (B). Cells were fixed with formaldehyde and incubated with polyclonal antibody raised against the pig liver ~107 kDa glucosidase II {alpha}-subunit, followed by incubation with an anti-rabbit IgG-antibody labeled with DTAF. For comparison, the immunofluorescence pattern for ER-glucosidase I from human hippocampus and for Golgi-resident Man9-mannosidase from human kidney are shown in (C) and (D), respectively.

 
Conclusions
This paper describes the cDNA cloning, COS-1 cell expression and functional characterization of the {alpha}- and ß-subunits of the hetero-dimeric glucosidase II complex from human liver. The enzymatic results show that cotransfection of COS-1 cells with both the HL-GII{alpha} and HL-GIIß vector resulted in a more than 4-fold overexpression of glucosidase II activity. By contrast, the {alpha}-subunit was found to be synthesized as a catalytically inactive protein when expressed alone. We conclude from these observations that both the {alpha}- and ß-subunits are required for expression of enzymatic activity. The {alpha}-subunit alone may be obtained in a catalytically active form without the ß-subunit, as previously shown for the enzyme purified from pig liver (Hentges and Bause, 1997Go). This suggests that the ß-subunit may, in some way, be involved in {alpha}-subunit maturation but is not necessary for catalytic activity once the {alpha}-subunit is mature. The failure to express catalytically active {alpha}-subunit in COS-1 cells in the absence of the ß-subunit contradicts recent observations by Flura et al. (1997)Go. These authors describe overexpression of glucosidase II activity in CHO cell clones after transfection with the {alpha}-subunit cDNA from pig liver, although at rather modest levels. Currently we are unable to offer any explanation for these different observations.

The antibody raised against the ~107 kDa {alpha}-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)Go. 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 {alpha}-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 {alpha}-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 {alpha}-subunit-specific cDNA lacking the 66 bp insert (HL-GII{alpha}) 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 {alpha}-subunit sequence variants, as well as those for the ß-subunit, remains unknown at present.

The immunofluorescence data show that the glucosidase II {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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)Go. This interpretation is, at least, not inconsistent with the observation that the {alpha}-subunit contains one N-linked sugar chain at Asn97.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Materials and chemicals were obtained from the following sources: human liver 5'-stretch plus cDNA library HL3006a in {lambda}gt10, E.coli C600 host strain, E.coli DH5{alpha} strain, human multiple tissue Northern blot (Clontec); oligo(dT)-cellulose, pUC BM20 vector DNA, SP6 Cap Scribe in vitro transcription system, reticulocyte in vitro translation kit, canine pancreas microsomes, FuGENE 6 transfection reagent (Boehringer); mammalian expression vector pcDNA3 (Invitrogen); restriction endonucleases, DNA-modifying enzymes, Endo H (New England Biolabs); Prime-a-Gene labeling system (Promega); T7Sequencing kit (Pharmacia); synthetic oligonucleotides, Taq polymerase, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (Gibco BRL); COS 1 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH); nitrocellulose membranes, goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma); goat anti-rabbit IgG 5-(4,6-dichlorotriazine-2-yl)aminofluoresceinhydrochloride (DTAF) conjugate (Dianova); [{alpha}-32P]dATP (specific activity 3000 Ci/mmol), [35S]dATP-{alpha}-S (specific activity >1000 Ci/mmol) (Hartmann Analytic); Hybond N membranes (Amersham); X-OMAT AR film (Kodak). 1-Deoxynojirimycin was a generous gift from Dr. D. Schmidt, Bayer AG. All other chemicals used were of analytical grade purity.

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 SDS–PAGE, 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., 1979Go). 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 SDS–PAGE-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, 1967Go). The sample was diluted with water and lyophilized. The peptide mixture was then dissolved in sample buffer and separated by SDS–PAGE using the high resolution gel-system previously described by Schägger and von Jagow (1987)Go. 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 SDS–PAGE 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 (0–70%) 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 {alpha}- 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 {alpha}-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., 1989Go). The PCR products were then purified by agarose gel electrophoresis and characterized by sequencing using the dideoxynucleotide chain termination method (Sanger et al., 1977Go).

Isolation of cDNA clones from the human liver cDNA library
The purified PCR-products were radio-labeled with [{alpha}-32P]dATP by primer extension (Feinberg and Vogelstein, 1983Go) and used to screen an oligo-(dT)/random-primed human liver cDNA library constructed in {lambda}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 {alpha}-subunit was reconstructed from overlapping {lambda}-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{alpha}) 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 {lambda}-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 {alpha}- and the ß-subunit (HL-GII{alpha}ß), HL-GII{alpha} and HL-GIIß were cleaved with BspDI and ScaI, respectively. Sticky ends in HL-GII{alpha} 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{alpha}, HL-GIIß, HL-GII{alpha}ß or HL-GII{alpha} plus HL-GIIß, using the FuGENE 6 transfection reagent as described in the supplier’s 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., 1984Go). 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, 1970Go; Saiki et al., 1988Go; Schweden and Bause, 1989Go; White, 1993Go). Immunofluorescence microscopy, in vitro translation/glycosylation and Endo H cleavage were done according to the procedures described by Bieberich and Bause (1995)Go. Nucleotide sequencing was performed according to Sanger using the Pharmacia T7Sequencing Kit and [35S]dATP-{alpha}-S as tracer (Sanger et al., 1977Go). [14C]Glc2,1Man9GlcNAc2 was synthesized as detailed in Hettkamp et al. (1984)Go. 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)Go and Sambrook et al. (1989)Go.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We are indebted to Dr. B.Schmidt (Universität Göttingen) for peptide sequencing and to Dr. R.A.Klein (Universität Bonn) for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 284) and the Fonds der Chemischen Industrie.


    Abbreviations
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ER, endoplasmic reticulum; CNBr, cyanogene bromide; dNM, 1-deoxynojirimycin; DTAF, 5-(4,6-dichlorotriazine-2-yl)aminofluoresceinhydrochloride; MOPAC, mixed oligonucleotide-primed amplification of cDNA.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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